Ketoreductase/Transaminase, One-Pot, Multikilogram Biocatalytic Cascade Reaction

Article abstract of DOI:10.1021/acs.oprd.0c00557

A biocatalytic cascade to produce tert-butyl ((2R,4R)-2-methyltetrahydro-2H-pyran-4-yl)carbamate 6 has been demonstrated at the multikilogram scale. In this reaction, a racemic ketone is resolved by reducing the undesired ketone using a ketone reductase (KRED). The reduction is stereospecific for the 2-position of substrate (2S)-ketone leaving the (2R)-ketone unreacted. After the (2S)-ketone has been depleted, a transaminase is added to catalyze the enantioselective transamination of the ketone, resulting in formation of the (2R, 4R)-amine 6. The product is recovered from the aqueous reaction after Boc protection.

Full text of DOI:10.1021/acs.oprd.0c00557

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Article
Ketoreductase/Transaminase, One-Pot, Multikilogram Biocatalytic
Cascade Reaction
Michael Burns,* Wenying Bi, Hui Kim, Manjinder S. Lall, Chao Li, and Brian T. O’Neill
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ABSTRACT: A biocatalytic cascade to produce tert-butyl ((2R,4R)-2-methyltetrahydro-2H-pyran-4-yl)carbamate 6 has been
demonstrated at the multikilogram scale. In this reaction, a racemic ketone is resolved by reducing the undesired ketone using a
ketone reductase (KRED). The reduction is stereospecific for the 2-position of substrate (2S)-ketone leaving the (2R)-ketone
unreacted. After the (2S)-ketone has been depleted, a transaminase is added to catalyze the enantioselective transamination of the
ketone, resulting in formation of the (2R, 4R)-amine 6. The product is recovered from the aqueous reaction after Boc protection.
KEYWORDS: cascade reaction, biocatalysis, transaminase, alcohol dehydrogenase, ketoreductase
Parkinson’s disease. Specifically, tert-butyl ((2R,4R)-2-methyl-
tetrahydro-2H-pyran-4-yl)carbamate 6 was produced using
ketoreductase (KRED) and transaminase enzymes (Scheme
1). The compounds in this scheme have previously been
described.8,9 This cascade was conducted in a sequential
manner due to the cross reactivity of the enzymes with 1.
INTRODUCTION
■
Biocatalysis has been gaining acceptance in the process
chemistry community over the past couple of decades. The
benefits of using enzymes as catalysts are well known.
Foremost is their exquisite chemo-, regio-, and stereo-
selectivity. Enzymes are also highly regarded as green reagents
since these catalysts may operate in aqueous solutions, are
readily produced from renewable starting materials, and are
biodegradable. Advances in the field of protein engineering
have resulted in the availability of stable, process ready
enzymes that are commercially available. Biocatalysis has been
demonstrated in the commercial scale production of several
blockbuster pharmaceuticals, such as Lipitor, Lyrica, Xalkori,
and Januvia.1−4
Scheme 1. Production of Tert-Butyl ((2R,4R)-2-
Methyltetrahydro-2H-Pyran-4-Yl)Carbamate 6 Using
Ketoreductase (KRED) and Transaminase Enzymes
One of the more powerful methods used in biocatalysis is
the emerging field of multistep enzyme reactions, particularly
those run in-situ and commonly referred to as performed in
cascade mode. Benefits of cascade reactions include the ability
to drive an unfavorable equilibrium and the use of unstable
substrates or intermediates. Alleviating the need to isolate
intermediates confers the advantage of cost savings associated
with the use of solvents and the time required to clean the
reaction vessel between steps. Challenges facing cascades
include cross reactivity of the enzymes and the compatibility of
the steps in terms of pH, temperature, and tolerance of
cosolvents.5
Cascades may be characterized as being simultaneous or
sequential. Simultaneous cascades have the advantage of
simplicity and the possibility of driving the thermodynamic
equilibrium. Sequential cascades may be preferred when the
cascade includes incompatible steps. The steps in a sequential
reaction may be compartmentalized using engineering controls
such as membrane reactors or flow techniques. Some recent
reviews offer an excellent description of the current state of the
field of biocatalysis and cascades.6,7
The initial approach to 6 was inspired by a literature report
of the synthesis of (S)-2-methyltetrahydro-4H-pyran-4-one 7
(Scheme 2). This process was attractive because it utilized
cheap starting materials and readily available lipase CalB
(Novozyme 435). The authors report that the synthetic route
in Scheme 2 was used to produce ∼100 kg of the (S)-2-
pyranone 7. This resolution could potentially afford the desired
(R)-2-pyranone as well.
Received: December 31, 2020
Published: February 14, 2021
This paper describes the synthesis of an intermediate in the
synthesis of a compound under development to treat
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amination of 2 to produce 4. Amine 4 is then Boc-protected to
enable isolation from the aqueous medium, facilitating a two-
enzyme, three-step one-pot biocatalytic cascade reaction to
afford carbamate 6.
Scheme 2. Literature Report of the Synthesis of 7 Using
Stereoselective Enzymatic Resolution
RESULTS AND DISCUSSION
■
KRED Screen of Racemic Ketone 1. A screen was
conducted to identify an enzyme that could selectively reduce
1. A total of 338 enzymes from vendors and from an internal
Pfizer collection were screened. Most of the enzymes exhibited
high selectivity at the 4-position but were not selective at the 2-
position, resulting in a mix of diastereomers. Four enzymes did
show the desired selectivity at both positions. The most
selective of these was ADH-097 from c-LEcta, and this enzyme
was chosen for further development (Table 1).
Table 1. KRED Screen for the Conversion of 1 to 3
There were some pros and cons for the synthesis using the
lipase resolution. The Prins reaction utilized inexpensive
starting materials but was more selective toward the cis
stereoisomer. The pyranol and butyrate were separated by an
aqueous/organic partition followed by chromatography. The
Jones oxidation was not favorable on environmental grounds
due to the use of chromium. The route developed utilized flow
chemistry for the acylation step. Although this route was
effective, we often found it to be unreliable when adapted to
flow so an alternative route was developed as described below.
The newly developed route is based on the literature report
and enhanced by the addition of a transaminase enzyme that
was used to make (2R,4R)-2-methyltetrahydro-2H-pyran-4-
amine 4 (Scheme 3).
Conditions: 1, 17.5 mM; NADP+, 0.1 mg/mL; NAD+, 0.1
mg/mL; glucose dehydrogenase, 0.1 mg/mL; glucose, 21 mM,
enzyme, 2 mg/mL; DMSO, 2%; 30 °C, 1000 rpm, 24 h. Values
are expressed as area %.
Scheme 3. Initial Medicinal Chemistry Route
Transaminase Screen of 1. A screen was conducted to
identify enzymes that could enantioselectivity perform a
transamination of 2R-1 to yield 4. When the screen was
performed, chiral 2 was unavailable, so the screen was
conducted on the racemate 1 (Table 2). The Pfizer enzymes
are wild-type enzymes with the following accession numbers:
AT045, XP_381942; AT041, XP_001209325.1; AT043,
XP_001818566; and AT042, CAP97304.
Table 2. Transaminase Screen for the Conversion of 1 to
Diastereomers 4
Initially, a dynamic kinetic resolution was envisioned using a
transaminase to selectively convert racemic 1 to 4. This
approach was unsuccessful due to the difficulty of epimerizing
the C-2 methyl group. Subsequent calculations using an
internal Pfizer tool, based on COSMO-RS (Cosmotherm
Implementation), predicted that the proton at the 2-position
had a pKa of 27. With this result in hand, the route in Scheme
1 was developed. This route relies on an enzyme that can
stereospecifically reduce 2S-1, leaving 2R-ketone 1 unreacted.
When all the 2S-1 is depleted, a transaminase enzyme is added
to the reaction vessel to perform a stereoselective trans-
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Conditions: 1, 10 mM; enzyme, 2 mg/mL; isopropylamine
hydrochloride, 20 mM; pyridoxal phosphate, 3 mM; DMSO,
2%; 100 mM potassium phosphate, pH 7.0, 0.5 mL; 40 °C;
1000 rpm; 18 h. Values are areas under the curve of the HPLC
peak.
The KRED, ADH-097, can perform substrate-coupled
recycling of NAD+. The cosubstrate is isopropanol, which is
oxidized to acetone. The concentration of isopropanol and a
comparison to enzyme-coupled cofactor recycling with glucose
dehydrogenase were investigated (Figure 3). The result with
glucose dehydrogenase was superior. The difference in
isopropanol concentration was minimal, with concentrations
of 2−4% being preferred.
Process Development. This project was in the early
stages of development, and very limited process development
was conducted when the project was halted. The KRED
enzyme, ADH-097, and transaminase enzyme, ATA-P2-B01,
were chosen based upon the screening results and availability.
This was a robust process, and when transferred to a third
party vendor, proprietary enzymes owned by that vendor were
used. Variables in the KRED reaction that were investigated
were enzyme loading, the choice of a cofactor recycling system,
and the concentration of 1. The concentration of racemic-1
was investigated in reactions with 2.5% enzyme loading
(weight lyophilized enzyme powder: weight 1), 5% isopropa-
nol, 100 mM potassium phosphate buffer, pH 7.5, and 30 °C
in 1 mL overnight reactions (Figure 1). The results were
Figure 3. Comparison of cofactor recycling systems.
Standard conditions that have been used for previous
transaminase reactions were employed with no process
development. These transaminases from Codexis are known
to be extremely stable to temperature and organic solvents. It
was also known that the preferred amine donor is isopropyl-
amine. In the transaminase reaction, the isopropanol is
converted to acetone and an accumulation of acetone can be
detrimental. A critical parameter for driving the reaction to
completion is in-situ product removal, which may be
accomplished by distilling the acetone under vacuum or by
means of a nitrogen sweep.
With this minimal amount of development, the process was
conducted as a one-pot sequential cascade at the gram scale
and then transferred to a third party where it was repeated at
the lab scale and then at the multikilogram scale. However, at
the multikilogram scale, the third party vendor used a higher
loading of their proprietary enzymes, and in order to lessen the
chance of emulsions in the final work-up, the decision was
made to remove the KRED enzyme following the first step and
conduct the reaction as a telescoped process.
Figure 1. Effect of concentration of 1 in the KRED reaction with
ADH-097 when sampled at 24 h.
judged by the %ee of R-1. At the highest substrate
concentration tested, 50 g/L, the %ee was slightly lower than
that at more dilute concentrations, but the result at this
concentration was deemed adequate as a starting point for
further optimization.
KRED with ADH-097 enzyme loading was investigated in
reactions with cofactor recycling with glucose dehydrogenase,
100 mM potassium phosphate buffer, pH 7.5, and 30 °C in 1
mL overnight reactions (Figure 2). The best result was
observed with a KRED loading of 2.5%, which was deemed
adequate to use for further process development.
CONCLUSIONS
■
The original route to 4 was long, low yielding, and difficult to
carry out on the kilogram scale, required multiple chromato-
graphic separations, and used a chromium oxidation step and
an inefficient enzymatic step that used vinyl butyrate as the
substrate lowering the atom efficiency. A novel three-step two-
enzyme one-pot biocatalytic cascade reaction was developed,
which was stereoselective and atom efficient. Chromatography
was eliminated, thus greatly decreasing the use of large
volumes of organic solvents and silica gel waste. Reactions
were run in aqueous buffer, and the heavy metal oxidation step
was removed. Although recent advances in biocatalysis using
KREDs and transaminases have enabled great many reactions,
the disclosure of multikilogram-scale processes using a cascade
Figure 2. Effect of enzyme loading in the KRED reaction.
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or telescoped reaction that involves a transaminase is
uncommon.
charged into a 2000 L glass-lined reactor at 10∼20 °C, and the
stirrer was started. A solution of sodium hydroxide (29.9 kg) in
purified water (116.7 kg) was added to adjust pH to 6∼7.
Then, ruthenium (iii) chloride trihydrate (4.5 kg, 0.05 equiv)
was added in one portion, and the mixture was cooled to 0∼10
°C. Sodium hypochlorite solution (634.0 kg, 2.5 equiv) was
added into the above mixture at 0∼10 °C. Then, the reaction
mixture was maintained at 0∼10 °C for 2∼3 h. Meanwhile, the
mixture was sampled for GC analysis every 0.5∼2 h. The
reaction was considered complete when the area% of 2-
methyltetrahydro-2H-pyran-4-ol was ≤1%. Dichloromethane
(217.1 kg) was added to the reaction mixture. Then, the
phases were separated, and the aqueous phase was re-extracted
with dichloromethane (106.1 kg). Then, the combined organic
phase was filtered with a nutsche filter, which was preloaded
with Celite (20.3 kg). The filter cake was soaked with
dichloromethane (45.9 kg). The rinsing liquor was combined
with the filtrate. A solution of sodium sulfite (11.5 kg) in
purified water (116.9 kg) was added to the combined filtrate at
15∼25 °C. Then, the phases were separated, and the aqueous
phase was re-extracted with dichloromethane (46.9 kg) at
15∼25 °C. The combined organic phase was concentrated at
≤25 °C under reduced pressure until no more distillate was
observed and DCM residual ≤10%. The resulting pale yellow
liquid (31.1 kg, assay by NMR with 85.4%) was obtained with
GC purity of 96% (GC a/a). This equates to a 68.3% yield
from 2-methyl-tetrahydro-pyran-4-ol. The liquid was directly
used in the next step. 1H NMR (400 MHz, CDCl3): δ
4.25∼4.30 (m, 1 H), 3.70∼3.76 (m, 1H), 3.64∼3.67 (m, 1H),
2.54∼2.62 (m, 1H), 2.36∼2.42 (m, 1H), 2.25∼2.33 (m, 2H),
1.31 (d, J = 6.4 Hz, 3H).
EXPERIMENTAL SECTION
■
Analytical Methods. Chiral GC for 1 and racemic 3.
Column, Supelco BetaDex 225; 30 m × 0.25 mm × 0.25 μm,
#24348. Constant flow with helium at 1.2 mL/min; gradient,
60 °C for 0.25 min.
Ramp to 150 °C at 7 °C/min; hold for 2 min; FID
detection.
UPLC Method for 1and 2. Before analysis, the quenched,
clarified samples were derivatized with Marfey’s Reagent (CAS
95713−52-3).10 A 50 μL sample was mixed with 10 μL of
aqueous 1 M sodium bicarbonate and 200 μL of Marfey’s
Reagent (5 g/L in acetonitrile) for 60 min at 40 °C. The
reaction was quenched with 10 μL of 1 M HCl and diluted
with 230 μL of acetonitrile. Column, Waters BEH C18, 2.1 ×
100 mm, 1.7 μm, part # 186002350; column temperature, 45
°C; flow rate, 0.5 mL/min; mobile phase, 0.1% aqueous TFA/
ACN; gradient, t = 0 min 95:5, t = 4 min 70:30; UV detection
at 340 nm.
Screening of KRED Enzymes. Screening of the enzymes
was carried out in a 96-well format. Plates contained 1 mg of
enzyme in 20 uL of phosphate buffer. Each well was charged
with 100 mM potassium phosphate, 2 mM MgCl2 buffer, pH
7.0 (500 uL); pyranone 1 (1 mg) in DMSO (10 uL); glucose
dehydrogenase Codexis CDX901 (0.05 mg); NADP+ (0.05
mg); NAD+ (0.05 mg); and glucose (1.9 mg). The plates were
incubated with shaking at 30 °C overnight and quenched with
1.0 mL of ethyl acetate. The organic phase was analyzed using
chiral GC.
Preparation of (R)-2-Methyltetrahydropyran-4-one
(2). Purified water (365.3 kg) was charged into a 1000 L
glass-lined reactor, and the stirrer was started. Disodium
phosphate dodecahydrate (7.4 kg) and dihydrogen phosphate
dihydrate (2.4 kg) were added into the mixture at 20∼25 °C
and stirred until solid was dissolved completely. Then,
isopropanol (42.0 kg), 1 (31.0 kg, 26.4 kg corrected by the
NMR assay with 85.4%), and β-dpnh dipotassium salt (0.26
kg) were added into the mixture at 20∼25 °C. Then, the
system pH was detected and ensured in the range from 6.5 to
7.5. KRED lysate (6.6 kg) was added into the mixture at
10∼25 °C. Then, the reaction mixture was heated to 23∼27
°C and stirred for 13∼14 h. Meanwhile, the mixture was
sampled for GC analysis every 0.5∼1.5 h. The reaction was
considered complete when the ee value of 2 was ≥98.0%.
Hydrochloric acid (3.6 kg) was added into the mixture at
10∼25 °C to adjust pH to 2∼3 and stirred for 0.5∼1 h.
Sodium hydroxide solution (6.5 kg, 20% w/v), which was pre-
prepared, was added dropwise into the mixture at 10∼25 °C to
adjust pH to 6∼8. Then, Celite (6.6 kg) was added and stirred
for 0.5∼1 h. The mixture was filtered with a stainless steel
centrifuge under the protection of nitrogen. The filter cake was
rinsed with purified water (26.5 kg). The filtrate was
transferred into a 1000 L glass-lined reactor and stirred until
it becomes homogeneous for temporary storage. The pale
yellow liquid (482.5 kg, assay by Wt % with 2.5%) was
obtained with an ee value of 98.0%. This equates to a 46.2%
yield from 2-methyldihydro-2H-pyran-4(3H)-one. The solu-
tion was directly used in the next step.
Screening of Transaminase Enzymes. Screening of the
enzymes was carried out in a 96-well format. Plates contained 1
mg of enzyme in 20 μL of phosphate buffer. Each well was
charged with 100 mM potassium phosphate buffer, pH 7.5
(500 uL); pyranone 1 (0.57 mg) in DMSO (10 uL); pyridoxal
phosphate (0.37 mg); and isopropylamine hydrochloride (0.96
mg). The plates were incubated with shaking at 40 °C
overnight and quenched with 0.5 mL of acetonitrile. The
mixtures were centrifuged to clarify and analyzed using uPLC
after derivatization with Marfey’s Reagent (CAS 95713−52-3).
Preparation of 2-Methyl-tetrahydro-pyran-4-ol. Puri-
fied water (121.5 kg) was charged into a 500 L glass-lined
reactor, and the stirrer was started. Then, sulfuric acid (30.5
kg) was added slowly at <40 °C and stirred for 0.5 h. (3-Buten-
1-ol) (31.0 kg, 1.0 equiv) was added and stirred for 0.5 h.
Then, the mixture was heated to 50∼60 °C. Paraldehyde (19.8
kg, 0.36 equiv) was added into the above mixture in three
portions. Then, the reaction mixture was maintained at 50∼60
°C for 3∼4 h. Meanwhile, the reaction was sampled for GC
analysis every 1∼2 h. The reaction was considered complete
when the area% of 3-Buten-1-ol was≤1%. The resulting
mixture was cooled to 10∼20 °C to give 201.6 kg (assay by
NMR with 19.7%) of yellow liquid with GC purity of 73%
(GC a/a). This equates to an 82.4% yield from starting 3-
buten-1-ol. The solution was directly used in the next step. 1H
NMR (400 MHz, DMSO): δ 3.74∼3.78 (m, 1H), 3.51−3.55
(m, 1H), 3.19−3.30 (m, 2H), 1.74−1.78 (m, 2H), 1.66−1.68
(m, 2H), 1.19−1.22 (m, 1H), 1.02−1.05 (m, 3H), 0.93−0.99
(m, 1H).
Preparation of 2-Methyldihydro-2H-pyran-4(3H)-one
(1). 2-Methyltetrahydro-2H-pyran-4-ol (200.5 kg, 39.5 kg
corrected by the NMR assay with 19.7%, 1.0 equiv) was
Preparation of (2R,4R)-2-methyltetrahydro-2H-
pyran-4-amine (4). Purified water (6.1 kg) and isopropyl
amine (25.3 kg) were charged into a 200 L glass-lined reactor
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1, and the stirrer was started. Hydrochloric acid (41.1 kg) was
added to adjust the pH of the mixture to 6∼8. The mixture was
stirred until it becomes homogeneous. 2 (482.3 kg, 12.2 kg
corrected by Wt % with 2.5%) was added into a 1000 L glass-
lined reactor 2 at 10∼25 °C. The solution prepared in reactor
1 was added into the mixture at 10∼25 °C. Then, pyridoxal-5-
phosphate monohydrate (0.5 kg) was added, and the mixture
was stirred until it becomes homogeneous. The pH of the
mixture was adjusted to 7.4∼7.6 with the sodium hydroxide
solution (25.3 kg, 4.4% w/v) at 10∼25 °C. ATA lysate (192.9
kg) was added into the reaction mixture. Then, the mixture
was reacted at 27∼33 °C under the protection of nitrogen for
40∼42 h. The mixture was sampled for pH and GC analysis
every 4∼8 h; the reaction was considered complete when pH =
7.4∼7.6 and the content of 2 was ≤2%.
Hydrochloric acid (11.2 kg) was added into the mixture at
27∼33 °C to adjust pH to 2∼3. Celite (18.4 kg) was added
into the mixture at 27∼33 °C. Under the protection of
nitrogen, the mixture was filtered with a SS1000 hastelloy
centrifuge in portions. The filter cake was washed with purified
water (71.2 kg). At 10∼25 °C, the filtrate was transferred to
reactor 2, and then, the pH was adjusted to 11.5∼12.5 with the
sodium hydroxide solution (61.9 kg, 40% w/v). The mixture
was concentrated at T ≤ 42 °C under reduced pressure. The
mixture was sampled every 3∼5 h for pH change and
derivatives of isopropylamine analysis until it was ≤0.2%. A
yellow clear liquid (711.1 kg) was obtained with an ee value of
evaporator and continued to concentrate at≤45 °C under
reduced pressure until 0.5∼1.0 vol left. n-Heptane (13.5 kg)
was added into the mixture, and then, the mixture was sampled
for methyl tert-butyl ether residual analysis until methyl tert-
butyl ether residual ≤0.5%. The mixture was concentrated at
≤45 °C under reduced pressure until 9∼18 L left. n-Heptane
(13.5 kg) was added into the mixture, and then, the mixture
was sampled for methyl tert-butyl ether residual analysis until
methyl tert-butyl ether residual ≤0.5%. The mixture was
cooled to 10∼25 °C. Then, the mixture was filtered with a 10
L filter flask; the filter cake was rinsed with n-heptane (8.2 kg),
which was pre-cooled to 0∼10 °C, and then, the filter cake was
sampled for purity analysis by HPLC until it was ≥98%. While
maintaining the temperature at 15∼30 °C, the filter cake was
swept with nitrogen at a tray dryer for 5∼8 h. The mixture was
sampled every 5∼10 h for solvent residual analysis until n-
heptane residual ≤0.5%. A white solid (13.3 kg, assay by Wt %
with 98.8%) was obtained with HPLC purity of 99.6%. This
equates to a 58.6% yield, which was calculated without
correcting by assay from (2R,4R)-2-methyltetrahydro-2H-
1
̈
pyran-4-amine. H NMR (400 MHz, DMSO-d6) a ppm 1.00
(d, J = 12.13 Hz, 1 H), 1.03−1.15 (m, 3 H), 1.17−1.34 (m, 2
H), 1.34−1.41 (m, 9 H), 1.55−1.79 (m, 2 H), 3.18−3.36 (m, 3
H), 3.38−3.48 (m, 1 H), 3.72−3.90 (m, 1 H), 6.79 (d, J = 7.83
Hz, 1 H).
ASSOCIATED CONTENT
■
98.1%. The solution was directly used in the next step.
sı
* Supporting Information
1
̈
H NMR (400 MHz, DMSO-d6) a ppm 1.00−1.24 (m, 5
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.oprd.0c00557.
H), 1.42 (qd, J = 12.19, 4.89 Hz, 2 H), 1.65−1.97 (m, 3 H),
2.18−2.39 (m, 4 H), 3.23 (br. s., 2H), 3.33−3.51 (m, 2 H),
3.74−4.04 (m, 2 H), 6.96−7.26 (m, 3 H), 7.47 (d, J = 8.22 Hz,
2 H), 7.61−8.03 (m, 4 H).
1HNMR results for isolated compounds described in the
Experimental Section (PDF)
Preparation of tert-butyl (2R,4R)-2-methyltetrahy-
dro-2H-pyran-4-ylcarbamate (6). The solution of 4 was
filtered with a stainless steel nutsche filter, and the filter cake
was rinsed with purified water (24.5 kg). The filtrate was
transferred into the 1000 L reactor, and the stirrer was started.
Methyl tert-butyl ether (161.4 kg) was added into the mixture
at 10∼25 °C. The di-tert-butyl dicarbonate solution prepared
with di-tert-butyl dicarbonate (17.3 kg, 0.9 equiv) and methyl
tert-butyl ether (18.3 kg) was added into the mixture at 10∼25
°C. Then, the reaction mixture was heated to 25∼30 °C for 44
h. Meanwhile, the mixture was sampled every 1∼2 h for GC
analysis. The reaction was considered complete when the di-
tert-butyl dicarbonate content was ≥0.5% and the difference
between two consecutive samples was ≤0.5%.
Then, the phases were separated. The aqueous phase was
extracted with methyl tert-butyl ether (161.7 kg) twice at
10∼25 °C. The organic phase was combined and filtered with
a stainless steel nutsche filter, and the filter cake was rinsed
with methyl tert-butyl ether (6.8 kg). The filtrate and the
rinsing liquor were combined, which was transferred into the
1000 L reactor and then settled for 0.5∼1 h before separation.
The organic phase left in the reactor was concentrated at ≤45
°C under reduced pressure until 90∼135 L left. Purified water
(437.4 kg) and sodium chloride (48.6 kg) were added into two
150 L drums. The mixture was stirred until the solid dissolved
completely. While maintaining the temperature at 10∼25 °C,
the mixture was washed three times with sodium chloride
solution (182.1 kg). The organic phase left in the reactor was
concentrated at ≤45 °C under reduced pressure until 45∼60 L
left. Then, the organic phase was transferred into a 20 L rotary
AUTHOR INFORMATION
■
Corresponding Author
Michael Burns − Pfizer Worldwide Research, Development &
Medical, Groton, Connecticut 06340, United States;
Email: michael.p.burns@pfizer.com
Authors
Wenying Bi − Asymchem Life Science (Tianjin) Co., Ltd.,
Tianjin 300457, P.R. China
Hui Kim − Pfizer Worldwide Research, Development &
Medical, Groton, Connecticut 06340, United States
Manjinder S. Lall − Pfizer Worldwide Research, Development
& Medical, Groton, Connecticut 06340, United States;
orcid.org/0000-0002-2882-8075
Chao Li − Pfizer Worldwide Research, Development &
Medical, Groton, Connecticut 06340, United States
Brian T. O’Neill − Pfizer Worldwide Research, Development
& Medical, Groton, Connecticut 06340, United States;
orcid.org/0000-0002-1928-4803
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.oprd.0c00557
Notes
The authors declare no competing financial interest.
REFERENCES
■
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