Synthesis of γ-Hydroxy-α-amino Acid Derivatives by Enzymatic Tandem Aldol Addition-Transamination Reactions

Article abstract of DOI:10.1021/acscatal.1c00210

Three enzymatic routes toward γ-hydroxy-α-amino acids by tandem aldol addition-transamination one-pot two-step reactions are reported. The approaches feature an enantioselective aldol addition of pyruvate to various nonaromatic aldehydes catalyzed by trans-o-hydroxybenzylidene pyruvate hydratase-aldolase (HBPA) from Pseudomonas putida. This affords chiral 4-hydroxy-2-oxo acids, which were subsequently enantioselectively aminated using S-selective transaminases. Three transamination processes were investigated involving different amine donors and transaminases: (i) l-Ala as an amine donor with pyruvate recycling, (ii) a benzylamine donor using benzaldehyde lyase from Pseudomonas fluorescens Biovar I (BAL) to transform the benzaldehyde formed into benzoin, minimizing equilibrium limitations, and (iii) l-Glu as an amine donor with a double cascade comprising branched-chain α-amino acid aminotransferase (BCAT) and aspartate amino transferase (AspAT), both from E. coli, using l-Asp as a substrate to regenerate l-Glu. The γ-hydroxy-α-amino acids thus obtained were transformed into chiral α-amino-γ-butyrolactones, structural motifs found in many biologically active compounds and valuable intermediates for the synthesis of pharmaceutical agents.

Full text of DOI:10.1021/acscatal.1c00210

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Research Article
Synthesis of γ‑Hydroxy-α-amino Acid Derivatives by Enzymatic
Tandem Aldol Addition−Transamination Reactions
⊥
⊥
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Carlos J. Moreno, Karel Hernández, Simon J. Charnok, Samantha Gittings, Michael Bolte, Jesus Joglar,
Jordi Bujons, Teodor Parella, and Pere Clapés*
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ABSTRACT: Three enzymatic routes toward γ-hydroxy-α-amino acids
by tandem aldol addition−transamination one-pot two-step reactions are
reported. The approaches feature an enantioselective aldol addition of
pyruvate to various nonaromatic aldehydes catalyzed by trans-o-
hydroxybenzylidene pyruvate hydratase-aldolase (HBPA) from Pseudo-
monas putida. This affords chiral 4-hydroxy-2-oxo acids, which were
subsequently enantioselectively aminated using S-selective transaminases.
Three transamination processes were investigated involving different
amine donors and transaminases: (i) L-Ala as an amine donor with
pyruvate recycling, (ii) a benzylamine donor using benzaldehyde lyase
from Pseudomonas fluorescens Biovar I (BAL) to transform the
benzaldehyde formed into benzoin, minimizing equilibrium limitations,
and (iii) L-Glu as an amine donor with a double cascade comprising
branched-chain α-amino acid aminotransferase (BCAT) and aspartate amino transferase (AspAT), both from E. coli, using L-Asp as a
substrate to regenerate L-Glu. The γ-hydroxy-α-amino acids thus obtained were transformed into chiral α-amino-γ-butyrolactones,
structural motifs found in many biologically active compounds and valuable intermediates for the synthesis of pharmaceutical agents.
KEYWORDS: biocatalysis, 2-oxoacid aldolase, transaminases, aldol addition, reductive amination, γ-hydroxy-α-amino acids
However, the number of examples is scarce and, although they
represent a remarkable achievement, they are often limited by
the poor stereoselectivity profile of pyruvate aldolases as
catalysts to generate the corresponding 4-hydroxy-2-oxo acid
precursors or the lack of transaminase selectivity toward the
aldol adduct.
Herein, we report three strategies for the biocatalytic
diastereoselective synthesis of γ-hydroxy-α-amino acids by
combining an enantioselective pyruvate-aldolase and
S-selective transaminases,8b starting from pyruvate and diverse
aldehyde substrates.
INTRODUCTION
■
γ-Hydroxy-α-amino acids represent compounds with relevant
biological and pharmacological importance.1 Examples include
antidiabetics such as (2S,3R,4S)-4-hydroxyisoleucine,
4-hydroxy-L-norvaline, and 4-hydroxypipecolic acid and nutri-
tional supplements in the food industry, e.g.
trans-4-hydroxy-L-proline.1a,2 Moreover, they constitute struc-
tural motifs of more complex naturally occurring and synthetic
molecules, namely antibiotics,3 antimitotics,4 and (bio)-
herbicides,5 as well as chiral building blocks for the production
of active ingredients, e.g. α-amino-γ-butyrolactones,
4,5-dihydroxynorvaline, and 4-hydroxypyroglutamic acid and
derivatives (Figure 1).2b,d,6
Therefore, substantial research efforts have been devoted to
their synthesis using multistep catalytic or stoichiometric
chemical approaches (Figure 2).1a,2g,7 Biocatalytic access to
these compounds is regarded as a powerful strategy because of
their simplicity and stereoselectivity starting from simple
achiral materials (Figure 2).2c,e,8
RESULTS AND DISCUSSION
■
Stereoselective Aldol Addition of Pyruvate to
Aldehydes Catalyzed by trans-o-Hydroxybenzylidene
Pyruvate Hydratase-Aldolase (HBPA). The trans-o-hydrox-
ybenzylidene pyruvate hydratase-aldolase (HBPA, EC
4.1.2.45) from Pseudomonas putida was discovered in the
Received: January 15, 2021
Revised: March 20, 2021
Published: April 2, 2021
In this sense, the sequential combination of carboligases and
transaminases in one-pot one-step or one-pot two-step
reactions with or without substrate recycling offers broad
synthetic possibilities. Using this route, chiral L- and
D-homoserine, D-anti-dihydroxynorvaline, 4-hydroxyisoleucine,
norpseudoephedrine, and norephedrine were prepared.2e,8b,e,9
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Figure 1. Examples of bioactive compounds bearing a γ-hydroxy-α-amino acid moiety or α-amino-γ-butyrolactone.
aldol addition and a subsequent dehydration, both steps being
catalyzed by the enzyme. Interestingly, it has been reported
that HBPA catalyzes the aldol addition of fluoropyruvate to
aromatic aldehydes with high stereoselectivity, in which the
dehydration products were not detected, likely because the
fluorine atom precludes the dehydration step by the enzyme.11
We envisioned that HBPA could catalyze aldol additions of
pyruvate to nonaromatic electrophiles, in which the
dehydration activity could be largely minimized or even
suppressed, rendering aldol adducts with high enantioselectiv-
ity. We assayed various nonaromatic electrophiles (1a−s;
Scheme 1) as substrates of HBPA in the aldol addition of
pyruvate.
Good to excellent conversions to 4-hydroxy-2-oxo acids 3
(70−95%) were achieved for most of the electrophiles,
indicating an excellent structural substrate tolerance of
HBPA (Scheme 2). A single residue mutation, the HBPA
H205A variant, greatly improved the catalyst efficiency toward
1c,k,o, yielding 3c,k,o, respectively, in 66−75% conversion.
Molecular models of the complex of pyruvate-enamine-
bound HBPA with aldehydes 1c,o suggest that the H205A
mutation generates a new cavity in the HBPA active site, which
reduces the steric hindrance. This cavity can be occupied by
the phenyl moiety of the incoming aldehyde (Figure 3 and
Figure S93), thus facilitating the interactions with residues
Trp224 and Phe269. Moreover, an analysis of the HBPA
structure shows that the carboxylic groups of Glu206, Asp207,
Asp208, and Asp265 are within 5 Å of the δ and ε-N atoms of
the imidazole group of His205, stabilizing its protonated state
Figure 2. Examples of strategies for the synthesis γ-hydroxy-α-amino
acids: (a) synthesis from D-ribonolactone as a chiral precursor and
stereocontrolled transformation;7d (b) aldol reaction with oxazoli-
donyl chromium carbene complex and photocyclization;7c (c) cyanate
to isocyanate rearrangement and enzymatic kinetic resolution;1a (d)
aza-Michael additions controlled by a crystallization-induced asym-
metric transformation;7f (e) Mannich condensation and catalytic
epimerization;7b (f) palladium(II)-catalyzed aza Claisen rearrange-
ments;7a (g) direct β or γ-hydroxylation of amino acids via α-
ketoglutarate-dependent dioxygenases;2c,8a (h) enzymatic synthesis
via carboligase-transaminase reactions.2e,8b,e,9
metabolic degradative pathway of naphthalene and naphtha-
lenesulfonates.10 In vivo, HBPA catalyzes the reversible aldol
condensation of pyruvate to salicylaldehyde in two steps: an
Scheme 1. Panel of Aldehyde Substrates 1 Assayed in the Aldol Addition of Pyruvate 2 Catalyzed by Wild-Type HBPA and Its
H205A Variant
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Scheme 2. HBPA Wild-Type and H205A Variant Catalyzed Aldol Addition of Pyruvate to Aldehydes 1 and Transformation of
f
Aldol Adducts 3 into 3-Hydroxy Ester Derivatives 4, for Enantiomeric Ratio Measurement
a
b
c
d
Conversions measured by HPLC. HBPA wild-type. Isolated yield. ee not determined; the material was submitted directly to the enzymatic
transamination reaction and the stereochemistry inferred from the corresponding 14c (for 3c), 15k (for 3k), 14n (for 3n), and 14o (for 3o)
e
f
derivatives; see Schemes 7 and 8. HBPA H205A variant. The ee values were determined by HPLC on a chiral stationary phase.
(Figure 4). This positive charge is also stabilized by a π-cation
interaction with the aromatic moiety of Trp224. Therefore,
removal of this protonated imidazole, which is ∼8 Å from the
ε-amino group of the essential Lys183, by the H205A mutation
modifies the electrostatic environment of the active site, which
could also contribute to the enhanced activity of this mutant
toward the selected substrates.
PheAc has the advantage that it can be removed by penicillin G
acylase, a mild and orthogonal protection compatible with
most common amino masking groups.13
Single-crystal X-ray diffraction studies of compounds 4e,f,j
indicate that HBPA renders aldol adducts having an R
configuration as the major products (Figure 5). This is
consistent with the reported stereochemical outcome of the
reactions between fluoropyruvate and (hetero)aromatic
aldehydes.11 Therefore, an R configuration may safely be
assumed for the major enantiomers of 3a−k (Scheme 2).
Molecular models of the complexes of HBPA were obtained
with (i) the covalently bound pyruvate enamine and the
electrophile molecules (Figures S88−S93) and (ii) the imines
derived from the aldol adducts (Figures S94−S99). These
models suggest that there is no steric restriction to the re- or si-
face approach of the electrophile to the enamine. Thus, it is not
clear why there is a preference for the re-face approach, which
would generate the R-aldol adducts as major products of the
reaction. A more in depth theoretical study would be required
to determine the source of this preference; however, this is
beyond the scope of this paper.
Enantiomeric ratios were determined by HPLC on a chiral
stationary phase after transforming the aldol adducts 3 into
3-hydroxy ester derivatives 4 (Scheme 2 and Figures S74−
S85). The corresponding authentic racemic samples were
produced by employing 2-oxo-3-deoxy-L-rhamnonate aldolase
(YfaU), which yielded aldol adducts 3 as racemic mixtures with
the selected electrophiles (see the Supporting Information).
Excellent levels of enantioselectivity were achieved for 3a,f−h
(96−90% ee), while they were good to moderate for 3b,e,i,j
(88−80% ee). This is significant considering the low
stereoselective profile of wild-type pyruvate aldolases toward
low-molecular-weight electrophiles.12 Low enantioselectivy was
attained with the methoxy derivative 3d (42% ee), whereas
racemates were obtained with phenylacetaldehyde (3l) and
3-(benzyloxy)propanal (3m). Cbz-, Boc-, and Ts-protected
aminoethanal compounds 1p,r,s, respectively, and
(S)-Cbz-2-aminopropanal (1q) were not converted. Thus,
PhAc was the amino protecting group of choice for
aminoaldehydes in the planned HBPA catalysis. Interestingly,
Synthesis of γ-Hydroxy-α-amino Acid Derivatives
Using a Biocatalytic One-Pot Cyclic Cascade Approach
with L-Ala as an Amine Donor. We began to screen a
transaminase panel (T001−T050) provided by Prozomix Ltd.
toward the selected aldol adduct examples 3a,b,e,g,h, produced
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Figure 3. Models of the prereactive complexes of wild-type HBPA (A, C) and HBPA H205A (B, D) with the covalently bound pyruvate enamine
(yellow C atoms) and aldehyde 1o (green C atoms). The mutated residue is highlighted in orange. Interactions are shown with dashed lines: H
bonds in yellow, π-stacking in magenta, and π-cation in dark green. A comparison of the surface of the active sites of both proteins (C, D)
(transparent cyan) reveals that the H205A mutation generates an expanded cavity near residue A205, which can be occupied by the phenyl moiety
of 1o. These models were obtained by QM/MM optimization of the structure of the complexes at the DFT (B3LYP/6-31G**) level of theory, as
detailed in the Supporting Information.
with good conversions and enantioselectivities by HBPA
catalysis (Figures S8−S12). We used L-Ala (5) as an amino
donor in a one-pot two-step reaction sequence (Scheme 3).
Thus, for the screening, the enzymatic aldol addition was run
first and, once the formation of 3 reached a maximum, L-Ala
(5) (500 mM, ≥ 10 equiv with respect to 3) and the
transaminase (T###) were added, allowing the reaction to
proceed for 24 h. Since unpurified aldol adducts 3 were
supplied for the screening reactions, HPLC analysis was the
method of choice to detect false positives caused by
transamination of the remaining unreacted aldehyde 1.
Compounds 6b,e, bearing aromatic moieties, were directly
detected by HPLC. The percentages of 6a,g,h formed were
estimated by measuring the consumption of 3a,g,h by HPLC,
after precolumn derivatization of the carbonyl group via oxime
formation (see the Supporting Information).
Roughly, 4 out of the 50 different transaminases were
selected as promising candidates for the transamination
reaction (Figures S8−S12). For scale-up experiments, we
capitalize on a strategy developed by our group consisting of a
Figure 4. Scheme showing the interactions established by the
one-pot reaction recycling of pyruvate 2, formed in the
imidazole group of residue His205 of HBPA. Distances (dashed lines)
transamination reaction, into the aldol reaction (Scheme 4).8b
to nearby residues are indicated.
This approach effectively shifts the equilibrium of the
transamination to the product, since the pyruvate is
continuously removed by the aldol reaction.
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Figure 5. X-ray structures of (R)-4e, (R)-4f, and (R)-4j as ORTEP-type plots displaying one molecule with 50% probability ellipsoids. The data
can be obtained free of charge from The Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.
(55 mM, 55% yield), with no effective pyruvate recycling.
Further increasing the initial pyruvate concentration (i.e., up to
100 mM) caused an increase in 3g production, but 6g
decreased, likely due to an equilibrium limitation of the
transamination. Using 1a (200 mM) and HBPA/T039, the
best initial pyruvate concentration was 100 mM (Figure 6B
and Figure S19). Under these conditions, 47 mM of 6a (47%
yield, with respect to the limiting substrate 5) was formed, but
no pyruvate recycling occurred. Aldol adduct 3a was most
probably in an equilibrium between the cyclic hemiketal and
the acyclic form. It is likely that the hemiketal was not a
substrate for the transaminase and its activity was limited by
the actual concentration of the acyclic adduct. Disappointingly,
this strategy failed to produce the corresponding
γ-hydroxy-α-amino acid derivatives 6 for the rest of the
aldehydes with the selected transaminases and aldol adducts 3
were the only species detected (see Figure S20 for an
example).
One problem that jeopardizes the pyruvate recycling strategy
was that the aldolase and transaminase were not sufficiently
active toward the aldehyde and aldol adduct, respectively, in
comparison with the system reported by us for the synthesis of
homoserine.8b This makes a rapid conversion difficult for both
the initial pyruvate and the aldol adduct formed, compromising
the efficiency of the recycling process. Therefore, the one-pot
two-step reaction sequence appears to be the most convenient
route in dealing with aldolases and/or transaminases with
kinetic and thermodynamic limitations. In this case, however,
an effective method to overcome the equilibrium limitations of
the transaminase must be implemented.
Scheme 3. One-Pot Two-Step Screening Reaction for the
Synthesis of γ-Hydroxy-α-amino Acid Derivatives 6, Using
L-Ala (5) as an Amine Donor for the Enzymatic
a
Transamination Reaction
a
The dotted line indicates an enzymatic transamination of the
aldehyde remaining in the system.
Scheme 4. One-Pot Biocatalytic Cascade Synthesis of
γ-Hydroxy-α-amino Acid Derivatives 6 Starting from
Aldehydes 1 and an Amine Donor 5 with Pyruvate (2)
Recycling
To successfully perform this strategy, the transamination of
the aldehydes 1 must be largely avoided or minimized. To
evaluate the degree of conversion for each aldehyde and
establish suitable reaction conditions for the one-pot cascade
process, we ran control experiments incubating aldehydes
1a,b,e,g,h (100 mM) with selected transaminases (Figures
S13−S18). Having established the suitable conditions, we
started testing a range of starting pyruvate (2) concentrations
between 5 and 100 mM, against 100 mM of L-Ala. Using 1g
(100 mM) as the electrophile and HBPA/T039 catalysts, 42
mM of 6g (42% yield) was formed at 5 mM pyruvate
concentration, indicating pyruvate recycling (Figure 6A and
Figure S21). At 50 mM of pyruvate, 6g reached a maximum
Synthesis of γ-Hydroxy-α-amino Acid Derivatives
Using a Biocatalytic One-Pot Two-Step Approach
Using Benzylamine as an Amine Donor. Another strategy
was devised for the synthesis of the selected target γ-hydroxy-
α-amino acid derivatives using benzylamine (7) as an
alternative amine donor (Scheme 5). The effective shift of
the equilibrium of the transamination was accomplished by
converting the benzaldehyde (8) formed into benzoin (9) by
benzaldehyde lyase from Pseudomonas fluorescens Biovar I
(BAL) (Scheme 5). BAL is highly selective toward
benzaldehyde, ensuring an almost quantitative conversion
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T039 was able to convert the aldol adducts 3a,b,e,g into the
corresponding products 6 (Figure S24). The reaction with
aldehyde 1h resulted in a false positive, because no product
could be detected in scale-up experiments.
The same two-step reaction sequence was then repeated
with and without removing HBPA, using T039 and including
aldehydes 1i,j (Figure 7A). The HBPA did not affect the yield
Figure 6. One-pot biocatalytic synthesis of 6g (A) and 6a (B),
starting from aldehydes 1g (100 mM), 1a (200 mM), and 5 (100
mM) in both experiments: concentration of the components after 24
h of reaction as a function of the initial concentration of pyruvate
using HBPA/T039 catalysts.
Figure 7. One-pot two-step stereoselective synthesis of γ-hydroxy-α-
amino acids (6a,b,e,g,i,j), using the HBPA/T039 system and
benzylamine (7) as amine donor: influence of the presence of
HBPA (A) and BAL (B) on the reaction yield of products 6. Error
bars are the values of the estimated standard error of the mean of two
independent experiments under the same reaction conditions.
into the mostly insoluble benzoin, which benefits the reaction
equilibrium.14 Benzylamine has been reported to be a suitable
amine donor, and different strategies to eliminate the
benzaldehyde were proposed: e.g. conversion into benzoic
acid under 1 atm of oxygen,15 extraction with hexane in an
aqueous−organic two-phase system,16 and reduction to benzyl
alcohol during a cascade synthesis of ω-amino fatty acids and
α,ω-diamines from ω-hydroxy fatty acids and α,ω-diols,
respectively.17
For this strategy, an extended panel of 194 transaminases
from Prozomix Ltd. was screened in a one-pot two-step
sequence (Figure S22 and Table S7) using the crude reaction
products of adducts 3a,b,e,g,h as starting materials.
Next, we rescreened 27 positive hits from the first screening
in the two-step sequence, removing the HBPA before starting
the transamination reaction aldolase to avoid retroaldolysis
(see the Supporting Information). In this case, the aldol
adducts 3, products 6, benzaldehyde (8), and benzoin (9)
were analyzed and quantified by HPLC. Only transaminase
of 6a,g,i,j, indicating that T039 was rather selective toward the
corresponding aldol adducts and that no retroaldolysis was
taking place. On the other hand, elimination of HBPA
benefited 6b,e in comparison with those products without
aromatic substituents. Next, we investigated the effect of BAL
on the yield of 6a,g,i,j (Figure 7B). The addition of BAL was
largely positive for 6a,i and less so for 6g,j. Overall, this
depends on each individual case and should be considered for
establishing the optimal operational conditions. Moreover, no
aldol condensation of pyruvate to benzaldehyde catalyzed by
HBPA was detected.
The low conversion of aldol adducts with aromatic
substituents, 3b,e, and the need for a highly selective
transaminase for the 4-hydroxy-2-oxo acids 3 prompted us to
Scheme 5. One-Pot Two-Step Stereoselective Synthesis of γ-Hydroxy-α-amino Acids 6, Using Benzylamine (7) as an Amine
a
Donor for the Enzymatic Transamination Reaction and BAL to Transform the Benzaldehyde Formed (8) into Benzoin (9)
a
Dotted lines indicate a transamination reaction of the aldehydes 1 and pyruvate 2, favoring the retroaldolysis of 3 catalyzed by the HBPA present
in the system.
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explore another methodology based on branched-chain
α-amino acid aminotransferases (BCATs).18
comparison to those with the benzylamine/T039 system
(Scheme 7). For instance, this reaction system converted the
aldol adduct 3k, which was not a substrate for T039 (Scheme
8). On the other hand, benzylamine/T039 afforded product
6n, whereas its precursor 3n was not converted by the BCAT/
AspAT system. Therefore, both methodologies are somehow
complementary. Despite the fact that pyruvate is released
because of the decarboxylation of oxaloacetate 13, attempts to
run the reaction with one-pot pyruvate recycling failed to
provide the corresponding γ-hydroxy-α-amino acids 6 (Figure
S25).
Finally, for the laboratory-scale preparation of
γ-hydroxy-α-amino acids 6 we chose a system with better
performance for conversion, isolation, and product purifica-
tion. Products 6 were converted into the corresponding
Cbz-Nα-γ-butyrolactone derivatives 14 (Scheme 7).
α-Amino-γ-butyrolactones are structural motifs found in
biologically active compounds in addition to being valuable
chiral intermediates for the synthesis of pharmaceutical
agents.6a,20 The optimal lactonization conditions were
established, and particular attention was paid to avoid eroding
the enantiopurity of 14 (Table S7). The R configuration for
aldol adducts 3b,c,e−g,i (Scheme 2) and the S configuration
generated by the transaminases8b,19c afforded the expected
2S,4R-configured hydroxy amino acid derivatives 6, which were
confirmed by a NMR diastereochemical analysis of products
14. Exceptions were the products 6n,o. In this case, the aldol
addition reaction was not stereoselective, yielding a mixture of
diastereoisomers (5R/5S)-14n and (5R/5S)-14o, respectively.
Interestingly, the enzymatic transamination reaction of aldol
a d d u c t s 3 k , h l e d t o t h e f o r m a t i o n o f
(2S,4R)-(−)-trans-4-hydroxyproline (15k) (Scheme 8 A) and
γ-hydroxypyroglutamic acid (15h) (Scheme 8 B), respectively.
The first product could be formed by an intramolecular
nucleophilic substitution of the terminal Cl at C5 by the amine
group after the transamination reaction.
Synthesis of γ-Hydroxy-α-amino acid Derivatives
Using a Biocatalytic One-Pot Two-Step Approach with
the PLP-Dependent Branched-Chain Amino Acid Ami-
notransferase (BCAT) from E. coli. The branched-chain
α-amino acid aminotransferase (BCAT) from E. coli was
selected to convert 3 into 6, employing L-Glu (10) as an amine
donor and delivering 2-oxoglutarate (11), which is a strong
inhibitor of BCAT (e.g., 10 mM of 11 reduces the activity up
to 80%).19 Thus, the regeneration of L-Glu was needed, which
was accomplished by coupling with aspartate aminotransferase
(AspAT) from E. coli that employed L-Asp (12) as the
substrate (Scheme 6). The resulting oxaloacetate 13
spontaneously decomposes into CO2 and pyruvate, shifting
the transamination equilibrium to the formation of γ-hydroxy-
α-amino acids 6.
Scheme 6. One-Pot Two-Step Stereoselective Synthesis of
γ-Hydroxy-α-amino Acids 6 using L-Glu (10) as an Amine
a
Donor
a
The 2-oxoglutarate (11) formed was transaminated to L-Glu (10) by
AspAT using L-Asp (12) as an amine donor. The oxaloacetate (13)
decomposes into CO2 and pyruvate, shifting the equilibrium of the
transamination to γ-hydroxy-α-amino acids 6.
The reaction system worked successfully with the aldol
adducts 3, with better performances for some examples in
Scheme 7. Synthesis of γ-Hydroxy-α-amino Acids 6 by Tandem HBPA/Transaminase Catalyzed Reactions and Conversion to
α-Amino-γ-butyrolactone Derivatives 14
a
b
c
d
e
Conversions determined by HPLC. Transaminase T039/BAL (see Scheme 5). BCAT/AspAT system (see Scheme 6). Isolated yield. dr >95:5
f
g
as measured by NMR; no other diastereomers were detected. dr 50:50 as measured by NMR. dr 60:40 (S:R) as measured by NMR.
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Scheme 8. (A) Formation of (2S,4R)-(−)-trans-4-Hydroxyproline Derivative 15k by Tandem Enzymatic Aldol Reaction/
d
Transamination and Intramolecular Nucleophilic Substitution with the Amine Group and (B) Formation of
γ-Hydroxypyroglutamic Acid (15h) by Intramolecular Aminolysis of the Ethyl Ester Group under Basic Conditions
a
b
c
Percentage of product formed determined by HPLC. Isolated yield from 1k. Isolated yield from 1h. The material contained L-Asp and L-Glu as
d
major impurities. The stereochemistry of 15k was established unequivocally by a comparison with authentic samples (see Figure S26).
The γ-hydroxypyroglutamic acid 15h was probably formed
by intramolecular aminolysis of the ethyl ester group during
the attempts to protect the amino group by Cbz. The basic
conditions necessary to conduct the reaction with CbzOSu
likely favored the reaction (Scheme 8 B). On the other hand,
the corresponding isopropyl ester derivative of
Cbz-Nα-γ-butyrolactone 14i could be isolated, most likely
due to the steric hindrance imposed by the isopropyl group,
which precluded an intramolecular aminolysis.
Deprotection of the Cbz group of butyrolactones 14 was
accomplished by catalytic hydrogenolysis with H2 in the
presence of Pd/C, while PheAc was removed by enzymatic
hydrolysis mediated by penicillin G acylase (PGA) (Scheme
9). Hydrogenolysis of (5R/5S)-14n and (5R/5S)-14o
γ-hydroxy-α-amino acids. The trans-o-hydroxybenzylidene
pyruvate hydratase-aldolase afforded chiral 4-hydroxy-2-oxo
acids with a remarkable efficiency, broad substrate tolerance,
and unparalleled stereoselectivity, far beyond those of the
pyruvate aldolases hitherto reported. Thus, the HBPA/
benzylamine/T039 and HBPA/Glu/BCAT/AspAT systems
are adequate complementary approaches for the asymmetric
synthesis of chiral γ-hydroxy-α-amino acids 6. Overall, the
HBPA/Glu/BCAT/AspAT system renders, in some instances,
better results mainly due to the selectivity of the
α-transaminase BCAT for the 2-oxo acids and its inability to
catalyze the transamination of the remaining aldehyde from the
aldol addition. Moreover, the reaction can be carried out in
whole cells. The HBPA/benzylamine/T039 system is more
unspecific, allowing the conversion of various structurally
different substrates, such as that of the aldehydes 1 into
primary amines. The HBPA/L-Ala/T039 approach with
pyruvate recycling is not straightforward and needs an
optimization of the activities of the aldolase and transaminase
involved to develop an effective process.
Scheme 9. Protection Group Removal of Selected α-Amino-
γ-butyrolactone Derivatives 14
Using the strategy developed in this work, an unprecedented
number of chiral γ-hydroxy-α-amino acids and the correspond-
ing α-amino-γ-butyrolactones were constructed in two steps
with high stereoselectivity from small functionalized aldehydes.
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acscatal.1c00210.
General methods and protocols for the screening and
synthesis, activity determinations, synthesis of starting
materials, synthesis of γ-hydroxy-α-amino acids and
α-amino-γ-butyrolactones, NMR spectra, and computa-
tion methods and modeling (PDF)
a
b
Isolated yield. Cation exchange chromatography (CEC) eluted with
an aqueous solution of NH4OH.
furnished the lactones (5R/5S)-17n and (5R/5S)-17o, which
upon treatment with PGA and purification by cation exchange
chromatography, with NH4OH as eluent, led to the
corresponding amide derivatives (4R/4S)-18n and carbox-
ylates (4R/4S)-18o.
AUTHOR INFORMATION
■
Corresponding Author
Pere Clapés − Institute for Advanced Chemistry of Catalonia,
Department of Biological Chemistry, IQAC-CSIC, Barcelona
08034, Spain; orcid.org/0000-0001-5541-4794;
Email: pere.clapes@iqac.csic.es
CONCLUSIONS
A tandem enantioselective aldol addition−transamination
approach was established for the production of chiral
■
4667
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ACS Catal. 2021, 11, 4660−4669

ACS Catalysis
pubs.acs.org/acscatalysis
Research Article
Combination of Organo- And Biocatalysis. Chem. Commun. 2014, 50,
15669−15672. (e) Guérard-Hélaine, C.; Heuson, E.; Ndiaye, M.;
Gourbeyre, L.; Lemaire, M.; Hélaine, V.; Charmantray, F.; Petit, J.-L.;
Salanoubat, M.; de Berardinis, V.; Gefflaut, T. Stereoselective
Synthesis of γ-Hydroxy-α-Amino Acids Through Aldolase-Trans-
aminase Recycling Cascades. Chem. Commun. 2017, 53, 5465−5468.
(f) Zhang, Y.; Farrants, H.; Li, X. Adding a Functional Handle to
Nature′s Building Blocks: The Asymmetric Synthesis of β-Hydroxy-α-
Amino Acids. Chem. - Asian J. 2014, 9, 1752−1764. (g) Enoki, J.;
Authors
Carlos J. Moreno − Institute for Advanced Chemistry of
Catalonia, Department of Biological Chemistry, IQAC-CSIC,
Barcelona 08034, Spain
Karel Hernández − Institute for Advanced Chemistry of
Catalonia, Department of Biological Chemistry, IQAC-CSIC,
Barcelona 08034, Spain
Simon J. Charnok − Prozomix Ltd. West End Industrial
Estate, Haltwhistle, Northumberland NE49 9HA, U.K.
Samantha Gittings − Prozomix Ltd. West End Industrial
Estate, Haltwhistle, Northumberland NE49 9HA, U.K.
Meisborn, J.; Muller, A.-C.; Kourist, R. A Multi-Enzymatic Cascade
̈
Reaction for the Stereoselective Production of γ-Oxyfunctionalyzed
Amino Acids. Front. Microbiol. 2016, 7, 1−8. (h) Bunch, L.; Pickering,
D. S.; Gefflaut, T.; Vinatier, V.; Helaine, V.; Amir, A.; Nielsen, B.;
Jensen, A. A. 4,4-Dimethyl- and Diastereomeric 4-Hydroxy-4-methyl-
(2S)-Glutamate Analogues Display Distinct Pharmacological Profiles
at Ionotropic Glutamate Receptors and Excitatory Amino Acid
Transporters. ChemMedChem 2009, 4, 1925−1929. (i) Alaux, S.;
Kusk, M.; Sagot, E.; Bolte, J.; Jensen, A. A.; Bräuner-Osborne, H.;
Gefflaut, T.; Bunch, L. Chemoenzymatic Synthesis of a Series of 4-
Substituted Glutamate Analogues and Pharmacological Character-
ization at Human Glutamate Transporters Subtypes 1−3. J. Med.
Chem. 2005, 48, 7980−7992. (j) Singh, A. B.; Khaliq, T.; Chaturvedi,
J. P.; Narender, T.; Srivastava, A. K. Anti-diabetic and anti-oxidative
effects of 4-hydroxypipecolic acid in C57BL/KsJ-db/db mice. Hum.
Exp. Toxicol. 2012, 31, 57−65.
Michael Bolte − Institut fur Anorganische Chemie, J.-W.-
̈
Goethe-Universität, Frankfurt/Main, Germany;
orcid.org/0000-0001-5296-6251
́
Jesus Joglar − Institute for Advanced Chemistry of Catalonia,
Department of Biological Chemistry, IQAC-CSIC, Barcelona
08034, Spain
Jordi Bujons − Institute for Advanced Chemistry of Catalonia,
Department of Biological Chemistry, IQAC-CSIC, Barcelona
08034, Spain; orcid.org/0000-0003-2944-2905
Teodor Parella − Servei de Ressonancia Magnetica Nuclear,
Universitat Autonoma de Barcelona, Bellaterra, Spain;
̀
̀
̀
orcid.org/0000-0002-1914-2709
(3) (a) Ohyama, T.; Kurihara, Y.; Ono, Y.; Ishikawa, T.; Miyakoshi,
S.; Hamano, K.; Araei, M.; Suzuki, T.; Igari, H.; Suzuki, Y.
Arborcandins A, B, C, D, E and F, novel 1, 3-β-glucan synthase
inhibitors: production and biological activity. J. Antibiot. 2000, 53,
1108−1116. (b) Sugawara, T.; Tanaka, A.; Tanaka, K.; Nagai, K.;
Suzuki, K.; Suzuki, T. YM-170320, a novel lipopeptide antibiotic
inducing morphological change of colonies in a mutant of Candida
tropicalis pK233. J. Antibiot. 1998, 51, 435−438.
Complete contact information is available at:
https://pubs.acs.org/10.1021/acscatal.1c00210
Author Contributions
⊥C.J.M. and K.H. contributed equally.
Funding
This project has received funding from the Ministerio de
(4) (a) Wang, X.; Wang, J.; Lai, D.; Wang, W.; Dai, J.; Zhou, L.; Liu,
Y. Ustiloxin G, a New Cyclopeptide Mycotoxin from Rice False Smut
Balls. Toxins 2017, 9, 54. (b) Lin, X.; Bian, Y.; Mou, R.; Cao, Z.; Cao,
Z.; Zhu, Z.; Chen, M. Isolation, Identification, and Characterization of
Ustilaginoidea Virens from Rice False Smut Balls with High
Ustilotoxin Production Potential. J. Basic Microbiol. 2018, 58, 670−
678. (c) Hunter, L.; McLeod, M. D.; Hutton, C. A. Synthesis of the β-
hydroxydopa-γ-hydroxy-δ-sulfinylnorvaline component of ustiloxins A
and B. Org. Biomol. Chem. 2005, 3, 732−734.
́
Ciencia e Innovacion (MICIN), cofinanced by the Fondo
Europeo de Desarrollo Regional (FEDER) (grants RTI2018-
094637-B-I00 to P.C. and PGC2018-095808-B-I00 to T.P.),
́
and Programacion Conjunta Internacional (PCI2018-092937),
through the EU initiative ERA CoBioTech under grant
agreement No [722361] to P.C and S.J.Ch.) (Tralaminol).
Notes
The authors declare no competing financial interest.
(5) (a) Bassarello, C.; Bifulco, G.; Evidente, A.; Riccio, R.; Gomez-
Paloma, L. Stereochemical studies on ascaulitoxin: a J-based NMR
configurational analysis of a nitrogen substituted system. Tetrahedron
Lett. 2001, 42, 8611−8613. (b) Vurro, M.; Andolfi, A.; Boari, A.;
Zonno, M. C.; Caretto, S.; Avolio, F.; Evidente, A. Optimization of
the Production of Herbicidal Toxins by the Fungus Ascochyta
Caulina. Biol. Control 2012, 60, 192−198.
(6) (a) Cavanaugh, C. L.; Nicewicz, D. A. Synthesis of α-
Benzyloxyamino-γ-Butyrolactones via a Polar Radical Crossover
Cycloaddition Reaction. Org. Lett. 2015, 17, 6082−6085. (b) Avenoza,
A.; Cativiela, C.; París, M.; Peregrina, J. M.; Saenz-Torre, B. Synthesis
of enantiomerically pure constrained γ-hydroxy-α-amino acids by
directed hydroxylation. Tetrahedron: Asymmetry 1997, 8, 1123−1129.
(c) Mondal, D.; Schweizer, F. Synthesis of Allose-Templated
Hydroxyornithine and Hydroxyarginine Analogs. Carbohydr. Res.
2010, 345, 1533−1540. (d) Ren, J.-L.; Zhang, X.-Y.; Yu, B.; Wang, X.-
X.; Shao, K.-P.; Zhu, X.-G.; Liu, H.-M. Discovery of Novel AHLs as
Potent Antiproliferative Agents. Eur. J. Med. Chem. 2015, 93, 321−
329. (e) Mattmann, M. E.; Geske, G. D.; Worzalla, G. A.; Chandler, J.
R.; Sappington, K. J.; Greenberg, E. P.; Blackwell, H. E. Synthetic
Ligands that Activate and Inhibit a Quorum-Sensing Regulator in
Pseudomonas Aeruginosa. Bioorg. Med. Chem. Lett. 2008, 18, 3072−
3075.
ACKNOWLEDGMENTS
■
The authors gratefully acknowledge the “Consorci de Serveis
Universitaris de Catalunya” (CSUC) for allowing the use of its
software and hardware resources.
REFERENCES
■
́
́
(1) (a) Szczesniak, P.; Pazdzierniok-Holewa, A.; Klimczak, U.;
Stecko, S. Synthesis of β- and γ-Hydroxy α-Amino Acids via
Enzymatic Kinetic Resolution and Cyanate-to-Isocyanate Rearrange-
ment. J. Org. Chem. 2014, 79, 11700−11713. (b) Plaza, A.; Viehrig,
K.; Garcia, R.; Muller, R. Jahnellamides, α-Keto-β-Methionine-
̈
Containing Peptides from the Terrestrial Myxobacterium Jahnella
sp.: Structure and Biosynthesis. Org. Lett. 2013, 15, 5882−5885.
(2) (a) Zafar, M. I.; Gao, F. 4-Hydroxyisoleucine: A Potential New
Treatment for Type 2 Diabetes Mellitus. BioDrugs 2016, 30, 255−
262. (b) Sun, D. Y.; Cheng, X. T.; Gao, D. K.; Xu, P. P.; Guo, Q. Q.;
Zhu, Z. L.; Zhu, M. L.; Wang, X. Y.; Qin, H. M.; Lu, F. P. Properties,
Biosynthesis, and Catalytic Mechanisms of Hydroxy-Amino-Acids.
IOP Conference Series: Earth and Environmental Science 2018, 188,
012084. (c) Jing, X.; Wang, X.; Zhang, W.; An, J.; Luo, P.; Nie, Y.; Xu,
Y. Highly Regioselective and Stereoselective Hydroxylation of Free
Amino Acids by a 2-Oxoglutarate-Dependent Dioxygenase from
Kutzneria albida. ACS Omega 2019, 4, 8350−8358. (d) Simon, R. C.;
Busto, E.; Schrittwieser, J. H.; Sattler, J. H.; Pietruszka, J.; Faber, K.;
Kroutil, W. Stereoselective Synthesis of α-Hydroxynorvaline Through
(7) (a) Swift, M. D.; Sutherland, A. Stereocontrol of palladium(II)-
catalysed aza-Claisen rearrangements using a combination of 1,3-
allylic strain and a solvent mediated directing effect. Org. Biomol.
Chem. 2006, 4, 3889−3891. (b) De Lamo Marin, S.; Catala, C.;
4668
https://doi.org/10.1021/acscatal.1c00210
ACS Catal. 2021, 11, 4660−4669

ACS Catalysis
pubs.acs.org/acscatalysis
Research Article
Kumar, S. R.; Valleix, A.; Wagner, A.; Mioskowski, C. A Practical and
Efficient Total Synthesis of Potent Insulinotropic (2S,3R,4S)-4-
Hydroxyisoleucine through a Chiral N-Protected γ-Keto-α-amino-
ester. Eur. J. Org. Chem. 2010, 2010, 3985−3989. (c) Schmeck, C.;
Hegedus, L. S. Synthesis of Optically Active 4-Substituted 2-
Aminobutyrolactones and Homoserines by Combined Aldol/Photo-
cyclization Reactions of Chromium Aminocarbene Complexes. J. Am.
̃
Chem. Soc. 1994, 116, 9927−9934. (d) Ariza, J.; Font, J.; Ortuno, R.
M. Enantioselective synthesis of hydroxy α-amino acids. (−)-erythro-
and (−)-threo-γ-hydroxynorvalines. Tetrahedron 1990, 46, 1931−
349, 817−821. (c) Woodhall, T.; Williams, G.; Berry, A.; Nelson, A.
Creation of a tailored aldolase for the parallel synthesis of sialic acid
mimetics. Angew. Chem., Int. Ed. 2005, 44, 2109−2112. (d) Schurink,
M.; Wolterink-Van Loo, S.; van der Oost, J.; Sonke, T.; Franssen, M.
C. R. Substrate Specificity and Stereoselectivity of Two Sulfolobus 2-
Keto-3-deoxygluconate Aldolases towards Azido- Substituted Alde-
hydes. ChemCatChem 2014, 6, 1073−1081. (e) Baker, P.; Seah, S. Y.
K. Rational Design of Stereoselectivity in the Class II Pyruvate
Aldolase Bphl. J. Am. Chem. Soc. 2012, 134, 507−513. (f) Archer, R.
M.; Royer, S. F.; Mahy, W.; Winn, C. L.; Danson, M. J.; Bull, S. D.
Syntheses of 2-Keto-3-deoxy-D-xylonate and 2-Keto-3-deoxy-L-
arabinonate as Stereochemical Probes for Demonstrating the
Metabolic Promiscuity of Sulfolobus solfataricus Towards D-Xylose
and D-Arabinose. Chem. - Eur. J. 2013, 19, 2895−2902. (g) Clapés,
P.; Garrabou, X. Current Trends in Asymmetric Synthesis With
Aldolases. Adv. Synth. Catal. 2011, 353, 2263−2283.
̌
̌
̌
1942. (e) Berkes, D.; Kolarovic, A.; Povazanec, F. Stereoselective
sodium borohydride reduction, catalyzed by manganese(II) chloride,
of γ-oxo-α-amino acids. A practical approach to syn-γ-hydroxy-α-
amino acids. Tetrahedron Lett. 2000, 41, 5257−5260. (f) Jakubec, P.;
̌
̌
̌
Berkes, D.; Kolarovic, A.; Povazanec, F. Asymmetric Synthesis of
Aliphatic α-Amino and γ-Hydroxy α-Amino Acids and Introduction of
a Template for Crystallization-Induced Asymmetric Transformation.
Synthesis 2006, 2006, 4032−4040.
(13) Pathak, T.; Waldmann, H. Enzymes and protecting group
chemistry. Curr. Opin. Chem. Biol. 1998, 2, 112−120.
̈
(14) (a) Demir, A. S.; S
̧
esȩ
noglu, O.; Eren, E.; Hosrik, B.; Pohl, M.;
nkelmann, P.; Muller, M.
(8) (a) Baud, D.; Saaidi, P.-L.; Monfleur, A.; Harari, M.; Cuccaro, J.;
Fossey, A.; Besnard, M.; Debard, A.; Mariage, A.; Pellouin, V.; Petit,
J.-L.; Salanoubat, M.; Weissenbach, J.; de Berardinis, V.; Zaparucha,
A. Synthesis of Mono- and Dihydroxylated Amino Acids with New α-
Ketoglutarate-Dependent Dioxygenases: Biocatalytic Oxidation of C-
H Bonds. ChemCatChem 2014, 6, 3012−3017. (b) Hernández, K.;
Bujons, J.; Joglar, J.; Charnock, S. J.; Domínguez de María, P.;
Fessner, W. D.; Clapés, P. Combining Aldolases and Transaminases
for the Synthesis of 2-Amino-4-hydroxybutanoic Acid. ACS Catal.
2017, 7, 1707−1711. (c) Hara, R.; Yamagata, K.; Miyake, R.;
Kawabata, H.; Uehara, H.; Kino, K. Discovery of Lysine Hydroxylases
in the Clavaminic Acid Synthase-Like Superfamily for Efficient
Hydroxylysine Bioproduction. Appl. Environ. Microbiol. 2017, 83,
e00693−e00617. (d) Hibi, M.; Kawashima, T.; Kodera, T.; Smirnov,
S. V.; Sokolov, P. M.; Sugiyama, M.; Shimizu, S.; Yokozeki, K.;
Ogawa, J. Characterization Bacillus thuringiensis L-Isoleucine
Dioxygenase for Production of Useful Amino Acids. Appl. Environ.
Microbiol. 2011, 77, 6926. (e) Smirnov, S. V.; Samsonova, N. N.;
Novikova, A. E.; Matrosov, N. G.; Rushkevich, N. Y.; Kodera, T.;
Ogawa, J.; Yamanaka, H.; Shimizu, S. A novel strategy for enzymatic
synthesis of 4-hydroxyisoleucine: identification of an enzyme
possessing HMKP (4-hydroxy-3-methyl-2-keto-pentanoate) aldolase
activity. FEMS Microbiol. Lett. 2007, 273, 70−77. (f) Ogawa, J.;
Yamanaka, H.; Mano, J.; Doi, Y.; Horinouchi, N.; Kodera, T.; Nio, N.;
Smirnov, S. V.; Samsonova, N. N.; Kozlov, Y. I.; Shimizu, S. Synthesis
of 4-hydroxyisoleucine by the aldolase-transaminase coupling reaction
and basic characterization of the aldolase from Arthrobacter simplex
AKU 626. Biosci., Biotechnol., Biochem. 2007, 71, 1607−1615. (g) Xu,
L.; Wang, L.-C.; Su, B.-M.; Xu, X.-Q.; Lin, J. Multi-Enzyme Cascade
for Improving β-Hydroxy-α-Amino Acids Production by Engineering
L-Threonine Transaldolase and Combining Acetaldehyde Elimination
System. Bioresour. Technol. 2020, 310, 123439.
Janzen, E.; Kolter, D.; Feldmann, R.; Du
Enantioselective Synthesis of α-Hydroxy Ketones via Benzaldehyde
Lyase-Catalyzed C-C Bond Formation Reaction. Adv. Synth. Catal.
̈
̈
2002, 344, 96−103. (b) González, B.; Vicuna, R. Benzaldehyde lyase,
̃
a novel thiamine PPi-requiring enzyme, from Pseudomonas fluorescens
biovar I. J. Bacteriol. 1989, 171, 2401. (c) Brandt, G. S.; Nemeria, N.;
Chakraborty, S.; McLeish, M. J.; Yep, A.; Kenyon, G. L.; Petsko, G.
A.; Jordan, F.; Ringe, D. Probing the Active Center of Benzaldehyde
Lyase with Substitutions and the Pseudosubstrate Analogue
Benzoylphosphonic Acid Methyl Ester. Biochemistry 2008, 47,
7734−7743.
(15) Cheng, Y.; Shan, Q.; Zhang, Y.; Quan, Z.; Zhang, K.; Wang, B.
A Highly Efficient One-Enzyme Protocol Using ω-Transaminase and
an Amino Donor Enabling Equilibrium Displacement Assisted by
Molecular Oxygen. Org. Chem. Front. 2018, 5, 1633−1638.
(16) Shin, J.-S.; Kim, B.-G. Transaminase-Catalyzed Asymmetric
Synthesis of L-2-Aminobutyric Acid from Achiral Reactants.
Biotechnol. Lett. 2009, 31, 1595−1599.
(17) Sung, S.; Jeon, H.; Sarak, S.; Ahsan, M. M.; Patil, M. D.;
Kroutil, W.; Kim, B.-G.; Yun, H. Parallel Anti-Sense Two-Step
Cascade for Alcohol Amination Leading to ω-Amino Fatty Acids and
α,ω-Diamines. Green Chem. 2018, 20, 4591−4595.
(18) Aki, K.; Ogawa, K.; Ichihara, A. Transaminases of branched
chain amino acids: IV. Purification and properties of two enzymes
from rat liver. Biochim. Biophys. Acta - Enzymology 1968, 159, 276−
284.
(19) (a) Seo, Y.-M.; Yun, H. Enzymatic Synthesis of L-tert-Leucine
with Branched Chain Aminotransferase. J. Microbiol. Biotechnol. 2011,
21, 1049−1052. (b) Hong, E. Y.; Cha, M.; Yun, H.; Kim, B.-G.
Asymmetric Synthesis of L-tert-Leucine and L-3-Hydroxyadamantyl-
glycine using Branched Chain Aminotransferase. J. Mol. Catal. B:
Enzym. 2010, 66, 228−233. (c) Xian, M.; Alaux, S.; Sagot, E.;
Gefflaut, T. Chemoenzymatic synthesis of glutamic acid analogues:
substrate specificity and synthetic applications of branched chain
aminotransferase from Escherichia coli. J. Org. Chem. 2007, 72, 7560−
7566.
(9) Sehl, T.; Hailes, H. C.; Ward, J. M.; Wardenga, R.; von Lieres, E.;
Offermann, H.; Westphal, R.; Pohl, M.; Rother, D. Two Steps in One
Pot: Enzyme Cascade for the Synthesis of Nor(pseudo)ephedrine
from Inexpensive Starting Materials. Angew. Chem., Int. Ed. 2013, 52,
6772−6775.
(20) (a) Galloway, W. R. J. D.; Hodgkinson, J. T.; Bowden, S. D.;
Welch, M.; Spring, D. R. Quorum Sensing in Gram-Negative Bacteria:
Small-Molecule Modulation of AHL and AI-2 Quorum Sensing
Pathways. Chem. Rev. 2011, 111, 28−67. (b) Annedi, S. C.; Biabani,
F.; Poduch, E.; Mannargudi, B. M.; Majumder, K.; Wei, L.; Khayat,
R.; Tong, L.; Kotra, L. P. Engineering D-amino acid containing novel
protease inhibitors using catalytic site architecture. Bioorg. Med. Chem.
2006, 14, 214−236.
(10) (a) Stolz, A. Degradation of substituted naphthalenesulfonic
acids by Sphingomonas xenophaga BN6. J. Ind. Microbiol. Biotechnol.
1999, 23, 391−399. (b) Eaton, R. W. trans-o-Hydroxybenzylidene-
pyruvate Hydratase-Aldolase as a Biocatalyst. Appl. Environ. Microbiol.
2000, 66, 2668−2672.
(11) Howard, J. K.; Muller, M.; Berry, A.; Nelson, A. An Enantio-
̈
and Diastereoselective Chemoenzymatic Synthesis of α-Fluoro β-
Hydroxy Carboxylic Esters. Angew. Chem., Int. Ed. 2016, 55, 6767−
6770.
(12) (a) Lamble, H. J.; Danson, M. J.; Hough, D. W.; Bull, S. D.
Engineering stereocontrol into an aldolase-catalysed reaction. Chem.
Commun. 2005, 124−126. (b) Lamble, H. J.; Royer, S. F.; Hough, D.
W.; Danson, M. J.; Taylor, G. L.; Bull, S. D. A thermostable aldolase
for the synthesis of 3-deoxy-2-ulosonic acids. Adv. Synth. Catal. 2007,
4669
https://doi.org/10.1021/acscatal.1c00210
ACS Catal. 2021, 11, 4660−4669