In Situ Acetaldehyde Synthesis for Carboligation Reactions

Article abstract of DOI:10.1002/cbic.201900666

The enzyme 4-oxalocrotonate tautomerase (4-OT) can promiscuously catalyze various carboligation reactions using acetaldehyde as a nucleophile. However, the highly reactive nature of acetaldehyde requires intricate handling, which can impede its usage in practical synthesis. Therefore, we investigated three enzymatic routes to synthesize acetaldehyde in situ in one-pot cascade reactions with 4-OT. Two routes afforded practical acetaldehyde concentrations, using an environmental pollutant, trans-3-chloroacrylic acid, or a bio-renewable, ethanol, as starting substrate. These routes can be combined with 4-OT catalyzed Michael-type additions and aldol condensations in one pot. This modular systems biocatalysis methodology provides a stepping stone towards the development of larger artificial metabolic networks for the practical synthesis of important chemical synthons.

Full text of DOI:10.1002/cbic.201900666

Accepted Article
Title: In Situ Acetaldehyde Synthesis for Carboligation Reactions
Authors: Lieuwe Biewenga, Andreas Kunzendorf, and Gerrit J.
Poelarends
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ChemBioChem
10.1002/cbic.201900666
In Situ Acetaldehyde Synthesis for Carboligation Reactions
†
1
†1
,1
Lieuwe Biewenga , Andreas Kunzendorf , and Gerrit J. Poelarends*
1Department of Chemical and Pharmaceutical Biology, Groningen Research Institute of
Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The
Netherlands.
†These authors contributed equally to this work.
*Address correspondence to Prof. Dr. Gerrit J. Poelarends. Tel: +31-50-3633354; Fax: +31-
5
0-3633000; E-mail: g.j.poelarends@rug.nl.
1
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ChemBioChem
10.1002/cbic.201900666
Abstract
The enzyme 4-oxalocrotonate tautomerase (4-OT) can promiscuously catalyze various
carboligation reactions using acetaldehyde as a nucleophile. However, the highly reactive
nature of acetaldehyde requires intricate handling, which can impede its usage in practical
synthesis. Therefore, we investigated three enzymatic routes to synthesize acetaldehyde in situ
in one-pot cascade reactions with 4-OT. Two routes afforded practical acetaldehyde
concentrations, using an environmental pollutant, trans-3-chloroacrylic acid, or a
biorenewable, ethanol, as starting substrate. These routes can be combined with 4-OT
catalyzed Michael-type additions and aldol condensations in one pot. This modular systems
biocatalysis methodology provides a stepping stone towards the development of larger
artificial metabolic networks for the practical synthesis of important chemical synthons.
Keywords
Acetaldehyde, carboligation, biocatalysis, cascade reactions
2
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10.1002/cbic.201900666
Introduction
The enzyme 4-oxalocrotonate tautomerase (4-OT) naturally catalyzes the tautomerization of
-hydroxy-2,4-hexadienedioate to 2-oxo-3-hexenedioate using the N-terminal proline as key
2
[
1]
catalytic base . In addition, 4-OT can promiscuously catalyze several carbon-carbon bond-
forming reactions, including Michael-type additions and aldol condensations, employing Pro-
1
as a nucleophile[2–4]. Most notably, 4-OT can catalyze Michael-type additions and aldol
condensations using the highly reactive acetaldehyde (5) as a nucleophile (Scheme 1).
However, the use of 5 in enzymatic reactions causes several practical challenges. Compound
5
is toxic, highly volatile and reactive, which requires intricate handling. Hence, in situ
generation of 5 from less reactive and less toxic compounds is an attractive concept to
combine with 4-OT in one-pot cascade reactions. Previous studies have primarily focused on
in situ generation of longer aldehydes, for instance from their corresponding carboxylic acids
or alcohols using carboxylic acid reductases (CAR) or alcohol dehydrogenases respectively[5–
8].
Here we investigate three enzymatic routes for the in situ generation of 5 in one-pot cascade
reactions with 4-OT, using 1, 3 or 4 as starting substrates (Scheme 1). Route I involves the
dehalogenation of 1 into 2, catalyzed by the enzyme chloroacrylic acid dehalogenase (CaaD),
followed by the decarboxylation of 2 into 5 by the enzyme malonate semialdehyde
decarboxylase (MSAD). Route II involves the oxidation of 3 into 5 catalyzed by the alcohol
dehydrogenase from Saccharomyces cerevisiae (ScADH), in combination with a
commercially available NADH oxidase, PRO-NOX(009), to shift the unfavorable reaction
equilibrium and to recycle the co-factor. Route III involves the decarboxylation of 4 using the
pyruvate decarboxylase from Zymomonas mobilis (ZmPDC).
3
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ChemBioChem
10.1002/cbic.201900666
Routes I and II proved to be effective routes for in situ generation of 5 and could be used in
combination with 4-OT to synthesize 7 and 9 in one pot. Interestingly, route II afforded a
mixture of 9 and 10. The presented work provides a stepping stone towards the construction
of artificial enzymatic metabolic routes for the synthesis of valuable chemical commodities.
Scheme 1. Three envisioned enzymatic routes for in situ generation of acetaldehyde (5). The
in situ synthesized 5 is used as substrate in a 4-OT L8Y/M45Y/F50A catalyzed Michael-type
addition and a 4-OT M45T/F50A catalyzed aldol condensation reaction in one pot.
Abbreviations: CaaD: chloroacrylic acid dehalogenase, MSAD: malonate semialdehyde
decarboxylase, ScADH: alcohol dehydrogenase, PRO-NOX(009): NADH oxidase, ZmPDC:
pyruvate decarboxylase.
Results
There are several enzymes that naturally produce 5 as their main product, as 5 is at the
crossway of several metabolic routes. We selected three complementary enzymatic routes
(route I, II and III, Scheme 1) for in situ generation of 5, using the readily accessible starting
materials 1, 3 and 4. We initially focused on the enzymatic synthesis of 5 via route I. The
release of HCl and carbon dioxide in the first and second step respectively, is expected to shift
4
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ChemBioChem
10.1002/cbic.201900666
the equilibrium towards the formation of 5. Indeed, initial experiments showed that a cascade
reaction with CaaD and MSAD resulted in near quantitative conversion of 1 into 5 (Figure 1).
Notably, no significant enzyme inhibition occurred at this concentration of 5. Optimizing
enzyme concentrations afforded 50 mM 5 in 90 minutes using 5 µM CaaD and 1 µM MSAD
(Figure 1).
Figure 1. Progress curves of enzymatic synthesis of 5 using route I and route II. (a)
Formation of 5 via route I. Assay conditions: 50 mM 1, 100 mM sodium phosphate (pH 7.3).
(
b) Formation of 5 via route II. Assay conditions: 100 mM sodium phosphate pH 7.3, 2
+
mg/mL PRO-NOX(009), 10 U/mL ScADH, 10% v/v 3, 2 mM NAD , 5 mL reaction volume,
under oxygen atmosphere, performed in a 25 mL flask.
We next focused on the enzymatic oxidation of 3 (Scheme 1, route II), which can be catalyzed
by both alcohol oxidases and alcohol dehydrogenases. Since alcohol oxidases are prone to
[
9]
over-oxidation of aldehydes into their corresponding carboxylic acids , we opted for the use
of an alcohol dehydrogenase instead. The commercial available Yeast alcohol dehydrogenase,
(ScADH, Saccharomyces cerevisiae) was selected as biocatalyst because of its high
selectivity towards short-chain primary alcohols[10,11]. The commercially available NADH
+
oxidase PRO-NOX(009) was selected to recycle NAD and to overcome the unfavorable
reaction equilibrium.
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ChemBioChem
10.1002/cbic.201900666
Concentrations of up to 10% v/v 3 were well tolerated by both enzymes, but higher
concentrations rapidly inactivated at least one of the enzymes. Vigorous stirring of the
reaction mixture under an oxygen atmosphere significantly improved the rate of 5 production
compared to an unstirred reaction (Figure 1). Reaction conditions were optimized to reach a
concentration of approximately 50 mM 5 after 150 minutes.
Lastly, we focused on the enzymatic decarboxylation of 4 to 5 (Scheme 1, route III). The
pyruvate decarboxylase from Z. mobilis (ZmPDC) was cloned, expressed and purified to
homogeneity. However, we were unable to find conditions that afforded concentrations of 5
above 30 mM, due to either inactivation or inhibition of the enzyme by 5. This concentration
was considered too low for effective catalysis with 4-OT and hence we abandoned this route.
Having established two effective enzymatic routes for the in situ synthesis of 5, we next
focused our attention on the combination of these routes with 4-OT in one-pot multistep
cascade reactions. We initially focused on the 4-OT catalyzed Michael-type addition of 5 to 6
yielding 7, an important precursor for the γ-aminobutyric acid analogue phenibut[4,12]. As a
catalyst, the previously engineered 4-OT L8Y/M45Y/F50A variant was selected[13]
.
Combination of 4-OT L8Y/M45Y/F50A and 6 with in situ synthesized 5 (via both route I and
II) afforded R-7 in high enantiopurity (e.r. up to 98:2) and good to excellent isolated yields
(up to 96% compared to 6) (Table 1).
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10.1002/cbic.201900666
Table 1. Stepwise enzymatic cascade synthesis of 7.
Reaction time
Step I (min)c
Step II (min)d
e.r.e
98:2
98:2
Isolated yield (%)f
Route I
Route II
150
90
75
85
62
96
aDehalogenation of 1 into 2 catalyzed by CaaD, followed by decarboxylation of 2 into 5
catalyzed by MSAD, followed by the Michael-type addition of 5 to 6 yielding 7 catalyzed by
4
5
-OT L8Y/M45Y/F50A. The reaction mixture consisted of 50 mM 1, 4 mM 6, 1 µM MSAD,
µM CaaD, 56 µM 4-OT L8Y/M45Y/F50A, 100 mM sodium phosphate pH 7.3, 10% v/v
b
ethanol. Oxidation of 3 into 5 catalyzed by ScADH, followed by the Michael-type addition
of 5 to 6 yielding 7 catalyzed by 4-OT L8Y/M45Y/F50A. PRO-NOX(009) was used for co-
factor recycling. The reaction mixture consisted of 10% v/v 3, 4 mM 6, 10 U/mL ScADH, 2
+
mg/mL PRO-NOX(009), 2 mM NAD , 56 µM 4-OT L8Y/M45Y/F50A, 100 mM sodium
c
d
e
phosphate pH 7.3. Monitored by HPLC. Monitored by UV spectroscopy. Determined by
GC with chiral stationary phase or derivatized into a cyclic acetal and determined by HPLC
with a chiral stationary phase. The absolute configuration was determined by comparison to
[
14,15] f
literature
. Isolated yield compared to 6.
Next, we focused on combining route I and II with the 4-OT catalyzed aldol condensation of 5
with 8, forming 9. The previously engineered 4-OT M45T/F50A was used as a catalyst for
this reaction[16]. Combination of route I with 8 and 4-OT M45T/F50A afforded 9 in good
isolated yield (56% compared to 8) (Figure 2 and Table 2). Interestingly, the combination of
route II with 8 and 4-OT M45T/F50A afforded a mixture of 9 (26% isolated yield compared
to 8) and cinnamyl alcohol 10 (28% isolated yield compared to 8) (Figure 2 and Table 2).
These results demonstrate some exciting opportunities available for the combination of
natural metabolic routes with unnatural carboligation reactions in one pot.
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ChemBioChem
10.1002/cbic.201900666
Figure 2. Progress curves of the enzymatic cascade synthesis of 9 and 10. (a) Progress curve
of the 4-OT M45T/F50A catalyzed aldol condensation of 5 with 8 using in situ synthesized 5
via route I. Assay conditions: 50 mM 1, 4 mM 8, 1 µM MSAD, 10 µM CaaD, 75 µM 4-OT
M45T/F50A, 150 mM sodium phosphate pH 7.3, 5% v/v ethanol, 17.8 mL reaction volume.
(
b) Progress curve of the 4-OT M45T/F50A catalyzed aldol condensation of 5 with 8 using in
+
situ synthesized 5 via route II. Assay conditions: 10% v/v 3, 5.7 mM 8, 2 mM NAD , 133 µM
4
-OT M45T/F50A, 10 U/mL ScADH, 2 mg/mL PRO-NOX(009), 100 mM sodium phosphate
pH 7.3, 30 mL reaction volume.
Table 2. Enzymatic cascade synthesis of 9.
Reaction time (h)
Conversion (%)c
Isolated yield (%)d
56
Route I
Route II
23.5
24
71
85
e
26
aDehalogenation of 1 into 2 catalyzed by CaaD, followed by decarboxylation of 2 into 5
catalyzed by MSAD, followed by the aldol condensation of 5 with 8 yielding 9 catalyzed by
4
-OT M45T/F50A. The reaction mixture consisted of 50 mM 1, 4 mM 8, 1 µM MSAD, 10
µM CaaD, 75 µM 4-OT M45T/F50A, 150 mM sodium phosphate pH 7.3, 5% v/v ethanol.
b
Oxidation of 3 into 5 catalyzed by ScADH, followed by the aldol condensation of 5 with 8
yielding 9 catalyzed by 4-OT M45T/F50A. The reaction mixture consisted of 10% v/v 3, 5.7
+
mM 8, 2 mM NAD , 133 µM 4-OT M45T/F50A, 10 U/mL ScADH, 2 mg/mL PRO-
c
d
e
NOX(009), 100 mM sodium phosphate pH 7.3. Determined by HPLC. Compared to 8. The
8
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ChemBioChem
10.1002/cbic.201900666
low yield is caused by partial reduction of 9 into 10 (Figure 2b). Cinnamyl alcohol 10 could
be obtained with 28% isolated yield compared to 8.
Discussion
4
-OT is unique in that it can use 5 as substrate for C-C bond-forming Michael-type additions
to nitroolefin acceptors and for aldol condensations with benzaldehydes[3,4]. Previously, we
have demonstrated that 4-OT can be combined with other biocatalysts and chemocatalysts to
convert 6 and 8 into GABA analogues using two complementary one-pot cascade
reactions[13,15]. However, both routes rely on 5 as a substrate. The inherent high reactivity and
toxicity of 5 makes it a less desirable substrate and special care has to be taken to prevent
polymerization or oxidation. A solution to this problem is the enzymatic in situ generation of
5
using less toxic and reactive starting substrates in multistep cascade reactions. To this end,
we have investigated three enzymatic routes for the in situ generation of 5, using different
starting materials.
The first route we investigated involves the conversion of soil pollutant 1 into 5. Recycling of
1
, a degradation product from the xenobiotic soil fumigant trans-1,3-dichloropropene[17], into
GABA precursors provides an interesting example of the first principle of circular chemistry
and as such might be desirable for future sustainable synthesis[18]. To this end, we selected the
enzymes trans-3-chloroacrylic acid dehalogenase and malonate semi-aldehyde decarboxylase,
which are part of the metabolic pathway for the degradation of trans-1,3-dichloropropene in
Pseudomonas cichorii 170[19,20]. The second route comprises of the oxidation of 3 into 5. As
an inexpensive biorenewable, 3 is an attractive precursor of 5 that can concurrently act as a
co-solvent to solubilize 6 and 8. To catalyze the oxidation of 3, we selected yeast alcohol
dehydrogenase, using an NADH oxidase for co-factor recycling and as a driving force to
overcome the unfavorable reaction equilibrium between 3 and 5. Thirdly, we investigated the
9
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ChemBioChem
10.1002/cbic.201900666
readily available substrate 4 as precursor for 5, using pyruvate decarboxylase from Z. mobilis
as biocatalyst.
Gratifyingly, concentrations of 50 mM 5 or higher could be generated in situ using both route
I and II. Route III only afforded lower concentrations of 5 and unfortunately, we were unable
to find conditions to improve this. We speculate that ZmPDC was either inactivated or
strongly inhibited by 5[21], and the effective use of route III for in situ generation of 5 awaits
the discovery of 4-OT mutants with a lower K for 5.
M
We demonstrated the modularity of our approach by showing that the in situ generated 5 can
be used as a substrate for both 4-OT catalyzed aldol condensations and Michael-type
additions. The Michael-type addition of 5 to 6 was catalyzed by the previously engineered 4-
OT L8Y/M45Y/F50A and afforded R-7 in good to excellent isolated yield (up to 96%
compared to 6) and with an excellent e.r. value of 98:2. The aldol condensation of 5 with 8
was catalyzed by the previously engineered 4-OT M45T/F50A. Route I afforded 9 in 55%
isolated yield, but route II afforded a mixture of 9 and 10. The reduction of 9 to 10 is slowly
catalyzed by ScADH and by an alcohol dehydrogenase present in the CFE of PRO-
NOX(009).
In summary, we have investigated three enzymatic synthesis routes, using the readily
accessible starting materials 1, 3 or 4, for in situ generation of 5 to be used as substrate by 4-
OT in one-pot cascade syntheses. Route I and route II afforded 5 in effective concentrations
for synthesis with 4-OT, but route III afforded 5 only in low concentrations. Route I and route
II are fully compatible with 4-OT, which was demonstrated by the one-pot cascade synthesis
of 7 and 9. These modular enzymatic cascades provide a stepping stone towards the
development of larger “artificial metabolisms” that could facilitate greener and more
sustainable synthesis[22]
.
1
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Acknowledgement
We acknowledge financial support from the Netherlands Organization of Scientific Research
VICI grant 724.016.002 and ECHO grant 713.015.003), and the European Union’s Horizon
020 Research and Innovation Programme under grant agreement No 635595 (CarbaZymes).
(
2
Conflict of Interest
The authors declare no conflict of interest.
1
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TOC graphic and text
Modular systems biocatalysis – Enzymatic carboligation reactions often involve toxic and
reactive aldehyde substrates such as acetaldehyde, which requires intricate handling. Here, we
have investigated three enzymatic routes to synthesize acetaldehyde in situ in one-pot
cacascade reactions using engineered 4-oxalocrotonate tautomerase variants as non-natural
carboligases in aldol and Michael-type additions.
1
4
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