Pyruvate Aldolases Catalyze Cross-Aldol Reactions between Ketones: Highly Selective Access to Multi-Functionalized Tertiary Alcohols

Article abstract of DOI:10.1021/acscatal.9b05512

Tertiary alcohols are widely represented in nature and among bioactive molecules. Their importance is attested by the continuous efforts to meet the challenge of their stereoselective synthesis. In this context, we propose an enzymatic approach, involving class II pyruvate aldolases. These enzymes are shown to catalyze selective cross-aldol reactions between pyruvic acid or derivatives as nucleophiles and a series of ketones as electrophiles. This catalytic activity is exemplified by the highly stereoselective preparation of seven branched ketols with good yields. One of them was readily converted into a constrained 4-hydroxyproline analogue in a multienzymatic one-pot one-step process.

Full text of DOI:10.1021/acscatal.9b05512

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Letter
Pyruvate Aldolases Catalyze Cross-Aldol Reactions between Ketones:
Highly Selective Access to Multi-Functionalized Tertiary Alcohols
Victor Laurent, Léa Gourbeyre, Alexandre Uzel, Virgil Hélaine, Lionel Nauton, Mounir Traikia, Véronique
De Berardinis, Marcel Salanoubat, Thierry Gefflaut, Marielle Lemaire, and Christine GUERARD-HELAINE
ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.9b05512 • Publication Date (Web): 03 Feb 2020
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ACS Catalysis
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Pyruvate Aldolases Catalyze Cross-Aldol Reactions between Ketones:
Highly Selective Access to Multi-Functionalized Tertiary Alcohols
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Victor Laurent,† Léa Gourbeyre,† Alexandre Uzel,† Virgil Hélaine,† Lionel Nauton,† Mounir
Traïkia,† Véronique de Berardinis,‡ Marcel Salanoubat,‡ Thierry Gefflaut,† Marielle Lemaire† and
Christine Guérard-Hélaine*†
† Université Clermont Auvergne, CNRS, SIGMA Clermont, Institut de Chimie de Clermont-Ferrand, 63000
Clermont-Ferrand, France
‡ Génomique Métabolique, Génoscope, Institut François Jacob, CEA, CNRS, Univ Evry, Université Paris-Saclay,
91057 Evry, France
ABSTRACT: Tertiary alcohols are widely represented in nature and among bioactive molecules. Their importance is
attested by the continuous efforts to meet the challenge of their stereoselective synthesis. In this context, we propose an
enzymatic approach, involving class II pyruvate aldolases. These enzymes are shown to catalyze selective cross-aldol
reactions between pyruvic acid or derivatives as nucleophiles and a series of ketones as electrophiles. This catalytic activity
is exemplified by the highly stereoselective preparation of seven branched ketols with good yields. One of them was readily
converted into a constrained 4-hydroxyproline analogue in a multi-enzymatic one-pot one-step process.
KEYWORDS: pyruvate aldolase, tertiary alcohol, proline analogue, cascade reaction, transaminase
Tertiary
alcohols
are
important
motifs
in
oxoadipate aldolases13,14 were the sole aldolases able to
catalyze the formation of tertiary alcohols. They naturally
catalyze the self-addition of pyruvate or the addition of
pyruvate onto oxaloacetate. Nevertheless, this type of
reaction is limited by the substrate scope of the
enzymes.13,14 The sole example of cross-coupling of -keto
acids at a preparative scale is the development of the
enzymatic synthesis of monatin, a natural sweetener.11,15,16
Recently, a collection of 21 class-II pyruvate aldolases from
biodiversity, including RhmA, GarL and HpcH from E. coli,
were shown to display high synthetic potential due to their
exceptional tolerance towards nucleophile substrates, such
as hydroxypyruvate or ketobutyrate.17,18 This property
remains rare as aldolases are generally named according to
their strict specificity for their nucleophilic substrate.
Here, we further explored the catalytic potential of this
enzyme set by testing aldol reactions with various ketones
as electrophiles in combination with pyruvic acid or
derivatives.
pharmaceuticals, as well as being fine and specialty
chemicals, such as chiral ligands,1–3 building blocks in
asymmetric synthesis,4–6 and solvents.6–9 Many bioactive
compounds with these moieties are known, such as
tramadol
(analgesic),
erythromycin
(antibiotic),
doxorubicin (antitumor agent), and ethynylestradiol
(estrogen), along with many ethers, esters, and acetals
derived from tertiary alcohols. However, the challenges to
surmount for their synthesis are as high as the demand for
enantiopure tertiary alcohols.7,9 Recently, DHAP-
dependent class II aldolases were shown to be active with
ketones as electrophile substrates, thus allowing the
stereoselective synthesis of a series of tertiary alcohols.10
Therefore, we examined whether other members of the
aldolase family display the same catalytic power. We
assumed that this exceptional activity could be shared with
other class II aldolases, as the enolate intermediate11
formed in the active site of class-II aldolases should be
more nucleophilic than the enamine intermediate
characteristic of class I aldolases. Among the aldolase
family, pyruvate aldolases may be good candidates for this
purpose, as some of them belong to the class II aldolases.
In addition, they have been well studied 11,12 because of their
role in metabolism and their ability to provide chiral
synthons with high potential in total synthesis. Until the
discovery of DHAP-dependent aldolases in 2018,10 4-
hydroxy-4-methyl-2-oxoglutarate/4-carboxy-4-hydroxy-2-
First, the enzyme collection was assayed under standard
conditions (1 mg/mL) with pyruvic acid 1 (20 mM) as the
unique substrate to assess the self-addition reaction. Aldol
formation was not observed with any pyruvate aldolase.
Subsequently, three simple ketones (80 mM) were
evaluated as potent electrophiles with 1 (20 mM) (Figure 1).
These ketones could be classified into two groups: not
activated (acetone: 4), activated by either one hydroxyl
(hydroxyacetone: 5) or two hydroxyls (dihydroxyacetone:
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ACS Catalysis
6). All tested enzymes displayed the same activity profile:
Page 2 of 6
of 1 with 9, complete conversion of 1 occurred in three
hours with four of the selected enzymes (all except
I7DKY0, Figure 2). The enzyme activity was lower when 2
was used as a nucleophile in combination with 9, for which
the conversion rate never exceeded 50% after 48 h (data
not shown).
1
2
3
4
5
6
7
8
an aldol adduct was exclusively formed with ketone 6, with
complete conversion of 1 within 2 to 5 h (see SI). The two
other less electrophilic ketones 4 and 5 did not undergo
any significant reaction under our analytical conditions.
Next, in order to select the most promising enzymes with
the largest application scopes, ketones 4 to 6 were assayed
with 2-oxobutyric acid 2 or hydroxypyruvic acid 3 as
nucleophile substrates (20 mM), as these enzymes are
known to accept them as nucleophiles with an aldehyde as
electrophile.17,18 Again, as described for 1, the self-addition
reactions of keto acids 2 and 3 was first assessed. Although
no reaction was observed with 2 as a unique substrate (see
SI), partial or complete conversion of 3 was observed with
several enzymes. Given that the hydroxyl group of 3
provides an activating effect, the self-addition of 3 is
consistent with the results obtained with ketones 4 to 6.
Consequently, 3 was not further used as a nucleophile in
this study. Substrate 2 was then combined with ketones 4
to 6 in the presence of the various aldolases. As already
observed with 1 as nucleophile, an aldolisation product was
obtained only with ketone 6. However, different reactivity
profiles were observed. Indeed, only six enzymes were able
to completely convert 2 into the corresponding tertiary
alcohol in a few hours (see SI): Uniprot Id. Q0K203,
B4XH86, A5VAX1, A9E0U5, A3SLS0, and I7DKY0 (deleted
entry in UniProtKB, currently given by the UniParc ID
UPI000160F944). Q0K203 was shown in a previous study
to display very low stereoselectivity with 218 and was
discarded for the following experiments. Hence, five
pyruvate aldolases were assayed in aldolisation reactions
using 1 and 2 as nucleophiles combined with three other
activated ketones as potent electrophile substrates: L-
erythrulose 7, D-fructose 8, and 1,3-difluoroacetone 9
(Figure 1).
9
10
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Figure 2. Conversion of nucleophiles 1 or 2 (20 mM) in the
presence of ketones 7 or 9 (80 mM) with the selected aldolases
To note, the best performing above-mentioned enzymes
were able to convert the considered electrophile ketones
with an acceptable rate relative to that of structurally close
aldehydes. Indeed, for example, DHA was converted only
two times slower than D-glyceraldehyde using B4XH86 and
2 as nucleophile.
The promising analytical data obtained with ketones 6,
7, and 9, prompted us to carry out preparative syntheses at
the 50-mg scale using a concentration of 20 mM for the
nucleophiles and 80 mM for the electrophiles, to isolate
aldol adducts for their full characterization and
stereoselectivity assignments.
No asymmetric carbon was formed for the aldol reaction
1 + 6 (Scheme 1), so all five enzymes were expected to give
the same aldol. B4XH86, one of the most promising
aldolases, was thus chosen for this synthesis. The achiral
aldol 11 was isolated with a 92 % yield after 8 h reaction and
anion exchange chromatography for purification. NMR
analysis showed that 11 exists in solution as an equimolar
mixture of furanose derivatives.
NaO2C
a)
NaO2C
NaO2C
OH
O
O
O
1
2
3
OH OH OH OH OH
OH OH OH
O
F
F
b)
OH
OH
O
4
O
5
O
6
O
7
O
OH
O
10
O
9
8
Figure 1. Nucleophiles a) and electrophiles b) evaluated with
class II pyruvate aldolases from biodiversity.
Aldolisation of 2 and 6 led to the formation of one
asymmetric center on carbon 3 of aldol 12 (Scheme 1). The
five selected enzymes were 3S specific when using 2 as
nucleophile (see previous study18 or below for aldol
reaction 2 + 7 concerning A3SLS0). Thus, the five enzymes
again yielded the same aldol. B4XH86 was again chosen as
catalyst and product 12 was obtained after 12 h with a yield
of 75%. Aldol 12 was observed in solution as two major
furanose forms with roughly the same ratio.
There was no aldol addition observed between 1 and 8,
regardless of the enzyme tested. Such lack of reactivity
with 8 may result from the accommodation limit of the
substrate binding sites or the favored cyclisation of 8,
protecting the ketone from nucleophilic addition. Ketone
2
was therefore not tested with this electrophile.
Conversely, the acyclic ketone 7 proved to behave as a
substrate of the tested aldolases, although activity was less
than that observed for the shorter ketone 6. Aldolases
B4XH86 and A5VAX1 were the most efficient catalysts for
the aldolisation between 1 and 7, with almost 80%
conversion in 2 h (Figure 2). There was an additional
decrease in aldolase activity when nucleophile 2 was used
in combination with 7. Nevertheless, approximately 80%
conversion could be achieved in 30 h by A5VAX1 and
B4XH86 with this substrate couple. For the aldol reaction
R1
R1
O
(S)
CO2NH4
OH
1-B4XH86
H4NO2C
+
NaO2C
OH
OH
HO
OH
HO
HO
(S)
HO
O
2-purification
O
O
R1
1
2
: R1=H
: R1=CH3
6
11
12
: R1=H : 92%
: R1=CH3 : 75%
1-B4XH86
NaO2C
+
NaO2C
F
F
F
HO
F
O
O
2-purification
O
1
15
: 60%
9
Scheme 1. Aldolase-catalyzed reactions of pyruvate 1
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ACS Catalysis
or 2-ketobutyrate 2 with dihydroxyacetone 6 and
A3SLS0 to assess its stereoselectivity at carbon 3, as no
previous studies had been conducted with this enzyme. An
identical NMR spectrum was obtained as that for A5VAX1,
also showing a 3S preference with 2. However, the reaction
did not exceed 50% conversion after 48 h.
1
2
difluoroacetone 9.
Aldol reaction 1 + 7 (Scheme 2) resulted in the formation
of only one chiral center at C4 of aldol 13. In theory, a non-
stereoselective enzyme could lead to the production of a
total of twelve cyclic aldols, i.e. two epimers in the fourth
position with three possible cyclic forms and potential
alpha/beta anomerism (Scheme 2). Therefore, DEPT 13C
NMR experiments were first performed to focus on the
tertiary carbon of the various forms (see SI). These analyses
revealed that the most active enzymes, B4XH86 and
A5VAX1, displayed poor stereoselectivity, with a mixture of
nine forms observed by NMR. Conversely, with the most
stereoselective aldolase A3SLS0, only six forms were
observed, suggesting a possible unique configuration at
C4. This assumption was confirmed by NMR studies
performed on the decarboxylated aldol product. This
decarboxylation reaction was run with H2O2 in a buffered
solution (pH 7.5 to avoid the lactonization observed
without buffer) and led to the formation of only one linear
stereoisomer (> 95% by 1H NMR). Subsequently, 2D
NOESY NMR experiments revealed the (4S) configuration
of 13, as shown by correlations between H3, H7, and H5 of
the major pyranose form (see SI). Finally, although A3SLS0
was analytically found to be the least effective aldolase for
catalyzing the coupling 1 + 7, the reaction was fast enough
(15 h) to selectively provide the aldol (4S, 5S)-13 at an 85%
3
4
5
6
7
8
9
O
CO2NH4
(S)
HO
HO
OH
(S)
14a
(S)
not observed
OH
+
OH
OH OH
(S)
O
(S)
HO
1-A5VAX1
CO2NH4
(S)
H4NO2
C
OH
OH
+
NaO2
C
(S)
(S)
(S)
O
HO
HO
OH
(S)
2-purification
HO
OH
14b
O
O
 and 
+
2
7
14 : 90%
CO2NH4
OH
OH
10
11
12
13
14
15
16
17
18
19
20
21
22
23
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O
OH
-
HO (S)
O3'
CO2
CH3
(S)
(S)
(S)
5'
(S)
HO
(S)
14c
 or 
7'
H
H
HO
OH
OH
Scheme 3. Aldolase-catalyzed reaction of 2-
ketobutyrate with L-erythrulose: 2 + 7.
The reaction of 1 with symmetrical difluoroketone 9 also
led to an achiral aldol product 15 (Scheme 1). The aldol
reaction 1 + 9 was thus also performed with B4XH86,
previously identified in analytical experiments as one of
the most active aldolases with this substrate couple.
However, the reaction and purification process were
hampered by the volatility of 9 and the limited stability
(degradation on ion exchange resin) of 15 (see SI).
Nevertheless, aldol 15 could be isolated at a yield of 60%
after 7 h.
We applied the methodology for the highly efficient
preparation of a new constraint dehydro-proline analogue,
to demonstrate the usefulness of this new route to tertiary
alcohols. L-proline is an amino acid of particular interest
due to its involvement in peptide and protein folding.
Many proline analogues have been included in peptides
and mimetics behaving as ligands for various therapeutic
targets in the field of cancer, neuropathic pain, and
autoimmune diseases.19 Furthermore, naturally occurring
4-hydroxy-L-proline is found in plant and animal proteins,
such as collagen and gelatin,20 or in peptide antibiotics
produced by several microorganisms.21 It has also been
shown to display antioxidant properties in halotolerant
and halophilic microorganisms,22 and has been used in
polyamides as a porogen filler for bone reconstruction.23
yield
after
purification
by
anion
exchange
chromatography.
O
CO2NH4
(S)
HO
HO
OH
(S)
OH
+
OH
OH OH
(S)
O
HO
1-A3SLS0
CO2NH4
(S)
H4NO2C
OH
OH
+
NaO2C
(S)
(S)
(S)
HO
HO
OH
2-purification
HO
OH
O
O
O
+
7
1
13 : 85%
CO2NH4
O
OH
OH
OH
HO (S)
O3' CO2NH4
(S)
5'
(S)
7'
HO
(S)
H
H
HO
major
OH
OH
Scheme 2. Aldolase-catalyzed reaction of pyruvate
with L-erythrulose: 1 + 7.
The new targeted constrained hydroxyproline analogue
17 was readily prepared in a one-pot one-step process,
including an enzymatic transamination of aldol 16 coming
from the aldolisation reaction 1 + 10 (Scheme 4). The
aldolisation step was preliminary studied with the five
previously selected aldolases. B4XH86 and A5VAX1 gave
the best results at an analytical scale (total conversion of 1
in less than half an hour), A3SLS0 being the sole inactive
enzyme under our experimental conditions. The aldol
reaction was first performed using A5VAX1 as catalyst. As
observed for 15, purification by anion exchange
chromatography led to partial degradation of 16.
Consequently, the excess diketone 10 was simply removed
by liquid extraction and aldol 16 was thus obtained at a
yield of 51% in a linear form 16a and a cyclic hydrate 16b in
the same proportion. Subsequently, aldol 16 was used as a
substrate for branched chain aminotransferase (BCAT)
from Escherichia coli in the presence of glutamic acid (Glu)
Aldol reaction 2 + 7, was also thoroughly examined
(Scheme 3). As observed at the analytical scale, reactions
with 2 as nucleophile were much slower than those run
with 1. Consequently, aldolases A5VAX1 and B4XH86,
which gave the best conversions in a reasonable time (24
h), were selected for preparative-scale syntheses. After
purification on an anionic exchange resin, aldol 14 was
isolated in 90% and 74% yields, respectively. From the
twelve theoretical cyclic forms that could be obtained
(strict 3S configuration), in contrast from 1, only three
appeared on the NMR spectra for both enzymes, 2
furanoses 14b and one pyranose 14c (Scheme 3), all in the
same proportion. Additional 2D NOESY NMR experiments
allowed to assign the configurations (3S, 4S, 5S) to the aldol
formed for both enzymes. As expected, the configuration
at C4 was the same for both aldols 13 and 14, resulting from
the similar addition mode on the re face of 7. The reaction
was also performed under the same conditions with
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as amino donor. BCAT is a well-known enzyme that has
Page 4 of 6
spectrum of these biocatalysts is more restricted than that
of the Rhamulose-1-phosphate aldolases previously
described,10 with selective activity towards the more highly
activated ketones. Nevertheless, compared to DHAP
aldolases, which are highly specific for their nucleophile
substrate, pyruvate aldolases display significant activity
with pyruvate analogues, thus opening access to structural
diversity. This wide scope of application was illustrated in
this study by the stereoselective preparation of seven new
compounds isolated in moderate to very good yields (60-
1
2
3
4
5
6
7
8
already been used in many studies to prepare various
amino acids of the L-series with very high
stereoselectivity.24–26 Thus, as expected, aldol 16 was
converted into the corresponding α-amino acid, which
spontaneously underwent cyclization to give a mixture of
17a and 17b (Scheme 4). Cyclic imines were the only forms
observed by NMR, suggesting that the cyclization could
afford an efficient equilibrium shift of the transamination,
as reported in similar cases using diamino or dicarbonyle
derivatives as transamination substrates.27–29 In our case, it
appeared that conversion of 16 did not exceed 60% (see SI).
Nevertheless, assuming that BCAT exclusively produces
amino acids of the L-series, the formation of 17a and 17b in
equimolar quantities indicates that aldolisation was not
stereoselective and led to racemic 16a.
9
92%). Finally,
a one-pot one-step cascade process,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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involving four enzymes, was designed for the highly
efficient and stereoselective synthesis of a new constrained
dehydro-proline analogue.
AUTHOR INFORMATION
Given that such a lack of enantioselectivity could be the
result of a thermodynamic control of the aldol reaction,
A5VAX1 and BCAT were combined in a one pot one step
Corresponding Author
* E-mail: christine.helaine@uca.fr.
process to favor kinetic control and
a
possible
improvement in stereoselectivity. Transamination was
thus made irreversible through its coupling with the
reduction of the coproduct 2-oxoglutaric acid (OG) formed
from Glu. This reaction was catalyzed by an OG-
reductase30,31 in the presence of NADH, which was itself
irreversibly regenerated using the formate/formate
dehydrogenase (FDH) system.32 These cascade reactions
were achieved by adjusting the relative amounts of A5VAX1
and BCAT to slow down the aldolisation reaction, while
trying to minimize the side transamination reaction
between 1 and Glu, leading to the formation of alanine.
Finally, the pyrroline 17a and 17b were isolated after 8 h at
a yield of 57% at a 25/75 ratio using an A5VAX1/BCAT ratio
of 1/40 w/w in this multi-enzyme one pot process, with 20,
80 and 20 mM 1, 10, and Glu, respectively. The use of
B4XH86 in the same one step process gave a 35/65 ratio of
17a and 17b (purification was not performed). The cis (S, S)
configuration of the major isomer 17b could be
ORCID
Christine Guérard-Hélaine : 0000-0002-2311-7514
Notes
The authors declare no competing financial interest.
ASSOCIATED CONTENT
Supporting Information.
The supporting information is available free of charge on the
ACS Publications Website at DOI:
Details on material, methods, experimental procedures and
spectral data.
ACKNOWLEDGMENTS
This work was supported by the French National Center for
Scientific Research (CNRS) and the University of Clermont
Auvergne.
1
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In conclusion, we highlight a second class II family of
aldolases, namely pyruvate-aldolases, which are able to use
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