Efficient, chemoenzymatic process for manufacture of the boceprevir bicyclic [3.1.0]proline intermediate based on amine oxidase-catalyzed desymmetrization

Article abstract of DOI:10.1021/ja3010495

The key structural feature in Boceprevir, Merck's new drug treatment for hepatitis C, is the bicyclic [3.1.0]proline moiety "P2". During the discovery and development stages, the P2 fragment was produced by a classical resolution approach. As the drug candidate advanced through clinical trials and approached regulatory approval and commercialization, Codexis and Schering-Plough (now Merck) jointly developed a chemoenzymatic asymmetric synthesis of P2 where the net reaction was an oxidative Strecker reaction. The key part of this reaction sequence is an enzymatic oxidative desymmetrization of the prochiral amine substrate.

Full text of DOI:10.1021/ja3010495

Article
pubs.acs.org/JACS
Efficient, Chemoenzymatic Process for Manufacture of the
Boceprevir Bicyclic [3.1.0]Proline Intermediate Based on Amine
Oxidase-Catalyzed Desymmetrization
,
†,§,∇
Jack Liang, Alexandre Ambrogelly, Tim Brennan, Guy Gloor, Gjalt Huisman,‡
‡,∇
†
†,∥
†,⊥
Tao Li,*
‡
†
‡
‡
‡
‡
James Lalonde, Azzeddine Lekhal, Ben Mijts, Sheela Muley, Lisa Newman, Matt Tobin,
George Wong, Aleksey Zaks, and Xiyun Zhang
†
†,#
‡
†Merck & Co., Inc., 126 East Lincoln Avenue, Rahway, New Jersey 07065, United States
‡Codexis, Inc., 200 Penobscot Drive, Redwood City, California 94063, United States
*
S Supporting Information
ABSTRACT: The key structural feature in Boceprevir,
Merck’s new drug treatment for hepatitis C, is the bicyclic
[
3.1.0]proline moiety “P2”. During the discovery and develop-
ment stages, the P2 fragment was produced by a classical
resolution approach. As the drug candidate advanced through
clinical trials and approached regulatory approval and
commercialization, Codexis and Schering−Plough (now
Merck) jointly developed a chemoenzymatic asymmetric synthesis of P2 where the net reaction was an oxidative Strecker
reaction. The key part of this reaction sequence is an enzymatic oxidative desymmetrization of the prochiral amine substrate.
INTRODUCTION
Imine preparation by amine oxidation is a highly desirable
synthetic tool, because it opens many strategic opportunities for
The 3-azabicyclo[3.1.0]hexane structure of P2 adopts a
constrained conformation, so that the gem-dimethyl group
has a fixed angle with respect to the bicyclic ring structure. The
incorporation of the 3-azabicyclo[3.1.0]hexane moiety results
in a 1000-fold increase in NS3 protease binding over proline in
a pentapeptide scaffold. When boceprevir binds to the NS3
protease, the P2 moiety interacts with four amino acid residues
■
1
,2
C−C bond formation. Selective, clean, and efficient amine
oxidations are very important for pharmaceutical process
development, because the economic and environmental
impacts of a commercial manufacture process usually last as
long as the patent life cycle of the drug. Among all the available
methods, biocatalysis with amine oxidases is particularly
attractive by virtue of its selectivity and environmental
sustainability: enzymatic oxidation is highly enantioselective,
it uses dioxygen as the oxidant, and the reaction is in water.
Still, this reaction suffers from problems including low enzyme
activity at practical substrate concentration, instability of imine
product, and fire hazard at large scale. In this article, we report
the development of a practical imine preparation method based
on biocatalysis with amine oxidases and the application of this
method in developing a commercial process to manufacture a
bicyclic [3.1.0]proline intermediate for boceprevir.
4
,5
at the active site.
1a
The starting material for the P2 moiety is the HCl salt of the
dimethylcyclopropylproline methyl ester 2. For medicinal
chemistry studies, it was prepared by cyclopropanation of a
3
6
Δ -pyrroline derivative of pyroglutamic acid. For large scale
preparation, caronic anhydride was selected as the starting
material. In the first generation process, desymmetrization of
7
the anhydride was used to set up the two chiral centers in 4.
The third chiral center was installed by a diasterespecific
cyanation of 3. Athough this approach is asymmetric, the
procedure requires eight additional linear steps after
desymmetrization. In the second generation method, the
synthesis was simplified by maintaining molecular symmetry
Boceprevir (Victrelis, 1, Scheme 1) is a newly approved, first
in class oral medication developed for treating chronic hepatitis
C infection. It is recommended as an addition to the current
standard regimen of PEG−interferon and ribavirin in treating
patients infected with HCV genotype 1. Phase III clinical trial
results showed that the addition of boceprevir improved the
8
to the last possible step. In two steps, caronic anhydride was
converted to 6,6-dimethyl-3-azabicyclo[3.1.0]hexane (6). The
amine oxidation with chlorination and elimination gave (±)-5.
The trans-carboxylate was installed by diastereoselective trans-
cyanation and then nitrile methanolysis under Pinner
conditions. In the final step, the desired enantiomer was
isolated by classical resolution. Although the second generation
synthesis is significantly shorter, it still suffers from at least 50%
3
sustained viral response from <30% to close to 70%. This new
therapy represents a major advance in treating HCV infection.
Boceprevir is a peptidomimetic protease inhibitor with four
moieties, P1−P3 and a Cap, where P1 is a racemic β-
aminoamide, P2 is a chiral dimethylcyclopropylproline analog,
P3 is (S)-tert-leucine, and Cap is a tert-butylcarbamoyl group.
Received: February 1, 2012
Published: March 12, 2012
©
2012 American Chemical Society
6467
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Journal of the American Chemical Society
Article
Scheme 1. Key Disconnections in Boceprevir and the Retro-
Synthesis of the Central P2 Moiety
as products. The reaction is catalyzed by amine oxidases that
1
0
are either copper-containing or FAD-dependent. In an earlier
report, Turner’s group described that directed evolution of a
FAD-containing monoamine oxidase (MAO) from Aspergillus
11
niger (MAOs) provided a N336S variant with a broadened
substrate range as well as high enantioselectivity for several α-
12a
methyl amines. In further enzyme evolution, an improved
variant was identified (N336S/I246M) for secondary amines.
For synthetic application, a secondary amine deracemization
12b
protocol was developed by using the N336S/I246M variant.
Intriguingly, both variants exhibited exceptionally high activity
for isoindoline, a 3,4-fused bicyclic pyrrolidine similar to
compound 6. MAON is a FAD-dependent enzyme that can
11
oxidize a wide range of amines. It is also related to MAOA
and MAOB, two of the most important mammalian MAOs
13
known for their promiscuity in oxidizing xenobiotic amines.
Inspired by these results, we embarked on the development of a
MAO-based desymmetrization process (Scheme 2).
Scheme 2. Mechanism for Monoamine Oxidase-Catalyzed
Oxidation of 6,6-Dimethyl-3-azabicyclo[3.1.0]hexane
yield loss in the resolution at the last step. This is especially
significant considering that the synthesis of the pyrrolidine 6
from ethyl chrysanthemate involves the use of excess of both a
metal-based oxidant (e.g., KMnO ) and reductant (e.g., BH or
The catalytic cycle of the desired reaction as shown in
Scheme 2 has significant implications for both process
chemistry and enzyme optimization. From a process develop-
ment standpoint, the key aspects of the reaction are the use of
molecular oxygen and the release of a proton per turnover. For
safety reasons, the use of molecular oxygen precludes the use of
most organic solvents and makes the process potentially
subjective to gas−liquid mass transfer limitations. The
hydrogen peroxide byproduct is to be decomposed to water
and oxygen by catalase, a highly efficient enzyme available in
bulk quantity. The release of protons provides an opportunity
to maintain the pH of the reaction by titration with substrate
free base and to monitor the progress of the reaction based on
the amount of added substrate. From an enzyme optimization
perspective, the high throughput screening of MAO libraries
could be performed by measuring the hydrogen peroxide
4
3
LiAlH ). That is, the material lost (i.e., the waste stream) upon
4
resolution is compounded by the reagents used in the
preparation of the starting material. Given the high cost and
large demand for this intermediate, there were strong
motivations to develop a more efficient approach.
One can envision a superior process where 6 is
desymmetrized directly to give the desired enantiomer of 5.
Indeed, asymmetric phase transfer catalysis has been applied to
the dehydrohalogenation of the corresponding chloroamine,
8c
but the enantiomeric excess (ee) was only 20%. A biocatalytic
approach was therefore explored to carry out the asymmetric
amine oxidation.
Biocatalytic desymmetrization of substituted pyrrolidines is
an attractive approach to access proline analogs. Recently,
9
a
Turner’s group demonstrated that a variety of 3,4-substituted
meso-pyrrolidines can be enantioselectively oxidized by the
14
byproduct in an amplex red-based assay.
1
A collection of amine oxidases was tested for oxidation of 6.
MAON from Aspergillus niger and Aspergillus oryzae showed
activity on 6 in the amplex red-based assay. Larger scale
reactions generated sufficient quantity of 5 for absolute
configuration verification. The product from MAON catalysis
was converted to compound 2 and found to be the desired
enantiomer with an ee >99%. The undesired enantiomer was
never observed throughout the course of the enzyme
optimization. Of the two active wild type MAOs, the A. niger
enzyme was more stable but less active compared to the A.
oryzae enzyme.
MAON D5 mutant. The Δ -pyrroline products were converted
to the amino acids through the corresponding nitriles. In
1
another application, the chiral Δ -pyrrolines were directly used
9b
to prepare prolyl peptides in a Ugi reaction. This reaction has
9c
been applied to an efficient synthesis of Telaprevir. However,
the amine oxidation was carried out with low substrate
concentrations (≤25 mM) in all the examples. Applying this
reaction for practical preparation remains to be a challenge.
RESULTS AND DISCUSSION
■
Amine oxidation is a common pathway in biogenic amine
metabolism. Typically, an amine is oxidized by molecular
oxygen to give the corresponding imine and hydrogen peroxide
Enzyme optimization was initiated using the A. niger enzyme
(MAON) as the parent (see the Supporting Information for
sequence). Initial libraries were generated via random muta-
6
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Article
genesis using error-prone PCR as well as via a homology
model-inspired approach since the crystal structure of MAON
was not available. Screening of these round 1 libraries yielded a
number of variants with improved activity on 6. The best
variant, MAON156, contained one targeted mutation K348Q
and one random mutation A289V. Its activity was 2.4-fold
higher than that of the wild type enzyme. MAON156 was
selected as the backbone for the next round of evolution, where
mutations from other round 1 hits provided new diversity for
recombination into this backbone. In addition, a parallel
strategy was performed by family shuffling of MAON156 with
the homolog from A. oryzae. Two improved variants were
identified from the second round evolution including
MAON274, a hybrid A. niger/A. oryzae variant from the family
shuffling approach, and MAON291 from a hit shuffling library.
MAON274 and MAON 291 provided a ∼3-fold and a ∼6-fold
improvement over MAON156, respectively. The MAON291
variant contained an S-to-G mutation at position 465 that
resulted in a significant change in enzyme solubility as observed
by SDS-PAGE analysis. Retrospective modeling studies (Figure
the activity and stability objectives. This final variant was a
hybrid enzyme with one additional F382L mutation and was
2.8-fold more active than MAON304. MAON401 was
completely stable upon storage under air at −20 °C (standard
storage) for 33 days followed by 10 days at room temperature.
When incubated under air at 40 °C for three days, lyophilized
MAON401 retained more than 85% of its initial activity. The
evolution of the natural MAOs is summarized in Table 1
(
Sequences of the variants are described in the Supporting
16
Information).
Table 1. Summary of MAON Improvement over the Four
Rounds of Evolution
MAON
round variant
mutation
diversity source
improvement
activity
wild
type
A. niger wild type
1
2
2
3
156
274
291
301/
A. niger + A289V,
K348Q
active site tar-
geted library
many (see the Support- A. niger and A.
ing Information)
thermal stabil-
ity at 40 °C
activity and
solubility
oryzae
1) performed after the enzyme evolution had been completed
150 + S465G
diversity from
round 1
many (see the Support- MAON274 and
thermal stabil-
3
3
04/
08
ing Information)
MAON291
ity at 50 °C
4
401
304 + F382L
active site library 8-fold increase
in activity
As the enzyme optimization proceeded, we evaluated process
development-related challenges. In the beginning, MAON291
was used in the development of a laboratory protocol for
preparative amine oxidation. When MAON401 became
available later, we characterized the enzyme comprehensively
and modified the reaction condition for large scale production
based on the underlying principles in reaction design.
The first issue we addressed was limitation of oxygen
availability in aqueous medium. Under 1 atm air at 25 °C the
solubility of oxygen in pure water is ∼250 μM and is expected
to decrease as the salinity in the reaction medium increases. For
practical synthesis, oxygen supply needs to be facilitated. In our
set up oxygen was introduced to the aqueous solution by
passing air through a gas dispersion tube to maximize mass
transfer. The off gas was led through a condenser (−10 °C) to
minimize loss of volatile product 5 and water. The flow rate was
adjusted to meet the expected reaction rate while minimizing
material loss (solubility of 5 is approximately 6 g/L). For easy
scale adjustment the flow rate is expressed as relative volume
(with respect to reaction volume) per unit of time.
Initial experiments were run at 22 g/L (200 mM) of 6 at an
air flow rate of 0.1 v/v per minute. Catalase was added to
decompose hydrogen peroxide byproduct. The reaction was
initiated by adding 5 wt % (1.1 g/L) of MAON291. The pH
was maintained at 7.4 by NaOH titration (instead of using free-
base of 6) with a pH stat. After 11 h, the reaction came to a
complete stop at about 50% conversion (estimated concen-
tration for compound 5 = 92 mM). Adding more MAON291
marginally increased conversion. Enzyme assays showed that
MAON291 was completely deactivated at this point. Further
analysis revealed that 5 was a strong inhibitor to MAON and
the inhibition was irreversible (see the Supporting Informa-
tion).
Figure 1. MAON active site contour with substrate (green), aromatic
cavity (purple; W430, F466), G465 (turquoise), and FAD (yellow)
indicated.
and when the crystal structure of the enzyme became
1
5
available suggest that this mutation in the active site loop
may lead to increased flexibility in the substrate binding site. As
a consequence, the “aromatic cage” might be reoriented for
binding the non-natural substrate and enhance the nucleophil-
icity of the amine moiety.
The optimization of MAON continued through two more
rounds of evolution. Screening strategies were adjusted to
identify variants with tolerance to substrate and product
inhibition as well as thermostability improvement. Thermo-
stability is a desirable characteristic in a biocatalyst as it enables
reactions at higher temperatures, typically at a higher reaction
rate. It also correlates with storage stability, a necessary feature
for industrial application (see below). The thermostability of
the most active round 2 variants, MAON291 and MAON274,
was evaluated over a range of temperatures. MAON291 lost
∼
80% activity when incubated at 40 °C overnight, whereas
MAON274 retained 100% activity at 40 °C and lost only ∼30−
5% activity at 50 °C. In round 3, family shuffling of
3
MAON274 and MAON291 produced improved hybrid variants
MAON301, MAON304, and MAON308 that were more stable
than their parents at 50 °C. In round 4, 12 positions in the
active site of backbone MAON304 were targeted for further
mutation. The final variant, MAON401, was identified that met
In addition to MAON inhibition effects, compound 5 also
has low solubility in aqueous medium and tends to vaporize. Its
6
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volatility posed a safety challenge as the vapor constitutes a fire
hazard when mixing with oxygen. Therefore, we explored
conversion of 5 into a water-soluble intermediate during the
enzymatic reaction. This would increase the volumetric yield in
the aqueous phase and also minimize the fire hazard by
reducing the concentration of 5 in the gas phase. We
considered a one step oxidative Strecker reaction involving
imine capture with a cyanation cascade but dropped the idea for
two reasons: cyanide is a strong inhibitor of catalase, and
aminonitrile 8 racemizes in aqueous medium. Bisulfite adducts
of 5 and a related des-methyl pyrroline were known to be
highly soluble in water. We hypothesized that the formation of
amino sulfonate would minimize the concentration of 5 under
oxidative conditions and product 7 (Scheme 3) would be stable
enough to accumulate as the reaction proceeds.
1 and 2 predominate over reaction 4. Our process design is
equipped with two variables to accommodate the need: the
ratio of bisulfite to 6 can be adjusted to compensate for bisulfite
oxidation, and the addition rate of the substrate can be fine-
tuned to match the rate of reaction 1. The understanding of
MAON kinetics, formation, and decomposition of amino
sulfonate 7 allowed us to identify the reaction condition space.
For laboratory protocol development, we characterized the
impact of substrate 6 and product 5 on the activity of
MAON291 under air (see the Supporting Information).
The kinetic features for MAON401 have been comprehen-
sively characterized. This enzyme was purified to homogeneity
by a protocol involving ammonium sulfate fractionation
followed by ion-exchange chromatography on a Q-Sepharose
fast flow column (GE Healthcare). The specific activity of the
8
b
17
purified enzyme for 6 was 17.7 μmol min− mg , representing
an 8.4-fold increase over wild type MAON. It exhibited an
optimal pH between 7.2 and 7.5. The reaction rate increased
significantly as the concentration of oxygen rose above 0.26
1
−1
Scheme 3. List of Reactions in Amine Oxidation and Imine
Sulfonations Cascade
mM ([O ] in water saturated with air at 1 atm). Fitting the data
2
to kinetic models indicated the reaction followed a Ping Pong
18
mechanism with kinetic parameters listed in Table 2. The kcat
Table 2. Summary of Kinetic Parameters for MAON401
parameter
value
standard error
1
Vmax
51.8 μmol min− mg−1
1.14
0.019
0.14
Km (6)
Koxygen
0.29 mM
0.21 mM
was estimated to be 48 s− assuming a molecular weight for
MAON of 55.6 kDa. Although MAON401 is a more active
variant with better thermal stability, it is still inhibited by 6 as
well as 5 just like its progenitor. At 0.8 mM, imine 5 reduces the
activity of MAON401 by 40%. The inhibition is irreversible
1
(
see the Supporting Information).
Since sulfonation is reversible and bisulfite is oxidized via
We tested the formation of the amino sulfonate in the
enzymatic reaction stream. Sodium bisulfite was added to the
reaction mixture after the concentration of 5 (∼100 mM)
reached the point where enzyme had been completely
reaction 4, we evaluated the formation and stability of 7 under
oxygen. Although sulfonate 7 is known to form from the
reaction of 5 with bisulfite under nitrogen, it had never been
8b
isolated and characterized. We prepared sulfonate 7 by
reacting (1S,5R)-5 with bisulfite in nitrogen saturated water.
The isolated product was a crystalline solid with very high water
solubility (200−250 g/L, 25 °C). The oxidation of 7 was
carried out by adding three equivalents of sodium bisulfite to a
solution of 5 at 25 °C with pH maintained at 7.4. The
concentration of 5 was measured over the course of 180 min.
This reaction was carried out under nitrogen or under pure
oxygen with 70 and 130 mmHg gauge pressure (1.4 and 2.5
Psig). The result showed fast sulfonate formation followed by
slow degradation (Figure 2) through the equilibrium shift due
to bisulfite oxidation. The degradation accelerated as the
pressure of oxygen increased. Under 130 mmHg oxygen more
than 99% of sulfonate degraded in 180 min. Since the
formation of the sulfonate is much faster than its degradation,
sulfonate 7 is expected to accumulate as long as adequate
bisulfite concentration is maintained. In preparative method
development we optimized the reaction condition by max-
imizing the kinetic advantage of the cascade involving reactions
1 and 2 over reactions 4 and 5 (Scheme 3).
1
inactivated. H NMR analysis showed that the formation of
the bisulfite adduct 7 was instantaneous and the reaction
resumed as soon as additional enzyme was added. In a reaction
with 22 g/L of 6, the conversion reached 100% after 3.3 g of
MAON291 was added in three equal portions (with additional
sodium bisulfite) over 29 h. The material balance was 94%,
suggesting very little loss of 5 in the gas phase. Based on this
result we also conclude that the amino sulfonate 7 does not
inhibit or inactivate MAON.
For preparative amine oxidation of 6 we designed a
streamlined process to maximize both volumetric yield of
sulfonate 7 and turnover of MAON. In this plan, bisulfite is
added to the enzyme solution as a mixture with 6 so that imine
5
is instantaneously converted to sulfonate 7 in a cascade of
reactions 1 and 2 (Scheme 3). This would maximize the
volumetric yield and also minimize the inhibition of MAON
(
reaction 5). Unfortunately, under oxidation conditions, the
thermodynamically favored bisulfite oxidation (reaction 4)
competes with sulfonation (reaction 2). When reaction 4 is a
major pathway, the concentration of imine 5 is expected to
increase and cause MAON inactivation. To suppress this
scenario, the reaction conditions have to be such that reactions
In the laboratory protocol we used MAON291 as the catalyst
and air as the oxidant. Typically the reaction was carried out by
adding a mixture of bisulfite and 6 to a solution of MAON291
6
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Journal of the American Chemical Society
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organic phase in about 90% yield from 6. In the next step the
nitrile was transformed to the methyl ester under Pinner
conditions. The product was extracted into methyl t-butyl ether
(
MTBE) after being converted to the free base 9. In the final
step the free base was crystallized as the HCl salt. The overall
yield of the process was 56%, giving 2 with an ee of >99%. The
conditions of the procedure were successfully applied to pilot
plant scale preparation with minor modifications.
The biocatalytic step significantly improved the performance
and sustainability of the manufacturing process of 9. Compared
with the resolution method the product yield was increased by
1
50%, raw material use was reduced by 59.8%, consumption of
water was reduced by 60.7%, and the overall process waste (E-
factor) was reduced by 63.1% (Table 3). The product from
19
Table 3. Improvement in Green Chemistry Metrics
resolution process (kg/ enzyme process (kg/ reduction
parameter
kg product)
kg product)
(%)
Figure 2. Formation and decomposition of sulfonate 7 under nitrogen
or under oxygen with 70 and 130 mmHg gauge pressure.
raw material
used
2.44
0.98
59.8
water used
E-factor
83.5
191
32.8
70.3
60.7
63.1
and catalase at a constant rate over 24 h. At the end of addition
(65 g/L of 6) the product was mostly sulfonate 7 and a small
amount of imine 5 (<10%). The combined concentration of the
products was equivalent to approximately 63 g/L of 5. The
solution yield was >95%, and the product enantiopurity was
this process meets the stringent specifications and is suitable for
API manufacture. This process has been used for commercial
production.
>
99% ee.
Our laboratory protocol should be useful for making the
sulfonates corresponding to all imines from oxidative
For large scale manufacturing it is preferred to run the
reaction in a closed reactor with pure oxygen in the head space
since MAON401 activity increases by up to 3-fold under
pressurized pure oxygen. We determined the solubility of
oxygen as a function of pressure in the head space under
9
desymmetrization by MAON. The sulfonate product is useful
in all applications from the imines: it can be converted to the
amino acid through cyanation, or it can also be converted back
1
to Δ -pyrroline by treating with a mixture of MTBE and
reaction condition and characterized K a as the function of
L
aqueous solution of NaOH.
oxygen pressure and agitation rate. The pressure and agitation
rate was set just enough to allow MAON401 to work at the
optimal rate. On scale-up we chose an agitation speed that gave
us similar calculated KLa.
We then developed the process for amine oxidation and
CONCLUSION
■
We have developed a practical, highly efficient method for
asymmetric oxidation of 6,6-dimethyl-3-azabicyclo[3.1.0]-
hexane. This reaction involves an amine oxidase-mediated
desymmetrization and a concurrent bisulfite adduct formation
cascade to give the corresponding amino sulfonate. Based on
this reaction, a process has been developed to prepare the P2
intermediate for boceprevir. The procedure features great
improvement in efficiency as well as green chemistry
performance.
adapted the downstream chemistry to the chiral sulfonate
(Scheme 4). The procedure began with oxidation of 6 under
Scheme 4. Preparation of the P2 Intermediate from 7
ASSOCIATED CONTENT
■
*
S
Supporting Information
Descriptions of experimental protocols, characterization of new
compounds, amine oxidase cloning and expression, enzyme
evolution by protein engineering, and enzyme characterization.
This material is available free of charge via the Internet at
http://pubs.acs.org.
pure oxygen at 4.0 psi (207 mmHg) gauge pressure using an
induction impeller for mixing. Under optimal condition, the
substrate/bisulfite mixture was added to a solution of 6 wt %
AUTHOR INFORMATION
■
Corresponding Author
li.tao@epa.gov
(
(
3.9 g/L) MAON401 and catalase over 20 h. All of substrate 6
final concentration 65 g/L) was converted to sulfonate 7 with
Present Addresses
Sustainable Technology Division, ORD, U.S. EPA, 26 Martin
L. King Dr. West, Cincinnati, OH 45268-0001
Hospira, 4876 Sterling Dr., Boulder, CO 80301
§
a small amount of 5 (<10%). The enzyme reaction stream was
telescoped for cyanation in a mixture with cyclopentylmethyl
ether (CPME). The reaction gave only trans-nitrile 8 in the
∥
⊥
Chesilton LLC, 3 Sunrise Ct., Colts Neck, NJ 07722
6
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Journal of the American Chemical Society
Article
#Akermin, Inc. 1005 N. Warson Road, Suite 101, St. Louis, MO
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(
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10) For a review on copper containing amine oxidase, see:
a) Brazeau, B.; Johnson, B. J.; Wilmot, C. M. Arch. Biochem. Biophys.
∇
These authors contributed equally to this work.
5
Notes
7
(
The authors declare no competing financial interest.
(
ACKNOWLEDGMENTS
■
2
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and plant operation; Dr. Wen-Chen Suen, Ms. Patty Cheung,
Ms. Feng Tan, and Dr. Xiaodong Bu for providing analytical
support; and Dr. Gregory Hughes for suggestions in manuscript
preparation.
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