Biocatalytic Friedel–Crafts Alkylation Using a Promiscuous Biosynthetic Enzyme

Article abstract of DOI:10.1002/anie.201814016

The Friedel–Crafts alkylation is commonly used in organic synthesis to form aryl–alkyl C?C linkages. However, this reaction lacks the stereospecificity and regiocontrol of enzymatic catalysis. Here, we describe a stereospecific, biocatalytic Friedel–Crafts alkylation of the 2-position of resorcinol rings using the cylindrocyclophane biosynthetic enzyme CylK. This regioselectivity is distinct from that of the classical Friedel–Crafts reaction. Numerous secondary alkyl halides are accepted by this enzyme, as are resorcinol rings with a variety of substitution patterns. Finally, we have been able to use this transformation to access novel analogues of the clinical drug candidate benvitimod that are challenging to construct with existing synthetic methods. These findings highlight the promise of enzymatic catalysis for enabling mild and selective C?C bond-forming synthetic methodology.

Full text of DOI:10.1002/anie.201814016

A Journal of the Gesellschaft Deutscher Chemiker
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Accepted Article
Title: Biocatalytic Friedel–Crafts Alkylation Using a Promiscuous
Biosynthetic Enzyme
Authors: Erica E. Schultz, Nathaniel R. Braffman, Michael U. Luescher,
Harry H. Hager, and Emily P. Balskus
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201814016
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COMMUNICATION
Biocatalytic Friedel–Crafts Alkylation Using a Promiscuous
Biosynthetic Enzyme
Erica E. Schultz,+[b] Nathaniel R. Braffman,+[a] Michael U. Luescher,[a] Harry H. Hager,[a] and Emily P.
Balskus*[a]
Abstract: The Friedel–Crafts alkylation is commonly used in organic
synthesis to form aryl-alkyl C–C linkages. However, this reaction lacks
the stereospecificity and regiocontrol of enzymatic catalysis. Here, we
describe a stereospecific biocatalytic Friedel–Crafts alkylation of the
conditions.[3] Previously reported enzymatic Friedel–Crafts
reactions include methyltransferases that use non-natural S-
adenosyl-L-methionine (SAM) derivatives as activated
electrophiles,[ prenyl transferases that employ non-natural arene
nucleophiles,[6] intramolecular squalene hopene cyclases,[7]
artificial metalloenzymes that alkylate indole derivatives with
moderate enantiomeric excess,[8] and a recent report of a
biocatalytic Friedel–Crafts acylation of resorcinol rings using a
variety of acyl donors including N-acylimidazole and 2,4-
5]
2-position of resorcinol rings using the cylindrocyclophane
biosynthetic enzyme CylK. This regioselectivity is distinct from that of
the classical Friedel–Crafts reaction. Numerous secondary alkyl
halides are accepted by this enzyme, as are resorcinol rings with a
variety of substitution patterns. Finally, we have been able to use this
transformation to access novel analogs of the clinical drug candidate
benvitimod that are challenging to construct with existing synthetic
methods. These findings highlight the promise of enzymatic catalysis
for enabling mild and selective C–C bond forming synthetic
methodology.
[
9]
diacetylphloroglucinol. As with the majority of two electron
biochemical C–C bond formations,[ all of these biocatalytic
Friedel–Crafts alkylations require electrophiles that are more
activated and often less readily accessible than the alkyl halides
used in the traditional reaction.
10]
Efficient and selective C–C bond forming reactions have
facilitated natural product total synthesis and chemical
manufacturing. Discovered over a century ago, the Friedel–Crafts
alkylation is a classic example of a C–C bond forming reaction
that is widely used to construct aryl-alkyl linkages in laboratory
and industrial settings.[1] This reaction forms a C–C bond between
an aryl ring and an alkyl halide or alkene, mediated by a Lewis
acid catalyst. Though much improved in efficiency and scope
since its discovery in 1877, the Friedel–Crafts alkylation suffers
from several limitations. In particular, regioselectivity is dictated
by the nature of the arene nucleophile and is hard to modulate.
Polyalkylation is common because the addition of each
consecutive alkyl substituent further activates the arene. Finally,
the mechanism of many Friedel–Crafts alkylation reactions
invokes alkyl carbocations that are generated using harsh
conditions and are prone to rearrangement, overalkylation of the
arene, and ablating the stereochemical configuration of the
starting alkyl halide. Thus, there is a need for improved synthetic
methodology that can address these shortcomings.
Figure 1. Friedel–Crafts alkylations mediated by CylK. A) Native reaction in the
[
11]
biosynthesis of cylindrocyclophane
F
B) Reaction with discrete alkyl
chloride/resorcinol substrate pairs. C) Pharmacologically active 2-
alkylresorcinol, benvitimod is currently in Phase 2 clinical trials.[
4]
Enzyme-based catalysts provide a potential solution to this
challenge. Biocatalysis can enable efficient, highly selective
transformations on multiple reaction scales and is growing in
popularity due to increasing access to powerful enzymes and
robust protein engineering platforms.[2] In particular, biocatalysts
are attractive alternatives to classic organic and organometallic
catalysts due to their minimal toxicity and the use of mild reaction
Recently, we identified an enzymatic Friedel–Crafts
alkylation in the biosynthesis of cylindrocyclophane F (1, Figure
1
A).[11] In the final stages of this pathway, CylK mediates the
head-to-tail dimerization of chlorinated alkyl resorcinol 2 by
sequentially forming two aryl-alkyl linkages. We have found that
CylK can accept discrete non-native substrate pairs with a variety
of secondary halides (3) and resorcinol derivatives (4) bearing
substituents at different positions. (Figure 1B). Notably, CylK uses
a chiral secondary alkyl chloride electrophile, which would
normally be prone to rearrangements and stereocenter
racemization via the traditional reaction. Resorcinol alkylation is
also stereospecific, occurring with inversion of configuration at the
alkyl chloride stereocenter to generate a single diastereomer of
[
a]
N. R. Braffman,[+] Dr. M. U. Luescher, H. H. Hager, Prof. E. P.
Balskus
Department of Chemistry and Chemical Biology, Harvard University
12 Oxford St., Cambridge, MA 02138 (USA)
E-mail: balskus@chemistry.harvard.edu
Prof. E. E. Schultz[
Department of Chemistry, Lake Forest College
+]
[
[
b]
+]
555 Sheridan Rd, Lake Forest, IL 60045 (USA)
These authors contributed equally to this work.
cylindrocyclophane
F (1). Additionally, CylK mediates an
alkylation at the 2-position of the resorcinol ring, a substitution that
Supporting information for this article is given via a link at the end of
the document.
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Angewandte Chemie International Edition
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COMMUNICATION
is found in pharmacologically active compounds including
benvitimod (6) (Figure 1C).[4] This contrasts with the
regioselectivity of the classic Friedel–Crafts reaction, which favors
alkylation at the 4-position due to probability and steric
crowding.[3,12] The many attractive features of this enzymatic
Friedel–Crafts alkylation inspired us to further explore the
substrate scope of wild-type CylK in order assess its potential as
a biocatalyst.
4-6). The final, optimal conditions of 0.25 M of CylK, 200 M of
7 and 8 at 37 °C for 22 h gave 98% conversion. The reaction could
be scaled ten times by increasing the enzyme loading to 0.5 M
(entries 7-11). On a closely related substrate pair, we found that
the reaction catalyzed by CylK yields product 11 in >99%
enantiomeric excess (e.e.) (Figure 2A, SI Figure 2F), suggesting
that the mechanism of CylK is stereospecific. Based on the native
activity of CylK, we infer this reaction proceeds with inversion of
configuration at the alkyl chloride stereocenter. Furthermore,
challenged with racemic electrophile 7, CylK yields product 11 in
88% e.e. (SI Figure 2G), suggesting that CylK is stereoselective,
preferring a single enantiomer of electrophile 7. In order to
determine these e.e. values, we chemically synthesized the
racemic standard of product 11 in six steps and 31% overall yield
from dimethoxy protected 10, further demonstrating how
challenging even non-stereospecific alkylation at the 2-position of
resorcinol can be (Figure 2B).
Table 1. Optimization of biocatalytic Friedel-Crafts reaction with
discrete substrate pairs.
HO
Me
HO
Me
Cl
7
HO
OH
HO
OH
CylK
HEPES (pH 8)
+
C9H19
Me
C9H19
Me
9
8
Entry
[CylK]
[7]
M
[8]
M
Time
(h)
Vol
L)
%[b]
TON[d]
[
a]

M
1[c]
2[e]
3[e]
4
1.0
200
200
100
200
200
200
300
400
500
200
200
100
100
200
200
200
200
300
400
500
200
200
24
24
24
24
24
24
22
22
22
17
17
100
100
100
100
100
100
100
100
100
1000
1000
51
--[f]
--[f]
97
97
98
90
72
63
20
98
51
1.0
--
1.0
--
1.0
194
388
784
1080
1152
1260
160
392
5
0.50
0.25
0.25
0.25
0.25
0.25
0.50
Figure 2.
(a) CylK-mediated alkylation of resorcinols proceeds with
stereospecificity. (b) Synthesis of racemic standard takes place over 6 steps
with 31% overall yield from dimethoxy-10.
6
7
We next examined the scope of the electrophile (Table 2).
We found that CylK tolerates a variety of functional groups in
place of the resorcinol ring present on the native substrate (2)
including amides and thioesters (12a–b, e–h), ketones (12c), and
esters (12d) up to four methylenes away from the alkyl halide.
CylK also accepts multiple alkyl halides (12e–h). Chloride 12e
and bromide 12f are converted to product 13 in 88% and 76%
conversion, respectively. The significantly larger electrophile,[
iodide 12g, is converted to product with 29% conversion. This
trend seems counterintuitive, as C–I bonds are generally better
electrophiles than C–Br and C–Cl bonds. However, the CylK
active site likely preferentially binds alkyl chlorides. Additionally,
8
9
1
0
1
1
13]
[
[
a] Temp of 37 °C unless noted. All experiments were performed once.
b] Percent conversion (%) determined by HPLC as described in the
Supporting Information. [c] Reaction run at 23 °C. [d] Turn over
number (TON) determined by [product] / [catalyst]. [e] Same batch of
enzyme as entry 1 but stored for ~3 months. [f] No product detected.
We began our investigation by optimizing the conditions for
the coupling of (S)-6-chlorodecan-1-ol (7) and resorcinol 8, which
resembles the natural resorcinol substrate but lacks the chloride
substituent, rendering it unable to dimerize (Table 1). Though a
certain halogenases show promiscuity toward the halide and
accept both bromide and chloride.[14] While the specificity of CylC,
the halogenase in cylindrocylclophane F’s biosynthetic pathway,
is not yet known, its potential promiscuity coupled with CylK’s
ability to accept alkyl bromides could explain why
cylindrocyclophane natural products are still produced in bromide-
2
:1 ratio of chloride 7 to resorcinol 8 gave 51% conversion to
1
1
alkylated resorcinol 9 at room temperature (entry 1), these
conditions failed to show any product formation at 37 °C (entry 2).
Switching the alkyl chloride to the limiting reagent and using a 1:2
ratio of 7:8, also gave no product – potentially due to competitive
binding of 8 with the alkyl chloride electrophile (entry 3). Additional
experimentation revealed that a 1:1 ratio of alkyl chloride to
resorcinol afforded 97% conversion at 37 °C (entry 3). We then
varied enzyme loading and found that the concentration of CylK
could be reduced to 0.25 M without a loss in conversion (entries
[15]
enriched growth media. Of note, CylK does not accept primary
chlorides, tertiary chlorides, hydroxyl groups, alkyl fluorides,
alkenes, tosyl or mesyl groups as electrophiles (SI Figure 3).
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Angewandte Chemie International Edition
10.1002/anie.201814016
COMMUNICATION
(
14d,j). These building blocks can serve as a synthetic handle
for subsequent coupling reactions. [
16]
Table 3. Resorcinol nucleophile substrate scope for CylK mediated Friedel–
Crafts alkylation.
Table 2. Alkyl halide substrate scope for CylK mediated Friedel–Crafts
alkylation.
O
Me
H
N
R
Me
Me
R
S
Me
Me
O
Me
X
O
(S,R)-12b
Cl
H
HO
OH
CylK (1.0 mM)
HO
OH
Me
N
CylK (1.0 mM)
S
Me
12a-h
+
+
HO
OH
R1 14a-l
O
HO
OH
HEPES (pH 8)
HEPES (pH 8)
37 °C, 24 h
15a-l
37 °C, 24 h
C9H19
Me
C9H19
R1
8
13 Me
Substrate[a]
Conv.
(
TON[c] Substrate
Conv.
(%)
TON
%)[
b]
Substrate[a]
Conv.
(
TON[c]
%)[
b]
HO
OH
HO
OH
O
O
O
Me
H
56
112
17
34
18
<2
14
44
--
Me
N
14a
Me
S
Me
Me
100
100
200
200
194
44
Me
14g
1
2a
O
Cl
Cl
Me
H
HO
OH
Me
N
HO
OH
S
O
12b
14b
OMe
51
34
102
MeO
9
<1
7
14h
O
Me
97
22
88
Me
Me
1
2c
Cl
HO
OH
HO
OH
O
O
O
68
O
MeO
S
Me
Me
Me
14c
14i
1
2d
12e
12f
Cl
Cl
Br
MeO
O
OMe
H
N
176
152
Me
Me
HO
OH
HO
OH
O
O
27
54
H
N
Br
76
29
14d
14j
S
Br
O
O
HO
OH
H
N
HO
OH
Me
Me
58
--
S
S
Me
Me
37
74
22
1
2g
2h
O
O
I
14e
14k
HO
HO
H
N
--[d]
1
F
OH
HO
OH
25
50
--[d]
14f
14l
[
a] 200 M of racemic alkyl halide and 100 M of resorcinol were used, all
CN
reactions preformed twice. [b] Percent conversion (%) determined by
HPLC. [c] Turn over number (TON) determined by [product] / [catalyst]. [d]
No product detected.
[
a] All reactions were preformed twice. [b] Percent conversion (%) determined
by HPLC. [c] Turn over number (TON) determined by [product] / [catalyst]. [d]
No product detected.
A survey of arene nucleophiles showed that CylK can
accept resorcinol substrates with a variety of substituents at
the 4- and 5-positions (Table 3). The highest turnovers are
observed with 5-substituted resorcinols, which have the same
substitution pattern as the native substrate. Substrates with
electron donating groups at this position (14a-b) gave higher
conversion than substrates bearing electron withdrawing
groups (14c-d,l), supporting a role for the arene ring as a
nucleophile in this reaction. A free hydroxyl group (14e) and a
fused aryl ring (14f) are also tolerated. CylK accepts 4-
substituted resorcinol rings (14g-l) with lower conversion.
Such substrates could be good starting points for enzyme
evolution to expand nucleophile scope. We find the
nucleophile is currently limited to resorcinol moieties; anilines
and nitrogen containing heterocycles were not accepted (SI
Figure 3). Notably, CylK can accept resorcinol derivatives
substituted with a bromide at both the 4 and 5-positions
Having assessed the scope of the aryl ring partner, we next
employed DFT calculations to investigate the inherent
nucleophilicity of 5-(heptan-2-yl)-resorcinol and assess the extent
to which CylK modulates its intrinsic reactivity. We found that
upon hydrogen bonding to, or deprotonation of a phenol
substituent, the HOMO of the resorcinol shifts from having a node
at the 2-position (Figure 3B, I) to having electron density at this
site (Figure 3B, II–III). This result suggests that CylK alters the
nucleophilicity of the resorcinol ring to facilitate alkylation, likely
through hydrogen bonding to or deprotonation of a phenol. Our
calculations may also explain why resorcinol is not alkylated at
the 2-position under the acidic reaction conditions of the classic
Friedel–Crafts reaction.[
3, 12]
The importance of a free phenol in
this transformation is further illustrated by CylK’s inability to
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Angewandte Chemie International Edition
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COMMUNICATION
alkylate methoxy resorcinol derivatives (SI Figure 3). We
therefore propose that CylK’s mechanism involves partial or full
deprotonation of a phenol on the resorcinol, followed by either a
proceeds with low conversion, and smaller alkyl halide
electrophiles were not accepted as electrophiles (SI Figure 3), this
result provides an exciting starting point for engineering CylK to
more efficiently access these and related bioactive scaffolds.[
22]
N N
concerted (S 2-like) or step-wise (S 1-like) nucleophilic
substitution reaction (Figure 3A). Interactions between the
enzyme and both substrates presumably control the orientation of
nucleophilic attack and explain the high degree of
stereospecificity. Alkylation likely also requires concerted
activation of the alkyl halide electrophile, potentially through
active site-mediated hydrogen bonding.[14]
HO
Me
HO
Me
HO
OH
HO
OH
HO
OH
4
1
% conv.
6 TON
1% conv.
4 TON
6
OH
Figure 4. Derivatives of bioactive 4-isopropyl-3,5-hydroxystilbene 6 accessed
from a CylK mediated biocatalytic Friedel-Crafts alkylation. Reaction mixtures
contained 0.5 M CylK, HEPES (pH 8), 37 °C, 24 h, 200 M of stilbene-diol,
800 M of alkyl chloride, percent conversion determined by integration on HPLC.
Turn over number (TON) determined by [product] / [catalyst].
In conclusion, we have demonstrated that CylK is capable of
mediating
a biocatalytic Friedel–Crafts alkylation between
numerous non-natural substrate pairs. This transformation has
features not associated with the classical organic reaction,
including stereospecificity, stereoselectivity, and regioselectivity
for alkylation at the 2-position of the resorcinol nucleophile. The
use of easily accessible secondary alkyl halides, mild reaction
conditions, and avoidance of protecting groups all represent
improvements over related non-enzymatic reactions. Future
studies will include enzyme engineering to further expand CylK’s
substrate scope and efficiency, as well as additional structural and
mechanistic studies.
Figure 3. Mechanistic and electronic considerations of resorcinol alkylation. A)
Proposed mechanism of resorcinol nucleophilic activation via hydrogen bonding
or deprotonation. B) HOMOs of (I) 5-(heptan-2-yl)-resorcinol (R-resorcinol), (II)
R-resorcinol hydrogen bonding to acetate, and (III) R-resorcinol with complete
deprotonation; calculated at B3LYP-GD3BJ/6-31G(d). Electron density is
present at the 2-position only when R-resorcinol is hydrogen bonded to acetate
(
II) or fully deprotonated (III). [17]
Experimental Section
The dialkylresorcinol scaffold accessed via the CylK mediated
General procedure for biocatalytic Friedel–Crafts alkylation. To an
Friedel–Crafts alkylation is found in many bioactive molecules.
Most notably, benivitimod, (6) is currently in clinical trials to treat
psoriasis.[18] Current methods to access 6 require at least five
steps and rely on harsh conditions, with methoxy protecting
groups on the resorcinol moiety.[19-21] In particular, the process
route to 6 involves a Friedel–Crafts reaction that limits the alkyl
substituent to the symmetric isopropyl group.[19] Larger and/or
asymmetric secondary alkyl groups could not be accessed via this
route due to the generation of carbocationic intermediates that
hinder regio- and stereospecificity. Envisioning that biocatalysis
could eventually streamline access to this scaffold, we tested the
reactivity of CylK towards 3,5-hydroxystilbene substrates. CylK
accepts resveratrol and 3,5-hydroxystilbene as nucleophiles
Eppendorf tube containing reaction buffer (20 mM HEPES, pH 8.0,
5
(
0 mM NaCl, 10 mM MgCl
1–5 L of 10 mM solution in DMSO, 100–500 M, 1 or 2 equiv)
and resorcinol (1–5 L of 10 mM solution in DMSO, 100–500 M,
equiv) to a final volume of 100 L. The reaction was started with
2 2
, 5 mM CaCl ) were added electrophile
1
addition of CylK (1 M). The reaction mixture was gently mixed
and allowed to incubate at 37 °C for 24 h, after which time the
reaction was quenched with the addition of 0 °C 1:1
methanol:acetonitrile (200 L) and centrifuged (16,100 x g for 10
min at 4 °C) twice. The supernatant of the sample was then
subjected to analytical HPLC and HRMS analysis to measure
percent conversion and ion abundance, respectively. As a control,
reactions without enzyme were performed. No product was
detected in reactions without enzyme.
(
Figure 4). Compared to the process chemistry route, which
requires cleavage of hydroxyl protecting groups and attachment
of the conjugated phenyl ring after alkylation of the resorcinol
moiety, CylK mediates the direct alkylation of deprotected 3,5-
dihydroxystilbene in a single step. Though the final alkylation
Acknowledgements
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Angewandte Chemie International Edition
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COMMUNICATION
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This work was supported by a CAREER award (no. 1454007)
from the National Science Foundation (USA) and the Cottrell
Scholar Award. M.U.L. is grateful for funding from the Human
Frontier Science Program fellowship (LT001561/2017-C). We
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Angewandte Chemie International Edition
10.1002/anie.201814016
COMMUNICATION
Entry for the Table of Contents (Please choose one layout)
COMMUNICATION
Friedel-Crafts
alkylation
Erica E. Schultz, Nathaniel R. Braffman,
Michael U. Luescher, Harry H. Hager,
Emily P. Balskus*
R1
X
CylK
R1
HO
OH
HO
OH
HO
OH
R1
rearrangement
loss of stereochemistry
over alkylation
selective for 2° alkyl halide
enantioselective
monoalkylation
R2
R2
R2
harsh acidic conditions
37 °C, pH 8
Page No. – Page No.
Biocatalytic Friedel–Crafts Alkylation
Using a Promiscuous Biosynthetic
Enzyme
Enzymatic alkylation: The cylindrocyclophane biosynthetic enzyme CylK is identified as a promiscuous catalyst for Friedel–Crafts
alkylation of resorcinol substrates with alkyl halide electrophiles. This transformation, which proceeds with exceptional regioselectivity
and stereospecificity, highlights the promise of enzymatic catalysis for enabling mild and selective C–C bond forming synthetic
methodology.
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