Photoenzymatic Generation of Unstabilized Alkyl Radicals: An Asymmetric Reductive Cyclization

Article abstract of DOI:10.1021/jacs.0c07918

Flavin-dependent "ene"-reductases can generate stabilized alkyl radicals when irradiated with visible light; however, they are not known to form unstabilized radicals. Here, we report an enantioselective radical cyclization using alkyl iodides as precursors to unstabilized nucleophilic radicals. Evidence suggests this species is accessed by photoexcitation of a charge-transfer complex that forms between flavin and substrate within the protein active site. Stereoselective delivery of a hydrogen atom from the flavin semiquinone to the prochiral radical formed after cyclization provides high levels of enantioselectivity across a variety of substrates. Overall, this transformation demonstrates that photoenzymatic catalysis can address long-standing selectivity challenges in the radical literature.

Full text of DOI:10.1021/jacs.0c07918

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Communication
Photoenzymatic Generation of Unstabilized Alkyl
Radicals: An Asymmetric Reductive Cyclization
Phillip D. Clayman, and Todd K. Hyster
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.0c07918 • Publication Date (Web): 28 Aug 2020
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Photoenzymatic Generation of Unstabilized Alkyl Radicals: An
Asymmetric Reductive Cyclization
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Phillip D. Clayman and Todd K. Hyster*
Department of Chemistry, Princeton University, Princeton, New Jersey 08544, United States
Supporting Information Placeholder
ABSTRACT: Flavin-dependent ‘ene’-reductases (ERED)
A. Enzymatic Charge-Transfer Complexes
can generate stabilized alkyl radicals when irradiated with
visible light, however, they are not known to form
unstabilized radicals. Here, we report an enantioselective
radical cyclization using alkyl iodides as precursors to
unstabilized nucleophilic radicals. Evidence suggests this
species is accessed by photoexcitation of a charge-
transfer complex that forms between flavin and substrate
within the protein active site. Stereoselective delivery of
a hydrogen atom from the flavin semiquinone to the
prochiral radical formed after cyclization provides high
levels of enantioselectivity across a variety of substrates.
Overall, this transformation demonstrates that
photoenzymatic catalysis can address long-standing
selectivity challenges in the radical literature.
acceptor
acceptor
Enzyme
donor
donor
Negligible Interact ions In Solution
(No CT Complex Format ion)
Enzyme-Stabilized CT
Complex Format ion
B. Radical Precursors for CT Complexes
Known Acceptor
Unknown Acceptor
O
Ep/2
-1.67 V v. SCE
O
Ep/2
-2.19 V v. SCE
Me
Me
Et
Et
N
R
N
R
I
Cl [known for EREDs]
[unknown]
▪ ︎Electrophilic Radical ▪︎Resonance Stabilized
▪ ︎Nucleophilic Radical ▪ ︎1° Alkyl Unstabilized
C. Asymmetric Addition of Radical to Michael Acceptors
EWG
EWG
EWG
EWG
H
Common
Elusive
Radicals are versatile intermediates that enable
retrosynthetic disconnections distinct from those
available to other reactive species. While the advent of
�
-Stereogenic
�-Stereogenic
Achiral Radical
Products
Products
1
D. Proposed - Photoenzymat ic Radical Cyclization Involving Unstabilized Radicals
O
modern methods of radical formation has spurred the
development of new chemical transformations, general
catalytic strategies for controlling the stereochemical
O
I
RO
RO
‘
Ene’-Reductase
Cyan Light
2
outcome of these reactions are underdeveloped. The lack
of control provided by small-molecule catalysts across a
range of radical elementary steps highlights the need for
new approaches to controlling this family of reactive
intermediates.
Photoinduced
Electron Transfer
Hydrogen
Atom Transfer
O
O
C– C Bond
Formation
RO
RO
FMNsq
FMNsq
Enzymes are attractive catalysts for asymmetric
synthesis because they can mediate reactions with
3
unparalleled levels of stereochemical control. This
Figure 1. Principals of Charge-Transfer Complexes and
Challenges Associated with Asymmetric Radical Conjugate
Additions.
feature has enabled them to address some of the most
4
daunting selectivity challenges in organic synthesis.
Despite significant recent progress, only a small number
of synthetic challenges have been tackled with enzymatic
catalysis due to a perceived inability to catalyze non-
In our previous studies, we found that flavin-dependent
ene’-reductases (EREDs) can catalyze radical reactions
with high levels of stereoselectivity using an electron
transfer mechanism that is distinct from the hydride
transfer responsible for their native reactivity.
‘
5
,6
natural reactions. Inspired by the selectivity challenges
inherent to non-natural radical reactions, our group has
sought to develop strategies for controlling radical
9
,10
7
,8
Specifically, we found that electron-deficient 훼-
chloroamides form charge-transfer (CT) complexes with
intermediates within enzyme active sites.
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the flavin hydroquinone (FMN ) within the ERED active
site. When this complex is irradiated with visible light, an
in order to avoid undesired reduction of the alkene of the
starting material, this tyrosine was mutated to
phenylalanine for a small panel of EREDs. When this
hq
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19
electron is promoted from flavin to the substrate,
generating an electrophilic radical. In general, the
strength of association in CT complexes is dependent on
the electron affinity of the acceptor, the degree of electron
transfer in the ground state, and a high degree of planarity
panel of mutated enzymes was provided 1a and irradiated
with a cyan LED (휆max = 497 nm), every enzyme afforded
the desired cyclized product 1b, demonstrating
promiscuous reactivity across a diverse set of EREDs
(Supplemental Figure 1). The variant from
Gluconobacter (GluER-Y177F) proved most effective,
providing 1b in good yield and enantioselectivity, and
with no observed hydrodehalogenated product (Table 1,
entry 1). Alternatively, the opposite enantiomer could be
obtained using OYE2-Y197F (Table 1, entry 2). No
reaction occurred in the absence of light and enzyme
(Table 1, entries 3-4).
in both the donor and acceptor to maximize orbital
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interactions.
Consequently, their use in organic
synthesis has been restricted to substrates with low
reduction potentials that often possess a large degree of
휋-conjugation, as these tend to favor the formation of CT
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complexes in solution. Our previous study suggested
that enzymes can overcome this requirement by forcing
molecular interactions between the electron donor
(
FMN ) and acceptor (substrate) that do not occur in
hq
solution (Figure 1A). In theory, this feature should enable
enzymes to use less activated substrates as radical
precursors while also providing a mechanism for
restricting radical formation to the protein active site.
Based on this understanding, we questioned whether a
similar mechanism could be employed to generate
nucleophilic radicals from unactivated alkyl iodides
TABLE 1. Asymmetric Reductive Radical Cyclization
entrya
variation
nonea
yield (%)
er
92:8
14:86
-
(Figure 1B).
1
2
56
41
0
The reaction of nucleophilic radicals with electrophilic
OYE2-Y197F
alkenes is one of the most widely utilized transformations
1
3
2
no GluER-Y177F
in the radical literature. While chiral Lewis acid and
amine catalysts have proven to be effective at setting the
훽-stereocenter through the controlled addition of achiral
radicals to the prochiral alkenes,14 they are largely
ineffective at setting the 훼-stereocenter via hydrogen
3
no light
0
-
4
FMN instead of GluER-Y177F
GluER instead of GluER-Y177F
freeze-pump-thaw degassing
degassing and 12 ºC
preparative scale reaction
0
-
5
47
90
92
90:10
92:8
92:8
92:8
6
1
5
atom transfer (HAT) (Figure 1C). In our previous
reports, we found that EREDs can catalyze a radical
hydrodehalogenation reaction with high levels of
enantioselectivity. Central to this reactivity is the
protein’s ability to control the stereoselective delivery of
7
8b
82
a
+
1
a (10 μmol), ‘ene’-reductase (0.15 μmol), NADP (0.2 μmol) GDH-105
(1 mg/rxn), glucose (60 μmol), KPi (100 mM, pH = 8.0, 900 μL), iPrOH
b
(
100 μL), 48 h, rt, anaerobic. Run on 100 mg scale.
a hydrogen atom from flavin semiquinone (FMN ) to
sq
2
prochiral 훼-acyl radicals. Inspired by the selectivity
In optimizing this reaction, several elements proved to
achieved in this step, we sought to develop a
photoenzymatic cyclization involving the addition of
nucleophilic radicals to Michael acceptors terminating in
enantioselective HAT.
be essential for achieving high and reproducible yields.
Notable is the conserved tyrosine mutation as wild type
GluER afforded product in lower yield and
enantioselectivity (Table 1, entry 5), consistent with
We targeted unactivated alkyl iodides as precursors to
8
results from our previous hydrodehalogenation.
1
6
nucleophilic alkyl radicals.
In contrast to 훼-
Moreover, oxygen provided to be detrimental to the
reaction, with degassed reactions providing product in
halocarbonyl compounds that possess low reduction
potentials and produce electrophilic radicals, unactivated
alkyl iodides are more challenging to reduce and have
20
improved yields (Table 1, entry 6). Finally, cooling the
reaction from room temperature to 12 ºC significantly
improved reproducibilityand provided a slight increase in
1
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lower electron affinities (Figure 1B). Consequently,
they are not known to form stable CT complexes with
21
yield (Table 1, entry 7). Under these conditions, the
1
8
common electron donors in solution. We reasoned that
substrates with alkyl iodides primed for intramolecular
Giese cyclizations would be capable of forming CT
complexes with flavin (Figure 1D).
reaction could be carried out on preparative scale and
isolated in nearly identical yields and enantioselectivity
(Table 1, entry 8).
Next, we conducted experiments to elucidate the
mechanism of this transformation. At the onset, we
hypothesized that electron transfer was occurring via
photoexcitation of a CT complex that forms between the
We initiated our studies by exploring the reductive
cyclization of alkyl iodide 1a to chiral lactone 1b
catalyzed by EREDs. Mutation of a conserved active site
tyrosine is known to inhibit ERED’s native reactivity, so
alkyl iodide 1a and FMN . To probe this possibility, we
hq
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analyzed our reaction mixture via UV-Vis. Under
isotopic labelling experiments. As a probe for the HAT
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reaction conditions, FMN is fully reduced to FMN , and
mechanism, flavin was labelled in situ using d glucose
hq
1-
in the absence of substrate, there is negligible absorption
around 500 nm. However, when substrate is added, a new
absorption feature appears with a maximum absorption at
and glucose dehydrogenase (GDH-105). When this
reaction was examined, the lactone product 1b was
observed in 90% yield with 92:8 er and 76% deuterium
incorporation, supporting HAT from flavin as the primary
mechanism of radical termination (Figure 2b).
Alternatively, when the standard reaction is carried out in
deuterated buffer, only 16% deuterium is observed in the
product (Figure 2b). Collectively, these results suggest
that 훼-stereocenter is set via HAT from flavin. Further, an
experiment with an 훼-cyclopropyl ester confirmed the
intermediacy of a radical while also suggesting that HAT
is rapid and competitive with opening of the cyclopropane
(Supplemental Figure 12). This contrasts the native
mechanism, where the 훼-stereocenter is set via
protonation by the conserved tyrosine following hydride
4
85 nm (Figure 2a), suggestive of the formation of a CT
complex. Importantly, the same spectral feature is not
observed in the absence of protein (Supplemental Figure
1
1), suggesting further that binding to the protein active
10,22
site is required for CT complex formation.
As no
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reaction is observed with a brominated analog of 1a, we
hypothesize that the CT complex forms between flavin
and the σ*C–I orbital rather than the 휋* of the 훼,훽-
unsaturated ester. Mechanistically, excitation of this
complex facilitates electron transfer from flavin to
substrate, which results in loss of iodide and the formation
of an unstabilized alkyl radical, which then cyclizes to
form a resonance stabilized 훼-acyl radical.
transfer from FMN . As the conserved tyrosine and FMN
hq
reside on opposite prochiral faces of the substrate, we
hypothesized that reduction of a structurally related
butenolide by wild-type enzyme should provide the
opposite enantiomer to the one obtained in the radical
cyclization. Consistent with this hypothesis, when the
butenolide reduction was conducted with wild-type
GluER, the reduced product was isolated as the (R)-
enantiomer (Figure 2c).
Figure 2. Mechanistic Experiments
We then conducted a series of experiments targeted at
determining the mechanism of termination for the 훼-acyl
radical. While we hypothesized that radical termination
occurs via HAT, we recognized that single electron
reduction to the corresponding enolate followed by
protonation would provide the same product. To
differentiate between these two pathways, we conducted
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a
Substrate (10 μmol), GluER-Y177F (0.15 μmol), NADP+ (0.2 μmol),
Moreover, while alkyl iodides were only modestly
effective for 6-exo-trig cyclizations, requiring high
catalyst loadings (3 mol %) to achieve acceptable yields,
the reaction can be accomplished using the corresponding
NHPI ester 21 at lower catalyst loadings with comparable
yields and levels of selectivity (Figure 4, 17b).
GDH-105 (1 mg/rxn), glucose (60 μmol), KPi (100 mM, pH = 8.0, 900 μL),
iPrOH (100 μL), 72 h, 12 °C, anaerobic. OYE3-Y197F used instead of
GluER-Y177F. ‘ene’-reductase (0.3 μmol. 3 mol %), NADP (0.4 μmol).
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+
Figure 3. Substrate Scope
Lastly, we questioned whether the 훼-acyl radical
intermediate formed during this reaction could be trapped
with alternative radical coupling partners to build further
molecular complexity. We recently demonstrated that
EREDs can catalyze reductive radical cyclizations using
훼-acyl radical intermediates. We reasoned that an enzyme
with a large active site could accommodate a substrate
primed for a polycyclization (Figure 4, 22a). Indeed,
morphinone reductase (MorB), can catalyze the
polycyclization of a substrate bearing a cinnamyl amide
in promising yields (Figure 4, 22b). This proof of concept
demonstrates the potential of EREDs to be applied to
complex polyene-cyclizations.
With a working understanding of the mechanism and
ideal conditions in hand, we explored the scope and
limitations of this reaction (Figure 3). We were pleased to
find that simple esters are effective substrates with
GluER-Y177F, providing product in good yields and
enantioselectivities (Figure 3, 2b-5b). In general, smaller
substituents (Figure 3, 2b-3b) afford product with slightly
attenuated enantiomeric ratios by comparison to larger
substituents (Figure 3, 4b-5b). Additional functional
handles such as an alkene and alkyl bromide are well
tolerated off the ester (Figure 3, 6b-7b). Importantly, all
these substrates lack secondary coordinating groups,
providing a synthetic benefit by comparison to traditional
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approaches using chiral Lewis acids. The reaction also
accommodates different substituents at the 훼-position.
Alkyl substituents provide product with good levels of
enantioselectivity (Figure 3, 8b-9b). An acetamide
functional group is well tolerated by the enzyme but
afford cyclized product with low levels of
enantioselectivity (Figure 3, 10b). Other heteroatoms are
well tolerated, providing product in good yields and
modest levels of enantioselectivity (Figure 3, 11b-13b).
Notably, valuable fluorinated 12b and chlorinated 13b
2
3
stereocenters can be obtained.
Next, we examined other carbonyl compounds in this
reaction. Ketone 14a is an effective substrate for the
desired cyclization, but product 14b is formed with low
levels of enantioselectivity due to competitive radical
termination mechanisms (Supplemental Figure 8). Less
electrophilic amide 15a was also effective but required
another ERED from our panel, OYE3-Y197F, to achieve
synthetically useful yield and enantioselectivity. A
secondary alkyl iodide was also effective, providing
product in good yield but with modest diastereoselectivity
and little enantioselectivity (Figure 3, 16b). Finally, we
explored a 6-exo-trig radical cyclization and the impact
that ring substituents have on the transformation. In
general, the 6-exo-trig substrates were less reactive than
the 5-exo-trig substrates, requiring increased enzyme
loading to achieve comparable yields (Figure 3, 17b-
2
4
19b).
Figure 4. Redox Active Esters and
Polycyclization
Having identified that alkyl iodides form CT
complexes with flavin, we questioned whether other alkyl
radical precursors would be compatible in this reaction,
and if selectivity would be maintained in the resulting
cyclization. N-Hydroxyphthalimide (NHPI) esters have
been demonstrated to produce alkyl radicals upon single
In conclusion, we have developed a reductive
radical cyclization of unactivated alkyl iodides to afford
chiral esters, amides, and lactones. Radical formation is
achieved via photoexcitation of a CT complex that forms
between flavin and the substrate. This report expands the
types of reductive radical cyclizations reactions available
to biocatalysts, enhancing their utility in solving long-
standing challenges in asymmetric chemical synthesis.
2
5,26,27
electron reduction.
Indeed, when the redox active
ester precursor 20 was prepared and subjected to the
reaction conditions, the expected ester product was
formed in 93% yield with 92:8 er (Figure 4, 2b).
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ASSOCIATED CONTENT
Supporting Information
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Experimental procedures and characterization data
Hydrogen Atom Transfer: An Invigorating Approach to Free-Radical
Reactions. Curr. Opin. Chem. Biol. 2019, 49, 16–24.
AUTHOR INFORMATION
Corresponding Author
(9) Sandoval, B. A.; Meichan, A. J.; Hyster, T. K. Enantioselective
Hydrogen Atom Transfer: Discovery of Catalytic Promiscuity in
Flavin-Dependent ‘Ene’-Reductases. J. Am. Chem. Soc. 2017, 139,
11313–11316.
*
thyster@princeton.edu
(10) 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.
(11) Goetz, K. P.’ Vermeulen, D.; Payne, M. E.; Kloc, C.; McNeil,
L. E.; Jurchescu, O. D. Charge-transfer complexes: new perspectives
on an old class of compounds. J. Mat. Chem. C. 2014, 2, 3065.
(12) (a) Crisenza, G. E. M.; Mazzarella, D.; Melchiorre, P.
Synthetic Methods Driven by the Photoactivity of Electron Donor-
Acceptor Complexes. J. Am. Chem. Soc. 2020, 142, 5461-5476. (b) Fu,
M.-C.; Shang, R.; Zhao, B.; Wang, B.; Fu, Y. Photocatalytic
decarboxylative alkylations mediated by triphenylphosphine and
sodium iodide. Science 2019, 363, 1429-1434. (c) Bahamonde, A.;
Melchiorre, P. Mechanism of Stereoselective 훼-alkylation of aldehyde
driven by the photochemical activity of enamines. J. Am. Chem. Soc.
Notes
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The authors declare no competing financial interest.
ACKNOWLEDGMENT
Financial support provided by the NIH (R01 GM127703),
the Searle Scholars Foundation, and the Sloan Research
Fellowship.
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the substrate alkylates the protein throughout the reaction. We
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