Enantioselective Total Synthesis of Nigelladine A via Late-Stage C-H Oxidation Enabled by an Engineered P450 Enzyme

Article abstract of DOI:10.1021/jacs.7b05196

An enantioselective total synthesis of the norditerpenoid alkaloid nigelladine A is described. Strategically, the synthesis relies on a late-stage C-H oxidation of an advanced intermediate. While traditional chemical methods failed to deliver the desired outcome, an engineered cytochrome P450 enzyme was employed to effect a chemo- and regioselective allylic C-H oxidation in the presence of four oxidizable positions. The enzyme variant was readily identified from a focused library of three enzymes, allowing for completion of the synthesis without the need for extensive screening.

Full text of DOI:10.1021/jacs.7b05196

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Communication
Enantioselective Total Synthesis of Nigelladine A via Late-
Stage C–H Oxidation Enabled by an Engineered P450 Enzyme
Steven A. Loskot, David K. Romney, Frances H. Arnold, and Brian M. Stoltz
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b05196 • Publication Date (Web): 19 Jul 2017
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Enantioselective Total Synthesis of Nigelladine A via Late-Stage C–H
Oxidation Enabled by an Engineered P450 Enzyme
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Steven A. Loskot, David K. Romney, Frances H. Arnold*, and Brian M. Stoltz*
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, California 91125
Supporting Information Placeholder
bridging 3° carbon (e.g., C10),3 since oxidation general-
ABSTRACT: An enantioselective total synthesis of the
ly occurs at the position where hydrogen abstraction is
norditerpenoid alkaloid nigelladine A is described. Stra-
most favorable.4 On the other hand, enzymes are well
tegically, the synthesis relies on a late stage C-H oxida-
known to catalyze oxidation reactions with regioselec-
tion of an advanced intermediate. While traditional
tivity that defies conventional trends of chemical reac-
chemical methods failed to deliver the desired outcome,
tivity. Despite this, enzymatic methods are often over-
an engineered cytochrome P450 enzyme was employed
looked in total synthesis efforts due to a typically narrow
to effect a chemo- and regioselective allylic C–H oxida-
substrate scope deriving from their exceptional specifici-
tion in the presence of four oxidizable positions. The
enzyme variant was readily identified from a focused
library of three enzymes, allowing for completion of the
ty. Fortunately, recent progress in the field of directed
evolution has greatly increased the viability of biocatal-
ysis in total synthesis by increasing the reactivity and
synthesis without the need for extensive screening.
selectivity of enzymes for non-native transformations.5,6
Biocatalytic C–H oxidations with engineered enzymes
thus present an alternative approach that can circumvent
the limitations of traditional chemical oxidations. None-
theless, even though there have been examples of the use
of engineered enzymes for the functionalization of com-
plex molecules,7 their application in total syntheses is
still very limited. Herein we report the first enantioselec-
tive total synthesis of tricyclic alkaloid 1, enabled by the
selective late-stage allylic oxidation of unsaturated imine
5 via an engineered P450 enzyme.
The field of organic synthetic chemistry has benefited
greatly from the many recent advances in selective C–H
functionalization.1 Indeed, chemical oxidations of C–H
bonds are some of the most important transformations in
synthetic chemistry because they allow for direct access
to oxidized intermediates, without the need for synthetic
handles or functional group interconversions. Despite
recent advances, there remain significant limitations
regarding the regioselectivity of non-directed C–H oxi-
dation reactions. We envisioned that the recently isolat-
ed norditerpenoid alkaloids nigelladines A–C (1–3) and
pyrroloquinoline alkaloid nigellaquinomine (4) (Figure
1)2 would present a challenging test bed to evaluate the
viability of late-stage C–H oxidation methods.
N
late-stage
enzymatic [O]
N
Suzuki-Miyaura
cross-coupling
imine formation
7
O
Nigelladine A (1)
N
N
N
N
5
O
Br
O
Tsuji–Wacker
aldol
+
NHBoc
10
7
N
O
BPin
O
O
O
Nigelladine
A (1)
Nigelladine
B (2)
Nigelladine
C (3)
Nigellaquinomine (4)
6
7
8
O
O
O
enantioselective
allylic alkylation
O
Figure 1. Structures of nigelladines A–C and nigel-
laquinomine.
9
10
With respect to nigelladine A, we believed that late-
stage installation of the C7 ketone would offer signifi-
cant flexibility in how we chose to construct the highly
conjugated core of alkaloid 1. However, despite the
abundance of chemical allylic oxidation conditions,
there are relatively few examples of selective oxidation
at a 2° carbon (e.g., C7) in the presence of a non-
Figure 2. Retrosynthetic analysis of nigelladine A (1).
Retrosynthetically, we surmised that selective late-
stage allylic oxidation of 5 at C7 would be essential to
the synthesis of enone 1. Imine 5 could be generated by
the cross-coupling of vinyl boronic ester 6 and bromide
dienone 7. Vinyl bromide 7 was envisioned to arise via
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annulation of enone 8, with the quaternary stereocenter
provide dienone 12 in good yield (76% yield over 2
steps).14
Enone 12 was then selectively mono-
1
2
3
4
5
6
7
8
constructed by an asymmetric allylic alkylation reaction
from β-ketoester 9.8 With this retrosynthetic analysis,
we recognized that the strategically planned C–H oxida-
tion would be particularly challenging due to issues of
site selectivity. Imine 5 contains three different allylic
carbon centers that could be susceptible to oxidation, a
1°, 2°, and 3°, in addition to a relatively acidic α–carbon
adjacent to the imine. Together, the natural product
combined with our strategic analysis unveiled a chal-
lenging problem for selective C–H functionalization that
would need to be addressed in a successful synthesis.
brominated to form vinyl bromide 7 in 83% yield on
gram scale.15 The cross coupling of 616 and 7 was next
evaluated for optimal reaction conditions. Ultimately,
we found that Buchwald’s 2nd generation precatalyst
with XPhos (XPhos Pd G2),17 allowed for the union of 6
and 7 in 88% yield using only a slight excess (1.1 equiv)
of 6. Furthermore, triene 13 provided crystals suitable
for X-ray diffraction and allowed us to ascertain abso-
lute stereochemistry.18
9
10
11
12
13
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With triene 13 in hand, we turned our attention to the
synthesis of the heterocyclic portion of the alkaloid and
the final stages of the synthesis. Toward that end, triene
13 was treated with trifluoroacetic acid (TFA) in CH2Cl2
to deprotect the amine, which spontaneously cyclized to
form the requisite imine upon quenching with aqueous
sodium bicarbonate (Scheme 2). Fortuitously, upon re-
moval of the aqueous layer, treatment with silica gel
promoted isomerization of the exocyclic olefin into the
ring, which, after purification, afforded the TFA salt of 5
Scheme 1. Synthesis of dienone 13
O
O
Cl
O
N
N
1. LDA, THF, –78 °C
2. 1M HCl, THF, 50 °C
Me2NNH2, TFA
3. MeI, NaH, DMF, 0 °C
73% yield over 3 steps
benzene, 80 °C
91% yield
1.7:1 E/Z
10
11
O
O
O
Pd2(dba)3 (2.75 mol %)
(R)-CF3-t-BuPHOX (6.0 mol %)
O
(2:1) hexanes:toluene, 40 °C
96% yield, 87% ee
19
in 75% yield (25% over 10 steps from enone 10).
9
8
Scheme 2. Condensation of imine 5
O
O
Br
1. PdCl2 (20 mol %)
CuCl (2 equiv)
DMF:H2O (9:1), 23 °C
Boc
N
H
NBS (1.1 equiv)
NH
OTFA
O
2. KOH, xylenes 110 °C
TFA, DCM, 23 °C;
MeCN, 0 °C
83% yield
76% yield over 2 steps
then NaHCO3 (aq); then SiO2
12
7
75% yield
Boc
CF3
13
TFA
5•TFA
(25% yield from10,
over 10 steps)
N
H
6 (1.1 equiv)
XPhos Pd G2 (3 mol %)
0.5M K3PO4
O
O
(4-CF3-C6H4)2P
N
THF, 50 °C
88% yield
H3N
NH
O
NaHCO3 (aq)
SiO2
(R)-CF3-t-BuPHOX
13
In the forward direction, known enone 10 can be syn-
thesized in excellent yield through a Stork-Danheiser
transposition from 1,3-cyclohexanedione.9,10 Unfortu-
nately, attempts to directly acylate the α-carbon of 10
resulted in low yields due to competing self-aldol addi-
tions. To avoid these issues, we chose to increase the
reactivity and steric environment of the enolate by syn-
thesizing the 1,1-dimethyl hydrazone 11.11 Hydrazone
11 was readily acylated then hydrolyzed, and the result-
ing β-ketoester was subsequently methylated, providing
our desired asymmetric allylic alkylation substrate (9) in
good yield (73% over 3 steps, Scheme 1). With β-
ketoester 9 in hand, we evaluated a variety of reaction
parameters for the key quaternary stereocenter-forming
asymmetric allylic alkylation and determined that the
use of (CF3)3-t-Bu-PHOX (L1)12 in 2:1 hexanes–toluene
with Pd2(dba)3 was optimal, generating desired enone 8
in 96% yield and 87% ee. Standard Tsuji–Wacker oxi-
dation of enone 8 using PdCl2 and CuCl under an at-
mosphere of oxygen generated the desired ketone,13
which subsequently underwent an aldol condensation to
With only an allylic oxidation separating tricycle 5
from nigelladine A (1), we initiated a broad exploration
of oxidation methods for installing an oxidation handle
at the desired C7 position. Unfortunately, the required
site-selective C–H oxidation of 5 or the intermediate
enone 13 proved intractable for traditional synthetic
methods (Table 1). While numerous conditions were
probed on both substrates,20,21 only a limited few effect-
ed oxidation at the desired position. These reactions
also exhibited poor selectivity, generating an inseparable
mixture of mono-oxidation products, along with over-
oxidized byproducts that constituted the majority of the
product mixture. We thus turned our attention to en-
zymes as a potential means to achieve the desired oxida-
tion.
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Table 1. Attempted chemical oxidationsa
The variant 2A1, in which leucine residues 75, 181, and
1
2
3
4
5
6
7
8
437 are mutated to alanine, exhibited a worse selectivity
compared to wild-type, favoring the isopropyl oxidation
product, as well as an unidentified oxidation product
(Table 2, entry 2). The two other variants, however, ex-
hibited a reversal in selectivity, favoring oxidation at the
desired position in excess of 2:1 (Table 2, entries 3–4).
The best variant proved to be 8C7, which is identical to
2A1, but lacks the L437A mutation. This variant favors
oxidation at the desired position in a 2.8:1 ratio (Table 2,
entry 4).
O
O
14
N
N
1
R'''
Oxidation Conditions
or
or
1
R'''
10
R'
7
R' = H, OH, or t-BuOO
R'' =H, OH, t-BuOO, or O
R''' =H, OH, t-BuOO, or O
R''
10
R'
7
R''
5
12
HPLC Yield of Oxygenation Products (%)b,c,d
Substrate
Conditions
SM
C1
C7
C10
Polyoxygenation
79 (57)e
–
–
3
2
–
–
–
15
46
4
5
12
5
SeO2, DCM, 40 °C
SeO2, DCM, 40 °C
21
5
9
26 (22)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
–
–
Pd/C (10 wt%), TPHP, 0 °C
Pd/C (10 wt%), TPHP, 0 °Cf
7
44
42
12
46
Cr(V) (1 equiv), MnO2
CF3C6H5, 15-crown-5, 80 °C
35g
55
3
–
5
6
Table 2. Screening of focused library for allylic oxidation
–
5
Rh2(esp)2, T-HYDRO, 23 °C
Rh2(esp)2, T-HYDRO, 23 °C
2
–
8
–
21(10)
15
34
43
Active-Site
Ala Substitutions
Desired/
Undesiredb
Catalysta
Entry
12
16
a See supporting information for reaction conditions and description of the
P450BM3
–
1
2
3
4
0.86
0.37
2.1
b
product distribution.
Approximated by separating the products on
c
2A1
4H5
8C7
L75A, L181A, L437A
L75A, L177A, L181A
L75A, L181A
UHPLC-MS and comparing ion count of the various oxidation products.
Remaining percent balance remaining were unidentified side products.
d
Numbers given in parentheses indicate isolated yields of the oxidized
2.8
product. e Protection of 5 to as an N-acyl enamide results in oxidation at
f
aSee supporting information for full list of mutations. bApproximated by
separating the products on UHPLC-MS and comparing ion count of C-7
oxidation product to all other mono-oxygenation products (m/z = 286).
See supporting information for more details.
C-14. Shortened reaction time used to observe inherent regioselectivity
without over oxidation. g Oxidized product S14 was isolated.
The cytochrome P450 enzyme from Bacillus mega-
terium (P450BM3) is a workhorse of protein engineering
studies because it is soluble and self-contained, exhibits
one of the fastest reaction rates of any P450-catalyzed
hydroxylation (17,000 min–1 for arachidonic acid),5a and
possesses good stability (t1/2 = 68 min at 50 °C).22 Since
the substrate scope of the wild-type protein is mostly
limited to long-chain fatty acids, many engineering ef-
forts have focused on expanding the substrate scope of
P450BM3 to accept larger and more complex substrates.
To this end, various approaches have been applied to
develop biocatalysts derived from P450BM3 that perform
regioselective C–H oxygenation of complex molecules
for which the wild-type enzyme exhibits minimal activi-
ty.6c,23 However, these approaches involved extensive
screening endeavors, often requiring high-throughput
screening and limiting them to substrates that were read-
ily available.
Since enzyme active sites are well known to bind sub-
strate enantiomers differently, we wished to test if the
stereochemistry of 5 would affect the activity or selec-
tivity of 8C7. Using the Pd-catalyzed alkylation, we
obtained both enantiomers of 5, which were then en-
riched to enantiopurity by chiral preparative HPLC.
Each enantiomer was then subjected to oxidation by cat-
alyst 8C7. Interestingly, at both short (15 min) and long
(12 h) reaction times, the enantiomers gave nearly iden-
tical results, although the R enantiomer exhibited a
slightly higher initial rate by a factor of 1.3.
Having achieved the desired regioselectivity, we then
needed to improve the yield. Furthermore, we wished to
supplant the stoichiometric use of NADPH, which is
required if oxygen is used as the terminal oxidant. For-
tunately, we could use NADPH in catalytic amounts by
regenerating it in situ with an alcohol dehydrogenase
(ADH) and isopropanol as the terminal reductant.24
With these new conditions, we conducted the oxidation
on a 160 mg scale, followed by subjecting the crude
product mixture to oxidation with Dess-Martin perio-
dinane, thus affording the natural product in 21% yield
over both steps (43% yield based on recovered 5,
Scheme 3). The recovered starting material (5) was
competent in the same two-step procedure with no loss
For biocatalysis to be a viable strategy for the late-
stage of a total synthesis, it is desirable to identify a syn-
thetically useful catalyst with minimal screening. Thus,
we elected to screen a focused library of variants that
had been previously engineered to accept large sub-
strates,23 but had otherwise never encountered substrate
5. To compare the catalysts, we chose to allow the reac-
tions to proceed to only low conversion and compare the
ratio of desired to undesired oxidation products (Table
2). Encouragingly, wild-type P450BM3 exhibited some
selectivity for C7, although oxidation of the isopropyl
C–H bond was favored 1.2:1 (Table 2, entry 1). We then
probed a small selection of engineered variants that all
derive from the same parent (12 mutations from
P450BM3). These enzymes were previously evolved for
regioselective oxidation of methoxymethyl-protected
glycosides, but are distinct in that different combinations
of active-site residues have been mutated to alanine.
25
in efficiency (26% yield). Under these conditions, we
observed up to 1700 turnovers to the C7 mono-
oxygenation product.
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Scheme 3. Completion of the synthesis of nigelladine Aa
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1
2
3
4
5
6
7
8
OTFA
1.
P450 8C7 (0.02 to 0.04 mol %)
NADP (10 mol %), i-PrOH, ADH
2.5% DMSO in KPi buffer (pH 8)
23 °C , 12h
NH
N
2.
DMP, CH2Cl2, 0 °C, 4h
(3) Nakamura, A.; Nakada, M. Synthesis 2013, 45, 1421–1451.
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McFadden, R. M.; Roizen, J. L.; Enquist, J. A., Jr.; White, D. E.;
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from commercially available isobutyric acid via propargylation, Cur-
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Information).
21% yield over 2 steps
43% yield
based on recovered 5
O
5•TFA
1
aADH, alcohol dehydrogenase; KPi, potassium phosphate;
DMP, Dess-Martin periodinane.
9
10
11
12
13
14
15
16
17
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19
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21
22
23
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60
In summary, we have completed the first total synthe-
sis of nigelladine A in an expedient 12 steps and 5%
overall yield (11% yield based on recovered 5). The
asymmetric allylic alkylation allowed for the construc-
tion of the quaternary center in high yield and enantiose-
lectivity. Expedient identification of engineered en-
zymes allowed for a site-selective 2° allylic oxidation
without the need for extensive generational screening or
reaction optimization. These results demonstrate that
enzymatic transformations are capable of defying stand-
ard chemical limitations and should be included in the
repertoire of reactions that are traditionally considered
for the late stages of total syntheses.
ASSOCIATED CONTENT
Supporting Information. Experimental procedures and charac-
terization data as Supporting Information is available free of
charge on the ACS Publications website at DOI:
10.1021/jacs.5b00000.
AUTHOR INFORMATION
Corresponding Authors
B.M.S.: stoltz@caltech.edu
F.H.A.: frances@cheme.caltech.edu
Funding Sources
No competing financial interests have been declared.
ACKNOWLEDGMENT
This work was supported by the NSF under the CCI Center for
Selective C−H Functionalization (CHE-1205646). D.K.R. was
supported by a Ruth Kirschstein NIH Postdoctoral Fellowship
(F32GM117635). Additional financial support was provided by
Caltech and Novartis. We thank Dr. Scott Virgil (CIT) and the
Caltech 3CS for access to analytical equipment. The authors
gratefully acknowledge Larry Henling and Dr. Michael Takase
(CIT) for X-ray crystallographic structural determination, Dr.
Mona Shahgholi and Naseem Torian (CIT) for mass spectrometry
assistance, and Dr. David VanderVelde (CIT) for NMR experi-
mental assistance and helpful discussions. The authors also
acknowledge Dr. Sabine Brinkmann-Chen and Dr. Stephan
Hammer (CIT) for helpful discussions regarding the enzymatic
oxidation system.
(17) Bruno, N. C.; Tudge, M. T.; Buchwald, S. L. Chem. Sci.
2013, 4, 916–920.
H2N Pd
Cl
P
i-Pr
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i-Pr
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XPhos Pd G2
(18) Crystals grown for X-ray crystallography were synthesized in
the enantiomeric series using (S)-CF3-tBu-PHOX for the asymmetric
allylic alkylation of 9.
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N
H
O
ent-13
ent-13
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8
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