Development of a One-Pot tandem reaction combining ruthenium-catalyzed alkene metathesis and enantioselective enzymatic oxidation to produce Aryl epoxides

Article abstract of DOI:10.1021/acscatal.5b00533

We report the development of a tandem chemoenzymatic transformation that combines alkene metathesis with enzymatic epoxidation to provide aryl epoxides. The development of this one-pot reaction required substantial protein and reaction engineering to improve both selectivity and catalytic activity. Ultimately, this reaction converts a mixture of alkenes into a single epoxide product in high enantioselectivity and moderate yields and illustrates both the challenges and benefits of tandem catalysis combining organometallic and enzymatic systems.

Full text of DOI:10.1021/acscatal.5b00533

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Article
Development of a one-pot tandem reaction combining
ruthenium-catalyzed alkene metathesis and enantioselective
enzymatic oxidation to produce aryl epoxides
Carl A. Denard, Mark J. Bartlett, Yajie Wang, Lu Lu, John F. Hartwig, and Huimin Zhao
ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.5b00533 • Publication Date (Web): 12 May 2015
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Development of a OneꢀPot Tandem Reaction
Combining RutheniumꢀCatalyzed Alkene Metathesis
and Enantioselective Enzymatic Oxidation to
Produce Aryl Epoxides
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Carl A. Denard,a‡ Mark J. Bartlett,b‡ Yajie Wang,a Lu Lu,c John F. Hartwig*,b and Huimin
Zhao*,a,d
aDepartment of Chemical and Biomolecular Engineering, University of Illinois at Urbanaꢀ
Champaign, Urbana, IL 61801
bDepartment of Chemistry, University of CaliforniaꢀBerkeley, Berkeley CA 94720
cDepartment of Molecular and Cellular Biology, University of Illinois at UrbanaꢀChampaign,
Urbana, IL 61801
dDepartments of Chemistry, Biochemistry, and Bioengineering, Institute for Genomic Biology,
University of Illinois at UrbanaꢀChampaign, Urbana, IL 61801
We report the development of a tandem chemoenzymatic transformation that combines alkene
metathesis with enzymatic epoxidation to provide aryl epoxides. The development of this Oneꢀ
pot reaction required substantial protein and reaction engineering to improve both selectivity and
catalytic activity. Ultimately, this reaction converts a mixture of alkenes into a single epoxide
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product in high enantioselectivity and moderate yields and illustrates both the challenges and
benefits of tandem catalysis combining organometallic and enzymatic systems.
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KEYWORDS: Chemoꢀenzymatic catalysis, cytochrome P450, organometallic catalysis,
biocatalysis, tandem catalysis, olefin metathesis, biocatalysis.
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Introduction
The utility of biocatalysis in the synthesis of fine chemicals and medicinal compounds has
grown significantly in recent times.1 Furthermore, the development of oneꢀpot multistep
reactions containing both transitionꢀmetal catalysts and enzymes has proven appealing in terms
of both selectivity and synthetic efficiency.2 These tandem reactions exploit the broad reactivity
of Transitionꢀmetal catalysts and the high selectivity of enzymes simultaneously. Work in this
area has resulted in several wellꢀestablished dynamic kinetic resolutions involving metal
catalysts and enzymes3, along with a number of sequential and Oneꢀpot cascade reactions.2a, 4
Nonetheless, combining Smallꢀmolecule organometallic catalysts and biocatalysts remains
challenging, in part because the milieu in which these catalysts operate are typically different.5
Besides dynamic kinetic resolutions, there have been few reports in which an Enzymeꢀ
catalyzed transformation occurs with one substrate of a dynamic equilibrium or a transient
product which is consumed in a subsequent side reaction. Yet, these scenarios could lead to
cooperative catalytic reactions in which one catalytic transformation aids the efficiency of the
other and provides a higher yield of the final product than would be obtained by two sequential
reactions. For such processes, cytochromes P450 are particularly appealing, due to their ability to
catalyze the oxidization of C–H and C=C bonds in substrates of a specific size and shape.6, 7
These transformations utilize molecular oxygen and NADPH to form a reactive Ironꢀoxo
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intermediate and often provide alcohols or epoxides with high regioꢀ and stereoselectivity.8 In
previous work, we developed a cooperative tandem reaction in which P450ꢀBM3 and an alkene
metathesis catalyst work together to convert an equilibrium mixture of alkenes into a single
epoxide on the basis of chain length (Scheme 1).9 The yields of this Oneꢀpot reaction were
higher than those obtained when the reactions were run sequentially. In the present study, we
sought to evolve this strategy to convert stilbene selectively to aryl epoxides. We show that
alkene metathesis and enzymatic epoxidation can be used in a tandem reaction to selectively
provide aryl epoxides in high enantioselectivity.
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Previous Work:
O
P450BM3,
2.5 mol %
NHC-Ru
Catalyst
HO2C
HO2C
PTDH, NADP+,
O
8
O
HO
8
Na2HPO3
HO
+
+
8
CO2H
8
1:4
90% yield
38% ee
>2500 turnovers
isooctane/
Tris-HCl
buffer
8
This Study:
3–6 mol %
NHC-Ru
Catalyst
Ph
Ph
R
R
Ph
Ph
+
O
R
P450BM3 Mutant,
GDH, NADP+,
glucose
2.5% DMSO/
KPi buffer
R
up to 94% ee
>2500 turnovers
[Ru]
R = Me, Et
Ph
Ph
Scheme 1. Alkene Metathesis and Oxidation in Cooperative Tandem Catalysis. The P450BM3
image is based on the crystal structure reported previously (P450 KT2 crystal structure PDB ID:
3PSX).7
Results and Discussion
We investigated the metathesis reaction of Zꢀstilbene and a symmetrical alkene as shown in
Scheme 1. NHCꢀbased ruthenium complexes are known to catalyze alkene metathesis in the
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aerobic aqueous conditions often required for enzymatic transformations.10 Three Ruꢀcatalysts
(Entries 3–5) were tested for the Crossꢀmetathesis of Zꢀstilbene 1 and Zꢀ2ꢀbutene (Zꢀ2a) (Table
1). In all three cases, only moderate yields (32–35%) of Eꢀβꢀmethylstyrene (Eꢀ3a) were obtained,
but with very high E/Z selectivity (>30:1). The same reaction catalyzed by C2 provided a low
yield of Eꢀstilbene 4 (Entries 4 and 8). The lower reactivity of C2, compared with that of C1 and
C3, is likely due to the greater steric encumbrance from the isopropyl groups in this catalyst.11
Control experiments revealed that Eꢀstilbene (4) does not undergo cross metathesis with Eꢀ3ꢀ
hexene (Eꢀ2b) in the presence of either Ruꢀcatalyst C1 or C2 (Entries 1 and 2).12 Furthermore,
the Selfꢀmetathesis of Zꢀstilbene (1) to form Eꢀstilbene 4 is slow. In contrast to our previous
work,8 these results suggest that an irreversible pathway leads to the conversion of Zꢀstilbene (1)
to βꢀalkylstyrene (3), and the conversion of 3 to Eꢀstilbene (4). The reaction of 1 with the liquid
alkene Eꢀ2b, as opposed to the gaseous Zꢀ2ꢀbutene, provided higher yield of βꢀalkylstyrene
(Entries 3 and 6). Reaction with 1 equiv. of Eꢀ2b instead of 3 equiv. resulted in lower yield of βꢀ
ethylstyrene (Eꢀ3b) while the amount of Eꢀstilbene side product was relatively unchanged (Entry
7). Similar results were observed when reactions between 1 and Eꢀ2b were run in an isooctane:
buffer biphasic, where in the metathesis reaction occurred in the organic phase (Table S2). The
formation of Eꢀstilbene, presumably, is slowed by competing unproductive metathesis between 3
and 2. Thus increasing the equivalents of 2b provides a higher ratio of alkylstryene 3b to stilbene
4 after 16 hours. Overall, these results suggest that in order to epoxidize 3 efficiently in a tandem
system, the selfꢀmetathesis of Z-stilbene 3 to produce Eꢀstilbene 4 must be mitigated. This
reduction of the formation of 4 could be achieved by using the selective, but less reactive, Ruꢀ
catalyst C2 or by maintaining a low concentration of alkylstyrene by the simultaneous use of an
epoxidation catalyst that causes this reaction to be faster than the metathesis reaction.
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Table 1. CrossꢀMetathesis of Stilbene on Water.
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Yielda
3a/bb
[Ru]
Catalyst
Entry
1c
Alkenes (equiv)
4
C1
C1
4 + E-2b (3)
1 (1)
0%
-
-
2
8%
3d
4d
5d
6c
7c
8c
C1
C2
C3
C1
C1
C2
1 + Z-2a (10, 8 psig)
1 + Z-2a (10, 8 psig)
1 + Z-2a (10, 8 psig)
1 + E-2b (3)
33%
35%d
40%
8%
32%
20%
47%
43%
4%
50%
1 + E-2b (1)
33%
1 + E-2b (3)
47%(32%)
a
Determined by GC analysis. Isolated yield shown in parentheses.
Remaining mass balance consists of unreacted 1. b E/Z>30:1. c Reactions
run for 16 h. c Reactions run in triplicate, average yields are reported. d
23% yield after 4 h.
To establish these relative rates, we investigated the activity and selectivity of three P450ꢀBM3
variants: RLYF,13 KT2,14 and RH4715 (Table S1), previously shown to catalyze the oxidation of
arenes.16 Each P450 variant was evaluated using a competition experiment between Zꢀstilbene
(1) and alkylstyrene 3a or 3b, while determining the ee of the resulting epoxides 5a/b (Table 2).
In addition, a glucose dehydrogenase (GDH) system was used to regenerate NADPH by cloning
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and expressing the gdhIV gene from Bacillus megaterium.17 This system is more productive than
the previously used phosphite dehydrogenase (PTDH) system.18 A reaction conducted with 3
nmol of a P450 KT2 lysate and a glucoseꢀdriven GDHꢀNADP+ system led to the selective
epoxidation of Eꢀ3a to form transꢀ5a in 41% yield with more than 4000 turnovers (Entry 3).
Less than 3% yield of Zꢀstilbene oxide (Zꢀ6) was observed. The aqueous solubility of Zꢀstilbene
is higher than that of Eꢀβꢀmethylstyrene; therefore, the observed selectivity does not result from
differences in aqueous solubility. Lower yields were typically obtained from reactions conducted
with purified P450, presumably due to the presence of stabilizing agents in the lysate.19
Interestingly, performing the bioepoxidation of Eꢀ3b in a biphasic isooctane: buffer system
proved to be massꢀtransfer limited and produced transꢀ5b in <2% yield, even in emulsions
created by sodium docusate salt20 or methylꢀβꢀcyclodextrin.21 All three P450 variants formed
epoxide (R,R)ꢀtransꢀ5a with >80% ee.11b Furthermore, initial rates revealed that the reactions of
3a catalyzed by all three metalloenzymes occurred with a high degree of coupling between
NADPH consumption and rate of product formation. The epoxidation of Eꢀβꢀethylstyrene
catalyzed by RLYF, KT2 and RH47 occurred in significantly lower yields and NADPH coupling
(Entries 7–10) than epoxidation of βꢀmethylstyrene. However, epoxide 5b was formed in >90%
ee with all three variants. In contrast to epoxidation of Zꢀ and Eꢀβꢀmethylstyrene, the epoxidation
of Eꢀβꢀethylstyrene has proven challenging for a number of asymmetric epoxidation catalysts.22
Furthermore, P450 metalloenzymes typically display only moderate ee in the epoxidation of aryl
alkenes.23
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Table 2. Enzymatic Epoxidation of βꢀAlkyl Styrenes.
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0.03 mol % P450,
R
O
R
0.2 mM NADP+,
Ph
Ph
Ph
5a/b
3b
+
1 U/mL GDH, glucose,
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+
O
600 U/mL catalase,
Ph
200 mM KPi buffer pH 8.1,
2.5 v/v% DMSO, rt, 12–16 h
Ph
1
Z-6 Ph
Yielda Initial Rate
Entry
P450
R
equiv 1
eec
Yielda6
5
(Coupling)b
1
2
1
0
1
0
19%
-
-
5%
-
18%d
(15%)e
83%
(R,R)
RLYF
Me
630 (98%)
3f
4
41%
-
-
3%
-
34%d
(38%)e
88%
(R,R)
KT2
Me
Me
815 (95%)
-
5
6
1
0
29%
-
3%
-
RH47
86%
(R,R)
94%
91%
26%d 960 (80%)
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9
RLYF
KT2
Et
Et
Et
1
1
0
14%
8%
11%d
311 (75%)
7%
3%
-
-
RH47
-
93%
a Yield determined by GC. Reactions performed on a 0.027 mmol scale. b Initial rate
in nmol•minꢀ1•nmol P450ꢀ1, see SI for details. c Enantiomeric excess determined by
chiral SFC analysis. d NMR yield. e Isolated yield, average yield of four 0.08 mmol
reactions. f Reaction performed with 0.01 mol % P450.
Having developed a single set of reaction conditions that accommodate both catalytic
reactions, we investigated the two reactions in Oneꢀpot. These tandem reactions required careful
layering of Zꢀstilbene and the Ruꢀcatalyst on top of the aqueous layer to ensure adequate contact
between the Ruꢀcatalyst and gaseous Zꢀ2ꢀbutene (Figure S3). In addition, the aqueous solubility
of Eꢀβꢀmethylstyrene and Zꢀstilbene were determined to be 0.9 and 5.2 mM, respectively.24 This
low aqueous solubility highlights the need for a highly active epoxidation enzyme. To maximize
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reaction yields and maintain reproducibility, several reaction parameters were investigated,
including substrate loading, organic cosolvent, loading of the regeneration enzyme, reaction
volume and buffer concentration (Table S4). To assess the possibility of mutual catalyst
inactivation, several control reactions were performed. These experiments showed that the
alkene metathesis was unaffected by the volume of the aqueous phase, the nature of the
cosolvent, or the presence of enzymatic components (Tables S6–S8). Furthermore, enzymatic
oxidation was not inhibited by the presence of 3 mol % of the Ruꢀcomplex C2 (Figure S2) or 8
psig of Zꢀ2a.
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Epoxide transꢀ5a was initially obtained in 13% yield in a tandem reaction with 0.06 mol %
(15 nmol) P450 RLYF (Table 3, Entry 2). However, decreasing the volume of the reaction from
5 mL to 3 mL improved the yield of transꢀ5a to 22%, with only 0.03 mol % (9 nmol) P450
(Entries 4 and 5). When KT2 was used as the epoxidation catalyst, the yield of transꢀ5a
increased to 40%, and over 2400 turnovers were achieved (Entry 6). Reactions conducted with
twice the loading of P450/GDH did not result in significantly higher yields of transꢀ5a (Figure
S4), however, the addition of a second batch of enzyme after the initial 16 h of reaction gave a
slight increase in the conversion of Eꢀ3a, providing transꢀ5a in 50% yield (Entry 7). Similar
results have been observed when increasing the loading of P450 to improve the yield of styrene
epoxidation.25
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Table 3. Tandem Metathesis–Oxidation with Zꢀ2ꢀButene.
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psig
P450
(nmol)
Reaction
Volume
Yielda
trans-5a
P450
TTN
Entry
Z-
2a
6
E-3a
4
1
2
-
3 mL
5 mL
37%
28%
-
-
RLYF
(15)
RLYF
(9)
6
6
6
13%
14%
936
3
3 mL
4 mL
24%
43%
22%
25% 2640
26% 2280
RLYF
(9)
19%
(23%)c
4b
RLYF
(9)
5
8
6
6
6
5 mL
6 mL
3 mL
3 mL
33%
21%
13%
15%
20%
41%e
50%
38%
15% 2400
10% 2460
6d
7d
8g
KT2 (9)
KT2
(27)f
KT2 (9)
9%
2000
10% 4560
a
Yield determined by GC and is an average of multiple experiments, see
Table S10 for details. Isolated yield in parentheses. Reactions performed on a
0.054 mmol scale. b Reaction performed with 5 mol % C2. c 83% ee. d 0.027
mmol 1 was used. e 87% ee. f After 16 h, 19 nmol P450 and 0.5 U/mL GDH
were added (24 h reaction). g Oneꢀpot sequential reaction (0.027 mmol). 10 h
alkene metathesis, 16 h epoxidation.
Performing the two transformations as a sequential Oneꢀpot reaction afforded transꢀ5a in 38%
yield (Entry 8), which is approximately the same yield as both the tandem reaction (Entry 6) and
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the isolated epoxidation of Eꢀ3a with KT2. These results indicate a lack of cooperativity between
the two catalytic cycles.26
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Several additional observations are noteworthy. First, there was increased consumption of Zꢀ
stilbene (1) in all of the tandem reactions compared to the isolated metathesis reaction (Entry 1).
These results suggest that the selective removal of 3a from the system by epoxidation is
compensated by further production of 3a. Although higher yields of Eꢀstilbene (4) were observed
in tandem reactions, compared to isolated metathesis reactions, this is attributed to longer
reaction times and the viscous P450 lysate. The viscous P450 lysate is believed to partially
inhibit contact between the Ruꢀcatalyst and gaseous Zꢀ2ꢀbutene, which in turn favors the selfꢀ
metathesis of Eꢀ3a to form Eꢀstilbene (Figure S3). In general, tandem reactions with higher
yields of transꢀ5a also produced less Eꢀstilbene (i.e. Entries 4 and 6). There was little or no
formation of products from epoxidation of Zꢀβꢀmethylstyrene, Zꢀstilbene or Eꢀstilbene (1 and 4,
respectively) in these tandem reactions. Yet, separate oxidations of Zꢀβꢀmethylstyrene showed
that this alkene undergoes epoxidation in >65% yield with RLYF (Table S9). Thus, the excellent
Eꢀselectivity of the metathesis catalyst leads to the absence cisꢀ5a. Overall, the selectivity of both
the ruthenium catalyst and P450 variant combine to make this tandem system highly selective for
the epoxidation of Eꢀβꢀmethylstyrene.
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Tandem reactions involving 1 and Eꢀ3ꢀhexene (Eꢀ2b) were also conducted (Scheme 2).
These reactions were conducted with RLYF and the desired epoxide transꢀ5b was formed in low
yield, with the majority of the mass balance consisting of Eꢀ3b and Eꢀstilbene (4). These results
highlight the lower activity of the P450 variants towards Eꢀβꢀethylstyrene. Furthermore, cisꢀ
stilbene oxide (6) was obtained in 7% yield (Entry 1), indicating only moderate selectivity for Eꢀ
βꢀethylstyrene over Zꢀstilbene in the alkene epoxidation. Nonetheless, the desired epoxidation
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occurred with more than 300 turnovers, providing transꢀ5b in up to 22% yield (Entry 1). Similar
yield was obtained in a sequential reaction (Entry 2) and also an isolated epoxidation (Entry 3),
once again suggesting that the enzymatic reaction is not adversely affected by the metathesis
reaction.
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Yielda
[Ru]
Catalyst
P450
TTN
Entry
5b
3b
4
6
1
C2
C2
-
22%
17%
26%
30%
21%
10%
7% 198
2b
3d
4e
-
153
225
270
c
25%
-
-
-
c
C3
10%
43%
40%
-
a Yield determined by GC. Reactions performed on a 0.027 mmol
scale. b Reaction was run sequentially in oneꢀpot. 10 h alkene
metathesis, 16 h enzymatic epoxidation. c Yield not determined. d
Epoxidation only. e 0.054 mmol reaction with 20 nmol P450 in 2
mL reaction volume.
Scheme 2. Tandem Metathesis/Oxidation with Eꢀ3ꢀHexene.
Conclusion
In summary, we have developed a Oneꢀpot alkene Metathesis–enzymatic epoxidation reaction
to convert a mixture of stilbeneꢀderived alkenes selectively into a single epoxide. Simultaneous
alkene metathesis and epoxidation has the potential to disfavor the irreversible formation of Eꢀ
stilbene and provide improved conversion of Zꢀstilbene. Ultimately, moderate yield and excellent
enantioselectivity were obtained in the formation of a number of aryl epoxides. Current efforts
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are directed towards developing P450 BM3 variants with improved catalytic activity and the use
of emulsions to increase the enzymatic reaction rates.
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Experimental Details
Expression and Purification of P450 Variants
The cytochrome P450 BM3 variants were expressed as follows. Overnight cultures of DH5αꢀ
pCWori+ꢀBM3 variant were inoculated in 500 mL TB medium supplemented with 100 µg/mL
ampicillin. After 12 h of growth at 30 ºC and 250 rpm, protein expression was induced with 0.5
mM δꢀaminolevulinic acid and 1 mM IPTG and allowed to grow for a further 24 h at 30 ºC and
180 rpm, after which the cells were harvested by centrifugation (6000 rpm, 4 ºC, 10 min). The
cell pellets were resuspended in 27–30 mL of 0.1 M phosphate buffer (pH 8.1) and 1 mg/mL of
lysozyme was added. After a freezeꢀthaw cycle at ꢀ80 ºC, the cells were disrupted by sonication
(5s on, 5 off, 40% amplitude) for 5 minutes, and the lysate was clarified multiple times by
centrifugation (20,000 rpm, 4 ºC, 15 min). The clarified lysate was filtered through a 0.22 µM
Amicon filter. The P450 concentration was measured by the carbon monoxide binding assay.27
Typical concentrations of 40 µM P450 were readily obtained. This lysate was used as is in
tandem reactions.
For purification of the P450 BM3 variants, the cell lysate was purified as follows. The lysate
was loaded onto a column packed with DEAE 650ꢀM resin (Toyopearl, Los Angeles, CA)
coupled to a fastꢀperformance liquid chromatography. A wash step with 15% NaCl in 25 mM
phosphate buffer, pH 8.1 was applied. The protein eluted at 25% NaCl in the phosphate buffer.
Purity of the protein was estimated to around 70% using SDSꢀPAGE. At this purity, the protein
was judged to be pure enough for biocatalysis.
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General Procedure for Enzymatic Epoxidation
To a solution of
ꢀglucose (300 ꢁL of 1M stock in 200 mM KPi), NADP+ (30 ꢁL of a 20 mM
D
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stock in 100 mM KPi), GDH (20 uL, 1 U/mL), and catalase (30 ꢁL, 600 U/mL) in 200 mM
phosphate buffer pH 8.1 (to 3 mL) was added Eꢀβꢀmethylstyrene (3.51 ꢁL, 3.19 mg, 27 ꢁmol) in
75 ꢁL DMSO. Freshly prepared P450 lysate (9 nmol, 0.033 mol %) was added and the reaction
was incubated at 25–27 ºC, 100 rpm, overnight (12–16 h). Reactions were performed in 27 mL
crimp cap vials to avoid the loss of volatile compounds. Final concentrations are as follows; 3.3
ꢁM P450, 100 mM glucose, 0.2 mM NADP+, 1 U/mL GDH, 600 U/mL catalase, 9 mM Eꢀβꢀ
methylstyrene. 3 mL of EtOAc (or Et2O) and 200 ꢁL of a dodecane stock (20 ꢁL/mL in EtOAc)
were added to the reaction and thoroughly mixed. An aliquot was removed for GC analysis.
Isolated yields were obtained by extraction with Et2O (x2), the combined organic layers dried
over MgSO4, filtered and concentrated in vacuo (>400 mbar, room temperature). The crude
product was purified as described below.
General Protocol for Tandem Metathesis–Epoxidation with Z-Stilbene (1) and Z-2-
Butene (Z-2a)
All reactions were set up open to air in 27ꢀmL headspace crimp cap vials (SigmaꢀAldrich). In
general, it was found that the minimum substrate loading required to obtain reliable and
reproducible results was 0.054 mmol. To a solution of Dꢀglucose (300 ꢁL of 1M stock in 200
mM KPi), NADP+ (30 ꢁL of a 20 mM stock in 100 mM KPi), GDH (20 uL, 1 U/mL), catalase
(30 ꢁL, 600 U/mL) and 75 ꢁL DMSO (2.5 v/v%) in 200 mM phosphate buffer pH 8.1 (to 3 mL)
was added freshly prepared P450 RLYF lysate (9 nmol, 0.017 mol %). ZꢀStilbene (1, 9.6 ꢁL, 9.7
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mg, 0.054 mmol, 1 equiv.) was added to the top of the aqueous reaction mixture (avoid vial
walls). The ruthenium catalyst C2 (2 mg, 0.0033 mmol, 6 mol %) was carefully added to the
small organic layer, avoiding agitation that might cause the catalyst solution to sink (see Figure
S3 in SI) or stick to the walls. The vial was quickly sealed with a crimp cap and pressurized with
Zꢀ2ꢀbutene gas, Zꢀ2a (6 psig, 7.4 equiv.). The reaction was run in a shaking incubator at 100 rpm,
27 °C for 16 h. It is important to note that, in order to keep enough oxygen in the system for the
epoxidation reaction, the vial was not evacuated prior to addition of Zꢀ2ꢀbutene. In addition,
using Eꢀ2ꢀbutene or a mixture of E- and Zꢀ2ꢀbutene affords low yields of Eꢀ3a, presumably
because these two gases contain traces of 1,3ꢀbutadiene, a compound that is poisonous to
alkylideneꢀbased metathesis catalysts.28 Final concentrations are as follows: 3.3 ꢁM P450, 100
mM glucose, 0.2 mM NADP+, 1 U/mL GDH, 600 U/mL catalase, 18 mM Zꢀstilbene, 6 psig Zꢀ2ꢀ
butene. Reactions were extracted with 9 mL of ethyl acetate containing 1 mM eicosane, dried
over Na2SO4, filtered and analyzed by GC. The crude products were purified as described below.
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General Protocol for Tandem Reaction Using Z-Stilbene (1) and E-3-Hexene (E-2b)
All reactions were set up open to air in either 10ꢀmL or 27ꢀmL headspace crimp cap vials. To a
solution of D
ꢀglucose (300 ꢁL of 1M stock in 200 mM KPi), NADP+ (30 ꢁL of a 20 mM stock in
100 mM KPi), GDH (20 uL, 1 U/mL), catalase (30 ꢁL, 600 U/mL), and DMSO (75 ꢁL, 2.5
v/v%) in 200 mM phosphate buffer pH 8.1 (to 3 mL) was added freshly prepared P450 RLYF
lysate (30 nmol, 0.1 mol %). Eꢀ3ꢀHexene (20 ꢁL, 13.6 mg, 0.16 mmol, 3 equiv) and ZꢀStilbene
(1, 9.6 ꢁL, 9.7 mg, 0.054 mmol, 1 equiv.) were carefully added to the top of the aqueous reaction
mixture (avoiding vial walls). The ruthenium catalyst C2 (1 mg, 0.0033 mmol, 3 mol %) was
carefully added to the small organic layer, avoiding agitation that might cause the catalyst
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solution to sink (see picture in SI) or stick to the walls. The vial was sealed with a crimp cap and
the reaction was run in a shaking incubator at 100 rpm, 27°C for 16 h. Reactions were extracted
with 9 mL of ethyl acetate containing 1 mM eicosane, dried over Na2SO4, filtered and analyzed
by GC.
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ASSOCIATED CONTENT
Supporting Information.
This material is available free of charge via the Internet at http://pubs.acs.org.
Supplemental tables and figures, and additional information of experimental procedures and
methods, characterization data, and NMR spectra of organic products.
AUTHOR INFORMATION
Corresponding Author
Huimin Zhao: zhao5@illinois.edu
John F. Hartwig: jhartwig@berkeley.edu
Present Addresses
Carl Denard: University of TexasꢀAustin, Chemistry Department, 2500 Speedway, Austin Texas,
78712
Mark J. Bartlett: Gilead Sciences, Inc., 333 Lakeside Dr., Foster City, CA 94404
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Lu Lu: Civil and Environmental Engineering, 4146 Newmark, 205 N. Mathews Ave. Urbana, IL
61801
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Author Contributions
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The manuscript was written through contributions of all authors. All authors have given approval
to the final version of the manuscript. ‡These authors contributed equally.
ACKNOWLEDGMENT
This work was supported by NSF under the CCI Center for Enabling New Technologies through
Catalysis (CENTC) Phase II Renewal, CHEꢀ1205189. Electronic Supplementary Information
(ESI) available: supplementary tables and figures, experimental procedures and product
characterization data. See DOI: 10.1039/c000000x/
References
1
2
3
Bornscheuer, U. T.; Huisman, G. W.; Kazlauskas, R. J.; Lutz, S.; Moore, J. C.; Robins,
K. Nature, 2012, 485, 185ꢀ194.
(a) Denard, C. A.; Hartwig, J. F.; Zhao, H. ACS Catal, 2013, 3, 2856ꢀ2864; (b) Wallace,
S.; Balskus, E. P. Curr Opin Biotechnol, 2014, 30, 1ꢀ8.
(a) Pamies, O.; Bäckvall, J. E. Chem Rev, 2003, 103, 3247ꢀ3262; (b)
Turner, N. J.
Curr Opin Chem Biol, 2004, 8, 114ꢀ119; (c) Engström, K.; Johnston, E. V.; Verho, O.;
Gustafson, K. P. J.; Shakeri, M.; Tai, C.ꢀW.; Bäckvall, J.ꢀE. Angew Chem Int Ed, 2013,
52, 14006ꢀ14010.
4
5
Tenbrink, K.; Seßler, M.; Schatz, J.; Gröger, H. Adv Synth Catal, 2011, 353, 2363ꢀ2367.
For representative examples, see: (a) Köhler V.; Wilson, Y. M.; Dürrenberger M.;
Ghislieri D.; Churakova E.; Quinto T.; Knörr L.; Häussinger D.; Hollmann F.; Turner, N.
J.; Ward, T. R. Nat Chem, 2013, 5, 93ꢀ99; (b)
Wang, Z. J.; Clary, K. N.; Bergman,
R. G.; Raymond, K. N.; Toste, F. D. Nat Chem, 2013, 5, 100ꢀ103; (c)
Umeano, A.; Balskus, E. P. Angew Chem Int Ed, 2013, 52, 11800ꢀ11803.
Lee,
Y.;
6
7
Whitehouse, C. J.; Bell, S. G.; Wong, L. L. Chem Soc Rev, 2012, 41, 1218ꢀ1260.
Whitehouse, C. J. C.; Yang, W.; Yorke, J. A.; Tufton, H. G.; Ogilvie, L. C. I.; Bell, S. G.;
Zhou, W.; Bartlam, M.; Rao, Z.; Wong, L.ꢀL. Dalton Trans, 2011, 40, 10383ꢀ10396.
For representative examples, see: (a) Kille, S.; Zilly, F. E.; Acevedo, J. P.; Reetz, M. T.
Nat Chem, 2011, 3, 738ꢀ743; (b) Lewis, J. C.; Bastian, S.; Bennett, C. S.; Fu, Y.;
Mitsuda, Y.; Chen, M. M.; Greenberg, W. A.; Wong, C. H.; Arnold, F. H. Proc Natl
Acad Sci U S A, 2009, 106, 16550ꢀ16555.
8
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1
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4
5
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Denard, C. A.; Huang, H.; Bartlett, M. J.; Lu, L.; Tan, Y.; Zhao, H.; Hartwig, J. F. Angew
Chem Int Ed Engl, 2014, 53, 465ꢀ469.
10
11
12
(a) Connon, S. J.; Blechert, S. Angew Chem Int Ed Engl, 2003, 42, 1900ꢀ1923; (b)
Binder, J. B.; Raines, R. T. Curr Opin Chem Biol, 2008, 12, 767ꢀ773.
(a) Stewart, I. C.; Douglas, C. J.; Grubbs, R. H. Org Lett, 2008, 10, 441ꢀ444; (b) Sanford,
M. S.; Love, J. A.; Grubbs, R. H. J Am Chem Soc, 2001, 123, 6543ꢀ6554.
Chatterjee, A. K.; Choi, T. L.; Sanders, D. P.; Grubbs, R. H. J Am Chem Soc, 2003, 125,
11360ꢀ11370.
Carmichael, A. B.; Wong, L. L. Eur J Biochem, 2001, 268, 3117ꢀ3125.
Li, Q. S.; Ogawa, J.; Schmid, R. D.; Shimizu, S. Appl Environ Microbiol, 2001, 67, 5735ꢀ
5739.
Kubo, T.; Peters, M. W.; Meinhold, P.; Arnold, F. H. Chemistry, 2006, 12, 1216ꢀ1220.
See the Supplementary Information for details of the point mutations that comprise these
P450ꢀBM3 variants.
Nagao, T.; Mitamura, T.; Wang, X. H.; Negoro, S.; Yomo, T.; Urabe, I.; Okada, H. J
Bacteriol, 1992, 174, 5013ꢀ5020.
(a) Johannes, T. W.; Woodyer, R. D.; Zhao, H. Appl Environ Microbiol, 2005, 71, 5728ꢀ
5734; (b) Johannes, T. W.; Woodyer, R. D.; Zhao, H. Biotechnol Bioeng, 2007, 96, 18ꢀ
26; (c) Liu, D. F.; Ding, H. T.; Du, Y. Q.; Zhao, Y. H.; Jia, X. M. Appl Biochem
Biotechnol, 2012, 166, 1301ꢀ1313; (d) McLachlan, M. J.; Johannes, T. W.; Zhao, H.
Biotechnol Bioeng, 2008, 99, 268ꢀ274; (e) Woodyer, R.; van der Donk, W. A.; Zhao, H.
Comb Chem High Throughput Screen, 2006, 9, 237ꢀ245.
Kühnel, K.; Maurer, S. C.; Galeyeva, Y.; Frey, W.; Laschat, S.; Urlacher, V. B. Adv
Synth Catal, 2007, 349, 1451ꢀ1461.
Ryan, J. D.; Clark, D. S. Biotechnol Bioeng, 2008, 99, 1311ꢀ1319.
Tee, K. L.; Dmytrenko, O.; Otto, K.; Schmid, A.; Schwaneberg, U. J Mol Catal B: Enz,
2008, 50, 121ꢀ127.
The absolute configuration of epoxide 5a was determined by chiral SFC coꢀinjection with
authentic (S,S)ꢀ5a.
Koya, S.; Nishioka, Y.; Mizoguchi, H.; Uchida, T.; Katsuki, T. Angew Chem Int Ed Engl,
2012, 51, 8243ꢀ8246.
(a) Huang, W. C.; Cullis, P. M.; Raven, E. L.; Roberts, G. C. Metallomics, 2011, 3, 410ꢀ
416; (b) Tee, K. L.; Schwaneberg, U. Angew Chem Int Ed Engl, 2006, 45, 5380ꢀ5383; (c)
Fruetel, J. A.; Mackman, R. L.; Peterson, J. A.; Ortiz de Montellano, P. R. J Biol Chem,
1994, 269, 28815ꢀ28821.
Lin, M.; Tesconi, M.; Tischler, M. Int J Pharm, 2009, 369, 47ꢀ52.
Alcalde, M.; Farinas, E. T.; Arnold, F. H. J Biomol Screen, 2004, 9, 141ꢀ146.
The yield obtained in the tandem reactions is significantly higher than the theoretical
yield from separate metathesis (37%) and epoxidation (34%) reactions. We hypothesize
that this is a result of the low aqueous solubility of Eꢀβꢀmethylstyrene (ca. 0.11 mg/mL),
which means that the epoxidation of 27 ꢁmol (3.19 mg Eꢀ3a) and 10 ꢁmol (37% of 27
ꢁmol) of Eꢀ3a have the same amount of substrate available for epoxidation at a given
time. This is also reflected in the higher yield obtained with KT32 on a smaller scale
(Table 3, Entry 6). However, in general it was found that the minimum substrate loading
required to obtain reliable and reproducible metathesis reactions was 0.027–0.054 mmol.
Omura, T.; Sato, R. J Biol Chem, 1964, 239, 2370ꢀ2378.
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Patel, J.; Elaridi, J.; Jackson, W. R.; Robinson, A. J.; Serelis, A. K.; Such, C. Chem
Commun (Camb), 2005, 44, 5546ꢀ5547.
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Insert Table of Contents Graphic and Synopsis Here
An organometallic catalyst and a metalloenzyme working together in one-pot
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