Application of Threonine Aldolases for the Asymmetric Synthesis of α-Quaternary α-Amino Acids

Article abstract of DOI:10.1002/cctc.201800611

We report the synthesis of diverse β-hydroxy-α,α-dialkyl-α-amino acids with perfect stereoselectivity for the α-quaternary center through the action of l- and d-specific threonine aldolases. A wide variety of aliphatic and aromatic aldehydes were accepted by the enzymes and conversions up to >80 % were obtained. In the case of d-selective threonine aldolase from Pseudomonas sp., generally higher diastereoselectivities were observed. The applicability of the protocol was demonstrated by performing enzymatic reactions on preparative scale. Using the d-threonine aldolase from Pseudomonas sp., (2R,3S)-2-amino-3-(2-fluorophenyl)-3-hydroxy-2-methylpropanoic acid was generated in preparative amounts in one step with a diastereomeric ratio >100 favoring the syn-product. A Birch-type reduction enabled the reductive removal of the β-hydroxy group from (2S)-2-amino-3-hydroxy-2-methyl-3-phenylpropanoic acid to generate enantiopure l-α-methyl-phenylalanine via a two-step chemo-enzymatic transformation.

Full text of DOI:10.1002/cctc.201800611

Accepted Article
Title: Application of Threonine Aldolases for the Asymmetric Synthesis
of α-Quaternary α-Amino Acids
Authors: Julia Blesl, Melanie Trobe, Felix Anderl, Rolf Breinbauer,
Gernot A Strohmeier, and Kateryna Fesko
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10.1002/cctc.201800611
ChemCatChem
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Application of Threonine Aldolases for the Asymmetric Synthesis
of α-Quaternary α-Amino Acids
Julia Blesl,[a] Melanie Trobe,[a] Felix Anderl,[a] Rolf Breinbauer,[a,b] Gernot A. Strohmeier,[a,b] Kateryna
Fesko*[a]
Keywords: biocatalysis, asymmetric synthesis, amino acids, threonine aldolase, quaternary stereocenter
Abstract: We report the synthesis of diverse β-hydroxy-α,α-dialkyl-
α-amino acids with perfect stereoselectivity for the α-quaternary
center through the action of L- and D-specific threonine aldolases. A
wide variety of aliphatic and aromatic aldehydes were accepted by
the enzymes and conversions up to >80 % were obtained. In the
case of D-selective threonine aldolase from Pseudomonas sp.,
generally higher diastereoselectivities were observed. The
applicability of the protocol was demonstrated by performing
enzymatic reactions on preparative scale. Using the D-threonine
methylphenylalanine ((αMe)Phe) are the most widely used
candidates in peptide research.[5] Moreover, peptides containing
(αMe)Phe residues are promising sweeteners, chemoattractants,
and candidates for molecular recognition studies. α-Methyl-
DOPA is known as an antihypertensive drug, but has also found
application as catalyst in asymmetric synthesis.[6]
The creation of α-quaternary amino acids remains an often
laborious task as protective steps need to be included and full
control of the stereochemistry must be considered.[7] The
classical approach towards the asymmetric synthesis of α-
quaternary amino acids involves a diastereoselective alkylation
of chiral lactim ethers or oxazolidinones.[8] However, the
application of these methods is limited due to substantial
number of reaction steps involved. The asymmetric phase
transfer-catalyzed alkylation of intermediate Schiff bases, such
as the one formed from benzaldehyde and alanine tert-butyl
ester, is a method to obtain α-quaternary amino acids with e.e.’s
up to 82%.[9] Enzymatic resolutions of chemically produced
quaternary amino acid amides or amino acid esters using the
amino amidase from Mycobacterium neoaurum ATCC 25795,[10]
respectively pig liver esterase,[11] were reported for the synthesis
of (S)-αα-disubstituted amino acids with almost 100% e.e. at
50% maximal conversion.[12]
Biocatalytic methods for the direct formation of quaternary α-
amino acids are still scarce. Among those, aldolases proved to
be efficient biocatalysts for the stereoselective carbon-carbon
bond formation,[13] however, they are known for their strict
specificity towards nucleophiles which limits the accessible
product range. Recently, as one of few examples shown so far,
the nucleophile specificity of D-fructose-6-phosphate aldolase
from Escherichia coli could be increased by enzyme
engineering.[14] Another prominent example where the
nucleophile specificity was broadened was shown for the
threonine aldolase type.[15] For many years, threonine aldolases
have been known for the asymmetric synthesis of α-amino-β-
hydroxy amino acids by coupling glycine with diverse sets of
aldehydes with the aid of pyridoxal-5’-phosphate (PLP) as
cofactor.[16] Whereas the aldehyde scope is generally broad, the
amino acid substrate of this enzyme class was strictly limited to
glycine. In recent years, threonine aldolase enzymes able to
accept alanine as a donor were found via screening and
database mining[15] or created via engineering of an alanine
racemase[17] or L-serine hydroxymethyltransferase,[18] and thus
make these enzymes ideal catalysts for the direct synthesis of α-
quaternary α-amino acids starting from aldehydes and amino
acids. In addition, threonine aldolases are known to be highly
stereospecific at the amino acids’ α-carbon and the available
enantiocomplementary forms provide access to L- and D-amino
acids with high stereoselectivity. Here, we present a more
thorough investigation of threonine aldolases with broad donor
aldolase
from
Pseudomonas
sp.,
(2R,3S)-2-amino-3-(2-
fluorophenyl)-3-hydroxy-2-methylpropanoic acid was generated in
preparative amounts in one step with a diastereomeric ratio >100
favoring the syn-product.
A Birch-type reduction enabled the
reductive removal of the β-hydroxy group from (2S)-2-amino-3-
hydroxy-2-methyl-3-phenylpropanoic acid to generate enantiopure L-
α-methyl-phenylalanine
via
a
two-step
chemo-enzymatic
transformation.
Introduction
Stereoselective syntheses of quaternary stereocenters are
amongst the most challenging endeavors in organic chemistry.[1]
Apart from all-carbon frameworks, heteroatoms are also present
in assemblies of such structural elements. Several natural
products such as sphingofungines E and F, lactacystin, as well
as biologically-active molecules for pharmaceutical applications
contain α-quaternary amino acids and, consequently, make
these moieties attractive targets for the development of reliable
synthetic strategies.[2] Recently, α,α-disubstituted amino acids
were studied as building blocks for peptidomimetics to design
novel short chain peptides useful for the treatment or prevention
of diseases.[3] The presence of α,α-disubstituted amino acids in
peptides generates specific conformational changes compared
to their α-hydrogen analogs and, in addition, their conformation
tends to be more defined.[3-4] Among them, α-methyl-substituted
amino acids such as α-aminoisobutyric acid (Aib) or α-
[a]
[b]
J. Blesl MSc, Dr. M. Trobe, F. Anderl MSc, Prof. Dr. R. Breinbauer,
Dr. G. A. Strohmeier, Dr. K. Fesko
Institute of Organic Chemistry
Graz University of Technology
Stremayrgasse 9, A-8010 Graz, Austria
E-mail: fesko@tugraz.at
Dr. G. A. Strohmeier, Prof. Dr. R. Breinbauer
Austrian Centre of Industrial Biotechnology (ACIB) GmbH
Petersgasse 14, 8010 Graz, Austria
Supporting information for this article is given via a link at the end of
the document.
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10.1002/cctc.201800611
ChemCatChem
FULL PAPER
specificity and show their applicability for the asymmetric
synthesis of (S)- and (R)-α,α-disubstituted α-amino acids by
evaluating the substrate scope, reaction conditions and
performing enzymatic reactions at larger scale. Additionally,
methods to remove the β-hydroxyl group in serine derivatives
were investigated.
28
29
30
31
32
33
34
r
60
27
60
58
44
41
30
1.1 (syn)
1.3 (anti)
1.2 (anti)
1.2 (anti)
1.9 (syn)
1.3 (anti)
1.3 (anti)
-
-
s
t
14
41
80
26
30
56
1.4 (syn)
1.5 (anti)
2.4 (syn)
3.1 (syn)
5.1 (syn)
1.8 (syn)
u
v
w
x
Results and Discussion
[a] Conversion after 24 h at 30 °C in the presence of 50 mM aldehyde and
500 mM D-alanine. [b] Diastereomeric ratio determined by rp-HPLC after
derivatization with o-phthaldialdehyde and various thiol reagents and given
as syn/anti ratio; e.e. > 99.5% (2S or 2R).
To explore the synthetic potential of the wild-type threonine
aldolases with broad donor specificity, a panel of aldehydes (Fig.
1) was screened in the aldol condensation reaction using D- or
DL-alanine in reactions with the L-threonine aldolase from
Aeromonas jandaei (L-TA) and D-threonine aldolase from
Pseudomonas sp. (D-TA) (Scheme 1, Table 1).
Forced by the often limited solubility of the aldehydes in water,
different cosolvents were screened to study their compatibility
with the aldolase-catalyzed reactions (see Supporting
Information). DMSO turned out as the best suited cosolvent and
can be used in concentrations up to 20 % v/v in the enzymatic
reactions. To compensate for the unfavorable reaction
equilibrium in the aldol addition, a 10-fold excess of a donor was
used.
Table 1. Synthesis of α,α-disubstituted α-amino acids with threonine
aldolases using alanine.
L-TA
D-TA
Entry
1
1
C [%][a]
25
d.r.[b]
C [%][a]
10
d.r.[b]
a
1.7 (syn)
1.2 (syn)
1.5 (syn)
1.3 (anti)
2.8 (syn)
1.4 (syn)
1.4 (syn)
2.0 (syn)
1.4 (syn)
1.6 (syn)
1.5 (syn)
1.5 (syn)
1.4 (syn)
n. d.
9.0 (syn)
9.0 (syn)
18 (syn)
1.2 (syn)
>100 (syn)
>100 (syn)
n. d.
2
(m)-b
c
60
85
25
34
28
24
40
50
20
4
40
58
<5
13
8
3
4
d
5
(o)-e
(m)-e
(p)-e
(o)-f
(m)-f
(p)-f
(o)-g
(m)-g
(p)-g
(o)-h
(m)-h
(p)-h
(m)-i
(p)-i
(m)-j
(p)-j
k
6
7
< 1
29
18
2
8
>100 (syn)
>100 (syn)
n. d.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
Scheme 1. Biocatalytic synthesis of β-hydroxy-α,α-dialkyl-α-amino acids using
L- and D-specific threonine aldolases.
< 1
< 1
< 1
-
n. d.
5
n. d.
15
< 1
< 1
< 1
74
74
53
44
42
23
58
27
25
<1
26
n. d.
-
n. d.
-
-
n. d.
-
-
1.3 (syn)
1.2 (syn)
1.4 (syn)
1.1 (syn)
4.4 (syn)
2.3 (syn)
2.1 (anti)
1.9 (anti)
1.2 (anti)
n. d.
8
5.1 (syn)
1.8 (syn)
11.5 (syn)
n. d.
2
2
< 1
45
38
37
14
63
<1
38
1.5 (syn)
1.6 (syn)
2.8 (syn)
2.6 (syn)
1.2 (anti)
n. d.
l
Figure 1. List of aldehydes used.
m
n
o
In view of the α-methyl-phenylalanine motifs used for studying
peptide-peptide or peptide-membrane interactions,[5a] we have
tested a range of substituted benzaldehydes as electrophiles in
enzymatic aldol condensations with D-alanine to evaluate the
p
q
1.1 (syn)
1.9 (anti)
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10.1002/cctc.201800611
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herein described method for the generation of α-quaternary
amino acids (Scheme 1). In all cases, only a single enantiomer
of the product amino acid was obtained, which is strictly
controlled by the type of enzyme used (e.g. e.e.S >99% for L-TA,
e.e.R >99% for D-TA). Superior conversions were obtained with
benzaldehydes bearing electron withdrawing groups, such as
nitro (1b), cyano (1c), or acetyl (1i), because of the increased
electrophilic reactivity. In general, the conversion and
diastereoselectivity varies dependent on the nature of the
substituent and its position at the aromatic ring.
Interestingly, meta-substituted benzaldehydes were often better
converted with L-TA, whereas the reactions with ortho-
substituted benzaldehydes usually gave higher conversions in
the presence of D-TA. Using 3-nitrobenzaldehyde (1b), a
conversion up to 60 % and mixture of (S)-syn- and (S)-anti-
diastereomers with a d.r.syn of 1.2 was obtained with L-TA. Under
the same conditions using D-TA as catalyst, the corresponding
(R)-product was produced with a lower conversion of 40% but in
significantly higher d.r.syn of 9 (Table 1, Entry 2). Other excellent
acceptors in the aldol addition are halogenated aromatic
aldehydes. Chloro- (1e) and fluorobenzaldehydes (1f) were well
accepted by L- and D-TA with D-alanine as a donor (Table 1,
Entries 5-10). However, the bromobenzaldehydes (1g) were
significantly lower converted by the enzymes, presumably
because of the larger size of the halogen atom which causes
increased steric hindrance. The aromatic aldehydes bearing a
ketone (1i) or a methyl ester function (1j) were converted by L-
TA with up to 74 % and 53% conversion, respectively. In
contrast, the reactions with D-TA gave conversions below 15 %
(Table 1, Entries 17-20). Increasing the length of aliphatic chains
between the aromatic ring and carbonyl function (e.g. aldehydes
1m, 1n, 1o) did not much influence the conversion in the L-TA
catalyzed reaction, but is beneficial in the case of D-TA. Bulky
substrates are well accepted by the investigated threonine
aldolases and inverted stereopreference at the product β-carbon
was obtained in the case of L-TA (Table 1, Entries 23-25).
Interestingly, the enzymes showed particularly high activity
towards heterocyclic aldehydes, e.g. 1k, 1l, 1q, 1r and 1s,
yielding α-quaternary α-amino acids with heterocyclic side
chains, which are important motifs in many biologically relevant
compounds. Pyridine-3-carbaldehyde (1k) and quinoline-2-
carbaldehyde (1r) were converted into the corresponding amino
acids with conversions up to 60 % for L-TA and slightly lower
conversion for D-TA (Table 1, Entries 21 and 28). The aliphatic
aldehydes 1t-1x were also preferred substrates for the tested L-
TA and D-TA (Table 1, Entries 30-34). Conversions up to 56 %
was achieved with N-Boc-2-aminoacetaldehyde (1w) and 3-
[(carbobenzyloxy)amino]propionaldehyde (1x) as acceptors and
D-alanine as donor with both L-TA and D-TA (Table 1, Entries 33
and 34). As a wide variety of aliphatic, aromatic and heterocyclic
aldehydes was accepted by the enzymes, non-natural amino
acids with diverse side chains and different functional groups
can be accessed.
Figure 2. List of amino acids and other donors investigated.
Table 2. Investigation of amino acid donor specificities
.
L-TA
D-TA
C
Entry
1[a]
2[a]
d.r.[a,b]
C [%][a]
d.r.[a,b]
[%][a]
1
a
a
24
35
10
27
2
3.2 (syn)
1.1 (syn)
3.2 (anti)
1.4 (anti)
n.d.
14
10
1
1.2 (anti)
9.0 (syn)
n.d.
2
a
b
a
c
3
a
d
33[c]
<1
10[c]
0
1.5 (anti) [c]
n.d.
4
a
e
5
a
f
7
n.d.
n.d.
6
a
g-m
a
0
-
-
7-13
14
15
16
17
18
19
20-26
(m)-b
(m)-b
(m)-b
(m)-b
(m)-b
(m)-b
(m)-b
50
60
15
30
2
3.7 (syn)
1.2 (syn)
4.7 (anti)
1.3 (syn)
5.1 (syn)
n.d.
25
36
5
1.7 (syn)
7.3 (syn)
1.6 (anti)
1.1 (syn) [c]
2.3 (syn)
n.d.
b
c
d
39[c]
4
e
f
<10
0
<10[c]
0
g-m
-
-
[a] Conversion after 24 h at 30 °C in the presence of 50 mM aldehyde and 500
mM 2a-m. [b] Determined by rp-HPLC after derivatization with o-
phthaldialdehyde and various thiol reagents and given as syn/anti ratio;.e.e. >
99.5% (2S or 2R). [c] Determined by 18F NMR.
Among all nucleophiles tested, both enzymes best tolerate
glycine (2a), D-alanine (2b), D-serine (2c), and D-cysteine (2d)
as amino acid donors in most enzyme-aldehyde combinations.
More sterically demanding or electronically different candidates
including 2e-k, ethyl glycinate (2l) or 2-aminoethanol (2m) are
little or not converted (Table 2).
To demonstrate the applicability of the described asymmetric
biocatalytic synthesis of β-hydroxy α-quaternary amino acids,
selected reactions were performed at preparative scale.[19] Most
reaction products were isolated with typically 10-30 % yield and
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10.1002/cctc.201800611
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perfect enantioselectivity using 100-250 mM concentrations of
the aldehydes (Table 3), demonstrating a straightforward one-
step synthesis of α,α-disubstituted non-natural amino acids. At
increasing aldehyde concentration, some inactivation of the
enzymes was observed, and thus continuous feeding of the
acceptor substrate turned out to be advantageous. Alternatively,
for tackling substrate inhibition in threonine aldolase catalyzed
reactions, slug-flow microfluidic system could be used.[20] In
general, diastereomeric mixtures of amino acid products with
moderate excess of the syn-isomers were isolated from L-TA-
catalyzed reactions. The diastereomers can be typically
provided recently by pointing at the function of two histidine
residues in the active site (His85 and His128’ for L-TA from
Aeromonas jandaei), which both can form hydrogen bonding
with the Cβ-oxygen from opposite sides.[21] Until now, attempts to
alter the stereoselectivity at the β-carbon by enzyme engineering
have failed mainly due to complex hydrogen bonding network in
the active site participating in the protonation step.[22] On the
other hand, D-specific TA usually give higher d.r.’s, which was
explained by a slow thermodynamic equilibration step between
syn- and anti-isomers in contrast to the analogous reactions with
the L-TA.[23] But this is only true for the β-phenylserine
separated by flash-chromatography on reversed-phase silica gel. derivatives, where a d.r. up to >100 can be reached with the D-
A single (R)-syn-diastereomer was isolated from the reaction
with 3-fluoro-benzaldehyde ((m)-1f) and DL-alanine catalyzed by
D-TA (Table 3, Entry 8).
TA.[16j]
The initially formed aromatic β-serine derivatives can be
reductively de-hydroxylated in order to obtain optically pure α-
tetrasubstituted amino acids. To demonstrate this, we have
performed a Birch-type reduction starting from a syn/anti
diastereomeric mixture of (2S)-2-amino-3-hydroxy-2-methyl-3-
phenylpropanoic acid (3a), which had been obtained from an L-
TA-catalysed reaction of benzaldehyde 1a and DL-alanine 2b.
The Birch-reduction is a particularly useful transformation for the
Table 3. Preparative scale synthesis of α,α-dialkyl α-amino acids with
threonine aldolases.
Entry
1[a]
Enzyme[b]
Yield
[%][c]
d.r. [%][c,d]
reduction of aromatic compounds using group
I metals,
2.2 (syn)
7.8 (syn)
2.3 (syn)
1.6 (syn)
1.2 (syn)
2.5 (syn)
3.5 (syn)
> 100 (syn)
1.1 (syn)
1.2 (syn)
1.1 (anti)
1
a
L-TA
D-TA
L-TA
L-TA
L-TA
L-TA
L-TA
D-TA
L-TA
L-TA
L-TA
L-TA
L-TA
L-TA
D-TA
D-TA
13
commonly sodium or lithium, in liquid ammonia and in the
presence of weak acids which act as proton source.[24] The
advantage of this method is that unprotected amino acids can be
directly used in the reduction reaction.[25]
2
a
12
14
(o)-b
3
(m)-b
(p)-b
(o)-e
(o)-f
14
7
4
5
16
20
27
21
22
28
6
7
(m)-f
(m)-i
(p)-i
8
9
Scheme 2. β-Dehydroxylation of (2S)-2-amino-3-hydroxy-2-methyl-3-
phenylpropanoic acid 3a via Birch reduction.
10
11
12
13
14
15
16
(m)-j
k
r
23
4
2.2 (syn)
1.4 (syn)
The corresponding (S)-α-methylphenylalanine 4a was isolated in
91% yield and an unchanged high optical purity of >99 %
(Scheme 2). Analogously, the (R)-isomer can be obtained from
(2R)-2-amino-3-hydroxy-2-methyl-3-phenylpropanoic acid 3a,
u
u
10
1.25 (anti)
27
32
2.7 (syn)
57 (syn)
synthesized by D-TA. Thus, (S)-α-methylphenylalanine,
a
x
valuable non-natural amino acid, which is used in
peptidomimetics and as sweetener, was produced using a
simple two-step chemo-enzymatic synthesis starting from
inexpensive benzaldehyde and alanine in an overall yield of
10 % and an e.e. > 99 %.
[a] 100-250 mM aldehyde, 1.0 or 1.5 M DL-alanine used [b] L-TA from
Aeromonas jandaei, respectively D-TA from Pseudomonas sp. used. [c] After
reaction times of 2-7 d at 30 °C; isolated yield after chromatography. [d]
Diastereomeric ratio determined by 1H NMR.
As the conditions of the Birch-reductions are not compatible with
several functional groups (see SI for some examples), we
investigated an alternative method of dehydroxylation which
involves first the conversion of the -OH into a benzylic chlorine
substituent followed by mild dehalogenation with Zn/AcOH.[26]
The application of this method for the removal of hydroxyl-group
from a syn/anti diastereomeric mixture of (2S)-(o)-3e led to the
(2S)-2-amino-2-methyl-3-(2-chlorophenyl)propanoic acid (S)-6
with 38% yield and e.e. > 99%.
While the stereoselectivity at α-quaternary center is stringently
controlled by the applied enzymes, the selectivity at the β-
carbon (Cβ) forming the hydroxyl group is moderate and
depends on the nature of the substrate aldehyde. In L-TA-
catalyzed reactions with aromatic substrates, the syn-products
were preferably obtained, whereas with long-chain aliphatic
aldehydes or with increasing distance between the aromatic ring
and the aldehyde group, the formation of anti-products is
favored. A plausible reason for the low diastereoselectivity was
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1H, -CCH3NH2, anti), 1.35 (s, 1H, -CCH3NH2, syn). 13C-NMR (75.53 MHz,
D2O): δ = 175.1 (Cq, -COOH), 173.4 (Cq, -COOH), 135.5 (Cq, CAr), 135.4
(Cq, CAr), 133.3 (Cq, -CCl), 132.7 (Cq, -CCl), 130.2 (CAr), 130.1 (CAr),
129.8 (CAr), 129.6 (CAr), 129.1 (CAr), 128.4 (CAr), 127.6 (CAr), 127.1 (CAr),
71.3 (-CHOH), 70.3 (-CHOH), 65.7 (Cq, -CCH3NH2), 65.1 (Cq, -CCH3NH2),
19.2 (-CCH3NH2), 18.3 (-CCH3NH2). HRMS (MALDI-TOF): Calcd. for
C10H12ClNO3H [M+H]+: 230.0584; found: 230.0585.
Scheme 3. β-Dehydroxylation of (2S)-2-amino-3-hydroxy-2-methyl-3-(2-
chlorophenyl)propanoic acid (o)-3e.
Acknowledgements
Conclusions
The activity leading to the present results has received funding
from the European Community´s Seventh Framework
Programme (FP7/2007-2013) and EFPIA companies' in kind
contribution for the Innovative Medicine Initiative under Grant
Agreement No. 115360 (Chemical manufacturing methods for
the 21st century pharmaceutical industries, CHEM21). The
authors would like to acknowledge the financial support from the
Austrian Science Fund FWF: Hertha-Firnberg project T735-B21.
In summary, threonine aldolases show a broad substrate scope
for the preparation of β-hydroxy α-quaternary α-amino acids with
various structural features. In the aldol addition, two new
stereogenic centers are formed with perfect enantiospecifity at
the α-carbon (> 99 % e.e.). Moreover, a strategy for the removal
of the β-hydroxy-group gives access to quaternary α-amino
acids with perfect stereoselectivity. The herein described
biocatalytic method provides a fast and green access towards
enantiopure non-natural quaternary amino acids in preparative
amounts. Although some wild-type threonine aldolases have the
unique property to accept few D-amino acids as donor, further
sequence search or enzyme engineering based on directed
evolution or rational design may lead to the development of
catalysts with broader donor specificity, as recently shown for
other aldolases. Combining threonine aldolases in cascades
with other reactions can be beneficial to overcome the intrinsic
limitations of the reaction equilibrium and, consequently, could
lead to improved conversions.
Keywords: Amino acids • Asymmetric catalysis • Enzyme
catalysis • Quaternary stereogenic centers • Threonine aldolases
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Experimental Section
Procedure for the synthesis of (2S)-2-amino-3-(2-chlorophenyl)-3-
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In a 500 mL round-bottom flask, DL-alanine (19.39 g, 218 mmol, 10.0 eq)
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and 2-propanol (4.0 mL). Under stirring, PLP (6.0 mg) was added. The
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silica gel 60 C18, 35-70 µm, Carl Roth, Karlsruhe, Germany, prod. no.
5504; column size: 11 × 3 cm) using a stepwise gradient starting with
H2O/methanol 99:1 + 0.1 vol% formic acid up to 85:15, the product was
isolated. All fractions containing the pure product were pooled and
concentrated. Yield: 807 mg (3.51 mmol, 16 %) of colorless solid. e.e. >
99.5 (2S), d.r. = 2.5 ((2S,3R)/(2S,3S)). Mp = 195-212 °C (dec.). [α]D25=-
9.1 (c=0.1 in 1 M HCl). 1H-NMR (300.36 MHz, D2O): δ = 7.61 - 7.34 (m,
4H, HAr), 5.73 (s, 1H, -CHOH, syn), 5.47 (s, 1H, -CHOH, anti), 1.54 (s,
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10.1002/cctc.201800611
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FULL PAPER
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Entry for the Table of Contents
FULL PAPER
Julia Blesl, Melanie Trobe, Felix Anderl,
Rolf Breinbauer, Gernot A. Strohmeier,
Kateryna Fesko*
Page No. – Page No.
Application of Threonine Aldolases
for the Asymmetric Synthesis of α-
Quaternary α-Amino Acids
Quaternary α-amino acids in one step: The direct biocatalytic assembly of β-
hydroxy-α,α-dialkyl-α-amino acids is outlined. Through the application of L- or D-
selective threonine aldolases with donor specificities beyond glycine, a wide variety
of amino acid products can be created in a single step.
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