Photoexcitation of flavoenzymes enables a stereoselective radical cyclization

Article abstract of DOI:10.1126/science.aaw1143

Photoexcitation is a common strategy for initiating radical reactions in chemical synthesis. We found that photoexcitation of flavin-dependent “ene”-reductases changes their catalytic function, enabling these enzymes to promote an asymmetric radical cycli

Full text of DOI:10.1126/science.aaw1143

RESEARCH
eral catalysts for asymmetric radical reactions
could be accessed. Moreover, by repurposing
known enzymes, desired biocatalytic functions
can be achieved while retaining characteristics
such as ease of handling, substrate promiscuity,
and evolvability already associated with these
catalysts (3).
BIOCATALYSIS
Photoexcitation of flavoenzymes
enables a stereoselective
radical cyclization
Photoexcitation is widely used in organic syn-
thesis to facilitate radical reactions (4) but is
less common in enzyme catalysis. Light has been
used to activate enzymes through conformational
changes (5), drive charge separation in multi-
protein systems (6), and facilitate cofactor turn-
over or substrate epimerization (7). Direct use
of photonic energy to drive native, biological re-
actions is much more limited but has been docu-
mented for protochlorophyllide reductase (8), fatty
acid photodecarboxylase (9), and DNA photolyase
(10). We recently found that nicotinamide-
dependent ketoreductases (KREDs) and double-
bond reductases (DBRs) can use photoinduced
electron transfer to effect asymmetric radical
hydrodehalogenation and hydrodeacetoxylation
reactions (11, 12). These examples illustrate that
irradiation of common oxidoreductases can draw
out a non-natural reaction mode for catalysis, a
feature we anticipate can be exploited to address
challenges in chemical synthesis.
Kyle F. Biegasiewicz1*, Simon J. Cooper1*, Xin Gao1*, Daniel G. Oblinsky1,
Ji Hye Kim1, Samuel E. Garfinkle1, Leo A. Joyce2, Braddock A. Sandoval1,
Gregory D. Scholes1, Todd K. Hyster1†
Photoexcitation is a common strategy for initiating radical reactions in chemical
synthesis. We found that photoexcitation of flavin-dependent “ene”-reductases changes
their catalytic function, enabling these enzymes to promote an asymmetric radical
cyclization. This reactivity enables the construction of five-, six-, seven-, and
eight-membered lactams with stereochemical preference conferred by the enzyme
active site. After formation of a prochiral radical, the enzyme guides the delivery
of a hydrogen atom from flavin—a challenging feat for small-molecule chemical
reagents. The initial electron transfer occurs through direct excitation of an electron
donor-acceptor complex that forms between the substrate and the reduced flavin
cofactor within the enzyme active site. Photoexcitation of promiscuous flavoenzymes
has thus furnished a previously unknown biocatalytic reaction.
adical enzymes catalyze radical-mediated
reactions with exquisite chemo-, regio-,
and enantioselectivity (1). These catalysts,
however, exploit strategies for radical for-
mation that do not translate well out-
tional simplicity, generality, and ease of reaction
execution are desirable (2). To achieve these
features, it would be attractive to develop bio-
catalytic reactions that use mechanisms of rad-
ical formation commonly used in chemical
synthesis. If these mechanisms were to occur
within active sites of established enzymatic plat-
forms known to be substrate promiscuous, gen-
The stereoselective coupling of electrophilic
radicals and unactivated alkenes enables the
1Department of Chemistry, Princeton University, Princeton,
NJ 08544, USA. 2Department of Process Research and
Development, Merck, Rahway, NJ 07065, USA.
*These authors contributed equally to this work.
†Corresponding author. Email: thyster@princeton.edu
R
side of the natural setting and thus do not lend
themselves readily to be harnessed for robust
synthetic organic chemistry, in which opera-
Fig. 1. Strategy for achieving a biocatalytic radical-mediated C–C bond formation. (A) Challenges associated with achieving a catalytic asymmetric
radical hydroalkylation. (B) Proposed catalytic cycle for the biocatalytic radical cyclization to set b- and g- positions. (C) Enzyme screen and optimization
by means of mutagenesis. aReaction run by using cell free lysate on 1-g scale. (D) Reaction conditions and results for running this reaction open to air.
Biegasiewicz et al., Science 364, 1166–1169 (2019)
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RESEARCH
| REPORT
preparation of structural motifs found in agro-
chemical and pharmaceutical agents (13). Catalytic
strategies for rendering this type of transforma-
tion stereoselective remain elusive (14). This is
primarily because of the challenge of maintaining
association between radical species and chiral cat-
alysts throughout the stereoselectivity-determining
step and the lack of reagents capable of supplying
a hydrogen atom to one face of a prochiral rad-
ical (Fig. 1A) (15). Given these challenges, we imag-
ined that enzymes would be an ideal catalyst
for this transformation because of their ability to
precisely control the conformational landscape
of reactive species. As a model for this family
of reactivity, we targeted the development of a
biocatalytic radical cyclization of a-chloroamides
to afford b-stereogenic lactams (Fig. 1B). The
lactam motif is prevalent in medicinally valu-
able molecules (16), and the proposed synthe-
sis would be distinct from existing biocatalytic
approaches for generating N-heterocycles (17).
Although this cyclization is well known in the
radical literature, it is plagued by preferential
formation of the hydrodehalogenated and oligo-
merized product, and there are no known cat-
alytic asymmetric variants (18, 19). New enzymatic
Csp3–Csp3 bond–forming reactions are desired
in biocatalysis (20, 21).
We looked to flavin-dependent “ene”-reductases
(EREDs) as a potential catalyst family for the
proposed cyclization of a-chloroamides. Because
EREDs have large, substrate-promiscuous active
sites, we hypothesized that these enzymes would
be able to bind the substrate in a conformation
to enable cyclization (22, 23). Moreover, our re-
cent studies demonstrated that these enzymes
are able to control the stereochemical outcome
of radical hydrodehalogenation reactions (24).
Unfortunately, flavin hydroquinone (FMNhq) is a
modest single-electron reductant [E1/2 = –0.45 V
this initial electron transfer (25). Previous work
has investigated the fundamental photophysics
of the FMNhq* in flavin-dependent enzymes
(26, 27). If this excited state could be used for
the envisioned chemistry, it would transform
flavin-dependent enzymes into chiral photo the
envisioned chemistry, substantially expanding
their synthetic utility.
We began by testing the cyclization of a-
chloroacetamide 1 to afford g-lactam 2 under
visible light irradiation (table S1). An ERED
from Gluconobacter oxydans (GluER) was effec-
tive when irradiated with near–ultraviolet (UV)
light (390 nm), furnishing the desired cyclization
product with modest yield and enantioselectiv-
ity (table S2). Optimization of the lighting source
revealed cyan light (497 nm) to provide product
with improved yield and enantioselectivity while
producing less than 2% of hydrodehalogenation
product (Fig. 1C, entry 1). The opposite enantiomer
is favored by Old Yellow Enzyme 1 (OYE1) (Fig.
1C, entry 2). Control experiments confirm that
light, ERED, nicotinamide adenine dinucleotide
versus saturated calomel electrode (SCE)], mak-
red
ing electron transfer to a-chloroamides (Ep/2
=
–1.65 V versus SCE) thermodynamically challeng-
ing (fig. S23). The excited state of the flavin hy-
droquinone (FMNhq*) (E1/2* = –2.26 V versus SCE),
however, should be capable of accomplishing
Fig. 2. Scope of the biocatalytic radical cyclization. Reaction conditions: GluER-T36A (0.5 mol %), NADP+ (1 mol %), GDH-105 (0.2 mg/mg starting
material), glucose (6 equiv.), KPi (pH = 8.0, 100 mM, with 10% glycerol), and substrate (1 equivalent). 50 W Cyan LED. Average temperature, 35°C.
aResults obtained by using NostocER and favors the opposite product enantiomer. b12% formation of the hydrodehalogenated product. c1 mol % enzyme
loading (18 hours). d2 mol % enzyme loading (18 hours). e4 mol % enzyme loading with 8 mol % NAD+ (72 hours).
Biegasiewicz et al., Science 364, 1166–1169 (2019)
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| REPORT
phosphate (NADP+), glucose, and glucose dehy-
drogenase (GDH-105) are required for reactivity
(table S1). To increase the activity of GluER for
this non-natural function, we conducted mutage-
nesis and found that mutation of T36, a residue
located at the surface of the protein, to alanine
(T36A) furnished improved yields of product and
only trace quantities of the hydrodehalogenated
product (Fig. 1C, entry 3). We solved crystal
structures of GluER and GluER-T36A and found
no differences in the structure that would explain
the improved yield (backbone root mean square
deviation of 0.53 Å) (figs. S48 to S52). This mu-
tation does not have a detrimental effect on the
native function of this enzyme (fig. S45) and
allows the catalyst loading to be decreased to
0.5 mole % (mol %) without compromising yield
or enantioselectivity (fig. S46). Moreover, this
reaction can be conducted with lyophilized cell-
free lysate. This feature enabled the model cy-
clization to be run on gram scale, providing
product with no change in yield or enantiose-
lectivity (Fig. 1C, entry 4). The reaction could be
run open to air with slow addition of glucose
over 8 hours, providing comparable yields and
enantioselectivities to reactions run under
inert atmosphere (Fig. 1D). Alternatively, GDH-
105, NADP+, and glucose could be replaced with
periodic addition of sodium dithionite and de-
gassed buffer; when run open to air, there was
no observable change in the reaction outcome
(Fig. 1D).
albeit with increased catalyst loadings. Last, an
8-endo-trig cyclization is also possible by using
GluER-T36A, affording product in high yields
but with essentially no enantioselectivity. An
ERED from Lactobacillus casei (LacER) provides
significantly improved levels of enantioselec-
tivity but slightly diminished yields, with the
remaining mass balance being unreacted start-
ing material (Fig. 2, 28). The latter examples
are particularly exciting because they are under-
represented cyclization modes in the small mol-
ecule literature (29).
Inspired by the selectivity of the HAT event,
we considered the possibility of ERED-controlled
hydrogen atom delivery to exocyclic prochiral
radicals. Because these radicals are conforma-
tionally flexible, controlling HAT is challenging.
We began by testing substrates that possess tri-
substituted alkenes for the 5-exo-trig cyclization
mode. Substrates that contain phenyl/methyl and
phenyl/ethyl substituents at the terminal posi-
tion of the olefin afforded lactams with excel-
lent enantio- and diastereoselectivities (Fig. 2,
30 and 32). Because these substrates provide
an equimolar mixture of diastereomers when
prepared with photoredox catalysts, there does
not appear to be a thermodynamic preference
for one diastereomer, demonstrating that the en-
zyme is responsible for controlling the delivery of
the hydrogen atom. The cyclohexyl/Et substituted
alkene substrate 33, which lacks an aromatic
group, was also a substrate for this reaction,
with diastereoselectivity only slightly diminished
(Fig. 2, 34). Substrates that contain substituents
with steric bulk may thus be ideal for achieving
high levels of diastereoselectivity.
We conducted a series of initial rate exper-
iments to better understand how the kinetic
profile of the reaction compares with native al-
kene reduction. We found that the model reac-
tion follows Michaelis-Menten kinetics with a
Michaelis constant (Km) = 9.7 mM, an apparent
unimolecular rate constant (kcat) = 0.012 s−1, and
a kcat/Km of 0.0012 mM−1 s−1 (fig. S27). On the
With optimized conditions in hand, we ex-
plored the scope and limitations of this reaction
(Fig. 2). Aromatic substituted alkenes are tol-
erated for 5-exo-trig cyclizations, affording product
in high yield and enantioselectivity with substit-
uents at the para-, meta-, and ortho positions
(Fig. 2, 8-12). The electronic characteristics of
these substituents had only a modest effect on
reaction efficiency. Alkyl substituents on the
olefin are also tolerated, affording product in
nearly quantitative yield in all cases, albeit with
diminished enantioselectivities (Fig. 2, 17-20).
Because this mode of reactivity should be ac-
cessible across the entire ERED family, we
suspected that other members might provide
improved levels of enantioselectivity. An ERED
from Nostoc sp. (NostocER) accordingly pro-
vided improved enantioselectivities for the more
sterically demanding alkyl-substituted substrates
(Fig. 2, 19, 20).
We next shifted our attention to other cycli-
zation modes. GluER-T36A catalyzes a 5-endo-trig
cyclization, in which the stereogenic center is
created by means of hydrogen atom transfer
(HAT) to yield the 5-substituted g-lactam (Fig. 2,
22). The ERED is thus capable of controlling the
delivery of a hydrogen atom to sites distal to the
carbonyl, a feat largely unknown in the small
molecule literature (10, 28). A 6-exo-trig cycliza-
tion to furnish d-lactams occurs with GluER-T36C
in modest yield and enantioselectivity. Improved
levels of enantioselectivity and yield can be
achieved by using a variant of Morphinone re-
ductase (MorB-Y72F) (Fig. 2, 24). GluER-T36A can
also catalyze a 7-exo-trig cyclization (Fig. 2, 26),
Fig. 3. Mechanistic experiments. (A) UV-visible spectrum of reduced GluER-T36A (FMNhq) in the
presence of substrate (FMNhq + 1) and in the presence of substrate and sodium benzoate (FMNhq
1 + NaOBz). (B) Isotope labeling. (C) Conformation-selective HAT.
+
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RESEARCH
| REPORT
basis of our observation that increased light
intensities afford increased rates and higher
yields, we suspect that the decreased kcat is
due to the reaction being photon limited (fig.
S28). We observed a quantum yield of 7.8%.
We conducted a series of mechanistic experi-
ments to better understand the nuances of this
reaction, including the superior reactivity with
cyan light-emitting diodes (LEDs) [peak max-
imum (lmax) = 497 nm]. Reduction of GluER
to the FMNhq oxidation state with dithionite
revealed formation of the diagnostic FMNhq
signature with minimal absorption at 497 nm,
suggesting that direct excitation of the cofactor
is not responsible for the observed wavelength
preference (Fig. 3A, FMNhq). Addition of 100
equivalents of chloroamide 1 resulted in for-
Transient absorption spectroscopy with olefin
isomers of 29 provides additional support for
this mechanistic hypothesis. Initial excitation
at 370 nm followed by a broad-band probe pulse
reveals underlying dynamics of the flavin redox
states. We used a sequential kinetic scheme to
fit the data through global analysis. We found
three time constants that describe the tempo-
ral evolution of the various flavin species through
evolutionary associated difference spectra (EADS).
When (E)-29 is used as the substrate, a signal
corresponding to the charge transfer state de-
cays in 10 ps to the FMNsq. This time scale is
consistent with previously reported rates of de-
composition for the a-chloroacetophenone ketyl
radical anion (30). The neutral FMNsq decays on
the time scale of 250 ps to the flavin quinone
(FMNox) (figs. S42 and S43). This decay likely
corresponds to the rate of cyclization and termi-
nation of the radical through hydrogen atom
transfer from FMNsq. When the same experiment
is conducted on (Z)-29, the FMNsq lifetime in-
creases to 700 ps (figs. S39 and S40). These data
are consistent with post-cyclization rotation of
the exo-cyclic radical to a conformation in which
HAT from the FMNsq to the substrate-centered
radical is kinetically facile.
We anticipate that the light-promoted reac-
tivity demonstrated here will be replicable
widely in EREDs, KREDs, and other classes of
oxidoreductases known in biocatalysis for their
promiscuity and adaptability. These flavoen-
zymes may serve as stereoselective catalysts for
unexpected radical transformations beyond
those demonstrated here, depending on the
active site organization, provided starting mate-
rial, and properties of the excited state flavin.
By more widely investigating and exploiting
photoexcited states of cofactors, it should be
possible to photoinitiate radical-based reactions
within enzymes that are otherwise inaccessible
in the ground state.
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500 nm (Fig. 3A, FMNhq + 1). Because this
absorption feature is not lost upon addition of
sodium dithionite, we do not attribute it to
FMNsq. It is lost, however, upon addition of
150 equivalents of sodium benzoate (Fig. 3A,
FMNhq + 1 + NaOBz). These data are consist-
ent with the formation of an electron donor-
acceptor complex between the substrate and
FMNhq within the enzyme active site. We pro-
pose that excitation of this charge transfer band
promotes the initial electron transfer from
FMNhq to 1.
Next, we sought evidence for the existence
of radical intermediates by preparing a sub-
strate containing a cyclopropyl ring. GluER-T36A
produced only products of cyclopropane ring
opening, supporting the existence of a radical
intermediate (fig. S9). To determine the terminal
hydrogen atom source, we used labeled FMNDhq
generated in situ from D-glucose-1-d1 (Fig. 3A).
Deuterium was incorporated exclusively at the
benzylic carbon, with a high level of diaster-
eocontrol. These experiments support a reac-
tion mechanism in which substrate is initially
reduced by one electron after irradiation of
the electron donor-acceptor complex formed
between the substrate and FMNhq within the
enzyme active site. The a-acyl radical can react
with the pendent olefin to afford an exocyclic
radical that is terminated through hydrogen
atom transfer from neutral flavin semiquinone
(FMNsq) to afford the product and oxidized
flavin (fig. S47).
On the basis of this mechanistic hypothesis,
we reasoned that the configuration of the alkene
may be responsible for the observed levels of di-
astereoselectivity if HAT is faster than rotation
of the exocyclic C–C bond. We thus performed
the reaction on starting material 29 with (Z)-
alkene geometry rather than the (E)-isomer.
Both alkene geometries favor the same diaster-
eomer and produce no change in the enantio-
selectivity (Fig. 3C). The enzyme thus favors HAT
from one rotamer of the prochiral radical at rates
that are competitive with bond rotation. The di-
minished levels of diastereoselectivity observed
with the (Z)-alkene isomer are presumably due
to a small degree of hydrogen atom transfer be-
fore bond rotation.
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ACKNOWLEDGMENTS
We thank M. Souza for preparing glassware for spectroscopy
studies, C. Kraml and L. Wilson at Lotus Separations for
compound purification, P. Jeffrey for assistance with x-ray
structure determination, K. Conover for assistance in photoNMR
data acquisition, and the staff of NSLS-II beamline FMX
(17-ID-2) for help with data collection. Funding: Research
reported in this publication was supported by the National
Institutes of Health (NIH) National Institute of General Medical
Sciences (NIGMS) (R01 GM127703), the Searle Scholars
Award (SSP-2017-1741), Sloan Research Fellowship, the
Princeton Catalysis Initiative, and Princeton University. D.G.O.
acknowledges support from the Postgraduate Scholarships
Doctoral Program of the Natural Sciences and Engineering
Research Council of Canada. D.G.O. and G.D.S. acknowledge
support from 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.
The AMX (17-ID) beamline of The Life Science Biomedical
Technology Research (LSBR) resource is primarily supported
by NIH, NIGMS through a Biomedical Technology Research
Resource P41 grant (P41GM111244), and by the DOE Office
of Biological and Environmental Research (KP1605010).
As a National Synchrotron Light Source II facility resource
at Brookhaven National Laboratory, work performed at LSBR is
supported in part by the DOE Office of Science, Office of Basic
Energy Sciences Program under contract DE-SC0012704
(KC0401040). Author contributions: T.K.H. conceived and
directed the project. T.K.H., K.F.B., S.J.C., X.G., and J.H.K.
designed the experiments. K.F.B., S.J.C., X.G., and J.H.K.
performed and analyzed results. B.A.S. cloned the improved
variant, and S.E.G. obtained x-ray quality crystals and solved
the crystal structures. L.A.J. determined the absolute
configuration. D.G.O. designed and conducted the spectroscopic
studies, and D.G.O. and G.D.S. analyzed the data. Competing
interests: The authors declare no conflicts of interest. Data
and materials availability: All data are available in the
main text or the supplementary materials. Crystallographic
models and structure factors have been deposited in the Protein
Data Bank with accession nos. 6O08 and 6MYW for GluER
and GluER-T36A, respectively. X-ray crystallographic data
for lactam 2 have been deposited in the Cambridge
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7 December 2018; accepted 29 May 2019
10.1126/science.aaw1143
Biegasiewicz et al., Science 364, 1166–1169 (2019)
21 June 2019
4 of 4

Photoexcitation of flavoenzymes enables a stereoselective radical cyclization
Kyle F. Biegasiewicz, Simon J. Cooper, Xin Gao, Daniel G. Oblinsky, Ji Hye Kim, Samuel E. Garfinkle, Leo A. Joyce, Braddock
A. Sandoval, Gregory D. Scholes and Todd K. Hyster
Science 364 (6446), 1166-1169.
DOI: 10.1126/science.aaw1143
Light teaches (co)enzymes new tricks
Light is widely used in organic synthesis to excite electrons in a substrate or catalyst, opening up reactive
pathways to a desired product. Biology uses light sparingly in this way, but coenzymes such as flavin can be driven to
excited states by light. Biegasiewicz et al. investigated this reactivity and found a suite of flavoenzymes that catalyze
asymmetric radical cyclization when exposed to light. ''Ene''-reductases, when reduced and illuminated, converted
starting materials containing an α-chloroamide and an alkene into five-, six-, seven-, or eight-membered lactams.
Different enzymes furnished different stereochemistry in the products, likely because of changes in active-site pocket
geometry.
Science, this issue p. 1166
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