Scalable, Telescoped Hydrogenolysis-Enzymatic Decarboxylation Process for the Asymmetric Synthesis of (R)-α-Heteroaryl Propionic Acids

Article abstract of DOI:10.1021/acs.oprd.0c00397

Enantiopure α-aryl propionic acids are useful building blocks for pharmaceutical research and can be accessed enzymatically using arylmalonate decarboxylases (AMDases) from the corresponding malonic acids. However, the intrinsic instability of malonic acids is a major drawback to this approach in which spontaneous decarboxylation can occur, subsequently eroding enantioselectivity and giving rise to racemic products. This was particularly evident for a panel of N-heterocyclic propionic acids that we wished to access using the approach. Herein, we describe a process to overcome the spontaneous decarboxylation problem in which hydrogenolysis of the corresponding dibenzyl malonates was performed in a biphasic toluene-basic aqueous buffer mixture and telescoped into the subsequent AMDase step. This procedure enabled compounds to be accessed in high enantioselectivities and was successfully demonstrated on 120 g with high yield (76%) and ee (98%).

Full text of DOI:10.1021/acs.oprd.0c00397

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Article
Scalable, Telescoped Hydrogenolysis−Enzymatic Decarboxylation
Process for the Asymmetric Synthesis of (R)‑α-Heteroaryl Propionic
Acids
Caroline A. Blakemore,*,¶ Scott P. France,*,¶ Lacey Samp,¶ Deane M. Nason, Eddie Yang,
Roger M. Howard, Karen J. Coffman, Qingyi Yang, Aaron C. Smith, Edelweiss Evrard, Wei Li, Linlin Dai,
Lixia Yang, Zhiguang Chen, Qingli Zhang, Fangyan He, and Jiesen Zhang
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ABSTRACT: Enantiopure α-aryl propionic acids are useful building blocks for pharmaceutical research and can be accessed
enzymatically using arylmalonate decarboxylases (AMDases) from the corresponding malonic acids. However, the intrinsic
instability of malonic acids is a major drawback to this approach in which spontaneous decarboxylation can occur, subsequently
eroding enantioselectivity and giving rise to racemic products. This was particularly evident for a panel of N-heterocyclic propionic
acids that we wished to access using the approach. Herein, we describe a process to overcome the spontaneous decarboxylation
problem in which hydrogenolysis of the corresponding dibenzyl malonates was performed in a biphasic toluene−basic aqueous
buffer mixture and telescoped into the subsequent AMDase step. This procedure enabled compounds to be accessed in high
enantioselectivities and was successfully demonstrated on 120 g with high yield (76%) and ee (98%).
KEYWORDS: biocatalysis, arylmalonate decarboxylase, hydrogenolysis, α-heteroaryl propionic acids
scope.28−30 This enzyme has high selectivity to form R-
INTRODUCTION
■
configured products; however, one of the key challenges in this
α-Aryl propionic acid derivatives are synthetically useful
approach is the preparation of the required malonic acid
building blocks and are common fragments in drug discovery.
substrates which are prone to spontaneous decarboxylation to
For example, the motif is a key part of the nonsteroidal anti-
give racemic propionic acids. Therefore, it is crucial to
inflammatory drug (NSAID) Profen class of molecules such as
minimize this spontaneous process to be able to generate
naproxen and ibuprofen. For the Profen family, only the S-
desired product in high enantiomeric excess.
enantiomer has the desired pharmacological effect,1 and, as for
A way to address this was reported by Gaßmeyer et al., in
any chiral fragment, much of the synthetic challenge in
which the malonic acid substrates were synthesized and
accessing the motif is in generating enantiopure material. A
isolated via hydrogenolysis of their dibenzylmalonate pre-
wide range of approaches have been developed to synthesize
cursors to avoid saponification conditions which can promote
optically pure α-aryl propionic acids2−6 including catalytic
the decarboxylation process.13 Therefore, we initially set out to
asymmetric hydrogenation of acrylic acid precursors,7,8 as well
apply this methodology to access a range of (R)-heteroaryl and
as biocatalytic,9−13 auxiliary-based,14 and resolution meth-
substituted aryl propionic acids, which have so far not been
ods.15,16
exemplified in the literature using the wild-type AMDase from
We were interested in synthesizing a range of heteroaryl and
B. bronchiseptica. Unfortunately, however, many of these
substituted aryl propionic acids and were particularly attracted
electron-deficient aryl malonic acids were too unstable to
by biocatalytic approaches to these compounds. Biocatalysis is
synthesize and isolate via this approach, prompting us to
becoming an ever more prominent enabling technology for
investigate their stability as well as their utility for enzymatic
chemical synthesis in the pharmaceutical industry due to the
decarboxylation. Based on this understanding, we developed a
high chemo- and enantioselectivity that is typically offered as
new telescoped protocol that enabled the synthesis of a range
well as often milder reaction conditions and environmental
of (R)-α-heteroaryl propionic acids in high ee and was
benefits compared to traditional chemical methods.17−19
amenable to scale-up and demonstrated on 120 g scale.
Of the enzymatic approaches available, arylmalonate
decarboxylases (AMDases) represent a direct way to access
enantiopure α-aryl propionic acids from the corresponding
malonic acids with only carbon dioxide as a byproduct (Figure
Special Issue: Celebrating Women in Process Chem-
istry
1A). One of the most well studied AMDases is from Bordetella
bronchiseptica, which was first reported by Miyamoto and
Ohta20 and subsequently investigated in terms of enzyme
structure,21,22 mechanism,22−24 engineering,25−27 and substrate
Received: September 1, 2020
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© XXXX American Chemical Society
A

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Figure 1. (A) AMDase-catalyzed enantioselective synthesis of (R)-α-aryl propionic acid derivatives. α-Aryl malonic acids can be prone to
spontaneous decarboxylation, resulting in racemic product which lowers the overall ee achieved in the process. (B) Previous work has shown
malonic acids can be synthesized by hydrogenolysis of their dibenzyl malonate precursors.13 This work describes a telescoped, hydrogenolysis−
AMDase process to (R)-α-aryl propionic acids, avoiding the isolation of unstable malonic acids, particularly when the aryl group is N-
heteroaromatic.
malonic acid 4a be isolated cleanly as a bench-stable solid
(Figure 3).
RESULTS AND DISCUSSION
■
We first set out to synthesize and isolate a panel of novel
heterocyclic and substituted phenyl malonic acids in order to
test them as substrates for the AMDase. The synthetic route
followed involved an initial cross-coupling reaction with a
range of substituted (hetero)aryl halides (1a−j) to give
dibenzyl malonate derivatives (2a−j). Subsequent methylation
generated substrates 3a and 3c−j in yields of 17−58% over the
two steps unoptimized and, in the case of 3b, 70% after
optimization (Figure 2). Hydrogenolysis and attempted
To further understand the driving force for decarboxylation,
computational calculations of the ΔE values for the
decarboxylation of malonic acids 4 were carried out. These
suggested that the ΔE(decarboxylation) values for the heteroaryl
compounds (4b−j) are all lower than those for the
experimentally stable nonheteroaryl compound 4a. Further-
more, in the case of 4i and 4j, negative ΔE(decarboxylation) values
were calculated, which correlated with the experimentally
observed propensity for decarboxylation of these compounds
(see Supporting Information, section 7).
With these results in hand, it was apparent that the
conditions were not viable for the synthesis of the
heteroaromatic malonic acids. This led to an investigation of
whether there were hydrogenolysis and enzymatic decarbox-
ylation conditions under which the rate of decarboxylation
could be minimized, and the isolation of the malonic acid
could be avoided by employing a telescoped procedure.
Choosing substrate 3b as a model case, we tested the
hydrogenolysis directly in aqueous buffer and charged AMDase
enzyme to aqueous hydrogenolysis product after Pd/C catalyst
filtration. Pleasingly, initial results showed that this was a
promising approach with enantioenriched product observed
(see Supporting Information, section 1.4).
Further understanding and optimization of the reaction
parameters ultimately resulted in a telescoped process that
achieved good yield and high ee of (R)-5b (Figure 4).
The key features of this procedure include the following:
Figure 2. Synthetic route toward a panel of N-heteroaryl and
substituted aryl methyl benzyl malonates 3a−j. aOptimized yield over
two steps from 1b. All other quoted yields are unoptimized over two
steps from 1.
(1) Minimizing spontaneous decarboxylation with careful
control of pH and temperature. A study of malonic acid
4b at various pH values (5−11) and temperatures (20,
40, 60 °C) showed that spontaneous decarboxylation
could be effectively mitigated at pH ≥8.0 and 20 °C
even after a 7 day time point (Figure 5 and Supporting
Information, section 1.4.2). Therefore, the composition
of the aqueous buffer was particularly important for the
success of the process. pH 8.0 Tris-HCl buffer was
employed so that the final AMDase step could proceed
near the optimal pH for enzyme activity,29 and crucially,
isolation of the malonic acids soon highlighted the instability
of the heterocyclic compounds (4b−j) despite using a
synthetic approach, which has previously been shown to
mitigate decarboxylation for related compounds.13 Sponta-
neous decarboxylation was observed during the hydrogenolysis
of 3b−f and 3h−j, and in the case of 3g, a complex mixture
resulted. Following catalyst filtration and concentration under
vacuum, either monodecarboxylation products (5b−f) or the
bisdecarboxylation products (6h−j) were isolated. Only in the
case of the dimethoxy phenyl ring system could the desired
B
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Figure 3. Hydrogenolysis of dibenzyl malonates 3a−j in attempts to access corresponding malonic acids 4. However, only 4a was stable and
isolable, with hydrogenolysis of 3b−j resulting in either spontaneous decarboxylation to racemic propionic acids 5b−f, to aryl ethanes 6h−j, or to a
a
b
complex mixture (from 3g). Pd(S)/C used instead of Pd/C. Hydrogenolysis carried out at 50 °C as the reaction at 20 °C gave incomplete
conversion and a complex product mixture.
Figure 4. Telescoped hydrogenolysis−AMDase process applied to the synthesis of substituted aromatic and N-heteroaromatic (R)-propionic acids
(R)-5. Reactions performed on 200 mg scale. aAn additional recharge of AMDase (5 wt %) was required for full conversion. bPd(S)/C used instead
c
of Pd/C to avoid dehalogenation (see Supporting Information). Unable to extract into organic layer following acidification during workup.
Pleasingly, when applied to the panel of aryl methyl dibenzyl
malonates (3a−g), this process enabled the synthesis of (R)-
5a, (R)-5c, (R)-5e, and (R)-5g in excellent ee (Figure 3).
Acids (R)-5d and (R)-5f were also accessed although with
modest ee due to a small degree of spontaneous decarbox-
ylation after the hydrogenolysis step, which was not completely
mitigated for these unstable compounds. The products were
isolated after acidification to pH 2.5 and extraction into
organic solvent, with higher isolated yields for the more
lipophilic acids. In the case of (R)-5f, high water solubility
precluded attempts to extract into the organic phase.
For substrate 3c, some dechlorination was observed during
the hydrogenolysis when Pd/C was used. Investigation of
alternative catalysts identified Pd(S)/C as a suitable alternative
in the telescoped process that completely avoided dechlorina-
tion (see Supporting Information, section 1.4.4).
Additionally, compounds 3h−j were subjected to the
telescoped process. Background decarboxylation was still
observed, but interestingly, the new biphasic hydrogenolysis
conditions were able to suppress the previously observed
second decarboxylation event and generate racemic aryl
propionic acids 5h−j in situ. However, these compounds
were not stable to acidification for subsequent isolation.
To demonstrate the scalability of the process, we performed
the telescoped procedure on 50 g of 3b with a decrease in
2 equiv of NaOH was charged before hydrogenolysis in
order to neutralize the 2 equiv of carboxylic acid formed.
Therefore, the initial pH of the aqueous reaction media
at the start of the hydrogenolysis is pH ∼11.0 but
decreases to pH 8.0 once the hydrogenolysis has gone to
completion. Neutralization in this manner employs a
similar concept to Lewin et al. who precipitated out α-
heteroaryl-α-hydroxy malonic acids from organic solvent
as their sodium salts in order to preserve them.30
(2) Ensuring substrate solubility using an organic cosolvent. The
hydrogenolysis step was operated as a biphasic reaction
mixture. Toluene (2 mL/g) was employed to fully
dissolve substrate 3b, which was otherwise poorly
soluble in the aqueous buffer alone.
(3) Pd/C filtration and organic cosolvent separation before the
enzymatic step. Upon completion of the hydrogenolysis,
the malonate dianion 4b resides entirely within the
aqueous phase. Subsequent filtration to remove the Pd/
C catalyst and separation of the toluene layer provided
an aqueous buffered malonate solution. To this was
simply added lyophilized, cell-free extract of the B.
bronchiseptica AMDase, and the reaction mixture was
stirred overnight at 20 °C to affect the asymmetric
decarboxylation.
C
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Figure 5. Stability profile of an aqueous solution of 4b at various pH values and temperatures after 7 days showing the percentage decomposition of
4b to 5b from spontaneous decarboxylation (for earlier time point data, see the Supporting Information, section 1.4.2).
Table 1. Hydrogenation−AMDase Telescoped Process on Scale
entry
scale (3b)
Pd/C (wt %)
buffer volume (mL/g)
buffer pH
yield (%)
ee (%)
1
2
3
4
5
50 g
63 g
120 g
3 g
10
5
5
2
2
10
10
10
10
6
8.0
8.0
8.5
8.0
8.0
80
85
76
86
83
99
99
98
98
99
5 g
AMDase loading (3.5 wt %). The reaction performed equally
well, generating desired (R)-5b in 80% yield and 99% ee
(Table 1, entry 1).
process was suitable for large-scale application and used to
process over 240 g of starting material. We hope that our
addition to the asymmetric decarboxylation methodology
increases its utilization through the applicability to a broader
range of heterocyclic propionic acid derivatives.
To further improve the process, we examined the effect of
lowering the Pd/C loading and buffer charges (Table 1, entries
2−5). Decreasing the loading of Pd/C was successful and
aided the filtration step compared to the original 10 wt %
conditions (Table 1, entries 2, 4, and 5). Using pH 8.5 Tris-
HCl as the main buffer component versus pH 8.0 had no major
effect on ee and validated a 0.5 pH unit acceptable range for
the buffer pH (Table 1, entry 3). Finally, intensifying the
reaction by reducing the volumes of buffer from 10 to 6 mL/g
was also successful, with good yield and high ee achieved,
enabling higher throughput (Table 1, entry 5). The largest
single run was on 120 g scale, and in total, over 240 g of
material was processed using this telescoped procedure.
EXPERIMENTAL SECTION
■
All reagents and solvents were obtained from commercial
sources and used without further purification. Hydrogenation
catalysts were purchased from Johnson Matthey (10% Pd/C
catalog no. A402028-10, and 5% Pd(S)/C catalog no.
A103023-5). The gene encoding the wild-type aryl malonate
decarboxylase from Bordetella bronchiseptica (accession number
Q05115)13,20,31 was prepared in pET28a, recombinantly
expressed in Escherichia coli, and purchased as a lyophilized
cell-free extract from Prozomix, Haltwhistle, UK. This enzyme
has a known strict selectivity for producing R-configured
products.13,28−30 See Supporting Information for further detail.
General Procedure for Telescoped Hydrogenolysis−
AMDase Process on Small Scale. The processes were
carried out in a parallel pressure screening reactor (Biotage
Endeavor). Hydrogenolysis: To a solution of listed substrate
CONCLUSIONS
■
A process was developed to overcome the instability of
malonic acid derivatives in order to access high enantiose-
lectivities and high yields of heteroaromatic propionic acids.
Our approach utilizing a telescoped hydrogenolysis−AMDase
D
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in toluene (2 mL/g) were added 0.5 M Tris-HCl(aq) buffer pH
as listed (10 mL/g) (containing NaOH(aq), 2.0 equiv relative to
the listed substrate) and the listed catalyst. The resulting
mixture was purged with N2(g) (three times) with stirring at
250 rpm and then with H2(g) (three times) with no stirring.
The mixture was heated to 22 °C and then pressurized to the
listed pressure. These conditions were held with stirring at
1000 rpm for 4 h, after which the mixture was purged with
N2(g) and filtered (eluent, water, 10 mL/g). The filtrate layers
were separated, the toluene organic phase was discarded, and
the aqueous phase was analyzed by 1H NMR (D2O).
Enzymatic decarboxylation: To the aqueous phase was
added lyophilized AMDase powder (5 wt % relative to the
dibenzyl malonate substrate or as stated), and the reaction was
shaken (250 rpm) at 20 °C overnight. The pH of the reaction
was carefully adjusted to pH 2.5 with dropwise addition of 6 M
HCl(aq), during which off-gassing occurred. Foaming was
controlled by vigorous stirring and slow pH adjustment. To
this mixture was added MTBE (10 mL/g), and the mixture
was filtered through diatomaceous earth (eluent, MTBE, 10
mL/g). The organic layer (containing desired compound) was
separated, and the aqueous layer was extracted with MTBE (10
mL/g). The combined organic layers were dried over Na2SO4,
filtered (eluent, MTBE 10 mL/g), and concentrated in vacuo
(<35 °C bath) to give the titled compound.
Aaron C. Smith − Medicine Design, Pfizer Inc., Groton,
Connecticut 06340, United States; orcid.org/0000-0001-
6796-5155
Edelweiss Evrard − Medicine Design, Pfizer Inc., Cambridge,
Massachusetts 02139, United States
Wei Li − BioDuro LLC, Shanghai 200131, China
Linlin Dai − BioDuro LLC, Shanghai 200131, China
Lixia Yang − BioDuro LLC, Shanghai 200131, China
Zhiguang Chen − BioDuro LLC, Shanghai 200131, China
Qingli Zhang − WuXi AppTec, Shanghai 200131, China
Fangyan He − WuXi AppTec, Shanghai 200131, China
Jiesen Zhang − WuXi AppTec, Shanghai 200131, China
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.oprd.0c00397
Author Contributions
¶C.A.B., S.P.F., and L.S. contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors would like to thank Xiaochun Wang for analytical
method development, James Bradow for purification, and Tim
F. Ryder and Kathleen Farley for NMR analysis. Finally, thanks
go to Daniel W. Widlicka and Christopher J. Helal for fruitful
project discussion.
ASSOCIATED CONTENT
■
sı
* Supporting Information
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The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.oprd.0c00397.
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AUTHOR INFORMATION
■
Corresponding Authors
Caroline A. Blakemore − Medicine Design, Pfizer Inc., Groton,
Connecticut 06340, United States; orcid.org/0000-0001-
7055-6379; Email: caroline.blakemore@pfizer.com
Scott P. France − Medicine Design, Pfizer Inc., Groton,
Connecticut 06340, United States; orcid.org/0000-0003-
1679-6165; Email: scott.france@pfizer.com
Authors
Lacey Samp − Chemical Research and Development, Pfizer Inc.,
Groton, Connecticut 06340, United States
Deane M. Nason − Medicine Design, Pfizer Inc., Groton,
Connecticut 06340, United States
Eddie Yang − Medicine Design, Pfizer Inc., Groton, Connecticut
06340, United States
Roger M. Howard − Medicine Design, Pfizer Inc., Groton,
Connecticut 06340, United States; orcid.org/0000-0001-
5884-4896
Karen J. Coffman − Medicine Design, Pfizer Inc., Groton,
Connecticut 06340, United States
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