Quaternary Charge-Transfer Complex Enables Photoenzymatic Intermolecular Hydroalkylation of Olefins

Article abstract of DOI:10.1021/jacs.0c11462

Intermolecular C-C bond-forming reactions are underdeveloped transformations in the field of biocatalysis. Here we report a photoenzymatic intermolecular hydroalkylation of olefins catalyzed by flavin-dependent 'ene'-reductases. Radical initiation occurs via photoexcitation of a rare high-order enzyme-templated charge-transfer complex that forms between an alkene, α-chloroamide, and flavin hydroquinone. This unique mechanism ensures that radical formation only occurs when both substrates are present within the protein active site. This active site can control the radical terminating hydrogen atom transfer, enabling the synthesis of enantioenriched γ-stereogenic amides. This work highlights the potential for photoenzymatic catalysis to enable new biocatalytic transformations via previously unknown electron transfer mechanisms.

Full text of DOI:10.1021/jacs.0c11462

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Quaternary Charge-Transfer Complex Enables Photoenzymatic
Intermolecular Hydroalkylation of Olefins
Claire G. Page, Simon J. Cooper, Jacob S. DeHovitz, Daniel G. Oblinsky, Kyle F. Biegasiewicz,
Alyssa H. Antropow, Kurt W. Armbrust, J. Michael Ellis, Lawrence G. Hamann, Evan J. Horn,
Kevin M. Oberg, Gregory D. Scholes, and Todd K. Hyster*
Cite This: J. Am. Chem. Soc. 2021, 143, 97−102
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ABSTRACT: Intermolecular C−C bond-forming reactions are underdeveloped transformations in the field of biocatalysis. Here we
report a photoenzymatic intermolecular hydroalkylation of olefins catalyzed by flavin-dependent ‘ene’-reductases. Radical initiation
occurs via photoexcitation of a rare high-order enzyme-templated charge-transfer complex that forms between an alkene, α-
chloroamide, and flavin hydroquinone. This unique mechanism ensures that radical formation only occurs when both substrates are
present within the protein active site. This active site can control the radical terminating hydrogen atom transfer, enabling the
synthesis of enantioenriched γ-stereogenic amides. This work highlights the potential for photoenzymatic catalysis to enable new
biocatalytic transformations via previously unknown electron transfer mechanisms.
ntermolecular C−C bond-forming reactions are essential
Itools for the construction of societally valuable organic
molecules.1 Enzymes are attractive catalysts for these trans-
formations because of their ability to control reactive
intermediates with unique precision. Unfortunately, the
scope of natural C−C bond-forming enzymatic reactions is
limited to relatively few retrosynthetic disconnections and
often displays limited substrate promiscuity.1d This substrate
specificity results from the requirement that two substrates
bind and form reactive intermediates simultaneously within a
protein active site. While some enzymes ensure colocalization
through intricate gating mechanisms,2 the most substrate
promiscuous enzymes form long-lived reactive intermediates or
assume reactive conformations with substrates displaying a
high affinity for the protein active site. This approach enables
coupling with a reasonably broad collection of substrates that
possess only modest active site affinities. This catalytic strategy
is central to many of the most commonly used C−C bond-
forming enzymes, such as aldolases (Figure 1A),1d,3
Figure 1. Mechanisms of enzymatic intermolecular C−C bond
formation.
carboligases,4 and artificial metalloenzymes.5 While long-lived
intermediates are amenable to this approach, it is incompatible
with more transient species.
engage in various intramolecular C−C bond-forming reactions,
often with high levels of enantio- and diastereoselectivity.
Organic radicals are versatile intermediates capable of
Given the variety of radical cyclizations available to these
undergoing a variety of synthetically valuable chemical
enzymes, we questioned whether they could also catalyze
reactions.6 However, these species can be challenging to
intermolecular reductive coupling with alkenes.9 Such a
utilize for radical biocatalytic reactions owing to their short
lifetimes. Our group has recently developed modular
coupling must compete with undesired hydrodehalogenation
mechanisms for forming radical intermediates within enzyme
Received: October 31, 2020
Published: December 28, 2020
active sites to address long-standing selectivity challenges in
the radical literature.7 During this time, we found that
organohalides will form charge-transfer (CT) complexes with
flavin hydroquinone (FMNhq) within the active sites of “ene”-
reductases (EREDs).8 Upon photoexcitation, both nucleo-
philic and electrophilic radical intermediates are generated that
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if radical formation occurs in the absence of the coupling
partner (Figure 1B). As EREDs are only known to possess a
single substrate-binding site, it was unclear at the onset how
gating of radical formation would occur.10,11
400 nm, consistent with the absorption features of FMNhq
(Figure 2A). Upon the addition of chloroamide 1, a new
We initiated our studies by exploring the coupling of N,N-
dimethylchloroacetamide 1 with three and a half equivalents of
α-methylstyrene 2 under cyan light irradiation (λmax = 497 nm)
with a small collection of EREDs (Table S1). In a concurrent
study by Huang et. al, this substrate was reported to be
unreactive.9 We found that many EREDs can catalyze the
coupling reaction with only trace formation of the undesired
hydrodehalogenated product 4. The ERED from Gluconobacter
oxidans, with a previously identified beneficial mutation
(GluER-T36A), afforded amide 3 in 80% yield with high
levels of enantioselectivity for the R-enantiomer (Table 1,
Table 1. Enzyme Screen
entry
ERED
yield (%)
enantiomeric ratio (e.r.)
a
1
GluER-T36A
NostocER
GluER-T36A
NostocER
GluER-T36A
GluER-T36A
80
50
99
96
97:3
10:90
99:1
10:90
99:1
99:1
a
2
b
3
b
4
c
d
5
77 (61)
e
6
87
a
1 (10.0 μmol, 2.0 mg), “ene”-reductase (0.050 μmol), NADP+ (0.10
μmol), GDH-105 (0.5 mg/rxn), glucose (60 μmol), Tris (100 mM,
b
pH = 9.0), 36 h, 25 °C. 1 (20.0 μmol, 4.0 mg), “ene”-reductase (0.10
μmol), NADP+ (0.10 μmol), GDH-105 (1.0 mg/rxn), glucose (60
μmol), Tris (100 mM, pH = 9.0), DMSO (10%(v/v)), 24 h, 25 °C
c
d
Optimized conditions with cell-free lysate. Preparative gram scale
e
reaction using cell-free lysate. Preparative scale reaction run on 0.2
Figure 2. Mechanistic information.
mmol scale, 40.0 mg using purified enzyme.
entry 1). The S-enantiomer of product 3 can be accessed using
the homologue from N. punctiforme (NostocER) (Table 1,
entry 2). Reaction optimization revealed that both enzymes
could catalyze the transformation in near quantitative yield by
adding 10% (v/v) DMSO (Table 1, entries 3 and 4). Under
these conditions, reactions could be carried out with as little as
0.5 mol % lyophilized enzyme with little change in the yield
(Table S3). The reaction can be run with either purified
protein or lyophilized cell-free lysate, providing identical levels
of enantioselectivity, with only a modest decrease in yield
when run on a preparative scale, highlighting the trans-
formation’s robust nature (Table 1, entries 5 and 6).12 Overall,
this reaction demonstrates the ability of EREDs to precisely
control hydrogen atom transfer to prochiral radicals, a distal
site that has proven challenging to control with conventional
catalytic methodologies.13
The most striking feature of this reaction was the lack of
hydrodehalogenated product 4, the expected product if alkene
is not present within the protein active site during radical
formation.14 A series of mechanistic experiments were
conducted to understand the origin of this selectivity. We
began by probing whether a CT complex was responsible for
radical formation. When GluER-T36A was reduced with
sodium dithionite, negligible absorption was observed above
absorption feature is observed at 500 nm. This feature is less
pronounced when compared to amides containing tethered
alkenes.8 Addition of α-methylstyrene 2 to the ternary CT
complex of GluER-T36A, FMNhq, and chloroamide 1
produced a new absorption feature at 380 and 500 nm.
When this mixture was passed through molecular weight cutoff
filters to remove all small molecules, the absorption feature is
lost, and FMNhq was reformed (Supplemental Figure 4).
Nearly identical spectral features were observed when the same
experiments were run with NostocER, and no CT complex was
observed in the absence of protein (Supplemental Figures 6
and 8).
These experiments provide strong evidence that a
quaternary CT complex between the protein, FMNhq,
chloroamide 1, and α-methylstyrene 2 is responsible for the
observed reactivity. This unexpected complex provides an
explanation for the lack of hydrodehalogenated product, as
radical formation is largely limited to the situation where both
substrates are present within the enzyme active site. Moreover,
the similarity between the absorption features of flavin
semiquinone (FMNsq) and the quaternary CT complex implies
a large degree of charge transfer in the ground state. Docking
both substrates into the enzyme active site provided a potential
binding pose, with TD-DFT calculations supporting the
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Figure 3. Substrate scope.
increased absorptivity of the quaternary complex by compar-
ison to the ternary one lacking alkene (Figure 2a and
Supplemental Figures 41 and 43). This implies that radical
formation is gated by a rare quaternary CT complex.15,16
Transient absorption spectroscopy was conducted to better
understand charge-transfer dynamics in the presence and
absence of styrene (Figure 2B). When GluER is reduced with
dithionite and chloroamide 1 is added to the mixture, we
observe a charge-transfer state with a lifetime of 9 ps that
decays to the flavin quinone with a quantum yield of 8%.17 In
contrast, when the same experiment is run in the presence of
α-methylstyrene, the lifetime of the charge-transfer state is 5 ps
but decays instead to the flavin semiquinone, which exhibits a
lifetime of 45 ps before ultimately forming flavin quinone on a
300 ps time scale. The quantum yield of this process was
significantly higher at 12%. These results suggest that α-
methylstyrene facilitates C−Cl mesolytic cleavage for produc-
tive chemistry.18
With an understanding of both the mechanism of radical
formation and the lifetime of various intermediates, we next
focused on the mechanism of radical termination.8 A series of
isotope incorporation experiments were conducted with both
GluER-T36A and NostocER. When reactions were run with
glucose-1-d1, leading to deuteration of the flavin N5-position,
with GluER-T36A, 93% deuterium incorporation was observed
at the γ-position of the product (Figure 2C). In contrast, when
reactions are run with glucose-1-H1 in deuterated buffer, to in
situ label tyrosine OH protons, <5% deuterium incorporation
is observed. These results suggest that with GluER-T36A,
radical termination occurs almost exclusively through hydrogen
atom transfer from FMNsq.
isotopically labeled flavin, only 75% deuterium incorporation
was observed at the γ-position of the product, while the
experiments with deuterated buffer afforded 21% deuterium
incorporation (Figure 2C). These experiments suggest radical
termination via hydrogen atom transfer from flavin and
tyrosine are competitive.19 These competing mechanisms of
radical termination potentially account for the lower levels of
enantioselectivity observed with NostocER. We hypothesized
that mutation of these tyrosines to phenylalanine would shut
down the tyrosine termination pathway to favor hydrogen
atom transfer from flavin. We selected two tyrosines within the
active site and found that mutation of Y219 to phenylalanine
(Y219F) afforded a variant that provided improved yields and
enantioselectivities (Table S5). Re-evaluating the degree of
deuterium incorporation for this mutant revealed the
undesired tyrosine termination pathway was significantly
decreased20 (Figure 2c).
With an improved mechanistic understanding in hand, we
explored the transformation’s scope and limitations (Figure 3).
A variety of tertiary amides are tolerated by the reaction, with
pyrrolidine, Weinreb, and benzyl amides affording products in
high yields and enantioselectivities (Figure 3, 16−18). In
general, GluER-T36A afforded products with higher levels of
enantioselectivity than NostocER. Pleasingly, α,α-difluoro-
chloroamides are tolerated and afford products in high yield
and enantioselectivity with NostocER with no observed
defluorination (Figure 3, 19). Secondary amides are effective
(Figure 3, 20), while primary amides were less reactive
(Supplemental Figure 2). Finally, α-chloroacetophenone was
an effective coupling partner, providing product in high yield
and selectivity with GluER-T36A (Figure 3, 21).21 UV−vis
experiments to probe the spectral features of a possible CT
complex were complicated by ground state oxidation of flavin
NostocER provided different results when the same
experiments were conducted. When reactions were run with
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hydroquinone (Supplemental Figure 6). As visible light
irradiation affords higher yields than reactions run in the
dark, it is possible that a CT complex is formed but is obscured
by competing ground state reduction of the substrate.
to provide other useful compounds. We found that when using
α-bromostyrene with N,N-dimethylchloroamide, a lactone is
formed in high yields with excellent levels of enantioselectivity
(Figure 5, 34). This likely forms via initial formation of the
Next, we explored the scope of the alkene coupling partner
with the α-chloroamide. Despite their differing impact on the
electronics of the styrenyl alkene, methoxy substituents at the
para-, meta-, and ortho- positions are well tolerated and afford
high yields and selectivity when using NostocER (Figure 3,
22a−e). Electron-withdrawing substituents, such as bromide
and trifluoromethyl, are also accepted with products formed in
excellent yields and selectivity.22 We found that larger aliphatic
groups at the α-position are accommodated, providing
products with excellent enantioselectivity (Figure 3, 23a,b).
Trifluoromethyl groups are also compatible, although yields
are more modest (Figure 3, 23c).23 Ester substituents are also
tolerated, affording products in high yields with good levels of
enantioselectivity (Figure 3, 23d). Finally, unsubstituted
styrene is reactive (Figure 3, 24).
Figure 5. Lactone and ketone synthesis.
Beyond simple styrenyl alkenes, we found that this chemistry
accommodates a broad range of alkenes (Figure 4). For
coupled γ-bromo product followed by intramolecular SN2
cyclization. We also found that electron-rich enamides can
function as coupling partners to generate in situ formation of
ketones, providing an effective method for preparing 1,4-
dicarbonyls (Figure 5, 35). This product is likely formed from
oxidation of the α-amido radical via the intermediate
semiquinone followed by hydrolysis.
In conclusion, we have discovered an effective method for
preparing γ-stereogenic amides using photoenzymatic catalysis
and other coupled products. This represents a non-natural
mechanism of intermolecular C−C bond formation that we
anticipate being useful in preparing various synthetically
valuable motifs. The unique quaternary CT complex provides
an unprecedented mechanism for gating radical formation.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/jacs.0c11462.
■
sı
Experimental procedures and characterization data,
including supplemental Figures 1−43 and Supplemental
Tables S1−S6 (PDF)
AUTHOR INFORMATION
Corresponding Author
■
Figure 4. Nonstyrenyl alkenes.
Todd K. Hyster − Department of Chemistry, Princeton
University, Princeton, New Jersey 08544, United States;
orcid.org/0000-0003-3560-355X; Email: thyster@
princeton.edu
instance, these enzymes are tolerant of electron-deficient
heterocycles such as pyridines and pyrazine (Figure 4, 25−27).
When using α-methylvinylpyridines, the product is afforded in
high yields with excellent levels of enantioselectivity.
Interestingly, NostocER can differentiate sterically similar
phenyl and pyridine rings to furnish products with promising
levels of enantioselectivity. Beyond aromatic substituents,
aliphatic alkenes are also tolerated. Protected allylic amines
and unprotected allylic alcohols are also reactive, with
NostocER providing the best yields (Figure 4, 28 and 29).
Finally, enamides, vinyl ethers, and vinyl acetates are
competent coupling partners (Figure 4, 30−33).
Authors
Claire G. Page − Department of Chemistry, Princeton
University, Princeton, New Jersey 08544, United States
Simon J. Cooper − Department of Chemistry, Princeton
University, Princeton, New Jersey 08544, United States;
orcid.org/0000-0003-1465-5879
Jacob S. DeHovitz − Department of Chemistry, Princeton
University, Princeton, New Jersey 08544, United States;
orcid.org/0000-0001-9994-6311
Daniel G. Oblinsky − Department of Chemistry, Princeton
University, Princeton, New Jersey 08544, United States;
orcid.org/0000-0001-7460-8260
To further demonstrate the synthetic utility of this reaction,
we explored intermediates or products that could be diverted
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Kyle F. Biegasiewicz − Department of Chemistry, Princeton
University, Princeton, New Jersey 08544, United States;
orcid.org/0000-0003-1905-1919
Alyssa H. Antropow − Bristol Myers Squibb, Cambridge,
Massachusetts 02140, United States
Kurt W. Armbrust − Bristol Myers Squibb, Cambridge,
Massachusetts 02140, United States
J. Michael Ellis − Bristol Myers Squibb, Cambridge,
Massachusetts 02140, United States
Lawrence G. Hamann − Takeda Pharmaceuticals,
Cambridge, Massachusetts 02139, United States
Evan J. Horn − Bristol Myers Squibb, San Diego, California
92121, United States
Kevin M. Oberg − Bristol Myers Squibb, San Diego,
California 92121, United States
Gregory D. Scholes − Department of Chemistry, Princeton
University, Princeton, New Jersey 08544, United States;
orcid.org/0000-0003-3336-7960
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Complete contact information is available at:
https://pubs.acs.org/10.1021/jacs.0c11462
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Reaction optimization and substrate scope explorations were
supported by the National Institutes of Health (NIH) National
Institute of General Medical Sciences (NIGMS) (R01
GM127703) and Bristol-Myers-Squibb as part of the Princeton
Catalysis Initiative (T.K.H.). Mechanistic experiments, includ-
ing and transient absorption and UV−vis spectroscopy, were
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.
We acknowledge the MacMillan Group for the use of their
instrumentation. D.G.O. acknowledges support from the
Postgraduate Scholarships Doctoral Program of the Natural
Sciences and Engineering Research Council of Canada. C.G.P.
acknowledges the NSF-GRFP for support. J.S.D. acknowledges
support from the Bristol-Myers-Squibb Graduate Fellowship.
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D.; Hyster, T. K. Photoexcitation of flavoenzymes enables a
stereoselective radical cyclization. Science 2019, 364, 1166−1169.
(b) Clayman, P. D.; Hyster, T. K. Photoenzymatic Generation of
Unstabilized Alkyl Radicals: An Asymmetric Reductive Cyclization. J.
Am. Chem. Soc. 2020, 142, 15673−15677.
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Biedermann, F.; Scherman, O. A. Cucurbit[8]-uril Mediated Donor-
Acceptor Ternary Complexes: A Model System for Studying Charge-
Transfer Interactions. J. Phys. Chem. B 2012, 116, 2842−2849.
(16) A competition experiment between p-Me-α-methyl styrene and
p-CF3-α-methyl styrene afforded almost exclusive formation of the p-
Me with both NostocER and GluER-T36A, suggesting more electron
rich alkenes more readily form quaternary CT complexes or that the
corresponding CT complex is more reactive. See Supplemental Figure
34.
(17) Experiments of irradiating the ERED with just chloroamide
show flavin alkylation (Supplemental Figures 38 and 39). When
alkene is added, the formation of alkylated flavin is limited. This
suggests this is a catalyst degradation pathway.
(18) Similar results were observed with NostocER (Supplemental
Figure 5).
(19) (a) Nizam, S.; Gazara, R. K.; Verma, S.; Singh, K.; Verma, P. K.
Comparative Structural Modeling of Six Old Yellow Enzymes (OYEs)
from the Necrotrophic Fungus Ascochyta rabiei: Insight into Novel
OYE Classes with Differ-ences in Cofactor Binding, Organization of
Active Site Resi-dues and Stereopreferences. PLoS One 2014, 9,
No. e95989. (b) Chaparro-Riggers, J. F.; Rogers, T. A.; Vazquez-
Figueroa, E.; Polizzi, K. M.; Bommarius, A. S. Comparison of Three
Enoate Reductases and their Potential Use for Biotransformations.
Adv. Synth. Catal. 2007, 349, 1521−1531.
(20) The lack of complete suppression of deuterium incorporation
when the reaction is run in D2O suggests additional active site
tyrosines also act as competitive hydrogen atom sources, likely with
the same facial selectivity as the flavin.
(21) Reaction in the dark with GluER T36A affords a product in 3%
yield with an additional hydrodehalogenated product being formed
(Supplemental Figure 3).
(22) Mutating tyrosine 219, the residue we hypothesize to function
as a competitive hydrogen atom source, to phenylalanine (NostocER-
Y219F) can provide improved enantioselectivity.
(23) A small amount of defluorinated product is observed,
potentially formed via a reduction/elimination mechanism.
102
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