Multi-Functional Oxidase Activity of CYP102A1 (P450BM3) in the Oxidation of Quinolines and Tetrahydroquinolines

Article abstract of DOI:10.1002/anie.201904157

Tetrahydroquinoline, quinoline, and dihydroquinolinone are common core motifs in drug molecules. Screening of a 48-variant library of the cytochrome P450 enzyme CYP102A1 (P450BM3), followed by targeted mutagenesis based on mutation-selectivity correlations from initial hits, has enabled the hydroxylation of substituted tetrahydroquinolines, quinolines, and 3,4-dihydro-2-quinolinones at most positions around the two rings in good to high yields at synthetically relevant scales (1.5 g L^?1 day^?1). Other oxidase activities, such as C?C bond desaturation, aromatization, and C?C bond formation, were also observed. The enzyme variants, with mutations at the key active site residues S72, A82, F87, I263, E267, A328, and A330, provide direct and sustainable routes to oxy-functionalized derivatives of these building block molecules for synthesis and drug discovery.

Full text of DOI:10.1002/anie.201904157

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Accepted Article
Title: Multi-function oxidase activity of CYP102A1 (P450BM3) in the
oxidation of quinolines and tetrahydroquinolines
Authors: Luet Lok Wong and Yushu Li
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10.1002/anie.201904157
Angewandte Chemie International Edition
COMMUNICATION
Multi-function oxidase activity of CYP102A1 (P450BM3) in the
oxidation of quinolines and tetrahydroquinolines
Yushu Li,[a] and Luet L. Wong*[a,b]
Abstract: Tetrahydroquinoline, quinoline and dihydroquinolinone are
common core motifs in drug molecules. Screening of a 48-variant
library of the cytochrome P450 enzyme CYP102A1 (P450BM3)
followed by targeted mutagenesis based on mutation-selectivity
correlations from initial hits, has enabled the hydroxylation of
substituted tetrahydroquinolines, quinolines and 3,4-dihydro-2-
quinolinones at most positions around the two rings in good to high
yields at synthetically relevant scales (1.5 g/L/day). Other oxidase
activities such as C–C bond desaturation, aromatisation and C–C
bond formation were also observed. The enzyme variants, with
mutations at the key active site residues S72, A82, F87, I263, E267,
A328 and A330, provide direct and sustainable routes to oxy-
functionalised derivatives of these building block molecules for
synthesis and drug discovery.
initial hits followed by iterative site saturation mutagenesis.[10]
Smaller libraries generated from 4–5 mutations at two active site
residues have shown high product selectivity for some
substrates.[11] We have adopted a similar approach based on
CYP102A1 variants found to have increased activity for unnatural
substrate oxidation as a result of the heme iron–axial water
interaction being weakened by a number of mechanisms.[12]
Further mutations were introduced at two to four active site
residues (S72, A74, V78, F81, A82, F87, A184, I263, E267, A328,
P329 and A330) in these base variants to afford a library of ~100
enzymes with diverse substrate pocket topologies. Screenings
have shown this library to be capable of C–H bond oxy-
functionalisation of different classes of organic compounds.[13] We
report here the oxidation of THQs and quinolines by CYP102A1
variants with the aim to functionalise these building-block
molecules selectively at as many positions as possible and thus
provide synthetic handles to compounds with biological activity. In
addition to C–H bond oxidation, we found that CYP102A1
displayed other oxidase activities including desaturation,[14]
aromatisation,[15] and C–C bond formation reactions.
Tetrahydroquinoline (THQ), quinoline and dihydroquinolinone are
ubiquitous core motifs in natural products with important
medicinal properties, e.g. alkaloids such as quinine, camptothecin
and integriquinolone,[1] and in synthetic drugs with varied
pharmacological activities including anti-viral, anti-microbial, and
anti-tumour compounds. A variety of routes have been developed
for the synthesis of compounds with these core motifs for use as
building blocks for generating biologically active compounds.
Strategies for accessing THQ derivatives include (a) reduction of
the heterocyclic ring in the corresponding quinoline derivative, (b)
construction of the heterocyclic ring by reactions that form one,
two, and three C–C bonds, and (c) heterocyclic ring contraction
and expansion.[2] Direct functionalisation of the heterocyclic core
is a less common strategy due to the modest product selectivity
and often more forcing conditions with transition-metal catalysts.
Screening scale reactions for an initial library of 48 variants
[Tables S1 & S2 in Supporting Information (SI)] were carried out
in 24-well plates at 25°C, shaking at 120 rpm. The 1 mL reactions
in 200 mM phosphate buffer (pH 8.4) contained 1 µM CYP102A1
enzyme, 40 µM NADP+ and 2 mM substrate. Glucose and glucose
dehydrogenase were used to recycle the NADPH cofactor.
Organics were extracted with ethyl acetate and analysed by gas
chromatography. Products were isolated from preparative scale
reactions (20–100 mg) by silica gel chromatography and
characterised by NMR and MS data. The variant library oxidised
1 to a mixture of products (Fig. 1), including 6-hydroxy-THQ, 2,
the aromatisation product quinoline, 3, 3,4-dihydro-2-quinolinone,
5, and the dimers 4, 10 and 11 (See Supporting Information). On
prolonged reaction, the THQ derivative 2 was fully converted by
most of the variants to the aromatised product, 6-quinolinol, 6.
In contrast to chemical systems, enzymatic C–H bond
activation could provide sustainable and more selective routes for
direct functionalisation. CYP102A1 (P450BM3) from Bacillus
megaterium,[3] is a good candidate system on account of its
catalytic self-sufficiency,[4] high-level expression in E. coli and the
excellent scalability of its reactions.[5] The enzyme has been
extensively engineered for C–H activation of unnatural substrates
for applications in synthesis.[6] Rational design,[7] random
mutagenesis,[7d, 8] site-saturation mutagenesis and combinatorial
active-site saturation test (CAST)[9] have been applied with great
success. Another approach is to screen a library of variants for
Mechanistically, 2 is formed via the NIH shift pathway
whereby the initially formed 5,6-arene oxide undergoes ring-
opening to form the C5 carbocation preferentially because of
stabilisation by the nitrogen lone pair (Scheme 1). Aromatisation
of 1 and 2 is likely to be initiated by hydroxylation at C2, the
activated α-position to the amine nitrogen. The carbinolamine A
loses OH– to give the iminium species which is in equilibrium with
the enamine tautomer, the C4–H bond of which is highly activated
to hydrogen atom abstraction by Compound I to generate the
stabilised allylic-benzylic radical. Aromatisation could occur by H-
atom abstraction from the enamine NH or spontaneous
elimination of water from the enamine alcohol formed by radical
rebound. The enamine can also undergo nucleophilic attack on its
iminium tautomer to form the dimeric iminium species B; under
the reaction conditions, the iminium/enamine half of the dimer is
[a]
[b]
Y. Li and Dr L.L. Wong
Department of Chemistry, University of Oxford, Inorganic Chemistry
Laboratory, South Parks Road, Oxford OX1 3QR, UK
E-mail: luet.wong@chem.ox.ac.uk
Oxford Suzhou Centre for Advanced Research, Ruo Shui Road,
Suzhou Industrial Park, Jiangsu, 215123, P.R. China
Supporting information for this article is given via a link at the end of
the document.
This article is protected by copyright. All rights reserved.

10.1002/anie.201904157
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COMMUNICATION
H
O
+
−
O
HO
aromatised to give 4. This 2,3¢-THQ-quinoline dimer is
hydroxylated at C4 of the THQ moiety to give 10 which is
aromatised to the 2,3¢-quinoline-quinoline dimer 11, most likely
via an unobserved enamine-alcohol intermediate.
O
N
H
N
N
N
H
H
H
2
P450
+
N
H
N
H
N
5
8
4
H
6
3
2
+
N
H
When 5 was screened with the library, we observed
hydroxylation at the a-position to give 12, desaturation of the Ca–
Cb bond to form 13, benzylic oxidation to give 14, and formation
of the 6-phenol 15 and 8-phenol 16 (Fig. 1). The greater product
diversity for 5 compared to 1 suggested that the NH group in THQ
might be involved in restricting substrate binding within the active
site. Therefore, the N-Boc and N-acyl derivatives of THQ (17 and
20) were tested with the library. We only observed the C4
hydroxylation products 18 and 21. On prolonged incubation, the
ketones 19 and 22 from further oxidation of these benzylic
alcohols were obtained in quantitative yield (Fig. 1, Tables 1, S11
& S12). Quinoline (3) was also a substrate for the variants, being
oxidised to 3-quinolinol, 7, and a stable 5,6-arene oxide, 8. Acid-
catalysed arene oxide ring-opening of 8 gave 5-quniolinol, 9.
N
N
1
N
H
7
H
B
4
FeIV
O
1
FeIV
OH
OH
H
4
H
P450
–H+
+H+
–
+
N
H
–OH
N
H
N
H
N
H
P450
P4
–H2O
5
0
N
H
OH
A
OH
N
H
N
H
N
O
N
O
H
5
3
H
FeIV
OH
Scheme 1. The proposed mechanistic pathways for the oxidation of 1,2,3,4-
tetrahydroquinoline (THQ, 1) by CYP102A1.
K19/A82M/F87A/E267F gave 85% of the aromatisation product 3
(TON = 1,600, 94% conversion, K19 = H171L/Q307H/N319Y),
whereas R19/F87A/A328I converted 1 to 67% of 4 (TON = 1,340,
100% conversion). The variant R19/F87A gave the highest
percentage (16%, TON = 320) of 3,4-dihydro-2-quinolinone, 5.
For quinoline (3), RT2/V78I/A184I/A330P gave 28% of 3-
quinolinol, 7 (TON = 170), and RP/H171L/I263G gave 91% of the
5,6-oxide, 8 (57% conversion, TON = 1,040). With 5, the 3-alcohol
12 was a minor product, the RP/H171L/I263G giving 6% (TON =
80, Table S10); the 6-phenol 15 was the major product for this
variant (48%, TON = 630). The RP variant gave the highest
Within the screening library, 29 of 48 variants gave >50%
conversion of 1 while the WT showed negligible activity. Of the
products, the variant RP/H171L/I263G (RP = R47L/Y51F/I401P)
gave the highest proportion of the 6-phenol, 2 (54%, TON = 1080,
Fig. 1, Tables 1 & S5), which was then fully converted to the
aromatised product 6 at longer reaction times. Unexpectedly, this
variant, and some others (Tables S5 & S6), did not show any
further oxidation of 2 until 1 had been fully consumed. When
purified 2 was used as substrate these variants fully converted it
to 6 with 100% selectivity (TON = 2,000). The variant
proportion of 8-phenol 16 (73%, TON
=
220). The
R19/F87A/A328I formed 65% (TON = 530) of the aromatised
product 2-quinolinol, 13, and RT2/S72W/A330W gave 65% of the
C4-hydroxylation product 14 (TON = 910).
β
α
HO
N
P450BM3
H
N
O
N
N
N
N
H
H
H
5
2
3
4
We then sought to improve product selectivity by comparing
the results for different mutation combinations. The data for K19,
K19/F87A, K19/A328I and R19/F87A/A328I indicated that the
native residue F87 disfavoured C2 oxidation and gave more
C5,C6 oxidation, likewise for the bulky substitution A328I. The
F87A and F87V mutations promoted C2 oxidation, leading to
aromatisation and formation of the lactam 5 and dimers. The
series RP, RP/H171L and RP/H171L/I263G revealed that the
I263G mutation promoted C5,C6 oxidation. Therefore, the I263G
and I263A mutations were combined with A328V, A328L, A328I
and A328F. These four A328 mutations were also combined with
F87A in the R19/FA variant. From this new set of variants (Table
S3), the R19/I263G showed 100% conversion of 1 to 80% of 2
(TON = 1,600) compared to 54% for RP/H171L/I263G (Tables 1
& S6). This was improved to 87% with the R19/I263G/A328L
(TON = 1,740, 100% conversion). The change from A328I to
A328L led to increased formation of the THQ-quinoline dimer 4
for the R19/F87A/A328L variant (78%, up from 67%). The new
variants also showed increased selectivity for the oxidation of 3,4-
dihydro-2-quinolinone 5, with R19/I263G giving an albeit low 12%
of the 3-alcohol 12 (TON = 170), and the R19/I263G/A328L
forming 100% of the 6-phenol 15 (95% conversion).
1
R19/F87A
16% TON 320
RP/H171L/I263G K19/A82M/F87A/E267F
R19/F87A/A328I
67% TON 1,340
54% TON 1,080
85% TON 1,600
HO
HO
P450BM3
N
H
N
2
6
RP/H171L/I263G
100% TON 2,000
OH
O
OH
P450BM3
+H+
N
N
N
N
3
7
8
9
RT2/V78I/A184I/A330P RP/H171L/I263G
28% TON 170
91% TON 1,040
OH
P450BM3
P450BM3
N
N
N
H
H
N
N
N
4
10
R19/F87A/A328I
100% TON 480
11
OH
β
OH
O
HO
α
P450BM3
N
H
O
N
H
N
O
N
H
O
N
O
N
O
H
H
H
OH
16
RP
73% TON 220
12
13
14
15
5
RP/H171L/I263G R19/F87A/A328I RT2/S72W/A330W RP/H171L/I263G
6% TON 80
65% TON 530
65% TON 910
48% TON 630
OH
O
P450BM3
P450BM3
N
R
N
R
N
R
17: R=Boc
20: R=Ac
18: R=Boc
21: R=Ac
19: R=Boc
22: R=Ac
RP/I263A/E267V RP/I263A/E267V
100% TON 1,200 100% TON 1,500
RP/I263A/E267V RT2/A82M/A330P
100% TON 1,200 100% TON 2,000
Next, we explored the effect of substituents on THQ
oxidation by both the initial library and 2nd-generation variants with
2-methyl-tetrahydroquinoline (2MTHQ, 23) and 8-methyl-
tetrahydroquinoline (8MTHQ, 31). Formation of the 6-hydroxy
Figure 1. Products from the CYP102A1-catalysed oxidation of 1,2,3,4-
tetrahydroquinoline (THQ, 1), showing the most selective variant in the
screening library, the proportion of each product and the turnover number (TON).
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Table 1. Activity and selectivity for the oxidation of tetrahydroquinolines and
quinolines by CYP102A1 variants. * denotes a second-generation variant.
derivatives was unaffected (Fig. 2). Gratifyingly, as with THQ, the
2nd-generation variant R19/I263G/A328L was also the most
selective for both, with 100% selectivity at 93% conversion for 24
(TON = 1,860, Table 1) and 84% for 32 (TON = 1,680). On
prolonged reaction both 24 and 32 were aromatised with total
conversion to the corresponding methylquinolinols 26 and 36. As
observed with 2, there was no further oxidation of 24 and 32 with
some variants until 23 and 31 had been fully consumed. F87-
based variants showed the highest aromatisation activity, at 66%
for the formation of 2-methylquinoline (25, TON = 420) and 84%
for 8-methylquinoline (33, TON = 1,010). It appears that
aromatisation of THQs to the quinoline analogues is a general
activity of CYP102A1 variants, which offers an unexpected route
for interconversion between these two classes of compounds by
hydrogenation/aromatisation. The K19/A82M/F87A variant gave
the most (77%, TON = 1,540) of the dimer 34. No dimer was
observed with 23, presumably because of steric hindrance by the
2-methyl substituent to nucleophilic attack by the enamine on the
iminium species. F87A and F87V-based variants gave higher
proportions of the dihydro-2-quinolinone 35 from 8MTHQ
(GVQ/A264G 27%, TON = 280).
Product
CYP102A1 Variant
Selectivity
TON
2
R19/I263G/A328L*
K19/A82M/F87A/E267F
R19/F87A/A328L*
R19/F87A/I263G*
R19/I263G/A328L*
RT2/V78I/A184I/A330P
RP/H171L/I263G
R19/F87A/A328L*
R19/I263G*
87%
85%
78%
20%
100%
28%
91%
100%
12%
65%
1,740
1,600
1,510
400
3
4
5
6
2,000
170
7
8
1,040
1,150
170
10
12
13
14
R19/F87A/A328I
530
RT2/S72W/A330W
65%
80%
910
380
A74G/F87L/L188Q/I263G/A328G*
15
16
18
19
21
22
24
25
26
27
28
29
32
33
34
35
36
37
38
39
40
R19/I263G/A328L*
RT2/S72H/A330W*
RP/I263A/E267V
RP/I263A/E267V
RT2/A82M/A330P
RP/I263A/E267V
R19/I263G/A328L*
K19/A82M/F87A/E267F
R19/I263G/A328L*
RP
100%
51%
1,900
730
100%
100%
100%
100%
100%
66%
1,200
1,200
2,000
1,500
1,860
420
The methyl substituent had greater effect on quinoline
oxidation. The 3-quinolinol derivatives were formed with moderate
selectivity: 56% (TON = 290) of 2-methyl-3-quinolinol, 27, by the
RP variant, and 38% (TON = 280) of 8-methyl-3-quinolinol, 37, by
R19/I263A/A328L. The 5,6-oxide 29 from the oxidation of 25 was
stable under turnover conditions and could be isolated; the
R19/I263G variant gave 29 with 81% selectivity (TON = 1,150).
Treatment of 29 with acid gave a mixture of 2-methyl-5-qunolinol
(30, 62%) and 2-methyl-6-quinolinol (26, 38%). Here, stabilisation
of the C5 carbocation from arene oxide ring opening by the 2-
methyl substituent altered the selectivity compared to
100%
56%
2,000
290
RP/H171L/I263G/A328L*
R19/I263G*
100%
81%
2,000
1,150
1,680
1,010
1,540
280
R19/I263G/A328L*
K19/V78I/F87V/E267V*
K19/A82M/F87A
GVQ/A264G*
84%
84%
77%
27%
R19/I263G/A328L*
R19/I263A/A328L*
R19/F87A/A328I
RP
100%
38%
2,000
280
HO
P450BM3
N
N
N
H
H
23
24
25
100%
100%
67%
1,820
2,000
930
2MTHQ
R19/I263G/A328L K19/F87A/A82M/E267F
100% TON 1,860
HO
66% TON 420
HO
P450BM3
RP/H171L/I263G
N
N
OH
H
26
24
R19/I263G/A328L
100% TON 2,000
unsubstituted quinoline. The 5,6-oxide of 33 was not observed,
with the RP variant forming 8-methyl-5-quinolinol (39) with 100%
selectivity (TON = 2,000). The 8-methyl group likely played a role
in this outcome by stabilising a C6 carbocation upon ring opening
of the 5,6-arene oxide. Interestingly, the RP/H171L//I263G variant
oxidised 33 to the 7,8-oxide 40 with 67% selectivity (TON = 930).
On treatment with acid, 40 gave 8-methyl-7-quinolinol, 41 (55%),
via an NIH shift after arene oxide ring opening to form the C8
carbocation but also the regioisomer 7-methyl-8-quinolinol, 42
(45%), from an NIH shift of the methyl group in a C7-cation
intermediate. Benzylic oxidation of both methylquinolines 25 and
33 was observed across the variant library with up to 100%
selectivity for the benzylic alcohols 28 (RP/H171L/I263G/A328L,
TON = 2,000) and 38 (R19/F87A/A328I, TON = 1,820).
O
N
H+
OH
P450BM3
30: 62%
OH
N
N
HO
N
N
25
27
RP
28
29
R19/I263G
RP/H171L/I263G/A328L
100% TON 2,000
N
56% TON 290
81% TON 1,150
26: 38%
HO
P450BM3
N
H
N
H
N
H
O
N
N
N
H
35
34
32
33
31
8MTHQ
GVQ/A264G
27% TON 280
K19/A82M/F87A
77% TON 1,540
R19/I263G/A328L K19/V78I/F87V/E267V
84% TON 1,680
84% TON 1,010
HO
HO
P450BM3
N
N
H
HO
N
N
32
36
OH
R19/I263G/A328L
100% TON 2,000
H+
42: 45%
41: 55%
OH
OH
P450BM3
With the 2nd-generation variants included, less than 80
CYP102A1 variants in total were screened and yet a good fraction
of the possible oxidation sites in the tested substrates, including
aromatisation of THQs to quinolines, were accessible with good
to high selectivity and TON (>1,000). Combinations of mutations
N
N
37
N
N
N
O
OH
33
38
R19/F87A/A328I
100% TON 1,820 100% TON 2,000
39
RP
40
R19/I263A/A328L
38% TON 280
RP/H171L/I263G
67% TON 930
Figure 2. The reaction scheme of P450BM3 oxidation of 2MTHQ and 8MTHQ.
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Wong, FEBS J. 2001, 268, 3117-3125; d) Q. S. Li, J. Ogawa, R. D.
Schmid, S. Shimizu, Appl. Environ. Microbiol. 2001, 67, 5735-5739; e) W.
C. Huang, A. C. Westlake, J. D. Marechal, M. G. Joyce, P. C. Moody, G.
C. Roberts, J. Mol. Biol. 2007, 373, 633-651; f) R. J. Sowden, S. Yasmin,
N. H. Rees, S. G. Bell, L. L. Wong, Org. Biomol. Chem. 2005, 3, 57-64;
g) R. J. Branco, A. Seifert, M. Budde, V. B. Urlacher, M. J. Ramos, J.
Pleiss, Proteins 2008, 73, 597-607; h) A. M. Sawayama, M. M. Chen, P.
Kulanthaivel, M. S. Kuo, H. Hemmerle, F. H. Arnold, Chem. Eur. J. 2009,
15, 11723-11729; i) A. Seifert, J. Pleiss, Proteins 2009, 74, 1028-1035.
a) Q. S. Li, U. Schwaneberg, P. Fischer, R. D. Schmid, Chem. Eur. J.
2000, 6, 1531-1536; b) E. T. Farinas, U. Schwaneberg, A. Glieder, F. H.
Arnold, Adv. Synth. Catal. 2001, 343, 601-606; c) A. Glieder, E. T. Farinas,
F. H. Arnold, Nat. Biotechnol. 2002, 20, 1135-1139; d) M. W. Peters, P.
Meinhold, A. Glieder, F. H. Arnold, J. Am. Chem. Soc. 2003, 125, 13442-
13450; e) B. M. Lussenburg, L. C. Babel, N. P. Vermeulen, J. N.
Commandeur, Anal. Biochem. 2005, 341, 148-155; f) B. M. van Vugt-
Lussenburg, E. Stjernschantz, J. Lastdrager, C. Oostenbrink, N. P.
Vermeulen, J. N. Commandeur, J. Med. Chem. 2007, 50, 455-461; g) J.
C. Lewis, F. H. Arnold, Chimia 2009, 63, 309-312.
at F87, A328 and I263 were particularly effective, with mutations
at S72, A82, E267 and A330 also playing a role. Interestingly, the
variant library catalysed the benzylic (C4) oxidation of the N-Ac
and N-Boc derivatives of THQ to the alcohol and then the ketone,
and near-total selectivity for benzylic oxidation of methyl-
quinolines was observed. Therefore, the combinations of
mutations in this collection of variants demonstrated good control
over the regioselectivity of attack on the two rings as well as the
partitioning between reaction pathways after initial H-atom
abstraction. The exceptions were the absence of C3, C7,C8 and
benzylic methyl oxidation of THQs and the low yields of the 3-
phenols from the quinolines. Further enzyme engineering is
required to achieve oxidation at these positions. The
biotransformation of THQ was scalable. For example, the
oxidation of 1 by RP/H171L/I263G at 100 mL scale in shake flasks,
without active aeration, agitation or pH control, afforded 100%
conversion of >20 mM of 1 with a TON >10,000 by sequential
addition of 4 mM aliquots of substrate over 2 days (1.5 g/L/day),
with a total product yield of 160 mg (60%).
[8]
[9]
a) S. Kille, F. E. Zilly, J. P. Acevedo, M. T. Reetz, Nat. Chem. 2011, 3,
738-743; b) C. G. Acevedo-Rocha, C. G. Gamble, R. Lonsdale, A. T. Li,
N. Nett, S. Hoebenreich, J. B. Lingnau, C. Wirtz, C. Fares, H. Hinrichs, A.
Deege, A. J. Mulholland, Y. Nov, D. Leys, K. J. McLean, A. W. Munro, M.
T. Reetz, ACS Catal. 2018, 8, 3395-3410.
[10] a) K. Zhang, B. M. Shafer, M. D. Demars, 2nd, H. A. Stern, R. Fasan, J.
Am. Chem. Soc. 2012, 134, 18695-18704; b) A. Rentmeister, F. H. Arnold,
R. Fasan, Nat. Chem. Biol. 2009, 5, 26-28; c) K. Zhang, S. El Damaty, R.
Fasan, J. Am. Chem. Soc. 2011, 133, 3242-3245; d) J. N. Kolev, K. M.
O'Dwyer, C. T. Jordan, R. Fasan, ACS Chem. Biol. 2014, 9, 164-173.
[11] a) A. Seifert, S. Vomund, K. Grohmann, S. Kriening, V. B. Urlacher, S.
Laschat, J. Pleiss, Chembiochem 2009, 10, 853-861; b) E. Weber, A.
Seifert, M. Antonovici, C. Geinitz, J. Pleiss, V. B. Urlacher, Chem.
Commun. 2011, 47, 944-946.
[12] a) C. J. C. Whitehouse, S. G. Bell, H. G. Tufton, R. J. Kenny, L. C. Ogilvie,
L. L. Wong, Chem. Commun. 2008, 966-968; b) C. J. C. Whitehouse, S.
G. Bell, W. Yang, J. A. Yorke, C. F. Blanford, A. J. Strong, E. J. Morse, M.
Bartlam, Z. Rao, L. L. Wong, Chembiochem 2009, 10, 1654-1656; c) C.
J. C. Whitehouse, W. Yang, J. A. Yorke, B. C. Rowlatt, A. J. Strong, C. F.
Blanford, S. G. Bell, M. Bartlam, L. L. Wong, Z. Rao, Chembiochem 2010,
11, 2549-2556; d) C. J. C. Whitehouse, W. Yang, J. A. Yorke, H. G. Tufton,
L. C. Ogilvie, S. G. Bell, W. Zhou, M. Bartlam, Z. Rao, L. L. Wong, Dalton
Trans. 2011, 40, 10383-10396.
In summary, CYP102A1 shows multi-function oxidase
activity in the oxidation of quinolines, tetrahydroquinolines and
3,4-dihydro-2-quinolinone. Substituents on both rings of these
core motifs were well tolerated by the CYP102A1 variants, and
there were good indications of the residues and mutations to be
targeted for the selective oxy-functionalisation of these important
building block compounds at specific positions.
Experimental Section
Experimental details are shown in Supporting Information.
[13] a) X. Ren, J. A. Yorke, E. Taylor, T. Zhang, W. Zhou, L. L. Wong, Chem.
Eur. J. 2015, 21, 15039-15047; b) X. K. Ren, J. A. O'Hanlon, M. Morris, J.
Robertson, L. L. Wong, ACS Catal. 2016, 6, 6833-6837; c) J. A. O'Hanlon,
X. Ren, M. Morris, L. L. Wong, J. Robertson, Org. Biomol. Chem. 2017,
15, 8780-8787.
Acknowledgements
[14] C. J. C. Whitehouse, S. G. Bell, L. L. Wong, Chem. Eur. J. 2008, 14,
YL is supported by a Vice Chancellor’s Scholarship from Oxford
University.
10905-10908.
[15] N. Beyer, J. K. Kulig, M. W. Fraaije, M. A. Hayes, D. B. Janssen,
Chembiochem 2018, 19, 326-337.
Keywords: P450 • Protein engineering • C–H activation •
Nitrogen heterocyclic compounds • alkaloids
References
[1]
a) A. A. L. Gunatilaka, in The Alkaloids: Chemistry and Biology, Vol. 52
(Ed.: G. A. Cordell), Academic Press, 1999, pp. 1-101; b) A. M. Patten, D.
G. Vassão, M. P. Wolcott, L. B. Davin, N. G. Lewis, in Comprehensive
Natural Products II: Chemistry and Biology, Vol. 3 (Eds.: H.-W. Liu, L.
Mander), Elsevier, 2010, pp. 1173-1296.
[2]
a) A. R. Katritzky, S. Rachwal, B. Rachwal, Tetrahedron 1996, 52, 15031-
15070; b) V. Sridharan, P. A. Suryavanshi, J. C. Menendez, Chem. Rev.
2011, 111, 7157-7259.
[3]
[4]
[5]
Y. Miura, A. J. Fulco, J. Biol. Chem. 1974, 249, 1880-1888.
L. O. Narhi, A. J. Fulco, J. Biol. Chem. 1986, 261, 7160-7169.
a) S. Pflug, S. M. Richter, V. B. Urlacher, J. Biotechnol. 2007, 129, 481-
488; b) J. Brummund, M. Muller, T. Schmitges, I. Kaluzna, D. Mink, L.
Hilterhaus, A. Liese, J. Biotechnol. 2016, 233, 143-150; c) I. Kaluzna, T.
Schmitges, H. Straatman, D. van Tegelen, M. Muller, M. Schurmann, D.
Mink, Org. Process Res. Dev. 2016, 20, 814-819.
[6]
[7]
a) C. J. C. Whitehouse, S. G. Bell, L. L. Wong, Chem. Soc. Rev. 2012,
41, 1218-1260; b) S. T. Jung, R. Lauchli, F. H. Arnold, Curr. Opin.
Biotechnol. 2011, 22, 809-817; c) R. Fasan, ACS Catal. 2012, 2, 647-666;
d) C. A. Denard, H. Ren, H. Zhao, Curr. Opin. Chem. Biol. 2015, 25, 55-
64.
a) W. L. Alworth, Q. W. Xia, H. M. Liu, FASEB J. 1997, 11, 9SS, 190; b)
T. W. Ost, C. S. Miles, J. Murdoch, Y. Cheung, G. A. Reid, S. K. Chapman,
A. W. Munro, FEBS Lett. 2000, 486, 173-177; c) A. B. Carmichael, L. L.
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10.1002/anie.201904157
Angewandte Chemie International Edition
COMMUNICATION
Entry for the Table of Contents
COMMUNICATION
Yushu Li and Luet L. Wong**
OH
O
HO
N
H
CYP102A1
Page No. – Page No.
N
H
O
N
R
N
N
N
H
N
R
N
R
Multi-function oxidase activity of
CYP102A1 in the oxidation of
R=H, Ac, Boc
R=Ac, Boc
quinolines and tetrahydroquinolines
A library of CYP102A1 (P450BM3) variants show desaturation, aromatisation and C–
C bond formation in addition to the normal C–H bond oxidation activity in the oxidation
of tetrahydroquinolines, quinolines and dihydroquinolinones with high selectivity and
turnover numbers.
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