Ground-State Electron Transfer as an Initiation Mechanism for Biocatalytic C-C Bond Forming Reactions

Article abstract of DOI:10.1021/jacs.1c04334

The development of non-natural reaction mechanisms is an attractive strategy for expanding the synthetic capabilities of substrate promiscuous enzymes. Here, we report an "ene"-reductase catalyzed asymmetric hydroalkylation of olefins using α-bromoketones as radical precursors. Radical initiation occurs via ground-state electron transfer from the flavin cofactor located within the enzyme active site, an underrepresented mechanism in flavin biocatalysis. Four rounds of site saturation mutagenesis were used to access a variant of the "ene"-reductase nicotinamide-dependent cyclohexanone reductase (NCR) from Zymomonas mobiles capable of catalyzing a cyclization to furnish β-chiral cyclopentanones with high levels of enantioselectivity. Additionally, wild-type NCR can catalyze intermolecular couplings with precise stereochemical control over the radical termination step. This report highlights the utility for ground-state electron transfers to enable non-natural biocatalytic C-C bond forming reactions.

Full text of DOI:10.1021/jacs.1c04334

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Ground-State Electron Transfer as an Initiation Mechanism for
Biocatalytic C−C Bond Forming Reactions
Haigen Fu, Heather Lam, Megan A. Emmanuel, Ji Hye Kim, Braddock A. Sandoval, and Todd K. Hyster*
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ABSTRACT: The development of non-natural reaction mecha-
nisms is an attractive strategy for expanding the synthetic
capabilities of substrate promiscuous enzymes. Here, we report
an “ene”-reductase catalyzed asymmetric hydroalkylation of olefins
using α-bromoketones as radical precursors. Radical initiation
occurs via ground-state electron transfer from the flavin cofactor
located within the enzyme active site, an underrepresented
mechanism in flavin biocatalysis. Four rounds of site saturation
mutagenesis were used to access a variant of the “ene”-reductase
nicotinamide-dependent cyclohexanone reductase (NCR) from
Zymomonas mobiles capable of catalyzing a cyclization to furnish β-chiral cyclopentanones with high levels of enantioselectivity.
Additionally, wild-type NCR can catalyze intermolecular couplings with precise stereochemical control over the radical termination
step. This report highlights the utility for ground-state electron transfers to enable non-natural biocatalytic C−C bond forming
reactions.
substrates and flavin hydroquinone (FMNhq) cofactors.16−18
This mechanism is effective for inducing electron transfer to
alkyl iodides and chloroamides whose low reduction potentials
INTRODUCTION
Radicals are versatile synthetic intermediates that enable
numerous fascinating processes and retrosynthetic disconnec-
■
tions.1−3 While the overall reactivity of radical intermediates is
make ground state electron transfer thermodynamically
well understood,4 catalytic strategies for controlling the chemo-
challenging (Figure 1A). However, as the energetics of CT
and stereoselectivity remain underdeveloped.5−7 Nature has
complexes are governed by both electrostatic attraction and
substrate electron affinity, it can be difficult to predict which
substrates will form such complexes.20 Ground-state electron
transfer is an attractive alternative to photoinduced mecha-
nisms because its viability is determined by the redox
potentials of substrate and cofactor (E1/2 = −0.45 V versus
saturated calomel electrode (SCE) for FMNhq21). We
previously demonstrated the viability of this mechanism for
the asymmetric hydrodehalogenation of electronically activated
α-bromophenylacetic esters.22 This mechanism has not,
however, been utilized for forming less stabilized radicals or
been deployed in C−C bond forming reactions.
As a model, we targeted the cyclization of α-bromoketone 1
to afford β-stereogenic cyclopentanone 2 (Figure 1B). As α-
bromoaliphatic ketones are more easily reduced in comparison
to chloroamides (E1/2 = −0.74 V versus SCE for α-
bromoacetophenone),23 we hypothesized that they would be
evolved various biocatalysts, such as cobalamin and radical S-
adenosylmethionine (SAM) enzymes, to catalyze the radical-
mediated transformations with unparalleled chemo-, regio-,
and enantioselectivity in the biosynthesis of complex natural
products.8−11 However, these enzymes are not readily
harnessed outside of their natural settings by organic synthetic
chemists, owing to either operational difficulties or lack of
catalytic versatility. We imagined that enzymes could be used
more broadly to solve selectivity challenges in the radical
literature by identifying and developing enzymes that can use
non-natural mechanisms for radical formation.12,13
Flavin-dependent “ene”-reductases (EREDs) naturally cata-
lyze the asymmetric reduction of electronically activated
alkenes and are among the most widely used class of
biocatalysts in chemical synthesis.14,15 Mechanistically, the
reaction occurs via a stereoselective hydride transfer from the
cofactor flavin hydroquinone (FMNhq) to the substrate’s
electrophilic position which is followed by protonation by a
conserved tyrosine residue in the enzyme active-site to give the
hydrogenated product.14,15 Our group recently demonstrated
that these enzymes can also catalyze asymmetric radical
cyclizations when irradiated with visible light.16−19 This
reaction occurs via photoexcitation of enzyme templated
charge transfer (CT) complexes formed between organohalide
Received: April 26, 2021
Published: June 11, 2021
https://doi.org/10.1021/jacs.1c04334
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© 2021 American Chemical Society
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Table 1. Screening an Initial Panel of EREDs for
e
Cyclization
a
Yield (%) of
Yield (%)
of 3
b
Entry
“Ene”-Reductase
2a
er
1
2
3
4
5
6
OPR1
NCR
PETNr
ERED-30
No enzyme
31
20
66
94
0
80:20
79:21
66:34
65:35
n.d.
n.d.
9
19
19
6
0
0
c
d
No NADPH regeneration
system
0
e
Reaction conditions: α-bromoketone (1 mg, 0.004 mmol), GDH-
105 (0.5 mg), NADP+ (0.5 mg), glucose (5 mg) and purified “ene”-
reductases (2 mol %) in 50 mM Tris-HBr buffer pH 8.0, with 5%
DMSO (v/v) as cosolvent, final total volume is 500 μL. Reaction
mixtures were shaken under anaerobic conditions at room temper-
ature for 24 h. Yield determined via LCMS relative to an internal
standard (1,3,5-tribromobenzene (TBB)). Enantiomeric ratio (er)
a
b
c
determined by HPLC on a chiral stationary phase. ERED-30 (1 mol
Figure 1. Strategies and challenges in using “ene”-reductases
(EREDs) for non-natural radical cyclization.
%) was screened from a genetically diverse EREDs library from
Prozomix. n.d., not determined.
d
amenable to reduction via ground state electron transfer. While
this represents an endergonic electron transfer, we reasoned
that subsequent fast and irreversible mesolytic cleavage of the
C−Br bond would enable the reaction to proceed. The
intermediacy of α-ketonyl radicals presents distinct challenges
in comparison to our previous biocatalytic cyclization involving
α-amidyl radicals.16 Specifically, α-ketonyl radicals have lower
reduction potentials and are more reactive, making them prone
to undesired electron and hydrogen atom transfer pathways
that ultimately lead to the formation of undesired hydro-
debrominated products (Figure 1B).23 These side reactions
also plague variants of this reaction mediated by small-
molecule reagents.24−26 Additionally, the enhanced reactivity
of this intermediate should also result in a transition state for
the C−C bond-forming step that more closely resembles the
starting material (per the Hammond Postulate), making it
harder to achieve high levels of enantioselectivity, potentially
accounting for the lack of general small molecule catalysts for
this reaction (Figure 1B).27
lectivity to OPR1 but with greater formation of 3a (Table 1,
entry 2). Interestingly, pentaerythritol tetranitrate reductase
(PETNr) afforded high yields for the desired product 2a, but
with lower levels of enantioselectivity (Table 1, entry 4).
Importantly, it is also found in commercial ERED collections.
For instance, ERED-30 from the Prozomix library affords
product 2a in 94% yield (65:35 er) and only 6% yield of the
linear dehalogenated product 3 (Table 1, entry 4). Control
experiments confirmed that both ERED and the NADPH
regeneration system were required for reactivity (Table 1,
entries 5 and 6).
As a wild-type enzyme could not be identified that afforded
high yields and enantioselectivities, we chose to develop an
improved catalyst from NCR. This enzyme was selected as a
starting point because it provided the appropriate balance of
product yield, enantioselectivity, and high expression levels in
96-well plates. EREDs have not previously been evolved for
radical biocatalytic reactions, so amino acid residues were
chosen for site saturation mutagenesis (SSM) that line the
active site pocket that are thought interact with either the
substrate or flavin cofactor (the docking model of substrate 1a
in wild-type NCR is shown in SI Figure 1).28 For each round
of SSM, enzyme libraries were coexpressed with the groES-
groEL chaperone using Escherichia coli cells and screened in
96-well plates in the form of cell-free extracts (CFEs). After
four rounds of SSM, we discovered four beneficial mutations
Y343W, F269W, W342A, and I231S, which collectively
furnished a notable 7.7-fold improvement in yield and further
enhancements in enantioselectivity; the undesired dehalogen-
ated linear product 3 was also effectively diminished to a
negligible level (Figure 2 and SI Tables 2−10). Using the final
quadruple mutant (Y343W/F269W/W342A/I231S), named
NCR-C9, as the biocatalyst under optimized conditions, the
(S)-configured cyclization product 2a was formed in 92% yield
and 95:5 er, with a 99:1 ratio of cyclized/linear (2a/3)
products (Figure 2). This reaction can be run on a preparative
RESULTS AND DISCUSSION
■
Discovery and Engineering of NCR for Radical
Cyclization. We began our studies by exploring the reductive
cyclization of α-bromoketone 1a to β-stereogenic cyclo-
pentanone 2a catalyzed by structurally diverse wild-type
EREDs under ambient conditions with an NADPH regener-
ation system (GDH/NADP+/glucose) (Table 1). Several
EREDs provided the desired cyclopentanone product 2a,
although significant quantities of the hydrodehalogenation
product 3 were observed in all cases. The ERED 12-
oxophytodienoate reductase 1 (OPR1) from Solanum
lycopersicum provided the highest enantioselectivity (31%
yield, 80:20 er) for product 2a, with 9% formation of the
undesired hydrodebrominated product 3 (Table 1, entry 1).
The nicotinamide-dependent cyclohexanone reductase (NCR)
from Zymomonas mobiliz afforded similar levels of enantiose-
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Figure 2. Directed evolution of NCR catalysts for non-natural asymmetric radical cyclization. (A) Chemoselectivity (cyclization versus
debrominative hydrogenation), stereoselectivity, and yields improvement of NCR variants over wild-type NCR. Standard reaction conditions: the
reaction mixture (500 μL) consisted of model substrate (1a, 0.004 mmol), GDH-105 (0.5 mg), glucose (5.0 mg), NADP+ (0.5 mg), and purified
NCR variants (1.0 mol %) in buffer (100 mM KPi buffer pH 8.0) with 5% DMSO as cosolvent at room temperature for 24 h. Yields were
determined via HPLC relative to an internal standard (average of triplicate). Enantiomeric ratio (er) was determined via HPLC on a chiral
stationary phase. (B) Crystal structure of wild-type NCR (PDB: 4s3u). Residues for substrate binding (H172 and N175) are shown as blue sticks.
Four beneficial sites Y343, F269, W342, and I231 are shown in orange sticks.
f
Scheme 1. Asymmetric Radical Cyclization Catalyzed by NCR-C9 Mutant
f
Standard reaction conditions: the reaction mixture (500 μL) consisted of substrate (1, 0.004 mmol), GDH-105 (0.5 mg), glucose (5.0 mg),
NADP+ (0.5 mg), and purified NCR-C9 mutant (1.0 mol %) in buffer (100 mM KPi buffer pH 8.0) with 5% DMSO as cosolvent at room
temperature for 24 h. aYields were determined via HPLC relative to an internal standard. bEnantiomeric ratio (er) was determined via HPLC on a
chiral stationary phase. Isolated yield was based on 0.08 mmol-scale reaction. 2 mol % of wild-type NCR was used. n.d., er not determined.
c
d
e
scale and afford product in 86% isolated yield with no changes
in enantio- or chemoselectivity (Scheme 1).
With the developed non-natural radical enzyme NCR-C9 in
hand, we explored the transformation scope and limitations
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(Scheme 1). The quadruple mutant NCR-C9 accommodated
various para-substituents of the aromatic ring, with electron-
donating (1e−f) and electron-withdrawing (1i) substituents
affording β-stereogenic cyclopentanones in high yields (74−
86%) and excellent enantioselectivities (>94:6 er, Scheme 1).
Notably, para-halogenated substrates (1g−h) were tolerated
providing a handle for potential downstream manipulation
(Scheme 1). Ortho-substituted (1b) and meta-substituted
(1c−d) substrates were also well accepted by the evolved
NCR-C9, giving the corresponding cyclized products 2c−d
with good yields (57−81%) and high levels of stereoselectivity
(>90:10 er, Scheme 1). The aromatic substituent proved
essential for the desired reactivity, with unsubstituted alkene or
enone affording the hydrodehalogenated products exclusively
(Scheme 1 and SI Figure 2).
mechanism, it was unclear whether radical formation would be
substrate gated.
We tested a panel of EREDs on the intermolecular
hydroalkylation of α-bromo acetophenone 6a with α-
methylstyrene 7a to provide γ-stereogenic ketone 8a (Table
2). GluER-T36A, the best variant for the coupling of
Table 2. Screening an Initial Panel of EREDs for
f
Intermolecular Hydroalkylation
a
b
Entry
X
“Ene”-Reductase
Loading
Yield (%)
er
1
2
3
Br
Br
Br
Br
Br
Cl
Br
GluER-T36A
NCR
NCR
NCR
YersER
NCR
2 mol %
2 mol %
1 mol %
1 mol %
1 mol %
2 mol %
1.5 mol %
62
99
61
64
44
97:3
97:3
97:3
97:3
17:83
97:3
99:1
Protein engineering campaigns result in catalysts that are
optimal for the reaction on which they were developed. To
probe the specialization in this protein engineering campaign,
we tested alternative substrates and cyclization modes. The
(Z)-isomer of the model substrate (Z)-1a was accepted by the
NCR-C9, although with diminished yield and enantioselectiv-
ity (62% yield, 69:31 er, S-isomer over access) in comparison
to those of the (E)-configured substrate (E)-1a, indicating the
importance of geometric configuration of the substrate during
the NCR-catalyzed radical cyclization reaction (Scheme 1). In
contrast, (E)-1a and (Z)-1a perform similarly to wild-type
NCR, indicating that the protein engineering campaign
enhanced the preference for the (E)-olefin isomer. In the
realm of alternative cyclization modes, the specialization was
less pronounced. While NCR-C9 was ineffective for a 6-exo-
trig cyclization, the wild-type enzyme could catalyze the
reaction in low yield, indicating the parent enzyme is more
promiscuous. In contrast, both variants afforded similar results
for 7-exo-trig cyclization (Scheme 1). In addition, both the
wild-type and evolved variants can catalyze the 6-endo-trig
cyclization providing γ-substituted cyclohexanone 2m in
moderate yield; however, 5-endo-trig cyclization was inacces-
sible to both enzymes, fitting to the Baldwin rules (Scheme 1
and SI Figure 3). Collectively, these results indicate that
specialization is apparent within individual cyclization modes
but does not necessarily hold when looking at other manifolds.
Intermolecular Radical C−C Bond Formation Cata-
lyzed by Wild-Type NCR. Having developed a selective
enzyme for a radical cyclization, we further questioned whether
EREDs could catalyze an intermolecular radical hydro-
alkylation. Zhao and co-workers have previously reported
that EREDs can catalyze the proposed reaction when irradiated
with blue LEDs.29 However, as α-bromoacetophenone has a
modest reduction potential (−0.74 V vs SCE),23 we
hypothesized that this reaction can occur without photo-
excitation. In a dark manifold, the central challenge is
identifying a gating mechanism to ensure that both the
bromoketone and alkene are bound within the protein active
site prior to electron transfer. In the absence of such a
mechanism, we would expect the formation of the hydro-
dehalogenated product primarily. In the photoenzymatic
coupling of chloroamides and alkenes, we observed an enzyme
templated ternary CT complex between FMNhq, chloroamide,
and α-methylstyrene.30 As this complex is more strongly
absorbing in comparison to the binary complex (FMNhq and
chloroamide), productive chemistry primarily occurs through a
CT complex that has both coupling partners bound within the
protein active site.30 In the absence of this photoregulation
c
4
5
6
7
49
99 (82 )
d
e
NCR
f
Reaction conditions: α-haloacetophenone (6, 0.015 mmol, 3 equvi),
α-methylstyrene (7a, 0.005 mmol, 1 equiv), NADP+ (0.2 mg), GDH-
105 (0.3 mg), glucose (5 mg) and purified “ene”-reductases in 50 mM
Tris-HBr buffer pH 7.5, with 10% DMSO as cosolvent, total volume is
500 μL. Reaction mixtures were shaken under anaerobic conditions at
room temperature for 36 h. Yield determined via LCMS relative to
an internal standard (TBB). Enantiomeric ratio (er) determined by
a
b
c
HPLC on a chiral stationary phase. Irradiation with a blue LED.
d
e
Total reaction volume is 1200 μL. Yield of isolated product from a
0.1 mmol-scale reaction.
chloroamides with alkenes, afforded product in 62% yield
with 97:3 er (Table 2, entry 1). Remarkably, wild-type NCR
catalyzed the desired transformation in a quantitative yield
(using alkene as the limiting reagent) with excellent
enantioselectivity (97:3 er) for the (R)-enantiomer, out-
performing other tested wild-type EREDs (Table 2, entry 2).
Decreasing the catalyst loading to 1 mol % afforded decreased
yields but with no loss in enantioselectivity (Table 2, entry 3).
Interestingly, irradiation with a blue Kessil lamp did not afford
an increase in yield (Table 2, entry 4). This result indicates
that a photoexcited electron transfer mechanism is not
available to this substrate/enzyme combination. The (S)-
enantiomer of 8a can also be accessed using Yersina bercovieri
alkene reductase (YersER) as the biocatalyst, albeit in
moderate yield and enantioselectivity (44% yield, 83:17 er)
(Table 2, entry 5). In all cases, <15% of hydrodehalogenated
product was observed, suggesting a mechanism for gating
radical formation. After the experimental parameters were
optimized, wild-type NCR (1.5 mol % loading) catalyzed the
non-natural intermolecular hydroalkylation providing the (R)-
8a in 99% yield (82% isolated yield) and excellent
enantioselectivity (99:1 er) (Table 2, entry 7). Control
experiments confirmed that both NCR and the NADPH
regeneration system are necessary for this biotransformation
(SI Table 12). Interestingly, α-chloroacetophenone is an
effective substrate under these reaction conditions (49% yield,
97:3 er), despite being less reducing by 600 mV (−1.44 V vs
SCE),23 indicating that photoinduced electron transfer is not
required for radical initiation (Table 2, entry 6).29
A variety of α-bromo aryl ketones are well tolerated by the
reaction (Scheme 2). α-Bromoketones possessing either
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d
Scheme 2. Asymmetric Intermolecular Radical Hydroalkylation Catalyzed by Wild-Type NCR
d
Standard reaction conditions: the reaction mixture (1.2 mL) consisted of substituted styrene (7, 0.005 mmol, 1 equiv), α-bromo carbonyl
(6,0.015 mmol, 3 equiv), GDH-105 (0.3 mg), glucose (5.0 mg), NADP+ (0.2 mg), and purified wild-type NCR protein (1.5 mol % to alkene) in
buffer (50 mM Tris-HBr buffer pH 7.5) with 10% DMSO as cosolvent at room temperature for 36 h. aYields were determined via HPLC relative to
b
c
an internal standard. Enantiomeric ratio (er) was determined via HPLC on a chiral stationary phase. An excess of styrene (7, 0.015 mmol, 3
equiv) to α-bromo carbonyl (6,0.005 mmol, 1 equiv) was used.
electron-donating (6b−c) or electron-withdrawing (6d−i)
substituents at the para-position were efficiently converted to
the desired enantioenriched γ-chiral ketones (8b−i) in yields
of 43−98% with excellent enantioselectivity (>97:3 er). The
meta-substituted substrates 6j−k were also well accepted by
the wild-type NCR, providing the corresponding products 8j−
k with good yields (72−96%) and high levels of
enantioselectivity (>97:3 er). Beyond acetophenone deriva-
tives, simple α-bromo alkyl ketone 6n was also successful in
the reaction, giving rise to the product 8n in 63% yield and
95:5 er, highlighting the generality of the ground-state electron
transfer mechanism. Unfortunately, α-bromo acetic acid and α-
bromo esters were unreactive (SI Figure 4).
The alkene scope is also broad for the NCR-catalyzed
intermolecular hydroalkylation (Scheme 2). Various ortho- and
meta-substituted α-methyl styrenes were well accepted by the
enzyme, providing the corresponding γ-chiral ketones 8o−r in
yields of 58%−88% with excellent enantioselectivity (up to
99:1 er, Scheme 2). Para-substituted α-methyl styrenes were
less tolerated in the reaction. The smaller para-F substituent
(8s, 99% yield, 95:5 er) outperformed larger groups such as
para-Me (8t) or para-Cl (8u) substituent, which afforded only
moderate yields (59−69%) and poor enantioselectivity
(Scheme 2). Beyond styrenyl alkenes, wild-type NCR could
also accept heterocycles, including an electron-deficient α-
methylvinylpyridine and electron-rich α-methylvinylthiophene,
providing the respective products 8w−x in 61−74% yield and
high enantioselectivity (up to 99:1 er). This enzyme, however,
was limited to small substituents at the α-position of styrene
(8y−z). Larger groups, such as i-propyl and n-propyl, were
poorly reactive (Scheme 2). We hypothesize that this
limitation could be overcome using protein engineering.
Finally, aliphatic alkenes, such as α-methylvinylcyclohexane,
were not reactive (Scheme 2 and SI Figure 4).
Mechanistic Experiments. To elucidate the mechanism
of termination for the benzylic radical in the NCR-catalyzed
intra- and intermolecular C−C bond-forming reactions, we
conducted a set of deuterium-labeling experiments (Figure 3
and SI Figures 6−11). For the cyclization mediated by NCR-
C9, when cofactor flavin was labeled in situ using d1-glucose
and glucose dehydrogenase (GDH), the product β-benzyl
cyclopentanone 2a was obtained in 83% yield and 95:5 er with
47% deuterium incorporation at the benzylic position (Figure
3A). Alternatively, when the model cyclization reaction was
performed in deuterated buffer, the product 2a was observed in
high yield and enantioselectivity (93% yield, 95:5 er) with only
11% of benzylic deuterium incorporation (Figure 3A).
Collectively, these deuterium-labeling data suggest that the
primary mechanism of quenching the benzylic radical would be
HAT from flavin (FMNsq), as proposed in SI Figure 5A. In the
experiment where in situ deuterium-labeled flavin was used, the
moderate deuterium incorporation (47 D%) of product 2a can
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with and without α-methylstyrene 7a. In the presence of 3
equiv of 7a, 6a was consumed at 7.6 mmol/hour. In the
absence of alkene, that initial rate decreased to 3.0 mmol/hour,
which was a 2.5-fold decrease to the initial rate (Figure 3C and
SI Figures 13−15). These experiments indicate that α-
bromoacetophenone consumption is enhanced by the presence
of α-methylstyrene, empirically suggesting a gating mechanism.
We hypothesize that an interaction between the π-bond of the
alkene and the π*C−Br could be responsible for the rate
enhancement. Electron transfer in these reactions is under-
stood to be endergonic and reversible. The first irreversible
step is mesolytic cleavage of the ketyl radical to form the α-acyl
radical. A hyperconjugative interaction between the alkene and
either α-bromoacetophenone or corresponding radical anion
would weaken the C−Br bond, making the mesolytic cleavage
more facile. Alternatively, the proposed interaction could
decrease the reduction potential of α-haloacetophenone by
polarizing the C−X bond causing a buildup of positive charge
on the α-carbon. This could potentially account for the ground
state reactivity observed with α-chloroacetophenone despite its
lower reduction potential.
CONCLUSIONS
■
In conclusion, we discovered that the flavin-dependent enzyme
NCR is capable of catalyzing C−C bond-forming radical
cyclization and the activity and selectivity of the reaction can
be significantly improved using directed evolution. The
engineered quadruple mutant NCR-C9 shows broad substrate
scope and excellent chemo- and stereoselectivity in the 5-exo-
trig cyclization reaction, allowing the enantioselective synthesis
of a variety of β-chiral cyclopentanones (Scheme 1). Moreover,
we also developed the challenging asymmetric intermolecular
radical C−C bond-forming reactions using wild-type NCR as
the biocatalyst, offering a valuable option for the efficient
preparation of γ-chiral ketones with high optical purity
(Scheme 2). While the β-chiral center of cyclopentanone
product was constructed by the C−C bond-forming step in the
cyclization catalyzed by NCR-C9, the γ-stereocenter of
intermolecular hydroalkylation products produced by wild-
type NCR was set via a stereoselective HAT process, thus
demonstrating the versatile roles of the enzymes with
unparalleled capability in controlling stereoselectivity (SI
Figure 5). Overall, our study showcases how the non-natural
reactivities of native EREDs can be exploited, which also can
be further enhanced by protein engineering, if needed, to
expand the biocatalyst toolbox and address the long-standing
selectivity challenge in radical chemistry. We foresee that
EREDs and other flavin-dependent enzymes can be further
leveraged to generate new synthetically applicable reactivities;
this is particularly true as other fields, such as photo-
catalysis,16−18,29,32 are increasingly merging with biocataly-
sis.33,34
Figure 3. Deuterium labeling experiments. (A) Deuterium labeling
experiments for the cyclization mediated by NCR-C9. (B) Deuterium
labeling experiments for the intermolecular hydroalkylation catalyzed
by wild-type NCR. (C) α-Methylstyrene (7a) affects the depletion of
α-bromoacetophenone (6a) in NCR-catalyzed reaction. For exper-
imental details, see SI Figures 6−15.
be rationalized by deuterium atom transfer being competitive
with the deuterium exchange at the flavin N5 position with
H2O buffer, suggesting a possible kinetic isotope effect in the
radical termination event (SI Figure 12).31
For the intermolecular hydroalkylation catalyzed by wild-
type NCR, when flavin was labeled in situ using d1-glucose and
GDH, the γ-chiral ketone product 8a was formed in 42% yield
with 99:1 er and 80% deuterium incorporation at the benzylic
position (Figure 3B). Additionally, when this reaction was
carried out in a deuterated buffer, the yield of product 8a
improved to 94% (99:1 er), but hardly any deuterium
incorporation at the benzylic position of 8a was observed
(Figure 3B). Collectively, these results suggest the benzylic
radical is primarily terminated via HAT from flavin (FMNsq),
which also set the γ-stereocenter of the hydroalkylation
products (SI Figure 5B).
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/jacs.1c04334.
Finally, we were interested in whether the presence of alkene
influenced radical formation. This was determined by
measuring the consumption of α-bromo acetophenone 6a
Detailed experimental procedures, characterization data
(NMR spectra and HPLC traces) (PDF)
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Reactions Involving Radical Intermediates. Nat. Chem. 2020, 12,
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AUTHOR INFORMATION
■
Corresponding Author
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Processes. Chem. Rev. 2003, 103, 3263−3295.
Todd K. Hyster − Department of Chemistry, Princeton
University, Princeton, New Jersey 08544, United States;
Department of Chemistry and Chemical Biology, Cornell
University, Ithaca, New York 14850, United States;
orcid.org/0000-0003-3560-355X; Email: thyster@
cornell.edu
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Electron Transfer Mechanisms to Access New Enzymatic Functions.
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Authors
Haigen Fu − Department of Chemistry, Princeton University,
Princeton, New Jersey 08544, United States; Department of
Chemistry and Chemical Biology, Cornell University, Ithaca,
New York 14850, United States
Heather Lam − Department of Chemistry, Princeton
University, Princeton, New Jersey 08544, United States;
Department of Chemistry and Chemical Biology, Cornell
University, Ithaca, New York 14850, United States
Megan A. Emmanuel − Department of Chemistry, Princeton
University, Princeton, New Jersey 08544, United States
Ji Hye Kim − Department of Chemistry, Princeton University,
Princeton, New Jersey 08544, United States
(13) Sandoval, B. A.; Hyster, T. K. Emerging Strategies for
Expanding the Toolbox of Enzymes in Biocatalysis. Curr. Opin.
Chem. Biol. 2020, 55, 45−51.
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Engineering, and Applications of Ene-Reductases for Industrial
Biocatalysis. ACS Catal. 2018, 8, 3532−3549.
(15) Winkler, C. K.; Faber, K.; Hall, M. Biocatalytic Reduction of
Activated C = C-Bonds and Beyond: Emerging Trends. Curr. Opin.
Chem. Biol. 2018, 43, 97−105.
Braddock A. Sandoval − Department of Chemistry, Princeton
University, Princeton, New Jersey 08544, United States
(16) Biegasiewicz, K. F.; Cooper, S. J.; Gao, X.; Oblinsky, D. G.;
Kim, J. H.; Garfinkle, S. E.; Joyce, L. A.; Sandoval, B. A.; Scholes, G.
D.; Hyster, T. K. Photoexcitation of Flavoenzymes Enables a
Stereoselective Radical Cyclization. Science 2019, 364, 1166−1169.
(17) Black, M. J.; Biegasiewicz, K. F.; Meichan, A. J.; Oblinsky, D.
G.; Kudisch, B.; Scholes, G. D.; Hyster, T. K. Asymmetric Redox-
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Complete contact information is available at:
https://pubs.acs.org/10.1021/jacs.1c04334
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
(18) Clayman, P. D.; Hyster, T. K. Photoenzymatic Generation of
Unstabilized Alkyl Radicals: An Asymmetric Reductive Cyclization. J.
Am. Chem. Soc. 2020, 142, 15673−15677.
This work was supported by the National Institutes of Health
(NIH) National Institute of General Medical Sciences
(NIGMS) (R01 GM127703) and the Princeton Catalysis
Initiative. H.L. acknowledges the BioLEC Distinguished
Postdoctoral Fellowship Program for support. The authors
thank Dr. Yucong Zheng for his insightful discussions and
assistance with docking. The authors also thank Josh Turek-
Herman for generously providing the NCR single-site library,
Jacob DeHovitz for sharing reference compounds, Jingzhe Cao
for sharing intermediates, and Simon Charnock (Prozomix) for
generously providing their ERED library. Dr. Ivan Keresztes,
Anthony M. Condo, and Dr. John Eng are acknowledged for
their help for measuring HRMS.
(19) Ye, Y.; Fu, H.; Hyster, T. K. Activation Modes in Biocatalytic
Radical Cyclization Reactions. J. Ind. Microbiol. Biotechnol. 2021, 021
DOI: 10.1093/jimb/kuab021.
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Methods Driven by the Photoactivity of Electron Donor-Acceptor
Complexes. J. Am. Chem. Soc. 2020, 142, 5461−5476.
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