Selective Light-Driven Chemoenzymatic Trifluoromethylation/Hydroxylation of Substituted Arenes

Article abstract of DOI:10.1021/acscatal.7b04160

The merging of photoredox trifluoromethylation with hybrid P450 BM3 variants has enabled the selective light-driven functionalization of several arenes. This approach capitalizes on the unique photochemical properties of the Ru(II)-diimine photosensitizer

Full text of DOI:10.1021/acscatal.7b04160

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Article
Selective light-driven chemoenzymatic
trifluoromethylation/hydroxylation of substituted arenes
Victor Sosa, Marya Melkie, Carolina Sulca, Jennifer Li, Lawrence Tang, Jeffrey
Li, Justin Faris, Bridget Foley, Tam Banh, Mallory Kato, and Lionel E Cheruzel
ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.7b04160 • Publication Date (Web): 31 Jan 2018
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Selective light-driven chemoenzymatic
trifluoromethylation/hydroxylation of substituted
arenes
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Victor Sosa,† Marya Melkie,† Carolina Sulca,† Jennifer Li,† Lawrence Tang,† Jeffrey Li,†
Justin Faris,† Bridget Foley,† Tam Banh,† Mallory Kato† and Lionel E. Cheruzel*,†
†San José State University, Department of Chemistry, One Washington Square, San José, CA
95192-0101
ABSTRACT: The merging of photoredox trifluoromethylation with hybrid P450 BM3 variants
has enabled the selective light-driven functionalization of several arenes. This approach
capitalizes on the unique photochemical properties of the Ru(II)-diimine photosensitizer to
initiate single electron transfer events. Under photoredox conditions, a CF3 radical promoted by
the d6 metal complex can add to arenes. In the hybrid P450 BM3 enzymes, the covalently
attached Ru(II)-diimine photosensitizer provides the necessary electrons to perform, upon visible
light activation, P450 oxyfunctionalizations on the trifluoromethylated substrates.
KEYWORDS
chemoenzymatic process, light-driven biocatalysis, trifluoromethylation/hydroxylation, Ru(II)-
diimine photosensitizer, electron transfer.
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Chemoenzymatic approaches offer the advantages of combining the potential of
chemocatalysis with biocatalysts selectivity and evolvability.1 Recent advances in C-H bond
activation have resulted in the expansion of the organic reaction toolbox and opened new
avenues for the diversification of building blocks and late-stage functionalization of various
compounds.2 Among the strategies employed for C-H functionalization, visible light photoredox
catalysis has emerged as a new and innovative approach.3 Among photosensitizers of choice, d6
metal complexes,3 e.g. Ru(II)-diimine, and more recently organic photosensitizers4 have been
shown to possess suitable excited state photochemical properties to initiate radical processes.
The Ru(II)-diimine complexes have also long been used in the study of long-range electron
transfer in modified metalloproteins5 and have found applications in the light-driven activation of
various enzymatic reactions.6 Our laboratory has developed hybrid P450 enzymes containing a
covalently attached Ru(II)-diimine photosensitizer able to deliver the necessary electrons to P450
BM3 heme domain enzymes7 and harness their synthetic potential8 upon visible light excitation.
Cytochrome P450s are heme thiolate enzymes that catalyze a breadth of chemical reactions9 such
as regio and stereoselective C-H oxyfunctionalization, O-dealkylation, heteroatom oxidation and
non-canonical reactions.10 Due to their mutational tolerance, they have been particularly suitable
for optimization by protein engineering.11
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With the recent advancement of photoredox trifluoromethylation,12 we sought to merge this
approach with the hybrid P450 BM3 enzymes in order to perform selective chemoenzymatic
trifluoromethylation/hydroxylation upon visible light irradiation. The introduction of
trifluoromethyl groups has surged in pharmaceuticals owing to their beneficial properties,13 and
is expected to keep increasing with the advancement of synthetic methodology. Selected
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examples of recently approved FDA drugs are shown in Figure 1 highlighting the diverse
scaffolds bearing CF3 groups.
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Figure 1. Selected examples of approved FDA drugs containing trifluoromethyl groups.
Pioneering work by Fasan and Arnold demonstrated the combination of P450-catalyzed
oxygenation with deoxyfluorination to achieve selective fluorinated organic compounds.14
Hartwig and Zhao merged an organometallic catalyst with the P450 BM3 enzyme to perform
selective epoxidation of the cross metathesis product.15 Recently, the Fasan group established a
chemo-biocatalytic approach for producing trifluoromethyl-substituted cyclopropanes using
engineered myoglobin variants.16
Herein, we focus on substituted arenes that could be trifluoromethylated using photoredox
catalysis and subsequently selectively functionalized by the light-driven hybrid enzymes
according to Scheme 1. Using the combined light-driven approaches, eight compounds (See
Table 1) were selectively functionalized with moderate to good yields by three novel hybrid
enzymes.
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Scheme 1. Merging of the photoredox and hybrid enzymes for the light-driven
trifluoromethylation/hydroxylation of substituted arenes taking advantage of the photochemical
properties of Ru(II)-polypyridyl complexes
The hybrid enzymes, F87V-Ru, A82F-Ru and D52-Ru, were generated as previously
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reported7a,
and feature a Ru(bpy)2PhenA (Ru) photosensitizer (bpy = 2,2’-bipyridine and
PhenA = 5-acetamido-1,10-phenanthroline) covalently attached to the non-native single cysteine
L407C. The covalent attachment of the photosensitizer at this position led previously to high
photocatalytic activity and initial reaction rates in the hydroxylation of long chain fatty acid
substrates.7c Subsequent characterizations revealed that the photosensitizer is ideally located
where the redox partner is thought to bind and that the photogenerated reductive species has the
appropriate driving force for fast electron delivery using highly conserved amino acid residues.7b
The F87V18 and the “gatekeeper” F87V/A82F19 mutations are known to enable the
oxyfunctionalization of several aromatic compounds. The D52-Ru mutant was identified via a
directed evolution approach, recently initiated in our laboratory, focusing on light driven
benzylic hydroxylation using the chromogenic 1-benzyl-4-nitrophenyl ether substrate.17 This
evolved mutant (R167L/K224N/S383N/L407C/D434A) displays enhanced photocatalytic
activity towards propylbenzene (3 fold) and naphthalene (10 fold) compared to the sL407C-Ru
parent (See Figure S1).
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Under Stephenson photoredox conditions using trifluoroacetic anhydride and pyridine N
oxide,12a all compounds listed in Table 1 could be trifluoromethylated in good yields. Often a
mixture of isomers is produced with the exception of 7-ethoxycoumarin (entry 7), which is
quantitatively converted to the 3-CF3 analog. The compounds were thoroughly characterized by
1H, 13C and 19F NMR as well as GC/MS (See Figures S2-13). In most cases, the isomers could be
separated by reversed-phase HPLC to unambiguously confirm the position of
trifluoromethylation and hence the substrate selectivity of the hybrid enzymes. All
trifluoromethylated compounds were subjected to the hybrid enzymes under the light-driven
reaction conditions.7a, 17 The products were characterized by HPLC and GC/MS after silylation of
the alcohol moiety.
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Figure 2. GC chromatograms of the products obtained from the light-driven chemoenzymatic
reactions; A) Example of selective hydroxylation by the F87V-Ru enzyme with the
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trifluoromethylated diphenylmethane mixture (1a-c) as well as with the separated meta
substituted isomer (1b) and ortho/para substituted isomers mixture (1a,c). The retention time for
the 2a and 2b products match those of authentic standards (black traces); Products obtained with
the various hybrid enzymes D52-Ru (blue), F87V-Ru (red) and A82F-Ru (black) with
trifluoromethylated indane, 3a,b (B); propylbenzene, 5a-c (C) and bromopropylbenzene, 8a,b
(D).
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Starting with diphenylmethane (entry 1, Table 1), the photoredox trifluoromethylation yielded
a mixture of three isomers (1a-c) that could be further separated into the ortho and para isomers
(1a and 1c, respectively) and the meta isomer (1b). When the mixture of 1a-c was submitted to
the light-driven enzymatic reactions, hydroxylated products were observed with the three hybrid
enzymes. As shown in Figure 2A for F87V-Ru, only the ortho (1a) and meta (1b) substituted
isomers are hydroxylated to the corresponding 2a and 2b products, respectively, based on mass
fragmentation and comparison with authentic standards. Furthermore, the compound 1a is
preferentially hydroxylated over the para isomer, 1c, for the A82F-Ru and F87V-Ru mutants,
while the D52-Ru mutant shows negligible activity (See Figure S14). The meta substituted
isomer (1b) is hydroxylated to 2b by all three variants with a 93% ee noted for the F87V-Ru.
We wanted to further investigate the isomer selectivity as well as the hydroxylation
regioselectivity of the hybrid enzymes using the trifluoromethylated compounds listed in Table
1. The trifluoromethylation of indane yielded a mixture of two isomers, 3a and 3b, in a 60:40
ratio, respectively (See Figure S4). The mixture was subjected to the hybrid enzymes and
hydroxylation was found to occur at the benzylic position with all enzymes (See Figure S15).
Substrate selectivity was achieved by varying the hybrid enzyme variant as shown in Figure 2B.
The D52-Ru and F87V-Ru mutants hydroxylated both CF3 isomers to the 4a,b products. The
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A82F-Ru, on the other hand, selectively hydroxylated only one of the two isomers to yield
primarily 5-CF3-1-indanol (4a) with a 62%ee. The wild-type P450 BM3 enzyme displays
minimal stereoselectivity in indane hydroxylation (16%ee), that could be enhanced using decoy
molecules (%50)20 or directed evolution (60-99%ee).21
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Upon trifluoromethylation of propylbenzene, three products (5a-c) were obtained in equal
amount with a slight preference for the meta position (See Figure S5). Different hydroxylated
products were obtained depending on the hybrid enzymes and the position of the CF3
substituents (See Figure S16). Using the evolved variant, two hydroxylated products were
observed and identified as the 3- and 4-CF3-phenyl-1-propanol (6b and 6c, respectively) when
compared to authentic standards. The D52-Ru mutant shows preference for the para and meta
position as no ortho product (6a) could be detected. The A82F-Ru mutant, however, shows
preference for the ortho/meta positions as no 4-CF3-1-phenylpropanol product (6c) was observed
(See Figure 2C). Interestingly, for the F87V-Ru, the hydroxylation occurs predominantly at the
least favored beta position on the propyl chain based on the mass fragmentation pattern observed
after silylation of the 7b product (See Figure S16). While some engineered P450 BM3 mutants
have shown unique selectivity for the beta position,22 the F87V mutant displayed only 20%
regioselectivity for the beta position of propylbenzene.18a The presence of the CF3 group at the
meta position in 5b promotes the regioselectivity of hydroxylation at this position
predominantly. Recent reports by Sherman23 and Bell24 elegantly demonstrated the directing role
of anchors and ester functionality on substrates to mediate selective P450 hydroxylation.
We expanded the scope of the light-driven reactions to probe the functional group tolerance
(with 8, 13 and 15, entries 4, 6 and 7, respectively), trifluoromethylation selectivity (15, entry 7)
and heteroarene functionalization (17, entry 8) as well as the various reactions catalyzed by P450
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enzymes including direct arene hydroxylation (11, entry 5), O-dealkylation (13 and 15, entries 6
and 7, respectively) and heteroatom oxyfunctionalization (17, entry 8).
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Trifluoromethylation of bromopropylbenzene (entry 4) yields a 65:35 mixture of 8a and 8b,
respectively, with the CF3 group adjacent to the electron withdrawing group on the arene of 8a.
Hydroxylation was noted with all three hybrid enzymes as shown in Figure 2D albeit with
different regioselectivities. The two isomers were separated by reversed-phase HPLC (Figures
S6-S7) and individually submitted to the enzymatic hydroxylation conditions to confirm the
isomer preference. All mutants selectively hydroxylate the major product 8a (Figure S17). The
D52-Ru variant hydroxylates at the benzylic position to form 9a while the F87V-Ru and A82F-
Ru mutants yield hydroxylation at the beta position of the propyl chain in 10a. Naphthalene,
entry 5, was primarily trifluoromethylated in high yield at position 1 (11a) as shown in Figure S8
and could solely be hydroxylated to 12 by the evolved variant, D52-Ru (Figure S18), which
showed enhanced activity towards naphthalene.
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The traditional substrates to probe O-dealkylation of P450 enzymes have involved the
chromogenic p-nitrophenoxy or fluorogenic ethoxycoumarin derivatives. The 1-octyl-4-
nitrophenyl ether compound was previously used to evolve P450 BM3 variants towards short
alkane functionalization.25 Its trifluoromethylation yielded, after purification, a major compound
(13, Figure S9) with the CF3 group adjacent to the electron withdrawing nitro group rather than
the electron donating alkoxy substituent. The trifluoromethylated substrate 13 could then be
dealkylated by the D52-Ru mutant to yield the yellow chromophore 3-CF3-4-nitrophenolate, 14,
displaying a 20 nm blue-shift in absorbance maximum compared to the well-known
nitrophenolate analog (See Figure S19).
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Table 1. Summary of the compounds selectively functionalized using the chemoenzymatic light-
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driven approach.
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Entry
1
Photoredox catalysisa
Major product
Overall yieldb Enzymec
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1a-c
2b
16%
F87V-Ru
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3a,b
5a-c
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15%
19%
A82F-Ru
F87V-Ru
F87V-Ru
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6%
D52-Ru
D52-Ru
11%
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39%
F87V-Ru
a) (*) represent the various sites of trifluoromethylation; b) determined HPLC yield over
two reaction steps; c) 400 μM substrate; 2 μM hybrid enzyme and 100 mM sodium
diethyldithiocarbamate.
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Interestingly, trifluoromethylation of the 7-ethoxycoumarin yielded solely the 3-CF3-7-
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ethoxycoumarin product (15, entry 7). The H, C and F NMR spectra in Figure S10 confirm
that compound 15 is an isomer of the commercially available 4-CF3-7-ethoxycoumarin used to
probe the activity of human P450 isoforms.26 Despite low activity noted with all current hybrid
enzyme mutants, the 3-CF3-7-ethoxycoumarin, 16, formed displays fluorescence emission
shifted compared to the 4-CF3- and 7-hydroxycoumarin counterparts (See Figure S20).
Heteroarene functionalization was probed with quinoline (entry 8). Under the
trifluoromethylation conditions, two isomers were obtained, which could be separated, revealing
that trifluoromethylation occurred primarily at the 5 and 8 positions, in compounds 17a and 17b,
respectively (Figures S11-12). The F87V-Ru mutant produced only one oxidized compound, in
78% yield, from the mixture of isomers (See Figure S21). From the mixture or the two
trifluoromethylated quinolines individually, only the 5-CF3-quinoline (17a) is being converted to
its N-oxide analog, which matches an authentic sample prepared independently by m-CPBA
oxidation27 (See Figure S21). The presence of CF3 group at the 5 position in 17a promotes N-
oxidation vs hydroxylation previously reported for quinoline.28
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In conclusion, this light-driven chemoenzymatic approach enables the selective
trifluoromethylation/hydroxylation of substituted arenes by capitalizing on the photochemical
properties of Ru(II)-diimine photosensitizers and hybrid P450 BM3 enzymes. This system also
benefits from the selectivity of the P450 BM3 mutants to differentiate between the
trifluoromethylated isomers and produce regio- and stereoselective products. Further engineering
of the hybrid enzymes could alleviate the need to purify the mixture of trifluoromethylated
products and yield highly selective hydroxylation of desired isomers “on demand”. This
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methodology could thus find applications in late-stage functionalization of target compounds3b, 29
and towards the tailored diversification of lead compounds.30
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ASSOCIATED CONTENT
AUTHOR INFORMATION
Corresponding Author
* lionel.cheruzel@sjsu.edu.
Author Contributions
L.C. and M.K. designed the experiments, analyzed the data and wrote the manuscript. V.S. and
M.M. performed the key experiments. All authors contributed significantly to the work presented
in this paper.
Notes
The authors declare no competing financial interests.
ABBREVIATIONS
CF3, trifluoromethyl; m-CPBA, meta-chloroperoxybenzoic acid; GC/MS, gas chromatography-
mass spectrometry; HPLC, high-performance liquid chromatography; D52-Ru, F87V-Ru;
A82F-Ru, hybrid P450 BM3 enzymes with a covalently attached Ru(II)-diimine photosensitizer
(Ru) at the non-native single cysteine L407C.
Supporting Information. The following files are available free of charge. Synthetic details and
general procedures; Characterization data including 1H, 19F, 13C NMR spectra; HPLC and
GC/MS chromatograms.
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Funding Sources
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This work was financially supported by the National Science Foundation through grant number
1509924 (L.C.). M.M. acknowledges financial support from National Institute of General
Medical Sciences of the National Institutes of Health under award number R25GM071381. C.S.
acknowledges financial support from CSU-LSAMP, funded through the National Science
Foundation under grant #HRD-1302873 and the Chancellor's Office of the California State
University.
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BRIEFS
Merging of photoredox catalysis with hybrid P450 enzymes enable selective light-driven
trifluoromethylation/hydroxylation of substituted arenes promoted by Ru(II)-diimine complexes.
TOC GRAPHIC
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