Ketone Reductase Biocatalysis in the Synthesis of Chiral Intermediates Toward Generic Active Pharmaceutical Ingredients

Article abstract of DOI:10.1021/acs.oprd.0c00120

A range of generic active pharmaceutical ingredients were examined for potential chiral alcohol motifs and derivatives within their structures that could be employed as key synthetic intermediates. For seven generic active pharmaceutical ingredients (APIs), eight precursor ketones were acquired and then subjected to reduction by >400 commercially available ketone reductases from different suppliers. Positive screening results were achieved for five ketones screened, with multiple ketone reductases available for each successful ketone. Selectivity was typically >99.5% ee in most cases, including for the opposite enantiomer. The three best examples were then optimized and quickly scaled up to 1 L scale in high conversion and isolated yield while retaining selectivity of >99.5% ee for the desired chiral alcohol enantiomer. This work illustrates that where a wide range of enzymes are available, productive enzymes to give either alcohol enantiomer can be readily identified for many ketones and rapidly scaled up to produce chiral alcohols. This approach is particularly applicable to generating chiral API intermediates.

Full text of DOI:10.1021/acs.oprd.0c00120

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Article
Ketone Reductase Biocatalysis in the Synthesis of Chiral
Intermediates Toward Generic Active Pharmaceutical Ingredients
̂
Marina Y. Raynbird, Joanne B. Sampson, Dan A. Smith, Sian M. Forsyth, Jonathan D. Moseley,*
and Andrew S. Wells
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ABSTRACT: A range of generic active pharmaceutical ingredients were examined for potential chiral alcohol motifs and derivatives
within their structures that could be employed as key synthetic intermediates. For seven generic active pharmaceutical ingredients
(APIs), eight precursor ketones were acquired and then subjected to reduction by >400 commercially available ketone reductases
from different suppliers. Positive screening results were achieved for five ketones screened, with multiple ketone reductases available
for each successful ketone. Selectivity was typically >99.5% ee in most cases, including for the opposite enantiomer. The three best
examples were then optimized and quickly scaled up to 1 L scale in high conversion and isolated yield while retaining selectivity of
>99.5% ee for the desired chiral alcohol enantiomer. This work illustrates that where a wide range of enzymes are available,
productive enzymes to give either alcohol enantiomer can be readily identified for many ketones and rapidly scaled up to produce
chiral alcohols. This approach is particularly applicable to generating chiral API intermediates.
KEYWORDS: active pharmaceutical ingredient (API), biocatalysis, chiral alcohol, ketone, ketone reductase (KRED)
INTRODUCTION
■
Biocatalysis has been recognized for some time as a viable
technology in the scale-up and manufacture of valuable chiral
intermediates in the chemical industry,1−3 especially pharma-
ceuticals.4,5 The benefits of their use have been well rehearsed,
notably their specificity and selectivity under moderate
reaction conditions in typically benign solvents. They are not
without their drawbacks however, which can include lack of
stability under productive reaction conditions, product
inhibition, low aqueous solubility for many substrates of
interest, and physical challenges such as causing emulsification
during isolation. Even so, biocatalysts can often be modified by
a variety of highly successful techniques derived from modern
molecular biology, such as directed evolution, random
mutagenesis, and protein engineering, which can overcome
these potential limitations.6 This provides almost infinite scope
for improvement and optimization of such processes.
Consequently, it is thought that for certain transformations,
biocatalysis is likely to provide the most sustainable long-term
manufacturing processes for overall efficiency, environmental
Figure 1. Reaction scheme for a generic KRED reduction of the
ketone to chiral alcohol with two typical co-factor recycling reactions
shown (IPA/KRED and glucose/GDH).
burden, energy usage etc., especially for higher-value products
such as active pharmaceutical ingredients (APIs).3,7,8
sulopenem,13 atorvastatin,14 rosuvastatin,15 ticagrelor16). Our
interest in KREDs was in their potential use as stereoselective
reductants in the synthesis of intermediates for APIs, whether
Ketone reductases (KREDs) (EC 1.1.1) are part of the class
of aldo-keto reductase enzymes that catalyze reduction−
oxidation (redox) reactions between ketones and alcohols
(Figure 1). Several examples of KRED reductions of prochiral
Received: March 24, 2020
ketones to chiral alcohols have been reported, including a
number on industrial scale.9−11 The synthesis of Montelukast
is a well-known example of the highly successful application of
a KRED to replace an existing (and expensive) chemical
reducing agent (DIP-Cl) in a current manufacturing process,12
but other potential examples have also been developed (e.g.
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Table 1. Background Details of APIs Investigated in This Project
patent
synthesis regulatory
major
patent
date
patent
expiry
ketone
API
therapy area
antiemetic
antiretroviral
anticancer (NSCLC) ALK and ROS1 inhibitor
COPD
asthma
asthma
thrombosis
COPD
mechanism
inventor
Merck
BMS
reference reference
approval
1
2
3
4
5
6
7
8
Aprepitant
Atazanavir
Crizotinib
Indacaterol
Montelukast
Montelukast
Ticagrelor
Vilanterol
NK1 substance P antagonist
HIV protease inhibitor
1995
1997
2007
2002
1996
1996
2000
2003
17a
18a
19a
20a
21a
21a
22a
23a
17b, 17c
18b
19b, 19c
20b
21b
21b
2003
2003
2011
2009
1998
1998
2011
2013
2015
2018
2027
2020
2012
2012
2019
2023
Pfizer
β2-adrenoreceptor agonist
LTD4 receptor antagonist
LTD4 receptor antagonist
P2Y12 receptor antagonist
β2-adrenoreceptor agonist
Novartis
Merck
Merck
AstraZeneca
GSK
22b
23b
patented or generic. Chiral alcohols can be derived as the
obvious targets directly from ketone reductions,13 but many
other chiral intermediates are also readily accessible from chiral
alcohols after further functionalization.
This work aimed to investigate the use of KREDs in the
reduction of prochiral ketones to generate chiral alcohol
intermediates (and by extension of their derivatives) for a wide
variety of APIs. We were especially interested in generic APIs
and patented APIs, which were approaching patent expiry, for
which biocatalytic methods might not have been considered in
the original manufacturing routes. Such new synthetic routes
using biocatalysts might well provide more efficient processes,
generate potential new intellectual property for commercial
advantage, and ultimately be more sustainable.
The APIs specifically chosen were as follows (generic rather
than branded trade names have been used in all cases):
Aprepitant, an NK1 substance P antagonist for use as an
antiemetic adjunct with chemotherapy treatments;17 Atazana-
vir, an HIV protease inhibitor for use as an antiretroviral
agent;18 Crizotinib, an ALK and ROS1 inhibitor for use as an
anticancer agent, predominantly against nonsmall cell lung
cancer (NSCLC);19 Indacaterol, an ultra-long-acting β2-
adrenoreceptor agonist for the treatment of chronic obstructive
pulmonary disease (COPD);20 Montelukast, an LTD4
leukotriene receptor antagonist for the treatment of asthma;21
Ticagrelor, a platelet aggregation inhibitor of the P2Y12
receptor for the prevention of thrombosis in patients with the
acute coronary syndrome;22 and Vilanterol, another ultra-long-
acting β2-adrenoreceptor agonist, also for the treatment of
COPD.23 These details, plus the innovator pharma company,
dates and reference for the first patent claim, reference for an
early medicinal or process chemistry synthesis route, and dates
of first major regulatory approval (FDA or EMA) and first
major market patent expiry, are listed in Table 1.
For this program, we wanted to demonstrate the broad
versatility of KREDs to access a range of chiral alcohols. We
judged the eight ketones selected to be sufficient to test this
general principle based on their degree of structural diversity. It
should be stressed that the primary aim of this project was to
demonstrate the generality of the application of KREDs in the
potential manufacture of generic APIs, not to fully optimize a
method for any specific target. The ketones chosen (1−8) are
shown in Figure 2.
Figure 2. Structures of ketones submitted to KRED reduction in this
project (the asterisk indicates the expected carbonyl group to be
reduced where more than one present).
assay; a positive response was indicated by the development of
a red color from the coupled biocatalytic reaction with
formazan.24,25 Plates were preloaded with KREDs, buffer, co-
solvent, and co-factor, to which the diluted test substrate was
added. Where chiral alcohols were available, the reverse
reaction (i.e., oxidation) was screened first, since a positive
response in such cases proved that the enzyme was competent
to produce the desired alcohol enantiomer. Where chiral
alcohols were not available, screening of the racemic alcohols
proved that at least one enantiomer could be obtained by
KRED reduction. Where alcohols were not commercially
available, chiral and racemic samples were prepared by
chemical reduction methods, as noted (see the Experimental
Section and the Supporting Information). This also provided
marker samples necessary to support analytical method
development. This initial screening assay yielded qualitative
results in which the response (intensity of the red color) was
indicative of some degree of reaction conversion. In the few
cases for which alcohols could not easily be prepared, initial
screening had to be conducted on the available ketones in test
tubes (3 mL scale), as described further below.
All of the positive responses from both sets of screening
reactions (reduction or oxidation) were then screened for the
desired (reduction) reaction on a 3 mL test tube scale to
confirm the results. This quantitative screening round
eradicated any false-positive results and also enabled weak
RESULTS AND DISCUSSION
■
Initial Enzyme Screening Studies. The initial qualitative
screening was based on a 96-well plate format and visualized
using a standard colorimetric NAD(P)H sensitive formazan
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responses against particular substrate/KRED combinations to
be evaluated for further study or eliminated from further
screening rounds. Over the course of the project, each ketone
was initially screened against over 400 KREDs from all
commercial sources.26−28
Confirmed positive results from the initial qualitative
screening rounds were obtained for ketones 1, 2, and 6−8
from a range of KREDs, with no positive results for ketones, 3,
4, or 5, as summarized in Table 2. Ticagrelor ketone (7) was
in the enzyme active site or their solubility in the reaction
medium was too low, since no conversion was detected. For
ketone 3, a benzophenone very similar in structure to the
successfully reduced ketones 1 and 7, it is suggested that the
two ortho-chlorine substituents effectively prevented reaction
at the ketone with the KREDs tested, since this was the
differentiating factor between 3 and the other two (1 and 7).
Two ortho-substituents are known to be problematic, although
it must be acknowledged that Codexis have achieved a KRED
reduction of 3 in excellent enantioselectivity (>99% ee) and
high yield (94%), albeit with a mutant KRED derived from an
alcohol dehydrogenase and after extensive optimization.32 This
does however illustrate well the previously stated advantage of
biocatalysis that excellent solutions to chemical transforma-
tions can often be improved despite initial poor or absence of
reactivity.
Finally, note that during the screening phase (up to 3 mL
scale), suitable control reactions were included in all reaction
screens to check for potentially reduced ee arising from achiral
background reduction from some other component or
contaminant and for inhibition of the enzyme. Specifically,
glucose dehydrogenase (GDH) (EC 1.1.99.10) (the most
likely source of background reduction in the reaction) was
shown to have no activity against any of the substrate targets.
KRED-free control reactions were conducted in duplicate on 3
mL scale with all other components present to determine the
presence of any background reduction and also with each
individual component missing (organic solvent, substrate, or
buffer).
The results from all of these control studies concluded that
as expected there was no background reduction within the
limit of detection and that ee’s determined by analytical LC
methods were therefore reliable. It was also confirmed that
sampling methods did not lead to contamination between
reactions or variability in the results.
Reaction Optimization Studies. For further develop-
ment, optimization, and scale-up studies, we chose to focus on
the ketone examples of Aprepitant (1), Atazanavir (2), and
Ticagrelor (7) (Figures 3 and 4), since these gave good
conversions in high ee’s and both enantiomers were produced
(Table 3).
Table 2. Screening Results by KRED Source With
Enantiomer Distribution and Highest ee for Each API
desired enantiomer
other enantiomer
series
total
highest ee (%)
total
highest ee (%)
overall total
1
2
3
4
5
6
7
8
24
9
0
0
0
5
20
6
99.5
99.5
2
1
0
0
0
1
14
2
99
50
26
10
0
0
0
6
36
8
95.4
99.5
99.8
71
99
99.4
unusual in having a high number of positive responses (36) for
both enantiomers in very high ee, although most published
results favor (S)-7. A successful KRED reduction has also been
achieved on a slightly larger ketone relevant to the synthesis of
Ticagrelor.29 Positive results were also obtained for ketones 1,
2, 6, and 8 in high ee for the desired enantiomer, with one or
two examples for the opposite enantiomer in each case. For
example, the opposite enantiomer to that required for
Aprepitant ((R)-1) is a key structural motif in at least two
other related pharmaceuticals, Casopitant30 and Vestipitant,31
the latter of which is still in development (Figure 3). Although
the same final (R)-stereochemistry is required to that of
Aprepitant, substitution by the nitrogen in Casopitant and
Vestipitant requires the opposite (S)-enantiomer as the
starting material to achieve this. In summary, positive results
were obtained for five out of eight ketones screened on a 3 mL
reaction scale (Table 2).
Table 2 illustrates the following key points. First, it was
possible to rapidly obtain multiple positive results in five out of
eight cases using KREDs on a range of pharmaceutically
representative ketones. Additionally, the opposite enantiomer
was also obtained in five cases and with high ee in three cases,
albeit by fewer enzymes. This again shows the power and
versatility of biocatalysis in that positive results could be
obtained in very high ee’s for both enantiomers.
Despite having found multiple active KREDs for most of the
ketones, a single KRED showed the greatest activity while
retaining high selectivity for all three ketones chosen for
further investigation. This greatly simplified further optimiza-
tion of the reaction conditions and aided finding a largely
common set of conditions.
Following these initial screening studies, the key factors to
investigate further were the co-factor recycling system and
solvent composition, along with continuous parameters such as
In the cases where no positive results were obtained, it is
suggested that the ketones 4 and 5 were either too bulky to fit
Figure 3. Structures of Aprepitant, Casopitant, and Vestipitant.
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Figure 4. Structures of Atazanavir and Ticagrelor.
Table 3. Conversion, Stereochemical Product, Enantiomeric Excess, and LC Methods Used for Selected Ketones 1, 2, and 7
After Initial Screening Studies
series
conversion at 24 h (%)
achiral LC method
stereochemical product
R
ee (%)
chiral LC method
C
compared to
1
2
7
>99
>88
>99
A
A
B
>99.5
>99.5
>99.5
Purchased marker
Supplied marker
Synthesis
a
b
2S, 3S
S
A
D
a
b
2-Position already set as (S), 3-position reduced by KRED. d.e. determined from the achiral LC method since diastereomers result in this case.
pH and temperature. Substrate concentration and KRED
loading were optimized during the scale-up studies.
Investigations into fine-tuning the solvent systems then largely
focused on reaction tolerance toward increasing IPA
concentration (to aid the substrate and product solubility).
Reactions of 1 and 7 showed that high conversion to the
desired product (>99%) could be achieved within 4 h until the
concentration of IPA had reached 20%; above this level of IPA,
the enzyme activity began to decline markedly, as Figure 5
The first key factor to finalize was the co-factor recycling
system, which was also linked to the solvent composition. For a
stoichiometric oxidation or reduction reaction, it is only the
recycling of the co-factor that is essentially catalytic. Recycling
of the expensive co-factor, NAD(P)H, as used here, is also
essential on cost grounds. Isopropanol (IPA) can be used as
the co-substrate in many cases, with KREDs and alcohol
dehydrogenases producing acetone as the co-product from the
reverse reaction (cf. Figure 1). Acetone is easily lost by
evaporation from the reaction medium, which helps to drive
the coupled forward reaction, promoting the reduction of the
target substrates (see screening procedure P).
Another commonly used co-factor recycling system is the
NAD(P)H/glucose dehydrogenase (GDH) (EC 1.1.99.10)/
glucose combination, which was also trialed in this study (see
screening procedure G). However, this combination requires a
second enzyme (GDH) and pH adjustment to compensate for
production of the acidic co-product (gluconolactone/gluconic
acid). A direct comparison of the IPA/KRED and glucose/
GDH co-factor recycling systems for these substrates
concluded that the glucose/GDH system was very marginally
faster; however, the simpler IPA/KRED system was retained at
this stage since it also greatly aided the solvent selection.
The identification of a single KRED as the preferred enzyme
for all three substrates greatly simplified further optimization of
the reaction conditions and aided finding a largely common set
of conditions. However, none of the ketones or alcohols were
water soluble so an organic solvent was required to help
solubilize them. The choice of solvent depended strongly on
the physical properties of the substrates and the tolerance of
the enzyme toward the organic component. To overcome the
solubility issues, particularly those posed by ketone 2, a range
of solvents were examined. The solvents trialed were acetone,
DMSO, hexane, isopropanol (IPA), methyl tert-butyl ether
(MTBE), THF, and toluene and in ratios of 1:9, 3:7, and 1:1
with water. As expected, more lipophilic solvents resulted in
phase separation with water. Water/IPA mixtures were found
to be best for ketones 1 and 7. IPA was an ideal co-solvent
since it was required as the co-substrate in screening procedure
P to enable regeneration of the NADPH co-factor.
Figure 5. Conversion at 4 h time points determined on ketone 7
showing reducing tolerance to increasing IPA concentration in water.
shows for ketone 7. A 4:1 ratio of water:IPA was also found to
be the most that could be tolerated for reduction of ketone 2
without reducing conversion below the maximum observed of
∼85−90%.
The important factor of pH was readily screened in the
range of 5.0−8.5 against ketone 1 under standard conditions.
Conversion was measured at an intermediate 2 h time point to
allow for some differentiation in the results. Figure 6 clearly
shows that the KRED is not sensitive to pH changes within the
range pH 6.0 and pH 8.0. Consequently, all subsequent
reactions in the study were performed at pH 7.0, which
allowed a tolerance of one pH log unit on either side of this
value. The stereoselectivity of the enzyme between pH values
5.0 and 8.5 was constant, and all reactions gave an
enantiomeric excess of >99.5%.
The optimum temperature was also easily determined in a
similar manner, again determined on ketone 1. As Figure 7
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A screening study for the reduction of ketone 2 under
conditions of screening procedure P at 40 °C was then
conducted on larger 10 mL scale so that physical effects could
more easily be observed. Ketone concentrations of 17, 20, 25,
and 33 mg/mL were trialed and found to give similar
conversions of ∼75−80% after 2 h and ∼85−90% at extended
reaction times. However, the most concentrated reaction
mixture had begun to solidify within 1 h. At the other
concentrations, the mixture was relatively mobile and stirring
was sufficient, despite some thickness developing. Interestingly,
this study showed that the concentration of the ketone in
solution, and hence the reaction mixture consistency, had
neither a beneficial nor adverse effect on the conversion to the
desired alcohol. Despite this, a compromise between the
reaction concentration and the consistency of the reaction
mixture needed to be reached, since 25 mg/mL was still
deemed dilute (40 relative volumes). Physical problems due to
agitation tend to increase on scale-up,33,34 despite often
improved stirring efficiencies when using larger (jacketed)
vessels equipped with mechanical stirrers. For the purposes of
this study, further optimization studies on ketone 2 were
performed at 25 mg/mL.
Figure 6. Conversion at 2 h time points determined on ketone 1
showing the pH tolerance range.
As noted, ketone 2 was much more soluble in IPA at 50 °C
than at 40 °C, and this factor was combined with portion wise
addition in 0.1 equiv doses to take advantage of improved
solubility. However, consistency worsened as the reaction
progressed until the reaction mixture eventually had the same
consistency as previous reactions at 40 °C added in a single
initial portion. As a result, neither change was implemented.
Each ketone reduction was then sequentially scaled up from
a 3 mL test tube through 10, 50, and 100 mL round-bottom
flasks with magnetic stirring, to 250, 500, and 1000 mL in
heated jacketed reactors with mechanical stirring. The purpose
of 10 and 50 mL reactions was to ensure that the optimized
conditions would prove to be robust as the reaction volume
increased. The stepwise increase in the reaction volume was
primarily to understand the potential effects of vessel size,
vessel type, and agitation, which can cause unexpected
problems.33,34 Reactions for ketones 1 and 7 scaled up with
no demanding issues, although conversions dropped slightly to
∼85 and ∼95%, respectively, from ≥99% at the 3 mL scale
with a 5 wt % loading of KRED. To retain the high level of
conversion previously achieved in screening studies, the
loading of KRED was increased from 5.0 to 8.0 wt %, as
noted above.
Reactions for ketone 2 gave a thick reaction mixture up to
∼100 mL. Scaling this to larger volumes seemed doubtful since
the reaction mixtures had become thicker with increasing
volume. However, conducting a 100 mL reaction in a 250 mL
jacketed vessel with a more efficient mechanical (turbine)
stirrer improved the consistency of the reaction mixture. A
relatively low rate of stirring at 130 rpm was used to avoid the
formation of emulsions or stable foams. A conversion of 85%
was achieved after 4 h, dropping only marginally to 79% when
scaled to 250 mL reaction volume in the same vessel.
Figure 7. Conversion against time determined on ketone 1 at
different temperatures.
shows, the highest levels of conversion were at approximately
40 °C, with the reaction reaching completion in <1 h under
standard screening conditions (i.e., high enzyme loading, low
substrate loading). Significant reaction progress was also made
at 50 °C (and slightly less so at 30 °C), with complete
conversion within 2 h. The ability of this KRED to withstand
higher temperatures was potentially advantageous when
addressing the solubility issues, which arose in the scale up,
especially for ketone 2.
Scale-Up Studies. Screening and optimization studies had
been conducted at high substrate dilutions, typically 10 mg/
mL ketone. Enzyme loadings were thus relatively high at this
scale (up to 3 mL). With a view to scale-up however, substrate
concentrations need to be much higher for manufacturing
efficiency (ideally >100 mg/mL), and enzyme loadings much
lower, to enable greater cost reduction.33 Consequently, initial
scale-up studies were conducted to determine what concen-
tration of ketone could be achieved in the reaction. For
ketones 1 and 7, a solubility of 100 mg/mL (10 relative
volumes) was readily achievable under the conditions of
screening procedure P at 5.0 or 8.0 wt % KRED loading with
no difficulties concerning substrate solubility or loss of
enzymatic efficiency. For the less soluble, more challenging
substrate ketone 2 however, it was immediately apparent that a
100 mg/mL solution was far too ambitious; even screening
reactions that had been conducted at 10 mg/mL became thick
slurries. It was speculated that this could be caused by the
alcohol product 2 precipitating from the reaction mixture.
However, on this scale (3 mL), mixing was still sufficient and
the reaction mixture did not solidify.
Reactions were also conducted on 250 mL for ketones 1 and
7 and on 500 mL for all three ketones. However, as the charge
of KRED increased, the enzyme tended to form a solid mass in
the reaction when added directly to the reactor to the ketone/
IPA/buffer mix. Although no reduction in the reaction rate was
observed at this scale, the formation of a solid mass of the
enzyme could result in an uneven distribution in the reaction
mixture and likely problems on further scaling. This was readily
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avoided by premixing the KRED in the buffered aqueous
reaction medium to give a homogeneous mixture and adding
to the 1000 mL reactor with stirring at 40 °C. The ketone (1
or 7) was dissolved in IPA and added to the reaction mixture,
which avoided the enzyme aggregating into a solid mass. This
reverse addition was not necessary with ketone 2, because
lower KRED loading and more dilute conditions were used.
Indeed, when added last in smaller-scale reactions, ketone 2
tended to form a solid layer above the solvent. This had to be
prevented since contact between the ketone and enzyme would
be reduced, potentially limiting the reduction reaction. KRED
was therefore mixed in the buffered aqueous medium to a
homogeneous mixture and then added to the ketone/IPA
slurry in the reactor. This ensured that the enzyme was well
dissolved and did not aggregate in the reaction.
chiral alcohols were obtained with high selectivity (>99.5% ee)
in good conversion and isolated yield. After further focused
reaction screening and some preliminary optimization, three of
the best examples were then scaled to 1 L scale.
Therefore, we conclude that KRED reductions can be
applied as a general strategy in the synthesis of key chiral
alcohols and their derivatives present in generic APIs and other
high-value materials. The successful scale-up to 1 L with only
minimal optimization so far demonstrates that these reductions
have the potential for successful application on manufacturing
scale, as has been shown elsewhere.9−12 There is clearly a
scope to apply this technology to many further examples.
Finally, in addition, the successful results were also obtained
in typically high selectivity (>99.5% ee) for the opposite
enantiomer in five examples. This further shows the versatility
of biocatalysis in general, and KREDs in particular, to produce
opposite enantiomers from the same prochiral precursors. It
should be noted that all KREDS were obtained from
commercial sources, which would not delay initial scale-up,
and provided both enantiomers without modification. This
powerfully demonstrates the utility of commercially available
KREDS in the potential manufacture of many other high-value
materials.
The reactions were scaled up for all three ketones in a 1000
mL jacketed reactor with mechanical stirring using the
optimized conditions developed so far. The finalized
conditions in all aspects, along with the comparative results
on 1000 mL scale, are summarized in Table 4. Mass recoveries
Table 4. Summary of Reaction Conditions and Results for
Reduction of Ketones 1, 2, and 7 by KRED on 1000 mL
Scale
ketone
Conditions
KRED loading (wt %)
concentration (mg/mL)
pH
temperature (°C)
agitation (rpm)
Results
conversion at 4 h (%)
conversion at 24 h (%)
mass recovery (%)
ee (%)
1
2
7
EXPERIMENTAL SECTION
■
General Procedures. Standard materials and reagents for
the synthesis of several ketones and their respective alcohols
were purchased variously from Sigma-Aldrich (Gillingham,
UK), Fisher Scientific (Loughborough, UK), Fluorochem
(Hadfield, UK), and TCI (Zwijndrecht, Belgium). Solvents
for reactions and HPLC grade solvents for analysis were
purchased from Fisher Scientific. All reagents and solvents
were purchased at standard grades and used as supplied except
where noted otherwise. Specifically, ketones 1, 3, and 7 were
purchased from Fluorochem; ketones 2, 4, and 5 were supplied
by industrial partners; and ketones 6 and 8 were prepared by
synthesis (see the Supporting Information). Marker samples of
racemic alcohols were prepared by standard reduction of the
relevant ketone with sodium borohydride or related reagents,
see Table 5, the representative example below, and the
Supporting Information.
8.0
5.0
25
7.0
40
130
8.0
100
7.0
40
100
7.0
40
230
230
77
90
91
>99.5
72
86
70
>99.5
77
96
94
>99.5
were high and gave product alcohols of quality, reflecting the
conversions achieved. Throughout the project, the preferred
KRED gave exceptionally high ee’s, and this was maintained at
1000 mL scale. This was pleasing and a good indication that
this key parameter would be maintained on further scale-up.
KREDs (EC 1.1.1.1 and EC 1.1.1.2, alcohol dehydrogenases
with NAD+ and NADP+ acceptors, respectively) were obtained
from Codexis (Redwood City, CA),26 Johnson Matthey
(Royston, UK),27 and Prozomix Ltd. (Haltwhistle, UK).28
Glucose dehydrogenase (GDH) (EC 1.1.99.10) and co-factors
NAD+ and NADP+ were also supplied by Prozomix.
CONCLUSIONS
■
In conclusion, KRED reductions have been identified for five
out of eight short-listed ketone precursors present in the
generic APIs chosen for investigation. In five cases, the desired
a b
,
Table 5. Summary of Material Sources, Alcohols, and Analytical Methods
ketone
source
rac-alcohol
reductions
chiral alcohol
source
analytical methods
chiral LC (C)
#
LC (A)
GC (B)
chiral LC (D)
(a)
1
2
3
4
5
6
7
8
purchased
supplied
purchased
supplied
NaBH4
NaBH4
NaBH4
n/a
Red-Al
NaBH4
NaBH4
NaBH4
purchased (R)
supplied (2S,3S)
n/a
n/a
n/a
n/a
yes
yes
yes
yes
yes
yes
yes
(a)
supplied
synthesized
purchased
synthesized
yes
synthesized (S)
n/a
yes
yes
yes
a
b
n/a: not attempted. Achiral LC method A also served to determine d.e. for series 2 compounds.
F
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Screening Procedures. Typical Tube-Scale Screening
With GDH/Glucose Recycle (Screening Procedure G). The
aqueous solvent mixture was prepared using 250 mM
potassium phosphate, 2.0 mM magnesium sulfate, 1.1 mM
NADP+, 1.1 mM NAD+, 80 mM D-glucose, and 10 units/mL
GDH adjusted to pH 7.0.26 Ketone (0.15 mmol) and the
relevant KRED (10 wt %) were added to a 9:1
aqueous:isopropanol reaction mixture (3.0 mL) in a test
tube and heated to 30 °C with magnetic stirring. Reactions
were sampled at intervals by taking aliquots (20 μL) diluted
into acetonitrile (800 μL) for LC-MS (method A) or methanol
(800 μL) for GC-MS (method B). Variations in screening
procedures included the stoichiometry of ketone and KRED,
choice of organic solvent, aqueous:organic solvent ratio,
concentration, pH, and temperature.
Typical Tube-Scale Screening With IPA/Co-factor Recycle
(Screening Procedure P). The aqueous solvent mixture was
prepared using 125 mM potassium phosphate, 1.25 mM
magnesium sulfate, and 1.0 mM NADP+ adjusted to pH 7.0.26
Ketone (0.15 mmol) and the relevant KRED (10 wt %) were
added to a 9:1 aqueous:isopropanol reaction mixture (3.0 mL)
in a test tube and heated to 30 °C with magnetic stirring.
Reactions were sampled at intervals by taking aliquots (20 μL)
diluted into acetonitrile (800 μL) for LC-MS (method A) or
methanol (800 μL) for GC-MS (method B). Variations in
screening procedures included the stoichiometry of ketone and
KRED, choice of organic solvent, aqueous:organic solvent
ratio, concentration, pH, and temperature.
(3H, m), 4.83−4.91 (1H, m), 3.71 (1H, dd, J = 3.5 and 11.5
Hz), 3.59 (1H, dd, J = 8.5 and 11.5 Hz), 2.89 (1H, br s).
Reduction of Ketone (2S)-2 to Chiral Alcohol (2S,3S)-2
With KRED on 1 L Scale. Isopropanol (200 mL) and ketone
(2S)-2 (25.0 g, 84 mmol) were added to a suitably serviced
1000 mL jacketed reactor and stirred at 130 rpm. Aqueous
buffer solution (800 mL) was prepared as described in
screening procedure P, KRED (1.25 g, 5 wt %) was added, and
the resulting mixture was stirred to achieve a homogeneous
solution. This was added to the reaction vessel and heated to
40 °C while stirring was maintained at 130 rpm. Reaction
progress was determined by LC (method A). Once reaction
had reached completion, agitation was increased to 250 rpm
for 15 min, and then, agitation and heating were stopped.
Work up and extractions were as for large-scale reduction of
ketone 1 above. Evaporation of the solvent extracts yielded the
crude desired chiral alcohol product (2S,3S)-2 as a yellow oil
(17.6 g, 70%). Achiral LC (method A, conversion 86%, Rt 2.55
1
min, (2S,3S)-2, >99.5% ee); H NMR (400 MHz, CDCl3) δ
7.29−7.34 (2H, m), 7.21−7.26 (3H, m), 4.58 (1H, br s),
3.79−3.97 (2H, m), 3.64−3.72 (1H, m), 3.56−3.63 (1H, m),
3.16 (1H, br s), 2.88−3.04 (2H, m), 1.38 (9H, s); 13C NMR
(100.6 MHz, CDCl3) 155.9, 137.3, 129.5, 128.6, 126.6, 80.0,
73.5, 54.4, 47.6, 35.8, 28.2.
Reference Marker Preparations. Typical Preparation of
Racemic Alcohols with Sodium Borohydride (NaBH4). A
solution of ketone 7 (100 mg, 0.53 mmol) dissolved in
methanol (4.0 mL) was cooled in an ice bath to 0 °C, and
sodium borohydride (12 mg, 0.32 mmol) was added in one
portion. The reaction mixture was stirred at 0 °C for 40 min
and then at room temperature for 1 h, after which the reaction
mixture was quenched with saturated aq. NH4Cl solution. The
methanol was removed by vacuum distillation, and the residual
aqueous phase was extracted three times with ethyl acetate (5
mL each). The combined organic extracts were dried (MgSO4)
and concentrated to dryness. The crude product was purified
by flash silica gel chromatography eluting with 10% ethyl
acetate in petroleum ether to yield the desired racemic alcohol
7 as a colorless oil (62 mg, 61%).35 TLC Rf 0.20 (20% EtOAc-
petrol). HPLC (method B, Rt 6.64 min, 93% purity); 1H NMR
(500 MHz, CDCl3) δ 7.23−7.28 (1H, m), 7.13−7.20 (1H, m),
7.08−7.12 (1H, m), 4.88 (1H, dt, J = 3.5 and 8.5 Hz), 3.72
(1H, dd, J = 3.5 and 11.5 Hz), 3.59 (1H, dd, J = 8.5 and 11.5
Hz), 2.71 (1H, d, J = 3.5 Hz); m/z (EI+) 141 (M+).
Scale-Up Preparations. Reduction of Ketone 1 to Chiral
Alcohol (R)-1 on 1 L Scale. Aqueous buffer solution (800 mL)
was prepared as described in screening procedure P and added
to a suitably serviced 1000 mL jacketed reactor. KRED (8.0 g,
8 wt %) was added, and the contents were mechanically stirred
at 230 rpm while heated at 40 °C until a colorless solution was
achieved. Isopropanol (200 mL) and ketone 1 (100 g, 390
mols) were added, and stirring was maintained at 230 rpm.
Reaction progress was determined by LC (method A). Once
the reaction had reached completion, agitation and heating
were stopped. The aqueous reaction mixture was extracted
three times with dichloromethane (400 mL each). The
aqueous phase was then filtered through Celite and extracted
twice more with dichloromethane (200 mL each). The
dichloromethane extracts were combined and filtered through
Celite, which was rinsed with further dichloromethane (200
mL). The combined dichloromethane filtrates were dried over
magnesium sulfate, filtered under gravity, and the solvent was
evaporated under vacuum to yield the desired chiral alcohol
product (R)-1 as a yellow solid (91 g, 91%). Achiral HPLC
(method A, Rt 3.76 min, conversion 90%); chiral HPLC
Reductions of other ketones to racemic alcohols using this
and related methods are noted in Table 5 and the Supporting
Information.
Determination of Absolute Stereochemistry for Scale-Up
Examples. Reference marker alcohols of known stereo-
chemistry were purchased for (R) and (S)-1 (Fluorochem)
and supplied for (2S,3S)-2, all of which could be compared to
the results of KRED reductions for ketones 1 and 2 using the
appropriate chiral LC method. Neither enantiomer of alcohol 7
could be purchased, so ketone 7 was reduced by a KRED using
general screening procedure G to give 7 in >99% ee. This was
in turn converted to its acetate ester and the sign of optical
1
(method C, Rt 7.53 min, (R)-1, >99.5% ee); H NMR (500
MHz, CDCl3) δ 7.84 (2H, s), 7.79 (1H, s), 5.01−5.07 (1H,
m), 2.01 (1H, d, J = 3.5 Hz), 1.55 (3H, d, J = 6.5 Hz).
Reduction of Ketone 7 to Chiral Alcohol (S)-7 With KRED
on 1 L Scale. The same procedure was used as for the large-
scale reduction of ketone 1 to chiral alcohol (R)-1 on the same
scale (100 g, 525 mmol) and volume (1000 mL), with all other
factors being identical, except as noted. Reaction progress was
determined by GC (method B). Evaporation of the solvent
extracts yielded the desired chiral alcohol product (S)-7 as a
dark yellow oil (94 g, 93%). Achiral GC (method B, Rt 6.64
min, conversion 96%); chiral HPLC (method D, Rt 12.28 min,
(S)-7, >99.5% ee); 1H NMR (400 MHz, CDCl3) δ 7.07−7.28
20
rotation ([α]D = +39.8 (c 0.7, CHCl3)) compared to the
known literature value ([α]D = +47.4 (c 0.7, CHCl3)),35
20
which identified this sample as having been derived from (S)-7.
Absolute stereochemistry was not formally determined on
other compounds that were not taken into the scale-up studies,
only racemic alcohol marker samples were needed in these
cases to determine if any stereoselectivity had been achieved.
G
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Org. Process Res. Dev. XXXX, XXX, XXX−XXX

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Article
Chromatographic Methods. Reaction Monitoring and
Analysis of Isolated Samples. KRED reduction reactions were
monitored by HPLC-MS (method A) or GC-MS (method B)
as appropriate. Synthetic chemistry reaction monitoring (for
the synthesis of ketones and alcohols) used the same or slightly
modified methods. ee’s were determined by either of two chiral
HPLC methods (C and D). Isolated samples were analyzed for
achiral purity (LC or GC) and enantiomeric purity (LC only)
using the same methods (A to D). Identification and purity by
1H NMR spectroscopy used standard methods on Bruker or
Varian 300, 400, or 500 MHz spectrometers in solvents, as
noted. Identification of isolated samples by mass spectrometry
used methods A or B.
Reaction Monitoring by HPLC-MS (Method A). Instru-
ments: Agilent 1100 with DAD and Waters ZQ (ESCI) MS;
column: Ace Ultra Core 2.5, Super C18, 75 mm × 3.0 mm
i.d.). Samples were prepared by taking 20 μL dissolved in 800
μL of acetonitrile and centrifuged at 3000 rpm for 4 min before
analysis. Injection volume was 2.0 μL, column temperature 40
°C, and detection at 220−300 nm. Eluent A was 10%
acetonitrile in water with 0.05% formic acid, eluent B was
100% acetonitrile with 0.05% formic acid. Run time was 5 min
with a flow rate of 1.25 mL/min. The starting eluent at 0.00
min was 80% eluent A with 20% eluent B rising linearly to 99%
eluent B at 4.00 min and then decreasing linearly to 20% eluent
B at 5.00 min, with a 1.00 min post-time. Mass spectrometry
ionization by ESI+ used 20 eV and by ESI− used 50 eV, both
with the m/z range of 100−1000 amu. Retention times: ketone
1 (3.95 min); alcohol 1 (3.76 min); ketone 2 (2.96 min); (S)-
alcohol (2S,3S)-2 (2.56 min); (R)-alcohol (2S,3R)-2 (2.80
min).
times: ketone 7 (9.16 min); alcohol (S)-7 (10.42 min); alcohol
(R)-7 (11.09 min).
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.oprd.0c00120.
Chiral LC and NMR spectra of alcohols 1, 2, and 7;
synthesis of ketones 6 and 8; preparation of additional
racemic alcohol marker samples (PDF)
AUTHOR INFORMATION
■
Corresponding Author
Jonathan D. Moseley − CatSci Ltd., CBTC2, Cardiff CF3 2PX,
U.K.; orcid.org/0000-0002-5730-051X;
Email: jonathan@scientificupdate.com,
jonathanmoseley15@gmail.com
Authors
Marina Y. Raynbird − CatSci Ltd., CBTC2, Cardiff CF3 2PX,
U.K.
Joanne B. Sampson − CatSci Ltd., CBTC2, Cardiff CF3 2PX,
U.K.
Dan A. Smith − CatSci Ltd., CBTC2, Cardiff CF3 2PX, U.K.
̂
Sian M. Forsyth − CatSci Ltd., CBTC2, Cardiff CF3 2PX, U.K.
Andrew S. Wells − Charnwood Technical Consulting Ltd., Leics
LE12 8AT, U.K.; orcid.org/0000-0003-0832-2410
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.oprd.0c00120
Reaction Monitoring by GC-MS (Method B). Instrument:
Varian Saturn 2000 GC-MS; column: J&W Scientific HP-1MS,
30 m × 2.5 mm with 0.25 μm film. Samples were prepared by
taking 20 μL dissolved in 800 μL of methanol and centrifuged
at 3000 rpm for 4 min before analysis. Injection volume was
1.0 μL, inlet temperature 300 °C, split ratio 50:1, and the
carrier gas helium. Total run time was 17.55 min with a
constant flow rate of 1.2 mL/min and the heating profile as
follows: 60 °C at 0.00 min, hold for 0.55 min, ramp at +15.0
K/min for 4.00 min to 120 °C and then ramp at +20.0 K/min
11.00 min to a final temperature of 300 °C, hold for 2.00 min
at 300 °C and then cool back to 60 °C. Mass spectrometry
ionization was by EI+ with the m/z range of 50−650 amu.
Retention times: ketone 7 (6.80 min); racemic alcohol 7 (6.64
min).
Chiral HPLC Method C. Instrument: Agilent 1100; column:
Diacel OD-H, 250 mm × 4.6 mm i.d., particle size 5.0 μm.
Samples were prepared by taking 20 μL dissolved in 1.0 mL of
heptane and centrifuged at 3000 rpm for 4 min before analysis.
Injection volume was 10.0 μL, column temperature 30 °C, and
detection at 220 nm. Run time was 11.0 min with a flow rate of
1.20 mL/min using an isocratic eluent of 2% ethanol in 98%
heptane. Retention times: ketone 1 (3.69 min); alcohol (S)-1
(6.72 min); alcohol (R)-1 (8.01 min).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
CatSci Ltd., CTC Ltd., and Prozomix gratefully acknowledge
Innovate UK for generous funding and support to this project
(Project no. TP 131918). CatSci thanks Nolwenn Derrien for
some preliminary screening studies; Cath Adam for additional
chemical synthesis; and Rob Jenkins (University of Cardiff) for
facilitating the polarimetry assay.
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