Selective biocatalytic hydroxylation of unactivated methylene C-H bonds in cyclic alkyl substrates

Article abstract of DOI:10.1039/c9cc02060h

The cytochrome P450 monooxygenase CYP101B1 from Novosphingobium aromaticivorans selectively hydroxylated methylene C-H bonds in cycloalkyl rings. Cycloketones and cycloalkyl esters containing C6, C8, C10 and C12 rings were oxidised with high selectively on the opposite side of the ring to the carbonyl substituent. Cyclodecanone was oxidised to oxabicycloundecanol derivatives in equilibrium with the hydroxycyclodecanones.

Full text of DOI:10.1039/c9cc02060h

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Selective biocatalytic hydroxylation of unactivated methylene C-H
bonds in cyclic alkyl substrates
Md Raihan Sarkar,a Samrat Dasgupta,a Simon Pyke a and Stephen G. Bell*a
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
The cytochrome P450 monooxygenase CYP101B1 from (TTN).14, 20-23 Therefore this system has all the desired attributes
Novosphingobium aromaticivorans selectively hydroxylated for the design of efficient biocatalytic reactions.
methylene C-H bonds in cycloalkyl rings. Cycloketones and We set out to assess if CYP101B1 was capable of acting as an
cycloalkyl esters containing C6, C8, C10 and C12 rings were oxidised biocatalyst for the selective hydroxylation of cycloalkanes and
with high selectively on the opposite side of the ring to the carbonyl their derivatives. All the substrates were tested in vitro for
substituent. Cyclodecanone was oxidised to oxabicycloundecanol substrate binding, turnover activity (NADH, product formation
derivatives in equilibrium with the hydroxycyclodecanones.
rate and coupling efficiency, Table 1). We initially tested
selected cycloalkane substrates ranging in size from C6 through
to C12. Cyclohexane was a poor substrate for the enzyme (Table
1, Figure S1 – S3) but cyclooctane and cyclodecane bound to the
enzyme with significantly higher affinity (Figure S1 and S2). Both
substrates were oxidised by CYP101B1 to the equivalent
cycloalkanol (>98% with the minor product being identified as
the ketone further oxidation product; Table1, Scheme 1, Figure
S3 and S4). The binding parameters, product formation activity
(PFR) and total turnover number (TTN; 3400) of cyclooctane
were better than that of cyclodecane (Table 1) highlighting that
it is a better substrate for the CYP101B1 enzyme.
The selective catalytic functionalisation of carbon-hydrogen
bonds in organic molecules would improve synthetic efficiency
but is a significant chemical challenge. The regio- and stereo-
selective insertion of an oxygen atom into the chemically inert
methylene carbon-hydrogen bonds of saturated cyclic
hydrocarbons is of interest due to the difficulties associated
with these reactions.1-4 Monooxygenase enzymes, such as the
cytochrome P450 superfamily of heme proteins, have the
potential to be utilised as biocatalysts for the oxidation of
organic molecules.5-10 However, the monooxygenase activity of
many CYP enzymes is often limited by low substrate affinity,
poor product selectivity for non-natural substrates or the lack
of suitable electron transfer proteins.11-14 As a result, extensive
modification of the enzymes is required before they are able to
catalyse the desired reaction. For example, CYP102A1, from
Bacillus megaterium, which is highly active for fatty acid
substrates, has been modified via protein engineering to
OH
CYP101B1
n
n
cycloalkanes n = 1, 3 or 5
C = 6, 8 or 10
C6-ol, C8-ol or C10-ol
all >99%
1,7-C12-diol
approx 90%
CYP101B1
OH
facilitate the oxidation of the CH bonds in many substrates.5,
12, 15-19
1,6-C12-diol
<10% minor
metabolite
HO
The bacterium Novosphingobium aromaticivorans DSM12444
contains the CYP101B1 encoding gene. This enzyme has been
shown to bind norisoprenoids, bicyclic terpenoid and tricyclic
hydrocarbon acetate esters with high affinity.14, 20-22 An electron
transfer system, comprising a ferredoxin reductase, ArR, and a
ferredoxin, Arx has been isolated from the same bacterium and
used to support the oxidation activity of CYP101B1 for these
substrates with high activity and total turnover numbers
cyclododecane
cyclododecandiols
Scheme 1. Oxidation of selected cycloalkanes by CYP101B1.
Cyclododecane bound to CYP101B1 with decreased affinity and
was oxidised at lower activity (Table 1). The product of the
enzyme catalysed reaction was not cyclododecanol (Figure S3).
GC-MS analysis highlighted the formation of double oxidation
metabolites as the major products (Figure S4). Cyclododecanol
was found to induce a higher spin state shift in CYP101B1 than
any of the cyclic alkanes tested above (Table 1, Figure S1 and
Department of Chemistry, University of Adelaide, Adelaide, SA, 5005, Australia
E-mail: stephen.bell@adelaide.edu.au
Electronic Supplementary Information (ESI) available: [experimental methods,
substrate binding data, gas chromatography, mass spectrometry and NMR data].
See DOI: 10.1039/x0xx00000x
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Table 1 Substrate binding and in vitro turnover data for CYP101B1 with different substrates. The spin state shift induced by substrate binding is given by the
percentage of high spin heme (%HS) and the binding affinity by the dissociation constant (Kd) in micromolar. The NADH oxidation rate is the frequency of NADH
oxidation. The coupling efficiency (coupling), is defined as the percentage of NADH utilised for the formation of products. The turnovers were measured using
a ArR:Arx:CYP101B concentration ratio of 1:10:1 (0.5 μM CYP enzyme, 50 mM Tris, pH 7.4). Rates (NADH; product formation rate, PFR) are reported as mean
 S.D. (n  3) and given in nmol.nmol-CYP-1.min-1. The selectivity of the oxidation is reported as the major product or site of hydroxylation and its percentage
(further details in Schemes 1-4). TTN is the total turnover number which is the number of turnovers catalysed per enzyme. – no product detected or not
measured due to low affinity (Kd) or levels of product formation (TTN).
%HS
heme
15
35
40
Coupling
Selectivity
Substrate
Kd (μM)
NADH
PFR
TTN
(%)
3
49
11
9
cyclohexane
cyclooctane
cyclodecane
cyclododecane
-
61 ± 9
391 ± 11
397 ± 15
174 ± 5
2 ± 1
209 ± 17
42 ± 34
15 ± 3
C6-ol (>98%)
C8-ol (>98%)
C10-ol (>98%)
-
31 ± 4
0.88 ± 0.04
0.16 ± 0.03
3400 ± 150
420 ± 75
-
30
C12-diols (90%)
-
cyclooctanol
cyclododecanol
10
90
-
158 ± 2
198 ± 7
-
-
21
-
1.5 ± 0.3
C12-diols
41 ± 1
1360 ± 90
-
cyclooctanone
cyclodecanone
cyclododecanone
10
50
90
-
93.4 ± 9
323 ± 38
391 ± 11
-
-
45
72
-
C5(60%)/C6(36%)00
C7 (96%)
46 ± 2
0.16 ± 0.03
149 ± 54
283 ± 38
1730 ± 50
5750 ± 280
C4 (>98%)
C4 (>98%)
C4 (>98%)
C4 (>98%)
C4 (>98%)
C5 (>98%)
C5 (95%)
C7 (76%)
cyclohexyl acetate
25
60
60
95
85
90
90
95
540 ± 40
110 ± 4
61 ± 5
31 ± 1.5
40 ± 1
1.6 ± 0.2
1.3 ± 0.2
0.05 ± 0.01
415 ± 56
603 ± 32
383 ± 26
736 ± 26
667 ± 29
722 ± 16
732 ± 11
394 ± 8
56 ± 6
14
51
57
34
42
31
75
23
910 ± 385
9580 ± 530
3230 ± 60
9460 ± 540
7250 ± 1720
8180 ± 1100
4660 ± 1700
2990 ± 773
cyclohexyl butyrate
cyclohexyl isobutyrate
methyl cyclohexylacetate
ethyl cyclohexylacetate
cyclooctyl acetate
310 ± 62
220 ± 62
261 ± 20
274 ± 15
223 ± 20
626 ± 30
94 ± 7
cyclooctyl isobutyrate
cyclododecyl acetate
S2). It was also more efficiently oxidised than cyclododecane mass spectrum was consistent with a monooxygenase product
(Table 1). Its oxidation resulted in mixture of with a mass, m/z 198.35 (Figure S4).
a
cycldodecanediols which coeluted with the major products
from cyclododecane oxidation. These included 1,7- and 1,6-
cyclododecanediol along with another minor product (Scheme
1, Figure S5). Cyclooctanol was also tested but was a poor
substrate both in terms of binding and productive activity (Table
1).
OH
O
O
OH
72%#
28%#
1-oxabicyclo[5.3.1]- 5-hydroxycyclodecanone
CYP101B1*
undecan-1-ol
60%
OH
As we have previously highlighted that substrates with a ketone
carbonyl group have enhanced affinity for CYP101B1 we next
tested cyclic ketones.22-24 Cyclooctanone like cyclooctanol did
not interact well with CYP101B1 but cyclodecanone and
cyclododecanone bound to the enzyme, inducing significant
shifts to the high spin form of the enzyme ( 50%, Table 1,
Figure S1 and S2). Both cyclodecanone and cyclododecanone
were efficiently oxidised by CYP101B1. Cyclododecanone was
oxidised with high selectivity generating a single product in
>95% yield which was identified as 7-hydroxycyclododecanone
(Scheme 2, Figure S3, S4 and S5). The major metabolites of the
turnovers could be generated in tens of mg quantities using a
whole-cell oxidation system (see ESI for further details).20 The
minor metabolite (4%) was unable to be generated in a large
enough quantity for characterisation by NMR. However, the
O
O
cyclodecanone
O
58%#
OH
42%#
1-oxabicyclo[6.3.1]- 6-hydroxycyclodecanone
undecan-1-ol
36%
O
O
CYP101B1*
OH
7-hydroxycyclododecanone
96%
cyclododecanone
Scheme 2. Oxidation of cyclodecanone and cyclododecanone by
CYP101B1. * 4% minor products. Distribution from NMR
#
(Figure S5).
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Cyclodecanone was oxidised to two major metabolites which fold greater (8180 versus 4660) and both were higVhieewrAtrhticalenOtnhlianet
were identified as arising from hydroxylation at the 5- and 6- achieved with cyclododecyl acetate (299D0O, IT:a10b.l1e0319)/.C9CC02060H
positions (60% and 36%, respectively) with a third minor Cyclohexyl acetate was oxidised by CYP101B1 with moderate
product making up the remainder. To complicate matters the activity but high selectivity to trans-4-hydroxycyclohexyl
two major compounds were present as mixtures of the acetate (Table 1, Figures S3, S4 and S5). However, the
hydroxycyclodecanone and the oxabicycloundecanol isomer dissociation constant was high and this substrate only induced
which presumably arises from intramolecular nucleophilic a 25% shift of CYP101B1 to the high spin form upon binding
attack of the alcohol on the electrophilic carbonyl group to form (Table 1, Figure S1 and S2). We hypothesised that the binding of
a cyclic hemiacetal (Scheme 2, Figure S5).25, 26 We measured the this ester was suboptimal, compared to the cyclooctyl and
TTN and, in line with the coupling efficiency and PFR, that of cyclododecyl equivalents due to the smaller size of the
cyclododecanone was greater than cyclodecanone (5750 versus substrate. Therefore we decided to assess the binding of a
1730) and any of the substrates investigated above.
range of other cyclohexyl derivatives with larger substituents to
The selective oxidation of cyclododecanone by CYP101B1 is an see if the turnover activity could be improved.
H
encouraging result for the application of this enzyme for the
oxidation of large ring cyclic alkyl derivatives. We next examined
CYP101B1
OH
R
the effect of adding an ester protecting group to improve the
activity of cycloalkanol oxidation. Ester protecting groups, such
as acetate and isobutyrate, could act as chemical auxiliaries and
directing groups by mimicking the butenone moiety of the
norisoprenoids. This would enable improved oxidation and
these functionalities were added to cyclooctanol and
R
H
cyclohexy
ester derivatives
4-hydroxycyclohexyl
products (all >98% selective for C4)
(R = O(CO)n-C3H7 and CH2(CO)OMe both >98% trans)
R = OAc, O(CO)n-C3H7, O(CO)iso-C3H7, CH2(CO)OMe, CH2(CO)OEt
H
CYP101B1
OH
RO
cyclododecanol using standard methods (ESI and Figure S6).23,
RO
H
24, 27-31
cyclooctyl esters
R = Ac and (CO)iso-C3H7,
5-trans-hydroxycyclooctyl products
R = Ac (>95%)and (CO)iso-C3H7 (90%),
Cyclododecyl acetate bound to CYP101B1 inducing a complete
shift in the spin state to the high spin form and bound to the
enzyme with nanomolar affinity (Table 1, Figure S1 and S2). It
was oxidised to one of the diastereomers of 7-
hydroxycyclododecyl acetate (with 74%, regioselectivity,
Scheme 3, Figure S3, S4 and S5). The coupling constants of JH1
and JH7 (JH1 = 5.5, 8.1 Hz, JH7 = 5.0, 8.0 Hz) inferred the major
diastereomer was the trans with both substituents in equatorial
positions. When isolated this was also found to contain a small
amount of the cis isomer (2%). One other minor product was
characterised as a stereoisomer of 5-hydroxycyclododecyl
acetate (10%; trans H1; JH1 = 6.4, 11.9 Hz and H5; JH5 = 6.4, 12.4
Hz) as both the C1 and C5 protons coupled strongly to C3 in the
HMBC NMR spectrum (Scheme 3, Figure S5).
OAc
OAc
OAc
CYP101B1
OH
OH
cyclododecyl
acetate
7-trans-hydroxy
cyclododecyl
acetate 74%*
5-trans-hydroxy
cyclododecyl
acetate 10%*
Scheme 3. The oxidation of cycloalkyl ester derivatives by
CYP101B1. * an additional 2% of the product was the cis isomer
of 7-hydroxy while the remaining 14% was another hydroxy
species not purified in sufficient quantity for characterisation
(possibly one of the other diastereomers of the 5-hydroxy).
We next assessed the turnover of the acetate and isobutyrate
esters of cyclooctanol. Both displayed enhanced binding and
turnover parameters with CYP101B1 compared to cyclooctanol
(Table 1, Figure S1 and S2). These esters were also efficiently
and selectively oxidised by CYP101B1 with the coupling
efficiency and product formation rate of the isobutyrate ester
being the greater (Table 1). Both were predominantly
hydroxylated to a single major product (Scheme 3 and Figure
Cyclohexyl-butyrate and -isobutyrate both displayed enhanced
binding parameters to CYP101B1 compared to cyclohexyl
acetate (Table 1, Figure S1 and S2). In addition methyl and ethyl
cyclohexylacetate, which are ester derivatives of
cyclohexanecarboxylic acid, also bound to CYP101B1 (Table 1,
Figure S1 and S2) whereas the parent acid did not (data not
shown). All of the esters were oxidised with higher product
formation activities and coupling efficiencies than cyclohexanol
and cyclohexane carboxylic acid, which were not substrates for
the enzyme, and cyclohexyl acetate (Table 1, Figure S3). They all
resulted in the formation of a single major metabolite in
significantly greater quantities (Figure S3). In all instances, this
was the 4-hydroxy metabolite (Figure S4 and S5). The CYP101B1
catalysed oxidations of cyclohexylbutyrate and methyl
cyclohexylacetate were the most selective (>98%) and
displayed the highest TTNs (>9000, Table1). The metabolites
were also identified as the trans-diastereomer (Figure S5).
1
S3). In each instance, this could be assigned from the H NMR
coupling constants as the trans-diastereomer of the 5-hydroxy
metabolite by NMR (Figure S4 and S5). Two minor products
were observed in the turnover of the isobutyrate ester one of
which was assigned as the other diastereomer of the 5-hydroxy
product (10%) based on the 13C NMR signals (Fig. S5). The
oxidation of cyclooctyl acetate by CYP101B1 was more selective
and generated trans-5-hydroxycyclooctyl acetate in >95% yield
with the other diastereomer making up the remainder (Figure
S5). Despite the higher product formation rate of the
isobutyrate ester, the TTN of the acetate ester was almost two-
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selectivity of P450 catalysed oxidations. For example, the
activity and selectivity of the CYP102A1 catalysed oxidation of
cembrenoid derivatives has been optimised with multiple
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H bonds but more saturated analogues displayed lower
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the optimal substrates (PFR > 200 min1 and TTN > 5000 in a 1
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favourable compared to other systems such as the
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Again the selectivity of these
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products were obtained with the protected cyclododecanol.29
In summary, we have identified that the CYP101B1 cytochrome
P450 monooxygenase when combined with its physiological
electron transfer partner Arx and ArR can efficiently oxidise
cyclic hydrocarbon derivatives. The C-H bond abstraction and
therefore selectivity of oxidation for a given methylene group
was high and could be made almost totally selective by
modifying the ester protecting group or functional group on the
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opposite side of the ring to the ketone or ester moiety. The
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Conflicts of interest
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“There are no conflicts to declare”.
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4 | J. Name., 2012, 00, 1-3
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The monooxygenase, CYP101B1, selectively hydroxylates undistinct methylene C-H bonds in
medium to large cycloalkyl rings and can generate oxabicycloundecanol derivatives.