Enantioselective Aldol Addition of Acetaldehyde to Aromatic Aldehydes Catalyzed by Proline-Based Carboligases

Article abstract of DOI:10.1021/acscatal.0c00039

Aromatic β-hydroxyaldehydes, 1,3-diols, and α,β-unsaturated aldehydes are valuable precursors to biologically active natural products and drug molecules. Herein we report the biocatalytic aldol condensation of acetaldehyde with various aromatic aldehydes to give a number of aromatic α,β-unsaturated aldehydes using a previously engineered variant of 4-oxalocrotonate tautomerase [4-OT(M45T/F50A)] as carboligase. Moreover, an efficient one-pot two-step chemoenzymatic route toward chiral aromatic 1,3-diols has been developed. This one-pot chemoenzymatic strategy successfully combined a highly enantioselective aldol addition step catalyzed by a proline-based carboligase [4-OT(M45T/F50A) or TAUT015] with a chemical reduction step to convert enzymatically prepared aromatic β-hydroxyaldehydes into the corresponding 1,3-diols with high optical purity (e.r. up to >99:1) and in good isolated yield (51-92%). These developed (chemo)enzymatic methodologies offer alternative synthetic choices to prepare a variety of important drug precursors.

Full text of DOI:10.1021/acscatal.0c00039

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Letter
Enantioselective Aldol Addition of Acetaldehyde to Aromatic
Aldehydes Catalyzed by Proline-based Carboligases
Mohammad Saifuddin, Chao Guo, Lieuwe Biewenga, Thangavelu
Saravanan, Simon J. Charnock, and Gerrit J. Poelarends
ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.0c00039 • Publication Date (Web): 28 Jan 2020
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Enantioselective Aldol Addition of Acetaldehyde to Aromatic
Aldehydes Catalyzed by Proline-based Carboligases
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Mohammad Saifuddin,a,† Chao Guo,a,† Lieuwe Biewenga,a Thangavelu Saravanan,a Simon J.
Charnock,b and Gerrit J. Poelarendsa,*
aDepartment of Chemical and Pharmaceutical Biology, Groningen Research Institute of
Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The
Netherlands.
bProzomix Ltd., Station Court, Haltwhistle, Northumberland NE49 9HN, U.K.
†M.S. and C.G. contributed equally to this work.
*Corresponding author. Tel.: +31503633354; E-mail: g.j.poelarends@rug.nl; Web:
http://www.rug.nl/staff/g.j.poelarends/
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ABSTRACT
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Aromatic β-hydroxyaldehydes, 1,3-diols, and α,β-unsaturated aldehydes are valuable
precursors to biologically active natural products and drug molecules. Herein we report the
biocatalytic aldol condensation of acetaldehyde with various aromatic aldehydes to give a
number of aromatic α,β-unsaturated aldehydes using a previously engineered variant of 4-
oxalocrotonate tautomerase [4-OT(M45T/F50A)] as carboligase. Moreover, an efficient one-
pot two-step chemoenzymatic route towards chiral aromatic 1,3-diols has been developed. This
one-pot chemoenzymatic strategy successfully combined a highly enantioselective aldol
addition step catalyzed by a proline-based carboligase [4-OT(M45T/F50A) or TAUT015] with
a chemical reduction step to convert enzymatically prepared aromatic β-hydroxyaldehydes into
the corresponding 1,3-diols with high optical purity (e.r. up to >99:1) and in good isolated yield
(51-92%). These developed (chemo)enzymatic methodologies offer alternative synthetic
choices to prepare a variety of important drug precursors.
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KEYWORDS
Biocatalysis, aldol reaction, aldolases, β-hydroxyaldehydes, carboligases
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The aldol reaction is one of the most important methodologies for the straightforward
construction of carbon-carbon bonds in synthetic organic chemistry.1 This reaction allows
complex chiral compounds to be rapidly assembled from simpler building blocks. Enzyme
catalysis by using native and engineered aldolases offers a powerful strategy to perform this
transformation.2 With the discovery and engineering of novel aldolase activities, an increasing
number of applications of aldolases in stereoselective synthesis have been reported.2e,2i Despite
the rapidly expanding biocatalytic toolbox, aldolases that can use various aromatic aldehydes
as acceptors for acetaldehyde addition are rare.2f-h Such aldolases would provide an attractive
synthetic tool for the preparation of chiral precursors to various biologically active compounds
and drug molecules.
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Our group has previously reported that the enzyme 4-oxalocrotonate tautomerase (4-OT)
from Pseudomonas putida mt-2 promiscuously catalyzes the cross-condensation of
acetaldehyde (1) with benzaldehyde (2a) to give cinnamaldehyde (4a) (Scheme 1).3 It has been
shown that 4-OT catalyzes both the initial aldol addition to yield 3-hydroxy-3-phenylpropanal
(3a) as well as the subsequent rapid dehydration of 3a to give 4a, preventing the accumulation
of chiral aldol adduct 3a in the reaction mixture.3a Rahimi and co-workers applied mutability
landscape-guided protein engineering to further improve this promiscuous aldolase activity of
4-OT. This resulted in the identification of a double mutant, 4-OT M45T/F50A, with a
remarkable 3300-fold improved catalytic efficiency (in terms of kcat/Km) over wild-type 4-OT.3b
OH
*
O
CH3CHO
CHO
CHO
1
4-OT (M45T/F50A)
4a
cinnamaldehyde
3a
2a
Scheme 1. 4-OT catalyzed aldol condensation of acetaldehyde (1) with benzaldehyde (2a) to
yield cinnamaldehyde (4a).
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Although cinnamaldehydes are valuable synthons in their own right, we were especially
interested in the enzymatic preparation of chiral aromatic β-hydroxyaldehydes, and the
corresponding 1,3-diols, which are important precursors to various natural products, bioactive
compounds and drugs (Figure 1).4,5 We envisioned that these chiral aldol adducts could be
constructed by the enzymatic addition of acetaldehyde to selected aromatic aldehydes, yielding
β-hydroxyaldehydes that are not (or slowly) dehydrated, allowing their accumulation in the
reaction mixture. Chemical reduction of the enzymatically prepared aromatic β-
hydroxyaldehydes would provide access to the corresponding aromatic 1,3-diols, which are key
intermediates in the synthesis of drugs such as Fluoxetine (Figure 1).5
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O
CF3
O
OH
O
OH
(S)
O
(S)
CHO
(S)
N
(R)
(R)
N
N
H
(R)
(S)
OH
H
R
Fluoxetine (ProzacR)
Tolterodine
Atomoxetine
diospongin B
H2N
H2N
H2N
COOH
CHO
COOH
COOH
(R)
(R)
(R)
R
MeO
Cl
Rolipram
OH
Baclofen
Phenibut
Figure 1. Chiral aromatic β-hydroxyaldehydes and cinnamaldehydes are valuable precursors
to biologically active compounds and drugs.
Herein we report the 4-OT(M45T/F50A)-catalyzed synthesis of various aromatic α,β-
unsaturated aldehydes, starting from acetaldehyde and a number of aromatic aldehydes, in good
isolated yields. We also report the one-pot, two-step chemoenzymatic asymmetric synthesis of
various aromatic 1,3-diols with high optical purity (e.r. up to >99:1) and in good isolated yield
(51-92%). This chemoenzymatic strategy highlights a highly stereoselective aldol addition of
acetaldehyde to diverse aromatic aldehydes, catalyzed by either 4-OT(M45T/F50A) or the
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newly identified carboligase TAUT015, yielding various enantioenriched aromatic β-
hydroxyaldehydes, which are chemically reduced into the corresponding 1,3-diols in the same
pot. The applied carboligases accept a broad range of aromatic aldehydes for acetaldehyde
additions, offering alternative synthetic choices to prepare important drug precursors.
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We previously reported that an engineered variant of 4-OT (mutant M45T/F50A) can
efficiently catalyze the aldol condensation of acetaldehyde (1) with benzaldehyde (2a) to yield
cinnamaldehyde (4a).3b This prompted us to start our investigations by testing a large set of
twenty-five aromatic aldehydes (2b-y and 2ac) as potential aldol acceptor substrates in the 4-
OT(M45T/F50A)-catalyzed aldol reaction with donor substrate 1 (Scheme 2). The results
demonstrate that the 4-OT(M45T/F50A) enzyme has a remarkably broad substrate scope,
accepting nineteen aromatic aldehydes with electron withdrawing substituents (2b-t) as aldol
acceptor substrates (Scheme 2, Figures S1-S19). On the contrary, aromatic aldehydes with
electron-donating substituents (2u-y and 2ac) were not, or very poorly, accepted by 4-
OT(M45T/F50A) (Scheme 2, Figures S20-S24, S28). While this may be attributed to
unfavorable equilibrium constants for these aromatic aldehydes in the aldol addition with
acetaldehyde, the electron donating properties of the substituents on the aromatic ring likely
also lead to weak activation of the carbonyl carbon for nucleophilic attack.2j,2l
Proline-based
CHO
tautomerase
R
CH3CHO
1
4b d 4f j 4t
- , - ,
OH
O
CHO
R
OH OH
*
Proline-based
tautomerase
R
*
2
3
R
NaBH4
5b d 5f-g 5k-n 5s
- ,
,
,
Electron deficient acceptors
2b 2c
Electron rich acceptors
2d
2e
2u
2v
2w
)
R = 2-Cl-Ph ( ), 3-Cl-Ph ( ), 2,4-di-Cl-Ph ( ), 3,4-di-Cl-Ph (
2f 2g 2h 2i
)
R = 4-OMe-Ph ( ), 4-Me-Ph ( ), 4-N,N-di-Me-Ph (
2x 2y
2-NO2-Ph ( ), 3-NO2-Ph ( ), 2-F-Ph ( ), 2-Br-Ph (
2j 2k 2l
)
2,6-di-OMe-Ph ( ), 3,4,5-tri-OMe-Ph (
2z 2aa
)
2ab
)
tetrafluoro-Ph ( ), 4-CN-Ph ( ), 4-CF3-Ph (
2m 2n
)
2-Me-Ph ( ), 3-Me-Ph (
2ac
), 2,4-di-OMe-Ph (
2o
)
2p
)
pentafluoro-Ph ( ), 3-CN-4-F-Ph ( ), 4-Br-Ph ( ), 4-Cl-Ph (
2q 2r 2s 2t
6-OMe-Napthyl (
)
4-F-Ph ( ), 4-NO2-Ph ( ), Pyridyl ( ), furanyl (
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Scheme 2. Enzymatic aldol reaction of acetaldehyde (1) with various aromatic aldehydes (2)
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using promiscuous proline-based tautomerases as carboligases.
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Out of the nineteen well-accepted aromatic aldehydes, fourteen substrates undergo an
aldol condensation reaction with formation of the corresponding α,β-unsaturated aldehydes,
whereas five substrates (2k-n and 2s, see below) undergo conversion without significant
formation of the corresponding α,β-unsaturated aldehydes (Figure S1-S19). To demonstrate the
synthetic usefulness of 4-OT(M45T/F50A) for the preparation of α,β-unsaturated aldehydes,
nine aromatic aldehydes (2b-d, 2f-j and 2t) that could be efficiently converted by 4-
OT(M45T/F50A) were selected and used in semi-preparative scale synthesis (Scheme 3). The
corresponding α,β-unsaturated aldehydes (4b-d, 4f-j and 4t) could be isolated in moderate to
good yields (Scheme 3, Figures S30-S47). Notably, ortho-substituted benzaldehydes (e.g. 2b,
2f, 2h and 2i) were better accepted by 4-OT(M45T/F50A) than para-substituted benzaldehydes
(2p, 2r, 2o and 2q).6 These results underscore the potential of 4-OT(M45T/F50A) for the
synthesis of versatile aromatic α,β-unsaturated aldehydes, which are important precursors to
various abundantly prescribed drugs (Figure 1).4f
CH3CHO
OH
*
O
1
CHO
CHO
R
R
4-OT(M45T/F50A)a
R
4b-d, 4f-j, 4t
2b-d, 2f-j, 2t
3b-d, 3f-j, 3t
NO2
Cl
Cl
CHO
O2N
CHO
CHO
Cl
CHO
CHO
Cl
b,c
b,c
b,c
b,c
b,c
4g
4f
(61%)
(73%)
4c
(70%)
4b
4d
(71%)
(81%)
F
F
F
Br
O
CHO
F
CHO
CHO
CHO
F
b,c
b,c
b,c
b,c
4i
(85%)
4h
4j
(85%)
(79%)
4t
(23%)
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Scheme 3. Synthesis of aromatic α,β-unsaturated aldehydes from simpler building blocks using
the carboligase 4-OT(M45T/F50A). Reaction conditions: reaction mixtures consisted of 100
a
mM 1, 3 mM 2 and 0.1 mg/mL 4-OT (M45T/F50A) in 20 mM sodium phosphate buffer, 5%
b
DMSO v/v, pH 7.3. The reaction volume was 60 mL. Reaction time (48 h, 72 h for 2t).
cPurified by silica gel column chromatography.
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Interestingly, for the 4-OT(M45T/F50A)-catalyzed aldol addition of 1 to 2k-n and 2s,
depletion of the starting substrates was observed without significant accumulation of the
corresponding α,β-unsaturated aldehydes (Figures S10-S13, S18). These results led us to
hypothesize that in these instances the initially formed aldol adducts, β-hydroxyaldehydes 3k-
n and 3s, were not (or slowly) dehydrated and accumulated in the reaction mixture. To confirm
this hypothesis, an analytical scale experiment was performed using 4-OT(M45T/F50A) in
NaPi buffer (0.3 mL, pH 7.3, 5% DMSO), containing 1 (50 mM) and 2k (2 mM). Reaction
progress was monitored by following the depletion of 2k by UV-VIS spectrophotometry, and
NaBH4 was added to the reaction mixture (after 1 h) to convert the aldol adduct into the more
stable 1,3-diol 5k. Analysis of chemoenzymatic product 5k by HPLC on a chiral stationary
phase, using authentic standards with known absolute configuration, revealed high
enantiocontrol at the site of addition with formation of the R enantiomer (e.r. = 94:6). These
results demonstrate that the enzyme 4-OT(M45T/F50A) can indeed catalyze enantioselective
aldol additions.
Having established that 4-OT(M45T/F50A) has potential for the asymmetric synthesis of
enantioenriched aromatic β-hydroxyaldehydes, we first optimized the reaction conditions by
varying the pH and the concentration of acetaldehyde, buffer and enzyme (Figure S88). Using
the optimized conditions, the one-pot two-step chemoenzymatic cascade synthesis of aromatic
1,3-diols 5k-n and 5s, via enzymatically prepared β-hydroxyaldehydes 3k-n and 3s, was
achieved with high enantioselectivity (e.r. up to 99:1) and in good isolated yield (63-92%)
(Table 1, Figures S48-S62).
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Table 1. Asymmetric chemoenzymatic synthesis of aromatic 1,3-diols using 4-
OT(M45T/F50A) as carboligase.a
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7
8
9
CH3CHO
OH OH
OH O
NaBH4
1
CHO
*
R
R
R
*
4-OT(M45T/F50A)
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2k-n, 2s
5k-n, 5s
3k-n, 3s
Reaction
time [h]b
Isolated
Entry Aldol acceptor
Product
e.r.d
91:9
Abs. Conf.
yield (%)c
OH OH
CHO
1
6
92
Re
Re
2k
NC
5k
NC
OH OH
CHO
2
7
63
88
90:10
2l
5l
CF3
CF3
F
F
OH OH
F
F
F
F
F
CHO
2m
3
2.5
90:10
Re
5m
F
F
F
OH OH
CHO
4
5
5
1
70
65
92:8
99:1
Rf
Rf
2n
F
5n
F
CN
CN
OH OH
N
CHO
N
2s
5s
aAssay conditions: acetaldehyde (1, 100 mM), aromatic aldehyde (2k-n, 2s, 2 mM) and purified 4-
OT(M45T/F50A) (0.125-0.475 mg/mL) in 20 mM sodium phosphate buffer (MOPS for 2k), with 5%
DMSO (v/v) as cosolvent, at pH 8.0 (pH 7.3 for 2s to increase its electrophilicity) and room temperature.
The reaction volume was 40 mL. To reduce the aldehyde functionality of the enzymatic products 3k-n
and 3s, NaBH4 (30 mM) was added to the reaction mixture followed by incubation for 3 h at room
temperature. Reaction time of the enzymatic step. Isolated yield of the aromatic 1,3-diol product.
dDetermined by HPLC analysis on a chiral stationary phase using chemically synthesized racemic
standards. The absolute configuration was determined by chiral HPLC using chemically prepared
authentic standards with known (R) or (S) configuration. fThe absolute configuration was assigned based
b
c
e
on analogy.
To expand the number of enzymatically accessible β-hydroxyaldehyde products (e.g. 3b-
d, 3f and 3g) in principle two different approaches can be used: (i) the use of protein engineering
to create new variants of 4-OT(M45T/F50A) that lack dehydration activity and possess
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unchanged or enhanced aldol addition activity, or (ii) the use of homologous enzymes that
posses the desired aldol addition activity but lack dehydration activity. To selectively eliminate
the dehydration activity of 4-OT(M45T/F50A), a structure-based approach is favored, which
awaits the determination of the enzyme crystal structure complexed with one of the β-
hydroxyaldehyde products (work in progress). In this study, we therefore have chosen to screen
a panel of fifty commercially available 4-OT homologues for enzymes with the desired aldol
addition activity to yield enantioenriched β-hydroxyaldehydes. For initial activity testing, we
used donor substrate 1 and aldol acceptor substrate 2b. Interestingly, out of the fifty tested 4-
OT homologues, three enzymes (TAUT015, TAUT021 and TAUT028) were found to show
significant activity towards the aldol addition of 1 to 2b to give the desired product 3b without
promoting significant dehydration leading to formation of 4b (Figure S29). Notably, the same
reaction catalyzed by 4-OT(M45T/F50A) resulted in the formation of α,β-unsaturated aldehyde
4b as the sole product, providing additional support that the β-hydroxyaldehyde dehydration
step is enzyme catalyzed.
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We next investigated the substrate scope of the best performing enzyme, TAUT015,
which shows 28% overall sequence identity to 4-OT (Figure S89), in additions using 1 as donor
substrate and a number of selected benzaldehyde derivatives as potential aldol acceptor
substrates. The results show that, like 4-OT(M45T/F50A), TAUT015 does not accept
benzaldehydes with electron donating substituents (2u, 2v, 2z, 2aa and 2ab) on the aromatic
ring (Figures S25-S27). However, several benzaldehydes with electron withdrawing
substituents (2b-d, 2f and 2g) were well accepted by TAUT015. Encouraged by these findings,
semi-preparative scale reactions were carried out under optimized reaction conditions. The
reaction progress (1 with 2b-d, 2f and 2g) was monitored by UV-VIS spectrophotometry and
the enzymatically prepared β-hydroxyaldehydes 3b-d, 3f and 3g were reduced to the
corresponding aromatic 1,3-diols (5b-d, 5f and 5g) using NaBH4. These one-pot, two-step
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chemoenzymatically prepared compounds 5b-d, 5f and 5g were obtained in good isolated yields
(up to 72%) and with high e.r. values (e.r. up to >99:1) (Table 2, Figures S63-S87). Note that
enzymatic access to these useful chiral synthons requires the use of TAUT015 as carboligase
and is not possible with 4-OT(M45T/F50A). Hence, the enzyme TAUT015 nicely supplements
the toolbox of biocatalysts for the production of important chiral aromatic β-hydroxyaldehydes
and corresponding 1,3-diols.
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Table 2. Asymmetric chemoenzymatic synthesis of aromatic 1,3-diols using TAUT015 as
carboligase.a
CH3CHO
OH OH
*
OH
O
NaBH4
1
CHO
R
R
R *
3b-d, 3f-g
TAUT015
5b-d, 5f-g
2b-d, 2f-g
Aldol
acceptor
Reaction
Isolated
Entry
Product
e.r.d
Abs. Conf.e
time [h]b yield (%)c
Cl
Cl
OH OH
CHO
(R)
1
4.5
8.3
3.3
72
51
62
>99:1
R
R
R
2b
5b
OH OH
Cl
CHO
Cl
(R)
2
3
4
94:6
99:1
99:1
2c
5c
Cl
OH OH
Cl
CHO
(R)
5d
2d
Cl
Cl
NO2
NO2 OH OH
CHO
2f
(R)
6.5
11
69
56
R
R
5f
OH OH
O2N
CHO
O2N
(R)
5
89:11
2g
5g
aAssay conditions: acetaldehyde (1, 100 mM), benzaldehyde derivative (2b-d, 2f-g, 2 mM) and enzyme
(crude cell extract, 0.33 mg/mL) in 20 mM sodium phosphate buffer, containing 5% EtOH (v/v) as
cosolvent, at pH 6.5 and room temperature. The specific enzyme activity was determined to be 0.044
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U/mg CFE. Unit definition: 1 U of enzyme converts 1 µmol of 2b in 1 minute; assay conditions: 20 mM
NaPi pH 6.5, 5% EtOH, 100 mM 1, 2 mM 2b. The reaction volume was 40 mL. To reduce the aldehyde
functionality of 3b-d and 3f-g, NaBH4 (30 mM) was added to the reaction mixture followed by
b
c
incubation for 3 h at room temperature. Reaction time of the enzymatic step. Isolated yield of the
aromatic 1,3-diol product. dDetermined by HPLC analysis on a chiral stationary phase using chemically
e
synthesized racemic standards. The absolute configuration was determined by chiral HPLC using
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chemically prepared authentic standards with known (R) or (S) configuration.
In conclusion, our results indicate that members of the 4-OT family of enzymes, which
possess an uncommon catalytic amino-terminal proline, can function as novel carboligases,
promoting the cross-aldol reaction of acetaldehyde with diverse aromatic aldehydes to give
access to a range of important α,β-unsaturated aldehydes as well as enantioenriched β-
hydroxyaldehydes. It is feasible that their promiscuous aldol addition activities, which are
beyond the synthetic scope of currently known aldolases, can be further optimized by directed
evolution or computational redesign. It is important to emphasize that the controlled cross-aldol
addition of acetaldehyde is a particularly challenging synthetic aim because acetaldehyde is a
relatively reactive and difficult to tame chemical.5,7 Remarkably, the promiscuous 4-OT
enzymes accept acetaldehyde as a nucleophile in aldol additions, which is unparalleled among
the aldolases, with the notable exception of 2‐deoxy‐D‐ribose‐5‐phosphate aldolase (DERA)
and d‐fructose‐6‐phosphate aldolase (FSA), which are also able to use acetaldehyde as a
nucleophilic substrate.2g,2k,2o As such, these proline-based 4-OT enzymes nicely complement
the toolbox of biocatalysts for (asymmetric) C-C bond-formation, and open up new
opportunities to develop practical enzymatic processes for the more sustainable and step-
economic synthesis of valuable drug precursors starting from simple building blocks. Further
screening of related proline-based enzymes might prove to be a valuable approach to discover
new aldolase activities that could be exploited to develop synthetically useful biocatalysts for
carbon-carbon bond formation.
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AUTHOR INFORMATION
5
6
7
Corresponding Author
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9
*Corresponding author. Tel.: +31503633354; E-mail: g.j.poelarends@rug.nl; Web:
http://www.rug.nl/staff/g.j.poelarends/
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Author Contributions
†These authors contributed equally: Chao Guo and Mohammad Saifuddin.
ASSOCIATED CONTENT
Supporting Information. Additional experimental procedures and characterizations of
compounds. This information is available free of charge on the ACS Publications website.
ACKNOWLEDGMENT
We acknowledge financial support from the Netherlands Organization of Scientific Research
(VICI grant 724.016.002), the European Research Council (PoC grant 713483), and the
European Union’s Horizon 2020 Research and Innovation Programme under grant agreement
No 635595 (CarbaZymes).
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