Biocatalytic Construction of Quaternary Centers by Aldol Addition of 3,3-Disubstituted 2-Oxoacid Derivatives to Aldehydes

Article abstract of DOI:10.1021/jacs.0c09994

The congested nature of quaternary carbons hinders their preparation, most notably when stereocontrol is required. Here we report a biocatalytic method for the creation of quaternary carbon centers with broad substrate scope, leading to different compound classes bearing this structural feature. The key step comprises the aldol addition of 3,3-disubstituted 2-oxoacids to aldehydes catalyzed by metal dependent 3-methyl-2-oxobutanoate hydroxymethyltransferase from E. coli (KPHMT) and variants thereof. The 3,3,3-trisubstituted 2-oxoacids thus produced were converted into 2-oxolactones and 3-hydroxy acids and directly to ulosonic acid derivatives, all bearing gem-dialkyl, gem-cycloalkyl, and spirocyclic quaternary centers. In addition, some of these reactions use a single enantiomer from racemic nucleophiles to afford stereopure quaternary carbons. The notable substrate tolerance and stereocontrol of these enzymes are indicative of their potential for the synthesis of structurally intricate molecules.

Full text of DOI:10.1021/jacs.0c09994

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Article
Biocatalytic Construction of Quaternary Centers by Aldol Addition
of 3,3-Disubstituted 2‑Oxoacid Derivatives to Aldehydes
⊥
⊥
́
́
Roser Marín-Valls, Karel Hernandez, Michael Bolte, Teodor Parella, Jesus Joglar, Jordi Bujons,
and Pere Clapes*
́
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ABSTRACT: The congested nature of quaternary carbons hinders their preparation,
most notably when stereocontrol is required. Here we report a biocatalytic method for
the creation of quaternary carbon centers with broad substrate scope, leading to
different compound classes bearing this structural feature. The key step comprises the
aldol addition of 3,3-disubstituted 2-oxoacids to aldehydes catalyzed by metal
dependent 3-methyl-2-oxobutanoate hydroxymethyltransferase from E. coli
(KPHMT) and variants thereof. The 3,3,3-trisubstituted 2-oxoacids thus produced
were converted into 2-oxolactones and 3-hydroxy acids and directly to ulosonic acid
derivatives, all bearing gem-dialkyl, gem-cycloalkyl, and spirocyclic quaternary centers. In
addition, some of these reactions use a single enantiomer from racemic nucleophiles to
afford stereopure quaternary carbons. The notable substrate tolerance and stereocontrol
of these enzymes are indicative of their potential for the synthesis of structurally
intricate molecules.
diversity besides the heteroaromatic “flat” chemistry, (ii)
enhance the binding selectivity with target proteins avoiding
unspecific interactions (e.g., π-staking), and (iii) have better
solubility and bioavailability.2
INTRODUCTION
■
Quaternary centers are important structural units that can be
found in naturally occurring, biologically active compounds
and pharmaceutical ingredients (Figure 1).1 Bioactive mole-
cules bearing this feature are becoming most appreciated in
medicinal chemistry because they (i) expand the chemical
Therefore, synthetic access to this type of compound can be
regarded as significant and a challenging task for organic
chemists. Indeed, creation of quaternary stereocenters is
elusive mainly due to the steric hindrance between the carbon
substituents, and consequently, considerable efforts have been
dedicated to implement chemical methodologies for this
purpose.1a,g,3 These encompass long-established approaches
such as Claisen rearrangement4 and Diels−Alder reaction5 up
to the most recent ones based on transition metal
catalysis1c,2b,3e,6 and organocatalytic reactions.3k,7 However,
innovative procedures expanding the range of structurally
diverse products with improved efficacy, simplicity, and
stereoselectivity are key for advancing the field of chemical
synthesis.6c,8
Biocatalytic creation of carbon−carbon bonds is widely
recognized as a useful synthetic technology particularly
because of its high selectivity, mild operation conditions, and
use of simple starting materials in a straightforward fashion.9
Received: September 18, 2020
Figure 1. Examples of bioactive molecules and pharmaceuticals
bearing quaternary centers.
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Furthermore, biocatalysis is accepted as a green and sustainable
technology.10 However, enzymatic methods to forge quater-
nary carbon stereocenters are scarce.11
Scheme 2. Selected Panel of 3,3-Disubstituted 2-Oxoacids
(1) and Aldehydes (2) as Substrates for KPHMT-Catalyzed
Formation of Quaternary Carbon Centers by Aldol
Addition Reactions
In this work, we explored the capabilities of metal-dependent
3-methyl-2-oxobutanoate hydroxymethyltransferase (KPHMT,
EC 2.1.2.11) from E. coli and its variants to catalyze the
formation of quaternary centers. In vivo, KPHMT catalyzes the
transfer of the hydroxymethyl group from N5,N10-methylene
tetrahydrofolate (MTHF) to 2-oxoisovalerate (2-OI) to
produce ketopantoate (KP), bearing a gem-dimethyl quater-
nary center and tetrahydrofolate (THF) (Scheme 1).12 It has
Scheme 1. 3-Methyl-2-oxobutanoate
Hydroxymethyltransferase (KPHMT) Catalyzed the
Synthesis of Ketopantoate (KP), a Precursor of Vitamin
a
B5
a
MTHF = N5,N10-methylene tetrahydrofolate; THF = tetrahydrofo-
late. CoASH = Coenzyme A.14
Figures S117−S128). Thus, seven KPHMT variants, i.e.,
I212A, I202A, V214G, I202A/I212A, I202A/V214G, I212A/
V214G, and I202A/I212A/V214G, were constructed and
tested as biocatalysts. Notably, the enzymes were obtained in
high yields after expression and purification (88−205 mg of
protein per liter of medium culture, see SI). Purified KPHMT
catalysts were stored and used in the reactions as glycerinate
preparations (i.e., a solution of the enzyme consisting of 20
mM MOPS buffer pH 7.0 containing NaCl (50 mM) and
glycerol (50%)). Furthermore, we previously found that
KPHMT catalysts with Co2+ as metal cofactor gave the best
activity.13b Therefore, we decided to employ Co2+ as a cofactor
for all KPHMT catalysts in this work.
Screening of KPHMT Catalysts and Substrates. We
began our investigation on the screening of the KPHMT
catalysts assaying the panel of nucleophiles and electrophiles
for measuring the formation of aldol products 3 and/or
consumption of substrates after 24 h (Figure 3, see SI) and
reaction rates (v0) (Figures S74 and S75) .
also been found that KPHMT can function as a metal-
dependent (Class II) aldolase, catalyzing the aldol addition of
2-oxoacids to methanal and operating under a tetrahydrofolate-
independent mechanism.13 Indeed, the enzyme is able to
catalyze the enolization of the 2-oxoacid via an α-proton
abstraction and formation of the corresponding stabilized
carbanion. This enolate can attack the methylene group of
MTHF, resulting in the formation of KP, in vivo activity, or
eventually on other electrophiles, e.g., aldehydes.13a
RESULTS AND DISCUSSION
■
In a previous work, we reported on the preparation of 2-
substituted 3-hydroxycarboxylic esters from 3-monosubstituted
2-oxoacids and methanal.13b Here, we investigated the aldol
addition of 3,3-disubstituted 2-oxoacids (1) to diverse
aldehyde (2), mediated by KPHMT catalysts, to generate
3,3,3-trisubstituted 2-oxoacids (3) bearing a quaternary
stereocenter (Scheme 2).
To establish optimal KPHMT catalysts, we capitalize on the
recently engineered KPHMT variants I212A and I202A, which
tolerate 3-monosubstituted 2-oxoacids with linear, branched,
or cyclic substituents.13b Furthermore, based on the KPHMT
structure (Figure 2A), we envisioned that residues I212, I202,
as well as V214 could also be involved in the accommodation
of the tertiary carbon nucleophiles (i.e., 3,3-disubstituted 2-
oxoacids).13b Molecular models of variants at these positions
showed larger active sites that could accommodate the
sterically hindered nucleophiles considered (Figure 2 and
Despite that wild-type KPHMT efficiently catalyzed the
aldol reaction in some cases, variants I202A, I212A, and the
double variant I202A/I212A gave the best (≥90%) con-
versions for most of the reactions and variant V214G
specifically for the addition of 1a to 2h. Several KPHMT
variants gave conversions ≥60% with combinations of
nucleophiles 1a, 1e, and 1f with electrophiles 2a−h.
Exceptions were 2b+1f (50%) and 2d+1f (28%). 2-Oxoacids
1g and 1h were substrates when methanal (2a) (20 and 40%
with variants I212A and I202A/I212A, respectively) and 2-
B
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Figure 2. (A) Structure of the active site of the ketopantoate complex
of wild-type KPHMT (chain A, PDB 1M3U).15 Ketopantoate is
shown with yellow C atoms, and the three residues proposed for
mutation are shown with orange C atoms. Models of the prereactive
complexes of (B) KPHMT V214G with (S)-1b and 2a, (C) KPHMT
I202A/V214G with (S)-1d and 2a, and (D) KPHMT I212A with 1f
and 2h. Nucleophiles 1b, 1d, and 1f are shown with yellow C atoms,
electrophiles 2a and 2h with green C atoms, and the mutated residues
with orange C atoms. Nucleophiles are shown in their enolate form
(1b and 1d as E-enolates) coordinated to the metal cofactor (purple
sphere), which is also coordinated by residues D45 and D84 and by
two water molecules. 2a and 2h are displayed with their carbonyl
oxygen atom forming hydrogen bonds with residue E181 and one of
the metal-coordinated waters. 2h is shown approaching the enolate
from its si-face. The surfaces of the active site cavities near the
nucleophile substrates are shown in cyan. Interactions are shown with
dashed lines: H-bond in yellow, salt bridges in magenta.
Figure 3. Screening of KPHMT-catalyzed aldol addition of selected
3,3-disubstituted 2-oxoacids 1 to aldehydes 2. Conversions to aldol
adducts (%) at 24 h of reaction measured by HPLC after
derivatization of the substrates and products via oxime formation
(see SI). Other nucleophile/electrophile combinations were not
performed due to the low activity in the screening with methanal as
electrophile (Figures S74 and S75). aNo product formation was
detected.
hydroxyethanal (2c) (50 and 42%, respectively, with I212A)
were used as electrophiles, whereas they were not converted
using 2b and 2e−h. Nucleophiles 1m−n and electrophiles 2i−
k were not converted by any KPHMT catalysts. The results are
consistent with the initial reaction rates (v0) of the
corresponding reactions (Figures S74 and S75). Notably,
KPHMT(Co2+) catalyst loading was 0.068 mol %. This
catalytic efficiency in addition to their high expression levels
make these enzymes highly valuable for preparative-scale
synthesis.
Preparative Synthesis. The positive reactions were run at
a preparative scale (2.5 mmol of the limiting 3,3-disubstituted
2-oxoacid (1) substrate), providing, after the adequate
treatment (see below), the corresponding 2-oxolactones
(4a−g) (Scheme 3A), 3-hydroxy acid derivatives (5a−j)
(Scheme 3B), bicyclic lactones (6) (Scheme 4A), and ulosonic
acid type products (7a−q) (Scheme 4B), all of them bearing
gem-dialkyl, gem-cycloalkyl, and spirocyclic quaternary centers,
with final product amounts ranging from 50 to 390 mg. The 2-
oxolactones4a−g were produced during workup (acidic
aqueous−organic solvent extraction). Thus, when quantitative
conversions were achieved, this methodology was enough to
afford pure material (Scheme 3A). Otherwise, an additional
extraction with aqueous buffer at neutral pH removed the
remaining starting 2-oxoacid 1, which was the main impurity
present. The 2-oxo group can be reduced,16 affording α-
hydroxy-γ-butyrolactones (e.g., (R)- or (S)-pantolactone and
analogues), chiral auxiliaries, and important precursors for the
synthesis of naturally occurring and synthetic biologically
active compounds.17 Elaborated chemical transformations on
the carboxylate group such as oxidative decarboxylation
followed by esterification lead to different 3-hydroxyesters
(5a−i and amide 5j, Scheme 3B) with application as building
blocks in the synthesis of pharmaceutical compounds18 and
polymers, coating oligomers, and dendrimers, e.g., 2,2-
bis(hydroxymethyl) propionic acid derivative 5g.19
Using 2-hydroxyaldehydes, 2c and 2e−h, as electrophiles,
the spontaneous formation of cyclic hemiketals effectively
shifted the reaction equilibrium to the aldol products 6a,b and
7a−q (Scheme 4A,B). The resulting adduct products were
easily purified by anion exchange chromatography, eluting with
formic acid. These ulosonic acid type products, bearing a
quaternary center at C3, have not been hitherto reported, and
their preparation using conventional chemical procedures may
be anticipated to be elusive because of the number of
functional groups and the intrinsic reactivity of the 2-oxoacid
moiety.20 Furthermore, KPHMT would facilitate access to a
great variety of ulosonic acids (e.g., sialic acid family), which
constitute important biologically active compounds involved in
cellular recognition and communication.21
The addition of 1j to 2c (Scheme 4A) gave a mixture of the
two diastereomeric lactones 6a and 6b (91:9, respectively),
identified by NMR after anion exchange purification. The
acidic elution conditions (i.e., HCOOH, 1 M) and the
C
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Scheme 3. General Routes for the Synthesis of Products 4 (A) and 5 (B)
a
b
c
d
e
I202A. Absolute stereochemistry assigned on the basis of crystal X-ray diffraction of compound 5c. I202A/V214G. I212A. Not determined.
For optical rotations see SI. Wild-type. Enantiomeric ratio determined by NMR using R-configured Pirkle’s alcohol.22 Enantiomeric ratio
f
g
h
i
j
determined by HPLC on the chiral stationary phase. V214G, The absolute stereochemistry was determined by single-crystal X-ray diffraction (see
a,c,d,f,i,k
Catalyzed by 0.068 mol % of KPHMT(Co2+) wild-type or variants thereof.
k
SI). I202A/I212A.
lyophilization of the fraction pool promoted the lactonization
of the hemiketal intermediate.
diffraction provided the absolute S-configuration of 5c (Figure
S79) that agrees with the S-5b stereochemistry.
The S-configured 5b and 5c could be rationalized based on
the molecular models shown in panels B and C of Figure 2. To
form the corresponding enolates, nucleophiles 1 should adopt
a bound orientation in the catalytic site with their H atom at
C3 oriented toward the proposed catalytic base (E181).15
Therefore, it could be reasoned that the S-enantiomer of 1b
can adopt an appropriate conformation with minimal steric
repulsion for its C3-ethyl substituent (Scheme 5). The
corresponding E-enolate would then expose its re face to the
electrophile (Figure 2B), providing the 3S-aldol adduct, a
precursor of S-5b. In contrast, R-1b has to adopt a different
bound conformation, with its C3-ethyl substituent pointing
toward residue V179, in order to place its α-proton within
reach of the catalytic base (Scheme 5). This conformation,
which is less favored because of the proximity of the ethyl
moiety to the side chains of V179 and E181, would generate a
Z-enolate that would expose its si-face to the electrophile,
providing the alternative 3R-aldol adduct. Similarly, each
enantiomer of nucleophile 1d can only adopt one reactive
conformation that would generate the corresponding E- and Z-
The absolute stereochemistry of 4f−g and 7a−e was
assigned by comparing the stereochemical outcome achieved
for 7f−q. Compounds 7f−q contain one or two chiral centers
of defined absolute configuration that act as a reference in the
determination of their overall relative configuration. In 7f−q
the absolute stereochemistry of the chiral center coming for
the electrophile was invariably S regardless of the aldehyde
substrate. This is also consistent with a previous study on the
steric course of KPHMT-catalyzed aldol reaction with
MTHF.23 Therefore, it may safely be assumed that the major
enantiomer of 4f−g, 7a−e, and 5h will be S, and that of 5j will
be R configured. The absolute stereochemistry of 5b was
assessed indirectly by analyzing the stereochemistry of the
unreacted 1b. This was determined to be R configured
according to chiral HPLC analysis against a standard (see
Figure S77). This indicated that only the corresponding
enantiomer S-1b was converted. From mechanistic consid-
erations of KPHMT catalysis (see below and Scheme 5), it
may be inferred that the S-1b furnished S-5b. The X-ray
D
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Scheme 4. General Routes for the Synthesis and Isolation of Products 6 and 7
a
b
c
d
Measured by NMR. ee not determined. For optical rotations, see SI. No other diastereomers were detected by NMR analysis. α:β anomeric
e-h
f
g
h
ratio. Catalyzed by 0.068 mol % of: wild-type KPHMT(Co2+), or variants I202A. I212A. V214G.
e
Scheme 5. Enolization Mechanism of Nucleophiles S-1b,d and R-1b,d
enolates (Figure 2C), from S- and R-1d, respectively. Due to
the larger size of the n-butyl C3-substituent, formation of the
E-enolate would be faster, and consequently, the 3S-aldol
adduct is formed preferentially. Similar reasoning could be
used to explain the steric outcome observed for the rest of the
products (see SI).
In some cases, minor cyclic structures such as 2-oxolactone 8
and the corresponding hydrated form 9 (Scheme 6 and spectra
in SI) were identified. It is likely that the purification process
under acid conditions and freeze drying favored the formation
of these side derivatives.
E
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2.1.2.11) (KPHMT), for the creation of quaternary carbon
centers using a structurally diverse array of 3,3-disubstituted 2-
oxoacid nucleophiles and aldehyde substrates. The biocatalytic
approach reported here benefits from a straightforward process
starting from diverse 2-oxoacid derivatives, obtained by well-
established and efficient chemical procedures. This method-
ology allows access to unprecedented 2-oxolactones, which can
be converted into α-hydroxy-γ-butyrolactones by reduction of
the carbonyl group, 3-hydroxy acid derivatives, and ulosonic
acid type products, all of them bearing gem-dialkyl, gem-
cycloalkyl, and spirocyclic quaternary centers. To the best of
our knowledge, the aldol additions of 3,3-disubstituted 2-
oxoacid derivatives to aldehydes have not been performed with
other catalysts. For example: the enamine activation of
branched α,α-disubstituted carbonyl compounds via organo-
catalysis (e.g., proline and derivatives using typically 1−20 mol
% compared with KPHMT(Co2+) catalysts employing 0.068
mol %) is much less reactive with respect to those derived from
α-monosubstituted carbonyl compounds, and therefore only
very strong electrophiles are able to react.3a Here, we are using
common aldehydes not specially activated and some even in
equilibrium with cyclic hemiketals in aqueous solution, e.g., D-
threose and D-erythrose.3a The present study expands the
portfolio of biocatalytic aldol reactions leading to unprece-
dented structures and offering new synthetic opportunities for
pharmaceutically relevant compounds and chiral precursors.
We anticipate a range of new applications by performing
further engineering of the KPHMT variants to render new
biocatalysts, efficient toward new substrate classes.
Scheme 6. Equilibrium between Lactones 8 and 9 and
Hemiketal 7 in Acidic Aqueous Medium
The addition of 1a, 1e, and 1f to D-threose was planned as a
simultaneous biocatalytic one-pot two-step cascade reaction
(Scheme 7A). To this end, D-threose (2g) was first produced
Scheme 7. One-Pot Two-Step Enzymatic Synthesis of
Products 7l (A) and the Diasteromeric Mixture 6a:6b (B)
a
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/jacs.0c09994.
Crystallographic data for structure 5c (CIF)
a
MBP-YfaU = maltose binding protein fused to YfaU.13b
Experimental details, NMR spectra, and computational
models (PDF)
by enzymatic homoaldol addition of hydroxyethanal (2c)
catalyzed by D-fructose-6-phosphate aldolase variant A129G
(FSA A129G) (Scheme 7A).24 Once 2g was produced, 1a and
KPHMT were added to the reaction mixture, yielding 7l.
When the second step was carried out without removal of FSA
A129G, a significant amount of 7a (∼27%), from the addition
of 1a and 2c, was detected. This side reaction was minimized
(∼5%) by removing FSA A129G from the medium. This result
suggested that 2c was a better substrate than 2g for KPHMT
and that it was transformed into 7a by KPHMT, via FSA-
catalyzed retro-aldol reaction of 2g. By analogy, the synthesis
of the diastereomeric mixture 6a:6b was also carried out by a
one-pot two-step cascade (Scheme 7B). This comprised the
aldol addition of 2-oxobutyrate 10 to 2a catalyzed by 2-keto-3-
deoxy-L-rhamnonate aldolase (YfaU, EC 4.1.2.53), generating
the nucleophile 1j.13b Then, 2c and wild-type KPHMT were
added to furnish the diastereomeric mixture 6a:6b after
purification. In this case, there was no need to remove the
YfaU since this pyruvate aldolase was not able to catalyze the
second aldol reaction, and the reaction with methanal was
virtually irreversible.
AUTHOR INFORMATION
■
Corresponding Author
́
Pere Clapes − Biological Chemistry Department, Instituto de
́
Quimica Avanzada de Cataluna, IQAC−CSIC, 08034
̃
Barcelona, Spain; orcid.org/0000-0001-5541-4794;
Email: pere.clapes@iqac.csic.es
Authors
Roser Marín-Valls − Biological Chemistry Department, Instituto
́
de Quimica Avanzada de Catalun
̃
a, IQAC−CSIC, 08034
Barcelona, Spain
́
Karel Hernandez − Biological Chemistry Department, Instituto
́
de Quimica Avanzada de Catalun
̃
a, IQAC−CSIC, 08034
Barcelona, Spain
Michael Bolte − Institut fu
̈
r Anorganische Chemie, J.-W.-Goethe-
Universitat, Frankfurt/Main, Germany; orcid.org/0000-
0001-5296-6251
̈
Teodor Parella − Servei de Ressonancia Magnetica Nuclear,
̀
Universitat Autonoma de Barcelona, 08193 Bellaterra, Spain;
orcid.org/0000-0002-1914-2709
CONCLUSION
́
Jesus Joglar − Biological Chemistry Department, Instituto de
■
́
In summary, we demonstrate the utility of the promiscuous 3-
methyl-2-oxobutanoate hydroxymethyltransferase (EC.
Quimica Avanzada de Catalun
̃
a, IQAC−CSIC, 08034
Barcelona, Spain
F
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Article
Quaternary Carbon Stereocenters via a Semipinacol Rearrangement
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(e) Sun, X.; Frimpong, K.; Tan, K. L. Synthesis of Quaternary Carbon
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P.; Marek, I. Four-Component Reactions for a New Diastereoselective
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(39), 11776−11777. (g) Li, Y.; Xu, S. Transition-Metal-Catalyzed C−
H Functionalization for Construction of Quaternary Carbon Centers.
Chem. - Eur. J. 2018, 24 (61), 16218−16245. (h) Cozzi, P. G.; Hilgraf,
R.; Zimmermann, N. Enantioselective Catalytic Formation of
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2001, 40 (24), 4591−4597. (j) Yamago, S.; Machii, D.; Nakamura, E.
Simple diastereoselectivity of the aldol reaction of persubstituted
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Chem. 1991, 56 (6), 2098−2106. (k) Fujii, K.; Mitsudo, K.; Mandai,
H.; Korenaga, T.; Suga, S. Enantioselective Acyl Migration Reactions
of Furanyl Carbonates with Chiral DMAP Derivatives. Chem.−Eur. J.
2019, 25 (9), 2208−2212. (l) Minko, Y.; Marek, I. Stereodefined
acyclic trisubstituted metal enolates towards the asymmetric
formation of quaternary carbon stereocentres. Chem. Commun.
2014, 50 (84), 12597−12611. (m) Das, J. P.; Marek, I.
Enantioselective synthesis of all-carbon quaternary stereogenic centers
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(4) Martín Castro, A. M. Claisen Rearrangement over the Past Nine
Decades. Chem. Rev. 2004, 104 (6), 2939−3002.
Jordi Bujons − Biological Chemistry Department, Instituto de
́
Quimica Avanzada de Catalun
̃
a, IQAC−CSIC, 08034
Barcelona, Spain
Complete contact information is available at:
https://pubs.acs.org/10.1021/jacs.0c09994
Author Contributions
⊥R.M.-V. and K.H. contributed equally. The manuscript was
written through contributions of all authors. All authors have
given approval to the final version of the manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This project has received funding from the Ministerio de
■
́
Ciencia e Innovacion (MICIN), the Fondo Europeo de
Desarrollo Regional (FEDER) (grant RTI2018-094637-B-I00
to P.C. and PGC2018-095808-B-I00 to T.P.), and Programa-
́
cion Conjunta Internacional (PCI2018-092937), through the
initiative ERA CoBioTech (Tralaminol). The authors grate-
fully acknowledge the “Consorci de Serveis Universitaris de
Catalunya” (CSUC) for allowing the use of its software and
hardware resources.
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