Photoenzymatic Reductions Enabled by Direct Excitation of Flavin-Dependent "Ene"-Reductases

Article abstract of DOI:10.1021/jacs.0c11494

Non-natural photoenzymatic reactions reported to date have depended on the excitation of electron donor-acceptor complexes formed between substrates and cofactors within protein active sites to facilitate electron transfer. While this mechanism has unlocked new reactivity, it limits the types of substrates that can be involved in this area of catalysis. Here we demonstrate that direct excitation of flavin hydroquinone within "ene"-reductase active sites enables new substrates to participate in photoenzymatic reactions. We found that by using photoexcitation these enzymes gain the ability to reduce acrylamides through a single electron transfer mechanism.

Full text of DOI:10.1021/jacs.0c11494

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Photoenzymatic Reductions Enabled by Direct Excitation of Flavin-
Dependent “Ene”-Reductases
Braddock A. Sandoval,§ Phillip D. Clayman,§ Daniel G. Oblinsky, Seokjoon Oh, Yuji Nakano,
Matthew Bird, Gregory D. Scholes, and Todd K. Hyster*
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ABSTRACT: Non-natural photoenzymatic reactions reported to date have depended on the excitation of electron donor−acceptor
complexes formed between substrates and cofactors within protein active sites to facilitate electron transfer. While this mechanism
has unlocked new reactivity, it limits the types of substrates that can be involved in this area of catalysis. Here we demonstrate that
direct excitation of flavin hydroquinone within “ene”-reductase active sites enables new substrates to participate in photoenzymatic
reactions. We found that by using photoexcitation these enzymes gain the ability to reduce acrylamides through a single electron
transfer mechanism.
hotoinduced electron transfer (PET) is a common
Pmechanism to facilitate electron transfer events that
would be inaccessible to molecules in their ground state. This
activation mode has been broadly applied in fields as impactful
and diverse as water splitting, organic synthesis, and photo-
voltaics.1 In the realm of biological systems, PET is responsible
for the activities associated with photosynthetic complexes and
light-oxygen-voltage sensing domains, yet it is rarely employed
in biocatalytic transformations. Indeed, only one of the three
classes of photoenzymes found in nature has shown any
synthetic potential.2,3
Our group has targeted PET as a mechanism to expand the
synthetic capabilities of substrate promiscuous enzymes.4 In
previous studies, we found that flavin-dependent “ene”-
reductases (EREDs) can catalyze radical dehalogenations and
cyclizations with high levels of stereoselectivity.5 In these
studies, radical formation occurred via photoexcitation of
ternary charge-transfer (CT) complexes formed between the
flavin hydroquinone (FMNhq) and electron deficient substrates
within the protein active site (Figure 1A).6 While this
mechanism of PET localizes the radical within the enzyme, it
also limits the types of substrates that can function as radical
precursors. Specifically, we have only observed radical formation
using alkyl chlorides, bromides, iodides, and redox-active esters.
To overcome this limitation, new photoenzymatic mechanisms
for radical formation need to be developed. For instance, direct
Figure 1. Photoenzymatic catalysis and biocatalytic reductions.
excitation of the flavin cofactor would enable high reduction
potentials to be accessed. DNA photolyase, a photoenzyme that
repairs pyrimidine dimer lesions formed on DNA, relies on the
photoexcited state of flavin hydroquinone to initiate DNA repair
via single electron reduction of its substrate. While this
mechanism is, in theory, available to all flavoproteins, no others
Received: November 1, 2020
Published: December 31, 2020
are known to utilize it. We recognized that if this mechanism
could be achieved with EREDs, it could function as a substrate
agnostic approach for generating a variety of radical
intermediates (Figure 1B).7
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The challenge inherent to all chiral photocatalysts is ensuring
that radical formation only occurs within the chiral environment
of the catalyst.8 To ensure binding, we targeted substrates that
closely resemble established substrates for EREDs. Inspired by
Pac et al.’s pioneering study of single electron reduction of
enones using photoredox catalysts,9 we questioned whether
EREDs could reduce α,β-unsaturated amides via direct
photoexcitation of FMNhq.10 While EREDs are known for
their ability to reduce electronically activated alkenes using a
hydride transfer mechanism, they are not known to reduce
moderately electrophilic amides (Figure 1C).11,12 We hypothe-
sized that a PET mechanism would unlock this elusive reactivity,
allowing for the generation of radical intermediates from simple
olefin precursors (Figure 1D).
Next, we irradiated the same panel of enzymes with a 52 W violet
LED whose emission window overlaps with the absorption of
FMNhq. Under these conditions, all enzymes provided the
reduced product, with OYE1 providing the highest levels of
enantioselectivity (Table 1, entry 5). While control experiments
confirmed that each component of the reaction was essential for
reactivity, they did reveal that FMN in the absence of protein
could catalyze the reaction, albeit in low yield and with no
enantioselectivity (Table 1, entry 7). Optimization of the
reaction with OYE1 resulted in improved yield upon addition of
FMN (1 mol%) in tricine buffer at pH 9.0 (Table 1, entry 8).
Site-saturation mutagenesis of residues lining the active site
revealed that mutation of phenylalanine 296 to glycine (OYE1-
F296G) increased catalyst efficiency, providing product in 97%
yield with 90:10 er (Table 1, entry 9).14
We conducted mechanistic experiments to gain a better
understanding of the reaction. The intermediacy of a CT
complex was ruled out due to a lack of spectral changes in the
UV−vis spectrum upon addition of 1a to reduced OYE1-F269G
(Supplemental Figure S7). Transient absorption spectroscopy
was conducted to probe the mechanism and determine the
lifetime of radical intermediates. These studies were conducted
using OYE1-F269G and allyl amide 7a due to the superior
solubility of this substrate. As an initial control, we tested
whether amide 7a quenched the excited state of free FMNhq in
the absence of protein. When FMN is reduced with sodium
dithionite and excited at 370 nm, the excited state hydroquinone
lifetime is 55 ps. This lifetime decreased to 50 ps in the presence
of substrate indicating that substrate reduction can occur in
solution, consistent with the control experiments (Supplemental
Figure S9).
We explored the viability of this mechanism on the
asymmetric reduction of acrylamide 1a with a panel of six
different EREDs (Table 1). In the absence of light, none of these
enzymes were able to reduce the amide, consistent with our
understanding of the substrate preference for these enzymes.13
Table 1. Enzyme Screen
a
b
c
c
entry
ERED
GluER
MorB
dark
violet LEDs
1
2
3
4
5
6
7
N.R.
N.R.
N.R.
N.R.
N.R.
N.R.
N.R.
N.R.
N.R.
93%, 27:73 er
96%, 31:69 er
92%, 81:19 er
79%, 80:20 er
68%, 91:9 er
66%, 57:43 er
19%, 50:50 er
90%, 89:11 er
97%, 90:10 er
NostocER
PETNr
OYE1
YqjM
FMN
Similar studies were conducted with OYE1-F269G. When
reduced with NADPH and excited, a biexponential decay of 9.9
and 323 ps is observed in the absence of amide 7a
(Supplemental Figure S16). In the presence of 7a, the initial
hydroquinone excited state decays within 6.6 ps to one of two
states shown in Supplemental Figure S18. Physically, these two
states most likely arise from enzyme with or without substrate
bound. The unbound protein decays to the ground state with the
same 323 ps lifetime as that observed without substrate, whereas
the substrate bound protein decays to a spectrum corresponding
to the neutral semiquinone (FMNsqH). This species further
decays with a lifetime of 47 ps to an initial combination of
d
8
d
OYE1+FMN
OYE1-F269G
9
a
1a (10.0 μmol, 2.0 mg), “ene”-reductase (0.050 μmol), NADP+
(0.10 μmol), GDH-105 (0.25 mg/rxn), glucose (40 μmol), KPi (100
mM, pH = 8.0, 470 μL), i-PrOH (30 μL), total volume (0.5 mL), 24
b
h, 25 °C. Purified “ene”-reductase, full sequence information found
c
in the Supporting Information. LCMS assay yield, enantiomeric ratio
d
determined by HPLC. Reaction run with FMN (1 mol%) in tricine
buffer (100 mM, pH 9.0).
Figure 2. Mechanistic studies.
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FMNsqH and anionic semiquinone (FMNsq). Finally, these
species decay over 2.5 ns into a long-time component that
represents an equilibration of flavin semiquinones (Supplemen-
tal Figures S22 and S25). This combination of the two radical
flavin species survives for a period greater than 80 ns.
Collectively, these experiments indicate that the excited state
hydroquinone is capable of electron transfer through PET
enabled by direct excitation of the flavin cofactor.
radiolysis measurements indicate the reduction potential of the
substrate to be approximately −2.7 V vs Fc/Fc+ (see
Supplemental Figures S27−S29 and discussion). Based on the
excited state potential of FMNhq in DNA photolyase, electron
transfer is likely modestly endergonic.17 Protonation of the
radical anion results in the formation of a stabilized α-acyl radical
which can undergo enantioselective hydrogen atom transfer
from FMNsqH to generate the reduced product and reform
oxidized FMN.11 Reduction of the cofactor with NADPH
returns flavin to the hydroquinone state and restarts the catalytic
cycle.
With an understanding of the reaction mechanism in hand, we
explored the scope and limitations of this novel activation mode.
We found that the reaction is tolerant to a variety of cyclic and
acyclic amide substituents affording products in good yields and
enantioselectivities (Figure 3, 1b−6b). A secondary allyl amide
was reactive but afforded product with more modest levels of
The results from the transient absorption spectroscopy
experiments suggest a unique radical mechanism. Electron
transfer is the only feasible mechanism for forming FMNsqH,
which simultaneously results in the formation of a substrate-
centered radical anion. We anticipate this species to be rapidly
quenched via regioselective protonation to afford an α-acyl
radical. Consistent with this mechanistic hypothesis, when we
run a reaction in buffer containing 95% D2O, we observe 90%
deuterium incorporation at the β-position (Figure 2A). Our
previous studies with these enzymes indicate that radical
termination of α-acyl radicals occurs via enantioselective
hydrogen atom transfer from flavin. Surprisingly, when flavin
is isotopically labeled using d7-glucose, only 4% deuterium
incorporation is observed at the α-position (Figure 2A). In
contrast, reactions run in 95% D2O provide product with 58%
deuterium incorporation at that position. These results suggest
two possible mechanistic scenarios: (i) radical termination
occurs via an electron transfer/proton transfer (ET/PT)
mechanism or (ii) radical termination occurs via HAT with
the isotopic label at the N-5 position being exchanged during the
reaction. Incomplete deuterium incorporation at the α-position
supports the second mechanistic hypothesis.15 The high degree
of enantioselectivity observed for this reaction suggests a single
reaction mechanism, as competing mechanisms would presum-
ably afford decreased selectivity. A decreased rate of conversion
to product when using d7-glucose is further evidence of a HAT
radical termination mechanism rather than ET/PT (Supporting
Information S6). Finally, when a reaction is run with a 1:1 ratio
of H2O/D2O and d7-glucose, 29% deuterium incorporation is
observed at the α-position. This experiment suggests that the
degree of deuterium incorporation at the α-position is a function
of kinetic effects in the HAT step rather than kinetic effects that
change the termination mechanism.
These experiments suggest that radical termination occurs via
hydrogen atom transfer from flavin, but that the isotopic label at
the N-5 position is lost over the course of the reaction. As
isotope exchange is slow in the hydroquinone oxidation state, we
hypothesize that exchange must be occurring at the semi-
quinone oxidation state. The N-5 position of FMNsqH is known
to be acidic. Reversible protonation of the FMNsq would provide
a mechanism for isotopic label exchange on flavin. In our
previous studies, we observed radical lifetimes of <250 ps,
providing insufficient time for complete label exchange. In this
reaction, spectroscopic evidence for FMNsq and FMNsqH,
coupled with radical lifetimes in excess of 80 ns, provides a
rationale for exchanging the label on flavin. We hypothesize that
this extended lifetime is due to the nature of the radical
intermediate being formed. Related species are known to have
persistent radical character, making them significantly less
reactive.16
In summary, we propose the following reaction mechanism.
Photoexcitation of FMNhq yields a highly reducing singlet
excited state of the anionic hydroquinone. Electron transfer to
the substrate occurs within picoseconds to generate a radical
anion with anionic character primarily at the β-position. Pulse
Figure 3. Scope exploration.
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enantioselectivity (7b). Trisubstituted alkenes were reactive, but
afforded products with poor enantioselectivities (8b−10b).
Interestingly, a tetra-substituted alkene (20a) provided only
trace product. The presence of an aryl substituent was essential
for achieving reactivity (19a), but the substituent could be
located at either the α- or β-position (11b and 12b).18 With
regard to aromatic substituents, the reaction is tolerant of
aliphatic substituents (13b−15b) and a surprising amount of
steric bulk (16b). However, heteroatom substituents signifi-
cantly decrease reactivity and selectivity (17b, 18b, and 21a).
Next, we explored whether this PET mechanism could be
applied to other types of difficult reductions. In particular, we
were interest in determining whether these enzymes could
catalyze defluorination reactions. C−F bonds represent some of
the strongest bonds in organic molecules, making them
challenging to cleave using traditional small molecule strategies.
Highly reducing photocatalysts have been demonstrated to
reduce these bonds, enabling them to function as radical
precursors.19 To explore whether EREDs could catalyze this
transformation, we prepared two α-fluoroamides. When
subjected to the photoenzymatic reaction conditions, these
amides were defluorinated in good yields and with enantiose-
lectivities that closely mirror those observed in the related alkene
reduction reaction (Figure 4). Interestingly, defluorination did
alized starting materials. Harnessing this neglected activation
mode allows enzymatic reduction of α,β-unsaturated amides,
which were previously considered to be an unreactive substrate
class for enzymatic reductions. We anticipate that this new
method for generating radical intermediates in ERED enzymes
could lead to a host of novel enzymatic transformations, thereby
fueling momentum in utilizing biocatalytic promiscuity for
organic synthesis.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/jacs.0c11494.
■
sı
Experimental procedures and characterization data,
including supplemental Figures S1−S29 (PDF)
AUTHOR INFORMATION
Corresponding Author
■
Todd K. Hyster − Department of Chemistry, Princeton
University, Princeton, New Jersey 08544, United States;
orcid.org/0000-0003-3560-355X; Email: thyster@
princeton.edu
Authors
Braddock A. Sandoval − Department of Chemistry, Princeton
University, Princeton, New Jersey 08544, United States
Phillip D. Clayman − Department of Chemistry, Princeton
University, Princeton, New Jersey 08544, United States
Daniel G. Oblinsky − Department of Chemistry, Princeton
University, Princeton, New Jersey 08544, United States;
orcid.org/0000-0001-7460-8260
Seokjoon Oh − Chemistry Division, Brookhaven National
Laboratory, Upton, New York 11973-5000, United States;
orcid.org/0000-0002-8980-5213
Yuji Nakano − Department of Chemistry, Princeton University,
Princeton, New Jersey 08544, United States
Matthew Bird − Chemistry Division, Brookhaven National
Laboratory, Upton, New York 11973-5000, United States;
orcid.org/0000-0002-6819-5380
Gregory D. Scholes − Department of Chemistry, Princeton
University, Princeton, New Jersey 08544, United States;
orcid.org/0000-0003-3336-7960
Figure 4. Defluorinations.
not require adjacent aromatic substituents, suggesting that
fluorine itself is sufficiently activating for reduction. As the
enantioselectivities closely mirror the selectivities observed for
related alkene reduction, we hypothesize that these reductions
similarly terminate via HAT from FMNsqH.
Finally, we looked to expand the synthetic applicability of this
reactivity to C−C bond formation. We previously found that
EREDs are capable of catalyzing cyclizations of α-chloroamides.
To our delight, α,β-unsaturated amides bearing a pendent
alkene can afford γ-lactams in good yields (Figure 5). These
Complete contact information is available at:
https://pubs.acs.org/10.1021/jacs.0c11494
Author Contributions
§B.A.S. and P.D.C. contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Figure 5. Radical cyclization.
This work was supported by the Division of Chemical Sciences,
Geosciences, and Biosciences, Office of Basic Energy Sciences of
the U.S. Department of Energy (DOE) through Grant DE-
SC0019370. D.G.O. acknowledges support from the Post-
graduate Scholarships Doctoral Program of the Natural Sciences
and Engineering Research Council of Canada.
types of highly substituted lactams are challenging to prepare
using traditional synthetic methods, particularly considering the
stability of the benzylic α-acyl radical. Moreover, this approach
avoids the need for organohalide functionalities that might be
unstable with certain substitution patterns.
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excitation of FMNhq in EREDs gives rise to a potent, enzyme-
bound, single electron reductant that can be used to carry out
asymmetric radical transformations using minimally function-
■
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