Investigation on the chemoenzymatic synthesis of threo- and erythro-β-hydroxy-L-glutamic acid derivatives

Article abstract of DOI:10.1016/j.molcatb.2011.11.004

A derivative of the malonic semialdehyde was transformed by means of a bioconversion catalyzed by the enzyme L-threonine aldolase from E. coli into a 6:4 epimeric mixture of two precursors of β-hydroxy glutamic acid. The enzyme was selective for the formation of the (S)-configuration at C-2, whereas the configuration at C-3 was not controlled. The two epimers were separated exploiting a diastereoselective acylation in organic solvent catalyzed by lipase PS. The relative and absolute configurations of the products were preliminarily assigned on the base of the model proposed by Kazslauskas for the stereopreference of lipase PS and by comparison of the chemical shifts of the H-2 and H-3 protons of the two homologues. The possibility of transforming the obtained products into β-hydroxy glutamic acid derivatives by conventional chemical reactions was demonstrated.

Full text of DOI:10.1016/j.molcatb.2011.11.004

Journal of Molecular Catalysis B: Enzymatic 75 (2012) 27–34
Contents lists available at SciVerse ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Investigation on the chemoenzymatic synthesis of threo- and
erythro-␤-hydroxy-l-glutamic acid derivatives
Francesca Saguia,b, Carlo De Michelib, Gabriella Rodab,∗, Pietro Magronea,
Rachele Pizzolia, Sergio Rivaa,∗
a Istituto di Chimica del Riconoscimento Molecolare, C.N.R., Via Mario Bianco 9, Milano, Italy
b Dipartimento di Scienze Farmaceutiche “Pietro Pratesi”, Università di Milano, via Mangiagalli 25, Milano, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 19 July 2011
Received in revised form 21 October 2011
Accepted 1 November 2011
Available online 7 November 2011
A derivative of the malonic semialdehyde was transformed by means of a bioconversion catalyzed by
the enzyme l-threonine aldolase from E. coli into a 6:4 epimeric mixture of two precursors of ␤-hydroxy
glutamic acid. The enzyme was selective for the formation of the (S)-configuration at C-2, whereas the
configuration at C-3 was not controlled. The two epimers were separated exploiting a diastereoselec-
tive acylation in organic solvent catalyzed by lipase PS. The relative and absolute configurations of the
products were preliminarily assigned on the base of the model proposed by Kazslauskas for the stere-
opreference of lipase PS and by comparison of the chemical shifts of the H-2 and H-3 protons of the
two homologues. The possibility of transforming the obtained products into ␤-hydroxy glutamic acid
derivatives by conventional chemical reactions was demonstrated.
Keywords:
␤-Hydroxy-l-glutamic acid derivatives
l-Threonine aldolase
Lipase PS
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
In this frame EAATs are considered as molecular targets
for the treatment of cerebral ischemia and neurodegenera-
l-Glutamic acid (Glu) is a nutrient involved in important bio-
chemical pathways (gluconeogenesis and ammonia detoxification)
[1]. In the mammalian central nervous system (CNS) it is consid-
ered to be the major mediator of excitatory signals, contributing
to brain development and to physiological functions such as cog-
nition, memory, learning and nociception [2–7].
Due to its potential neurotoxicity when accumulating in the
synaptic cleft, the extracellular concentration of glutamate is
controlled under physiological conditions by membrane-bound
proteins which act as high affinity excitatory amino acid trans-
porters (EAATs) [6–8]. In pathological conditions (e.g. ischemia,
neurotrauma, neurodegenerative diseases), EAATs release addi-
tional Glu in the synapsis through the reversed mode of operation,
leading to an overstimulation of glutamate receptors which
causes a massive calcium influx responsible for neuronal cell
death [8].
tive pathologies. In particular, attention is focused on EAAT
non-transportable inhibitors (blockers) which, in pathological
conditions, could reduce EAAT-mediated glutamate release into
the synaptic cleft, thus preventing excitotoxicity and the sub-
sequent cell death [9–11]. Initial synthetic and biochemical
studies, focusing on the modification of the structure of the
endogenous substrates, namely aspartate (Asp) and glutamate
(Glu), led to the identification of potent substrate inhibitors
and uptake blockers [12,13]. The strategies used to find out
EAAT inhibitors were based on two approaches: (i) the con-
formational blockade of the neurotransmitters skeleton [12]
and (ii) the insertion into their structure of further functional
groups [13]. The outcome of these investigations is exemplified
by 3-hydroxy-4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4-d]isoxazole-4-
carboxylic acid (HIP-A, 1) and 3-hydroxy-4,5,6,6a-tetrahydro-3aH-
pyrrolo[3,4-d]isoxazole-6-carboxylic acid (HIP-B, 2), which are
non-competitive inhibitors, and l-threo-␤-benzyloxyaspartate (l-
TBOA, 3). In particular, l-TBOA, a non-transportable blocker of
EAATs, is widely recognized as the gold standard compound for
the biological screening of novel derivatives as well as the tool to
investigate the physiological roles played by the different subtypes
of this set of proteins [13].
∗
Corresponding authors. Tel.: +39 2 50319328; fax: +39 2 50319359.
E-mail addresses: gabriella.roda@unimi.it (G. Roda), sergio.riva@icrm.cnr.it
(S. Riva).
1381-1177/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2011.11.004

28
F. Sagui et al. / Journal of Molecular Catalysis B: Enzymatic 75 (2012) 27–34
COOH
NH
HO
N
HO
H
H
H
NH
N
O
O
H
COOH
HIP-B:2
HIP-A:1
NH2
O
NH2
HO
HO
O
9
HOOC
CHO
OH
OCH2Ph
n COOH
CHO
n
O
O
OH
4 , n = 0
5 , n = 2
O
3
6 , n = 0
7 , n = 1
8 , n = 2
Formulae 1-9
Structural modification of glutamate chain also produced
contaminants in our aldolase preparation [23]. Moreover, tert-butyl
esters, if needed, can generate the corresponding carboxylate group
under nonracemizing mild acidic conditions.
intriguing biological results [14]. Particularly, in a recent work,
␤- and ␥-benzyloxy-l-glutamic acid derivatives have been syn-
thesized and their activity as EAAT blockers has been evaluated
[15].
In this frame we became interested in investigating a biocat-
alytic approach to obtain ␤-hydroxy-l-glutamic acid derivatives,
specifically by exploiting the lyase l-threonine aldolase (l-TA) from
E. coli [16]. The advantages of an enzymatic strategy over conven-
tional methodologies rely on the mild conditions and the minimal
need for substrate protection [17].
l-Threonine aldolases (l-TA) are pyridoxal-5-phosphate (PLP)-
dependent enzymes that catalyze either the cleavage of l-threonine
(or l-allo-threonine) into glycine and acetaldehyde, or the reverse
aldol reaction [18,19]. There is a strict requirement for glycine as
a donor, but a wide range of aliphatic and aromatic aldehydes as
electrophilic substrates can be accepted [20,21]. The aldol reaction
proceeds with complete control of the stereoselectivity at the ␣-
carbon, since the outcome is the sole (l)-epimer. On the other hand,
the stereochemistry at the ␤-carbon is not totally controlled and a
mixture of diasteroisomers is generally formed (Fig. 1).
In a previous work [22] we have demonstrated that a mixture
of stereoisomeric ␤-hydroxy-l-aspartates (4) and 2-amino-3-
hydroxyhexanedioic acids (5) can be obtained by l-TA-catalyzed
condensation of glycine with the commercially available glyoxylic
acid (6) and succinic semialdehyde (8), respectively. In this paper
we report the results of the investigation on the related synthesis
of ␤-hydroxy-l-glutamic acid derivatives through the biocatalytic
condensation of glycine with analogues of the malonic semialde-
hyde 7.
As shown in Fig. 2, the synthesis of substrate 9 was carried out
starting from Meldrum’s acid 10 which, after a condensation with
triethyl orthoformate followed by a treatment of the intermedi-
ate 11 with hydrochloric acid, gave aldehyde 12, depicted in Fig. 2
as the enol tautomer [24]. The protected semimalonic aldehyde 9
was then obtained by reacting derivative 12 with tert-butyl alco-
hol and subsequently purified by distillation. It was mixed with an
excess glycine and submitted to the action of the l-TA from E. coli,
produced and purified as previously described [22]. To our disap-
pointment, no significant product formation was detected by TLC.
A conceivable explanation could be related to the presence in the
aqueous solution of a substantial percentage of the enolic form 9a
which is unsuitable to undergo a nucleophilic attack (Fig. 2).
A different synthetic strategy based on aldehyde 13, an homol-
ogous of benzyloxyacetaldehyde known to be a good substrate for
l-TA [20–22] was subsequently investigated.
Compound 13 could be easily obtained by oxidation with
TEMPO, NaOCl and NaBr of the corresponding commercially avail-
able alcohol 14 (Fig. 3). The l-TA-catalyzed condensation of
aldehyde 13 with glycine gave a product of intermediate polar-
ity, whose TLC spot was positive to the ninhydrin test. This
compound was purified with an hydrophobic ion exchange resin
(SEPHABEADS SP20755-Resindion) suitable to separate amino
acids on the base of their different hydrophobicity, retaining the
more lipophilic ones. The excess of glycine could be easily washed
out, since it did not interact with the resin, whereas the expected
product was retained and it was eluted by increasingthe percentage
of ethanol in the solvent.
As expected, the enzymatic condensation showed complete
stereocontrol in the formation of the ␣-carbon centre but it was
not stereoselective at the ␤-carbon. The isolated compound, tagged
as (t,e)-15, was a mixture of diastereomers (l-absolute configura-
tion, >98% e.e.), as evident from the NMR spectrum of the crude
product. The threo isomer (t)-15 was characterized by a doublet at
3.11 ppm (H-2) and a multiplet at 3.95 ppm (H-3), both integrating
0.6 H, while the erythro isomer (e)-15 showed a doublet at 3.14 ppm
(H-2) and a multiplet at 3.90 ppm (H-3), both integrating 0.4 H
(Fig. 4).
2. Results and discussion
At variance with compounds 6 and 8, aldehyde 7 is not commer-
cially available, probably due to its instability. Taking into account
its tendency to decarboxylate to acetaldehyde, it became neces-
sary to develop a synthetic strategy to prepare a suitable derivative
in which the carboxylic moiety, if present, was masked. Aldehyde
9, carrying a tert-butyl ester, was identified as a suitable target
since we had previously verified that, due to its steric hindrance,
this alkyl moiety was not recognized by the hydrolases present as
NH2
R
OH
L-TA
RCHO
H2N
COOH
+
O
R = alkyl , aryl
OH
Fig. 1. l-TA-catalyzed condensation of a generic aldehyde and glycine to give ␤-hydroxy-␣-l-amino acids.

F. Sagui et al. / Journal of Molecular Catalysis B: Enzymatic 75 (2012) 27–34
29
O
OH
O
O
OEt
O
O
OEt
OEt
OH
EtO
O
O
9
HCl
O
O
CHO
O
O
O
O
O
11
10
12
O
glycine
L-TA
X
O
O
.
.
.
.
H
9a
Fig. 2. Synthesis of the malonic semi-aldehyde tert-butyl ester 9 and its attempted l-TA-catalyzed condensation with glycine.
NaOCl, TEMPO
NaBr, NaHCO3
CHO
O
O H
O
0°C to RT, 4h.
L
-TA
13
(47%)
14
glycine
DTT, PLP
RT, 20h.
O H
OH
a) SOCl2 , MeOH
0°C to RT, 12h.
COOH
COOMe
O
O
NH2
NHBoc
b) Boc2O, EtOAc-H2O
0°C to RT, 12h.
(t,e)-15
(t,e)-16
(60%)
(12%)
20% d.e., >98% e.e.
20% d.e., >98% e.e.
Fig. 3. Chemoenzymatic synthesis of (t,e)-16. Yields refer to isolated products after chromatographic purification.
Fig. 4. Portion of the 1H NMR spectrum of (t,e)-15.

30
F. Sagui et al. / Journal of Molecular Catalysis B: Enzymatic 75 (2012) 27–34
Fig. 5. Synthesis of (t,e)-18.
OAc
COOMe
NHBoc
O
(t)-19
(45%)
OAc
>98% d.e., >98% e.e.
O
COOMe
Ac2O, THF
O
O
(t,e)-16
+
NHBoc
lipase PS
MTBE, RT, 72h.
TEA, DMAP
RT, 24h.
(t,e)-19
OH
(63%)
COOMe
O
NHBoc
(e)-16
(29%)
>98% d.e., >98% e.e.
Fig. 6. Aspecific chemical acetylation and enzymatic diastereoselective acetylation of (t,e)-16. Yields refer to isolated products after chromatographic purification.
In order to evaluate the biological activity of the target com-
pounds, it is necessary to synthesize them in an enantiomerically
and diastereomerically pure form. Since in a previous work [22]
we were able to separate the two epimers of 4 by a simple silica
gel chromatography of their N-Boc methyl ester derivatives, the
same approach was attempted with the epimeric mixture (t,e)-15.
Accordingly, they were transformed into the corresponding deriva-
tives (t,e)-16 (Fig. 3), which, however, turned out to be unseparable
by silica gel chromatography.
In a further attempt the mixture (t,e)-16 was transformed into
the corresponding acetonides (t,e)-17, which subsequently under-
went a catalytic hydrogenation to remove the benzyl group and an
oxidation by means of the Corey–Schmidt reagent (PDC in DMF)
to give a mixture of the protected glutamate derivatives (t,e)-18
(Fig. 5). Unfortunately, even this strategy did not allow the separa-
tion of the two epimeric derivatives by silica gel chromatography,
despite the fact that they had been transformed into more rigid and
polar derivatives (t,e)-17 or (t,e)-18.
In a third attempt to separate the two epimers (t,e)-15, it was
then investigated the well-known ability of lipases to catalyze
stereoselective acylations in organic solvents [17d,25]. Preliminar-
ily the mixture (t,e)-16 was transformed into the corresponding
Fig. 7. (a) Chiral HPLC separation of (t,e)-19 and (t,e)-16 on a CHIRALPAK IA column;
(t)-19: 16.167 min, (e)-19: 24.608 min, (t)-16: 41.167 min, and (e)-16: 44.475 min.
(b) Chromatogram obtained after the enzymatic kinetic resolution of (t,e)-16.
Fig. 8. Stereopreference of lipase PS (Kaslauskas’s rule) [26].

F. Sagui et al. / Journal of Molecular Catalysis B: Enzymatic 75 (2012) 27–34
31
OH
OH
OH
COOMe
COOMe
O
O
COOMe
O
NHBoc
NHBoc
(t,e)-16
NHBoc
(t,e)-21
(t,e)-20
Fig. 9. “Inferior” (t,e)-20 and “superior” (t,e)-21 homologues of (t,e)-16.
acetyl derivatives (t,e)-19 by a standard procedure (Fig. 6). The mix-
tures (t,e)-16 and (t,e)-19 were analyzed by chiral HPLC and it was
found that both the epimers (t,e)-16 and (t,e)-19 could be separated
with a CHIRALPAK IA (Daicel) column; the acetylated derivatives
(t,e)-19 being the first to be eluted at 16.1 and 24.6 min versus 41.2
and 44.5 of (t,e)-16 (Fig. 7a).
Seven commercially available lipases preparations were tested
for their ability to catalyze the diastereoselective acetylation of
(t,e)-16 in methyl tert-butyl ether as a solvent and in the presence of
vinyl acetate. Three lipases were able to accept this epimeric mix-
ture as a substrate, but only a lipase from Pseudomonas resulted to
be diastereoselective.
formation of the (S)-stereocentre in position C-2 and gave two C-
3 epimers in a 6:4 ratio, followed by an esterification catalyzed
by a lipase from Pseudomonas, which selectively acylated the 3R-
epimer. The relative and absolute configurations of the products
were preliminarily assigned considering the model proposed by
Kazslauskas on the stereopreference of lipase PS and confirmed by
1H NMR. The possibility to transform the derivatives obtained with
the present enzymatic methodologies into stereomeric ␤-hydroxy
glutamic acids by means of conventional chemical reactions should
contribute to deepen their intriguing pharmacological profile.
4. Experimental part
The preparative enzymatic resolution of (t,e)-16 was then car-
ried out and, after 72 h the non-acetylated epimer corresponding
to the peak at 41.2 min was completely transformed into the
fastest eluted acetyl derivative with a retention time of 16.1 min
(conversion (t)-16 → (t)-19 was observed to be >98%). The acetyl
derivative and the residual alcohol were separated by silica gel
chromatography and isolated in 45% and 29% yield, respectively.
Their diastereomeric excesses, evaluated by chiral HPLC analysis,
resulted to be >98%. 1H NMR spectra of the two compounds con-
firmed, in both cases, the presence of a single epimer.
Relative and absolute configurations at the stereocenters of the
two isolated epimeric compounds were preliminarily assigned on
the basis of the well-known chiral preference of lipase from Pseu-
domonas. A model proposed by Kazslauskas [26] assesses that in
the case of a secondary alcohol with two substituents of different
steric hindrances, this enzyme preferentially acylates the stereoiso-
mer with a R configuration. As shown in Fig. 8, by applying this rule
to our substrates, and by considering that l-TA leads exclusively to
the formation of the 2S isomers, we propose the structure of (t)-19
and (e)-16 for the two isolated products, with the absolute config-
urations 3R,2S and 3S,2S, respectively. Additionally, for the sake of
comparison, (e)-16 could be easily acetylated to the corresponding
(e)-19.
The proposed relative configuration of the isolated (e)-16 and
(t)-16 could be confirmed by comparison of their 1H NMR spec-
tra with the literature data reported for their one carbon lower
homologues, (t,e)-20 [17,27] and one carbon higher homologues,
(t,e)-21 [28,29] (Fig. 9). As regards the signals of the H-3 proton in
this series, it could be noted that in the erythro epimers these sig-
nals resulted upshielded with respects to the corresponding signals
of the threo counterpart. In fact in (t,e)-21 the H-3 signal of the ery-
thro isomer fell at 3.89 ppm, while in the threo isomer it was found
at 4.05 ppm. Similarly, in (t,e)-20 the H-3 proton of the erythro iso-
mer was at 4.25 ppm, whereas in its threo counterpart it resonated
around 4.40 ppm. In analogy, it sounds reasonable to propose that
even in the case of the mixture (t,e)-16, the H-3 signal at higher
fields (4.08 ppm) due to the isomer that was not acylated by lipase
PS, could be associated to (3S,2S)-erythro epimer, thus confirming
the assignments based on the Kaslauskas’ rule.
4.1. Materials and methods
All the reagents and solvents were purchased from commer-
cial suppliers and were used without further purification unless
otherwise stated. The coding sequence of the ltaE gene, encoding
the E. coli low-specificity l-threonine aldolase (eTA) was previ-
ously obtained from the genomic DNA of the E. coli K-12 strain
as described in the literature [16]. The host strain for recombinant
expression was HMS174(DE3) [F− recA1 hsdR(rK12 mK12+) (DE3)
−
(RifR)].
Lipase form Candida rugosa, porcine pancreatic lipase and sub-
tilisin were purchased from Sigma–Aldrich, lipase PS from Amano,
Novozym 435 and lipozyme were from Novozymes, lipase from
Chromobacterium viscosum from Finnsugar.
Fermentations were performed with a 1 L fermentor Inova
4230, sterilizations were performed using autoclave Steristeam
CDL. Enzymatic activities were monitored using a Jasco V-530
UV/vis spectrophotometer. Enzyme purification and enantiomeric
excess determinations were performed by HPLC, using a JASCO
880-PU instrument equipped with a Jasco 870 UV detector. Mea-
surements of pH were carried out with a pH-meter Crison micropH
2000. TLC: silica plates (Merck 60 F254) or reverse phase silica
plates (Merck RP-18254). Substrates and products were visualized
at 254 nm and/or by treatment with the ninhydrin reagent (ninhy-
drin, 2 g, in 100 ml MeOH). Flash chromatography silica gel: Merck
60, 40–63 ␮m.
1H and 13C NMR spectra were recorded on a Varian Mercury 300
(300 MHz) or on a Bruker AC400 (400 MHz) or on a Bruker AC500
(500 MHz). MS spectra were recorded on Bruker Esquire 3000 Plus
spectrometer.
4.2. Production and purification of l-TA from E. coli
l-TA was expressed from E. coli HMS 174 (DE3)/pET 22 b(+)(l-
TA) constructs containing the DNA sequence. The cells were
cultivated on LB media (Trypton 10 g/L, yeast extract 5 g/L, NaCl
10 g/L) supplemented with 100 mg/L ampicillin and 30 mg/L of B6
vitamins. Overnight cultures (100 mL) in 500 ml flasks were inoc-
ulated with single colonies and grown at 37 ◦C. The 500 mL main
cultures in 2000 mL flasks were inoculated with 3 ml of the pre-
culture and grown at 37 ◦C to an OD600 of 0.3 and then induced
with IPTG at a final concentration of 0.05 mM. Cultivation was
continued overnight at 37 ◦C, and the cells were harvested by cen-
trifugation for 20 min at 4500 rpm. After resuspension of the pellets
(10 mL/g cell) in a lysis buffer (10 mM Tris/HCl, 1 mM NaCl, 1 mM
3. Conclusions
Two enantiomerically and diastereomerically pure precursors
of ␤-hydroxy glutamic acid were synthesized exploiting two selec-
tive enzymatic transformations: an aldol reaction catalyzed by a
l-threonine aldolase, which was completely stereoselective in the

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F. Sagui et al. / Journal of Molecular Catalysis B: Enzymatic 75 (2012) 27–34
EDTA, pH 7.6), the cells were disrupted using a solution of lysozyme
(1 mg/g cell) followed by the precipitation of the DNA by adding
streptomycine sulfate (10 g/L dissolved in the minimum amount
of the same buffer) and leaving the suspension at 0 ◦C for 20 min.
The crude lysate was cleared by centrifugation at 12,000 rpm for
30 min. The enzyme was precipitated from the supernatant (cell
free extract) by adding ammonium sulfate (80% saturation) and par-
tially purified using an ion-exchange column (DEAE 650-S, Merck)
where the dissolved pellet was loaded in a phosphate buffer 20 mM
pH = 7.5 and the enzyme eluted with a linear gradient of NaCl, up
to 0.4 M. The partially purified enzyme was stored as a precipitate
in ammonium sulfate at 4 ◦C.
1 h under inert atmosphere. The solvent was evaporated in vacuo at
room temperature. Distillation of the residue under reduced pres-
sure gave a mixture of products 9 and 9a in 28% yield (478 mg).
The formation of 9 was followed by TLC (CHCl3/MeOH 97:3; Rf:
12 = 0.23; 9 = 0.37).
1H NMR 9 (300 MHz, dmso-d6) ı (ppm): 1.45 (s, 9H, CH3);
3.28 (br s, 2H, CH2COO); 9.61 (br s, 1H, HCO). 1H NMR 9a
(300 MHz, dmso-d6) ı (ppm): 1.45 (s, 9H, CH3); 5.02 (d, 1H, CHCOO,
J = 12.2 Hz); 7.04 (m, 1H, HOCH ); 11.51 (d, 1H, HOCH , J = 7.8 Hz).
4.7. Synthesis of 3-(benzyloxy)propanal (13)
In order to improve the yields of recovered l-TA, cell growth was
performed in a 1 L capacity fermentor (INFORS AG CH-4103), fol-
lowing the same protocol developed for the suspended cell systems,
monitoring and actively controlling oxygen supply, pH, temper-
ature, and stirring at 220 rpm. This experiment gave satisfactory
results, as – following cells lyses, ammonium sulfate precipitation
and subsequent dialysis – 688 U of l-TA were obtained, to be com-
pared with the 125 U obtained with the previously described flask
cultures.
A solution of NaHCO3 (9.87 g) in NaOCl aq (2.5% in water,
130 mL) was added dropwise (3 mL/min) at 0 ◦C to a stirred mixture
of 3-(benzyloxy)propan-1-ol (14, 4.3 ml, 27 mmol), NaBr (2.77 g,
27 mmol) and TEMPO (0.8 g, 5.2) in AcOEt, toluene and water
(150 mL, 4:4:1). After the addiction, the mixture was stirred at
room temperature for 4 h and the reaction was monitored by TLC
(petroleum ether/EtOAc 8:2; Rf: 13 = 0.42). After the reaction, the
two phases were separated, the aqueous phase was extracted with
EtOAc (3× 15 mL) and the collected organic layers were washed
firstly with a solution (2× 15 mL) of KI (10%) in KHSO4 (aqueous
saturated solution), then with a solution (2× 15 mL) of Na2S2O3
(10% in water), then with phosphate buffer (pH 7, 2× 15 mL) and
finally with brine (2× 15 mL). The organic phase was dried over
anhydrous Na2SO4 and concentrated under reduced pressure. The
crude product was then purified by flash silica gel column chro-
matography (petroleum ether/EtOAc 8:2) obtaining the aldehyde
13 (2.07 g) in 47% yield.
4.3. l-TA activity assay
l-allo-Threonine (50 mM), sodium phosphate buffer (20 mM,
pH 7), PLP (50 ␮M), NADH (200 ꢀM), yeast alcohol dehydroge-
nase (32 U) were added in cuvette to a final volume of 990 ␮L.
The reactions were started by adding 10 ␮L of purified l-TA and
monitored by measuring the decrease of absorbance at 340 nm in
a spectrophotometer (ε = 6.2 × 103 l × mol−1 × cm−1) at 30 ◦C.
One unit of l-TA is the amount of enzyme that catalyzes the
formation of 1 ␮mol of acetaldehyde (1 ␮mol of NADH oxidized)
per minute at room temperature.
1H NMR (500 MHz, CDCl3) ı (ppm): 2.62 (dt, 2H, J = 1.8, 6.1 Hz,
H-2); 3.74 (t, 2H, J = 6.1, 6.1 Hz, H-3); 4.46 (s, 2H, H-4); 7.21–7.32
(m, 5H, ArH); 9.73 (t, 1H, J = 1.8, 1.8 Hz, CHO).
4.8. Synthesis of (2S)-3(t,e)-2-amino-5-(benzyloxy)-
3-hydroxypentanoic acid ((t,e)-15)
4.4. Synthesis of 5-(ethoxymethylene)-2,2-dimethyl-
1,3-dioxan-4,6-dione (11)
To a solution of glycine (2.57 g, 34.3 mmol), dithiothreitol
(DTT, 79 mg), pyridoxal phosphate (PLP, 9 mg, 0.125 mM) in water
(35 mL), NaOH (1 M) was added up to pH = 7.5. Then, l-TA (46 U)
and aldehyde 13 were added and the reaction mixture was stirred
at r.t. for 20 h and monitored by TLC (n-BuOH/H2O/AcOH 4:2:1;
Rf: 15 = 0.5, detection was carried out with ninhydrin reagent).
The mixture was extracted with EtOAc (60 mL) and the aque-
ous phase was loaded on a hydrophobic chromatographic column
[regenerated (15%, v/v MeOH) phenylic resin: Sepabeads SP20755
Resindion]. The resin was initially eluted with water to remove
the excess of glycine and then washed with solutions of ethanol
in water (up to 30%) to recover the product. The fractions con-
taining the product were collected and lyophilised to give the
epimeric mixture (t,e)-15 (324 mg) in 12% yield. The product was
characterized by 1H NMR which also allowed the evaluation of the
diastereoisomeric threo/erythro ratio (6:4) of (t,e)-15 (with a d.e. –
value of 20% in favor of (t)-15; e.e. – values resulted to be >98% for
both epimers by chiral HPLC analysis).
A mixture of Meldrum’s acid 10 (2,2-dimethyl-1,3-dioxan-
4,6-dione, 4.0 g, 27.77 mmol) and triethylorthoformate (13.35 g,
90.08 mmol) was stirred at 85 ◦C for 2 h. When TLC analysis
(CHCl3/MeOH 8:2; Rf: 11 = 0.67) indicated the complete consump-
tion of Meldrum’s acid the solvent was evaporated in vacuo to
obtain the crude product 11 as a viscous yellow oil.
The product was used for the following reaction without any
further purification and characterization.
4.5. Synthesis of 5-(hydroxymethylene)-2,2-dimethyl-
1,3-dioxan-4,6-dione (12)
The crude compound 11 (695 mg, 3.47 mmol) was stirred for
30 min in a solution of HCl 2 N (12 mL) until an orange precipitate
was formed. The suspension was then dissolved in brine and the
water phase extracted with diethyl ether (3× 10 mL). The organic
phase was washed with brine (1× 10 mL), dried over Na2SO4 and
the solvent removed in vacuo to give the product 12 as a yellow
solid in 69% yield (793 mg). The formation of 12 was followed by
TLC (CHCl3/MeOH 97:3; Rf: 12 = 0.23).
1H NMR (500 MHz, DMSO-d6) ı (ppm): 1.50–1.65 (m, 1H, H-
4erythro/threo); 1.74 (m, 0.4H, H-4erythro); 1.85 (m, 0.6H, H-4threo); 3.11
(d, 0.6H, J = 4.5 Hz, H-2threo); 3.14 (d, 0.4H, J = 5.8 Hz, H-2erythro); 3.53
(m, 2H, H-5); 3.90 (m, 0.4H, H-3erythro); 3.95 (m, 0.6H, H-3threo); 4.44
(s, 2H, H-6) 7.25–7.32 (m, 5H, ArH).
1H NMR (400 MHz, dmso-d6) ı (ppm): 1.51 (s, 6H, CH3); 8.53 (s,
1H, C–H); 12.72 (bs, 1H, C–OH).
13C NMR (75 MHz, CDCl3)
ı (ppm): 27.52 (CH3); 95.71
(C C–OH); 107.38 (C–CH3); 168.32 (C O); 171.75 (C O); 177.31
(C C–OH).
4.9. Synthesis of tert-butyl (S)-1-(methoxycarbonyl)-
4-(benzyloxy)-2-hydroxybutylcarbamate ((t,e)-16)
4.6. Synthesis of tert-butyl 3-oxopropanoate (9)
(A) Epimeric mixture (t,e)-15 (230 mg, 0.96 mmol) was added
portionwise at 0 ◦C to a solution of SOCl2 (0.21 mL, 2.88 mmol) in
methanol (2.5 mL) under nitrogen. After 1 h at 0 ◦C, the reaction
A solution of formyl Meldrum’s acid (12, 2.0 g, 11.6 mmol) and
tert-butyl alcohol (1.3 mL) in dry benzene (23.2 mL) was refluxed for

F. Sagui et al. / Journal of Molecular Catalysis B: Enzymatic 75 (2012) 27–34
33
mixture was stirred at r.t. overnight and monitored by TLC (n-
BuOH/H2O/AcOH 4:2:1; Rf: (t,e)-15a = 0.33). The mixture was then
concentrated under reduced pressure and the obtained crude mate-
rial (t,e)-15a was used in the next step without further purification.
(B) To a biphasic solution of (t,e)-15a (230 mg, 0.962 mmol) in
water (1 mL) and EtOAc (4 mL) at 0 ◦C under stirring, Boc2O (252.
mg, 1.154 mmol) was added. The pH of the mixture was monitored
and adjusted to a value ≥7 by adding a few drops of a saturated solu-
tion of NaHCO3. After 2 h at 0 ◦C the reaction mixture was stirred at
r.t. overnight and monitored by TLC (petroleum ether/EtOAc 6:4; Rf:
(t,e)-16 = 0.41). After the reaction, the aqueous phase was separated
and extracted with EtOAc (3× 2 mL), the collected organic layers
were dried (Na2SO4) and concentrated under reduced pressure. The
crude product was purified by flash silica gel column chromatog-
raphy (CH2Cl2/MeOH 9.9:0.1) obtaining (t,e)-16 (203 mg) in 60%
yield over two steps (starting from (t,e)-15). HPLC: Column CHIRAL-
PAK IA (Daicel); eluent: petroleum ether, isopropanol 9.5/0.5, flow:
0.6 mL/min; ꢁ = 254 nm. (t)-16: 41.167 min, (e)-16: 44.475 min; the
mixture showed a d.e. – value of 20% in favor of (t)-16; e.e. – values
resulted to be >98% for both epimers.
chromatography (CHCl3/MeOH 9.5:0.5) obtaining (t,e)-17a (77 mg)
in 85% yield.
1H NMR (500 MHz, CDCl3) ı (ppm): 1.41–1.49 (2s, 9H, CH3Boc);
1.52–1.73 (4s, 6H, CH3); 1.85–2.06 (m, 2H, H-4); 3.76–3.77 (2s, 3H,
OCH3); 3.82 (t, 2H, J = 6.5 Hz, H-5); 4.08 (d, 1H, J = 7.7 Hz, H-2); 4.23
(m, 1H, H-3). m/z (ESI) = 326 Da (M+Na+).
4.12. Synthesis of 2-((S)-3-(tert-butoxycarbonyl)-
4-(methoxycarbonyl)-2,2-dimethyloxazolidin-5-yl)acetic acid
((t,e)-18)
To a stirred solution of pyridinium dichromate (PDC, 400 mg,
1.06 mmol) in DMF (2 mL) (t,e)-17a (25 mg, 0.0824 mmol) was
added and the mixture was stirred at r.t. for 4 days. The reaction
was monitored by TLC (CHCl3/MeOH 9.5:0.5; Rf: (t,e)-18 = 0.52) and
when no starting material could be detected, NaHCO3 saturated
aqueous solution (5 mL) and petroleum ether (5 mL) were added to
the reaction. The organic phase was separated. The aqueous phase
was washed with EtOAc (5 mL), carefully acidified with HCl to a
value of pH ≤ 3, and extracted with EtOAc (3× 5 mL). The com-
bined organic phases were dried over Na2SO4 and concentrated.
The residue was purified by flash silica gel column chromatography
(CHCl3/MeOH, 9.5:0.5) giving (t,e)-18 (10 mg) in 38% yield.
1H NMR (500 MHz, CDCl3) ı (ppm): 1.44 (s, 5.5H, CH3Bocthreo);
1.45 (s, 3.5H, CH3Boceryithro); 1.74–1.98 (m, 2H, H-4erythro/threo);
2.90 (bs, 1H, OH); 3.61–3.74 (m, 2H, H-5erythro/threo); 3.75 (s, 2H,
OCH3threo); 3.76 (s, 1H, OCH3erythro); 4.08 (m, 0.4H, H-3erythro); 4.28
(bs, 0.6H, H-3threo); 4.32–4.38 (m, 1H, H-2erythro/threo) 4.51 (2s, 2H,
H-6erythro/threo); 5.37 (bs, 0.6H, NHthreo); 5.45 (bs, 0.4H, NHerythro);
7.23–7.36 (m, 5H, ArH). 13C NMR (125 MHz, CDCl3) ı (ppm):
28.62 (CH3Bocerythro/threo); 33.42 (C-4erythro); 33.49 (C-4threo); 52.63
1H NMR (400 MHz, CD3OD) ı (ppm): 1.40–1.49 (2s, 9H, CH3Boc);
1.51–1.60 (4s, 6H, CH3); 2.50 (2d, 1H, J = 4.3, 6.6 Hz, H-4); 2.69 (d,
1H, J = 6.6 Hz, H-4); 3.74 (2s, 3H, OCH3); 4.14 (m, 1H, H-2); 4.43 (m,
1H, H-3). m/z (ESI) = 340 Da (M+Na+), 318 Da (M+H+).
(erythro); 52.74 (threo); 58.29 (threo); 58.70 (erythro); 68.81 (erythro);
4.13. Synthesis of tert-butyl (2S)-1-(methoxycarbonyl)-
69.36 (threo); 72.34 (threo); 72.65 (erythro); 73.70 (erythro); 73.85 (threo);
80.23 (threo); 80.55 (erythro); 128.00 (C–Arerythro); 128.05 (C–Arthreo);
128.11 (C–Arerythro); 128.22 (C–Arthreo); 128.78 (C–Arerythro);
128.83 (C–Arthreo); 137.85 (C–ipsothreo); 138.10 (C–ipsoerythro);
156.04 (C–Oerythro); 156.43 (C–Othreo); 171.37 (C–Oerythro); 171.96
(C–Othreo). m/z (ESI) = 376 Da (M+Na+), 354 Da (M+H+).
2-acetoxy-4-(benzyloxy)butyl carbamate ((t,e)-19)
To
a solution of (t,e)-16 (10 mg, 0.028 mmol) in THF
(0.5 mL), triethylamine (TEA, 0.04 mL, 0.238 mmol), acetic anhy-
dride (0.016 mL, 0.283 mmol) and dimethylaminopyridine (DMAP,
0.35 mg, 0.002 mmol) were added. The reaction was stirred at r.t.
for 24 h and monitored by TLC (petroleum ether/EtOAc 6:4: Rf:
(t,e)-19 = 0.69). The mixture was then concentrated in vacuo and
the residue taken up in EtOAc and washed (3× 0.5 mL) with HCl
(1 M). The organic layer was dried (Na2SO4) and concentratedunder
reduced pressure. The crude residue was purified by flash silica
gel column chromatography (petroleum ether/EtOAc 7:3) giving
(t,e)-19 (7 mg) in 63% yield. HPLC [column: Chiralpack IA, elu-
ent: petroleum ether/i-PrOH 9.5:0.5, flow: 0.6 mL/min, ꢁ = 254 nm,
retention times: (t)-19 = 16.167 min; (e)-19 = 24. 608 min].
4.10. Synthesis of (S)-3-tert-butyl 4-methyl
5-(2-(benzyloxy)ethyl)-2,2-dimethyloxazolidine-
3,4-dicarboxylate ((t,e)-17)
To a solution of (t,e)-16 (140 mg, 0.396 mmol) in acetone (2 mL)
and 2,2-dimethoxypropane (DMP, 0.43 mL), BF3·Et2O (3 ␮L) was
added. The resulting solution was stirred at r.t. for 2 h and mon-
itored by TLC (petroleum ether/EtOAc 6:4; Rf: (t,e)-17 = 0.82). The
solvent was removed under reduced pressure, the residual oil taken
up in CH2Cl2 (20 mL) and the resulting solution washed with a mix-
ture of a saturated NaHCO3 solution and water (1:1, 10 mL), then
brine (10 mL), dried (Na2SO4) and the solvent evaporated in vacuo
to give the product (t,e)-17 as a pale yellow oil (118 mg) in 76%
yield, used in the next step without any further purification.
1H NMR (500 MHz, CDCl3) ı (ppm): 1.32–1.40 (2s, 9H, CH3Boc);
1.42–1.54 (4s, 6H, CH3); 1.90 (m, 2H, H-4); 3.54 (m, 2H, H-5); 3.65 (s,
3H, OCH3); 4.07–4.34 (m, 2H, H-2, H-3); 4.43 (s, 2H, H-6); 7.23–7.36
(m, 5H, ArH). m/z (ESI) = 416 Da (M+Na+), 394 Da (M+H+).
1H NMR (500 MHz, CDCl3) ı (ppm): 1.44 (s, 3.5H, CH3Bocerythro);
1.45 (s, 5.5H, CH3Bocthreo); 1.86–1.99 (m, 2H, H-4erythro/threo); 1.97
(s, 1.8H, CH3COthreo); 1.98 (s, 1.2H, CH3COerythro); 3.44–3.55 (m, 2H,
H-5erythro/threo); 3.72 (s, 1.8H, OCH3threo); 3.74 (s, 1.2H, OCH3erythro);
4.42–4.51 (m, 2.6H, H-2threo, H-6erythro/threo); 4.65 (bs, 0.4H, H-
2erythro); 5.18 (d, 0.6H, J = 9.5 Hz, NHthreo); 5.28 (m, 0.4H, H-3erythro);
5.41 (bs, 0.4H, NHerythro); 5.54 (m, 0.6H, H-3threo); 7.27–7.35 (m, 5H,
ArH). m/z (ESI) = 418 Da (M+Na+), 396 Da (M+H+).
4.14. Screening of commercially available lipase preparations for
the diastereoselective acetylation of (t,e)-16
4.11. Synthesis of (S)-3-tert-butyl 4-methyl
5-(2-(hydroxyethyl)-2,2-dimethyloxazolidine-
3,4-dicarboxylate ((t,e)-17a)
To a solution of (t,e)-16 (5 mg, 0.041 mmol) in methyl tert-butyl
ether (MTBE, 1 mL) and vinyl acetate (100 ␮L) seven commer-
cially available lipase preparations (lipase PS from Pseudomonas,
lipase from Chromobacterium viscosum, lipase from Candida rugosa,
porcine pancreatic lipase, lipozyme, subtilisin and Novozym 435)
were added. The reaction was shaken at 45 ◦C and 250 rpm and
monitored by TLC and by HPLC (column: Chiralpack IA, eluent:
petroleum ether/i-PrOH 9.5:0.5, flow: 0.6 mL/min). Lipase from
Pseudomonas, lipase from Candida rugosa and Novozym435 were
able to accept the epimeric mixture as a substrate, but only the
To a solution of (t,e)-17 (120 mg, 0.3 mmol) in MeOH (15 mL),
Pd/C-10% (40 mg) as catalyst was added. The resulting suspension
was stirred under H2 (1 atm) at r.t. for 4 h and the reaction was mon-
itored by TLC [petroleum ether/EtOAc 7:3; Rf: (t,e)-17a = 0.28]. The
mixture was filtered on celite and the filtrate concentrated in vacuo.
The crude residue was then purified by flash silica gel column

34
F. Sagui et al. / Journal of Molecular Catalysis B: Enzymatic 75 (2012) 27–34
lipase from Pseudomonas resulted to be diastereoselective trans-
forming the epimer at 41.176 min of (t,e)-16 into the acetylated
derivative with a retention time of 16.167 min.
Acknowledgments
We thank Dr. Martino Luigi di Salvo (Rome University), Dr.
Daniela Monti, Dr. Lara Baratto and Dr. Federica Loiacono (ICRM-
CNR) for their valuable technical assistance in the production of
l-TA and in the work-up of the biocatalyzed reactions. Thanks are
also due to Mr. Walter Panzeri (ICRM-CNR) for recording the NMR
spectra.
4.15. Immobilization of lipase from Pseudomonas
The enzyme (Lipase PS from Amano, 3 g) was carefully mixed
with celite (HyfloSuperCel, 10 g) and phosphate buffer 0.1 M pH = 7
was added (10 mL). The mixture was vigorously stirred and air dried
at room temperature for three days.
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To a solution of (t,e)-16 (100 mg, 0.283 mmol) in methyl tert-
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(on celite) lipase from Pseudomonas (2 g) were added. The reac-
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and by HPLC (column: Chiralpack IA, eluent: petroleum ether/i-
PrOH 9.5:0.5, flow: 0.6 mL/min, ꢁ = 254 nm, retention times:
(t)-19 = 16.167 min, (e)-16 = 44.475 min). Conversion (t)-16 → (t)-
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>98%) in 45% yield and (e)-16 (29 mg, d.e. and e.e. >98%) in 29%
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1.80–1.92 (m, 2H, H-4); 3.63–3.73 (m, 2H, H-5); 3.75 (s, 3H, OCH3);
4.08 (m, 1H, H-3); 4.34 (bs, 1H, H-2); 4.51 (s, 2H, H-6); 5.45 (bs, 1H,
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13C NMR (e)-16 (125 MHz, CDCl3) ı (ppm): 28.62 (CH3Boc);
33.42 (C-4); 52.63; 58.70; 68.81; 72.65; 73.70; 80.55; 128.00
(C–Ar); 128.11 (C–Ar); 128.78 (C–Ar); 138.10 (C–ipso); 156.04
(C–O); 171.37 (C–O).
1H NMR (t)-19 (500 MHz, CDCl3) ı (ppm): 1.45 (s, 9H, CH3);
1.93–1.96 (m, 2H, H-4); 1.97 (s, 3H, CH3CO); 3.48–3.55 (m, 2H, H-
5); 3.72 (s, 3H, OCH3); 4.45–4.51 (m, 3H, H-2, H-6); 5.18 (d, 1H,
J = 9.5 Hz, NH); 5.54 (m, 1H, H-3); 7.27–7.35 (m, 5H, ArH).
1H NMR (t)-19 (500 MHz, d6-benzene) ı (ppm): 1.41 (s, 9H, CH3);
1.54 (s, 3H, CH3CO); 1.83–1.95 (m, 2H, H-4); 3.22–3.37 (m, 2H, H-5);
3.30 (s, 3H, OCH3); 4.19–4.27 (2d, 2H, J = 12.1 Hz, H-6); 4.75 (dd, 1H,
J = 1.4, 9.7 Hz, H-2); 5.38 (d, 1H, J = 9.7 Hz, NH); 5.80 (m, 1H, H-3);
7.06–7.27 (m, 5H, ArH).
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4.17. Synthesis of tert-butyl (1S,2S)-1-(methoxycarbonyl)-
2-acetoxy-4-(benzyloxy)butyl carbamate ((e)-19)
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To a solution of (e)-16 (10 mg, 0.028 mmol) in THF (0.5 mL),
triethylamine (TEA, 0.04 mL, 0.238 mmol), acetic anhydride
(0.016 mL, 0.283 mmol) and dimethylaminopyridine (DMAP,
0.35 mg, 0.002 mmol) were added. The reaction was stirred at r.t.
for 24 h and monitored by TLC (petroleum ether/EtOAc 6:4: Rf:
(e)-19 = 0.69). The mixture was then concentrated in vacuo and
the residue taken up in EtOAc and washed (3× 0.5 mL) with HCl
(1 M). The organic layer was dried (Na2SO4) and concentratedunder
reduced pressure. The crude residue was purified by flash silica gel
column chromatography (petroleum ether/EtOAc 7:3) giving (e)-19
(7 mg) in 63% yield. HPLC (column: Chiralpack IA, eluent: petroleum
ether/i-PrOH 9.5:0.5, flow: 0.6 mL/min, ꢁ = 254 nm, retention times:
(e)-19 = 24.608 min).
(b) C. Dallanoce, M. De Amici, G. Carrea, F. Secundo, S. Castellano, C. De Micheli,
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1H NMR (e)-19 (500 MHz, CDCl3) ı (ppm): 1.44 (s, 9H, CH3);
1.86–1.99 (m, 2H, H-4); 1.98 (s, 3H, CH3CO); 3.44–3.54 (m, 2H, H-
5); 3.74 (s, 3H, OCH3); 4.42–4.50 (2d, 2H, J = 11.9, 11.9 Hz, H-6); 4.65
(bs, 1H, H-2); 5.28 (m, 1H, H-3); 5.41 (bs, 1H, NH); 7.28–7.35 (m,
5H, ArH).
[28] M. Alonso, A. Riera, Tetrahedron-Asymmetry 16 (2005) 3908–3912.
[29] J.-C. Lee, G.T. Kim, Y.K. Shim, S.H. Kang, Tetrahedron Lett. 42 (2001) 4519–4521.