Novel chemo-enzymatic route to a key intermediate for the taxol side-chain through enantioselective O-acylation. Unexpected acyl migration

Article abstract of DOI:10.1016/j.molcatb.2015.03.015

A new enzymatic strategy has been devised for the preparation of a key intermediate for the taxol side-chain from N-hydroxymethylated cis-3-acetoxy-4-phenylazetidin-2-one. Burkholderia cepacia (PS-IM)-catalyzed acylation of the primary OH group with an excess of vinyl butyrate (10 equiv.) furnished the corresponding diester and unreacted monoester with excellent enantiomeric excess values (ee ≥ 98%). Traces of undesirable enantiomeric diacetylated product and diol due to intramolecular acyl migration (～6% mole fractions) were also detected. Finally, (2R,3S)-3-phenylisoserine hydrochloride (ee = 99%), a key intermediate for the taxol side-chain, was prepared from the corresponding diester enantiomer through acidic hydrolysis.

Full text of DOI:10.1016/j.molcatb.2015.03.015

Journal of Molecular Catalysis B: Enzymatic 116 (2015) 101–105
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Novel chemo-enzymatic route to a key intermediate for the taxol
side-chain through enantioselective O-acylation. Unexpected acyl
migration
Eniko˝ Forróa,∗, Zsolt Gallaa, Zala Nádasdia, Judit Árvaa, Ferenc Fülöpa,b,∗
a Institute of Pharmaceutical Chemistry, University of Szeged, Eötvös u. 6, H-6720 Szeged, Hungary
b Stereochemistry Research Group of the Hungarian Academy of Sciences, University of Szeged, Eötvös u. 6, H-6720 Szeged, Hungary
a r t i c l e i n f o
a b s t r a c t
Article history:
A new enzymatic strategy has been devised for the preparation of a key intermediate for the taxol side-
chain from N-hydroxymethylated cis-3-acetoxy-4-phenylazetidin-2-one. Burkholderia cepacia (PS-IM)-
catalyzed acylation of the primary OH group with an excess of vinyl butyrate (10 equiv.) furnished the
corresponding diester and unreacted monoester with excellent enantiomeric excess values (ee ≥ 98%).
Traces of undesirable enantiomeric diacetylated product and diol due to intramolecular acyl migration
(∼6% mole fractions) were also detected. Finally, (2R,3S)-3-phenylisoserine hydrochloride (ee = 99%), a key
intermediate for the taxol side-chain, was prepared from the corresponding diester enantiomer through
acidic hydrolysis.
Received 17 February 2015
Received in revised form 13 March 2015
Accepted 16 March 2015
Available online 23 March 2015
Keywords:
O-Acylation
Acyl migration
Enzyme catalysis
Remote stereocentre
Taxol
© 2015 Elsevier B.V. All rights reserved.
1. Introduction
antitumour activity of taxol, there is a need for the development
of efficient processes for the preparation of (2R,3S)-3-amino-3-
Enantiomeric ˇ-amino acids and their derivatives are of great
interest from both pharmaceutically and chemical aspects [1–11].
Thus, in recent years, acyclic ˇ-amino acids have been recognized
as an important class of compounds in the design and synthesis
of potential pharmaceutical drugs; for example, they can serve as
essential building blocks for medicinally important molecules such
as Taxol® and its analogue Taxotère®, currently considered to be
among the most efficient drugs in cancer chemotherapy [12,13].
Taxol, isolated initially from the bark of the Pacific yew (Taxus
brevifolia), is a potent cytotoxic microtubule-stabilizing agent that
is used efficaciously in the treatment of a number of human
cancers, including ovarian, breast and non-small-cell lung cancer
[12–14]. To satisfy taxol needs, chemists have been working on
semisynthetic methods involving synthetic side-chain coupling to
the C(13)–O of the more readily available baccatin III derivatives
[from the needles of various Taxus species (e.g. Taxus baccata)].
Since a 3-phenylisoserine-derived side-chain is essential for the
phenyl-2-hydroxypropionic acid or its direct enantiopure sources.
A relatively large number of such syntheses have been developed
[15–19], and new synthetic strategies appear continuously [20,21].
The total synthesis of taxol has also been achieved, but is too com-
plex to serve as a commercial source of the drug [22,23].
We have also published some very efficient enzyme-catalyzed
direct and indirect strategies for the preparation of (2R,3S)-
3-phenylisoserine, either through Burkholderia cepacia lipase
(PS-IM)-catalyzed hydrolysis of the ester function of racemic ethyl
3-amino-3-phenyl-2-hydroxypropionate with H2O as nucleophile
in t-BuOMe or iPr2O at 50 or 60 ◦C (E > 200) [24] or through Candida
antarctica lipase B (CAL-B)-catalyzed ring cleavage of racemic cis-
3-hydroxy-4-phenylazetidin-2-one with added H2O in t-BuOMe at
60 ◦C (E > 200) [25]. A new type of enzymatic two-step cascade reac-
tion has also been devised and used successfully for the synthesis of
(2R,3S)-3-phenylisoserine. In this latest strategy, the hydrolysis of
racemic cis-3-acetoxy-4-phenylazetidin-2-one was performed in
the presence of CAL-B with H2O in iPr2O at 60 ◦C, and the hydrolysis
of the ester at C(3) (the first enzymatic step, with relatively low E)
was followed by a rapid ring cleavage of the corresponding lactam
enantiomer (the second, highly enantioselective enzymatic step).
At 50% overall conversion of the starting racemate, there were two
enantiopure products, cis-(3S,4R)-3-hydroxy-4-phenylazetidin-2-
one and (2R,3S)-3-phenylisoserine with ee ≥ 98% [25].
∗
Corresponding authors at: Institute of Pharmaceutical Chemistry, University of
Szeged, Eötvös u. 6, H-6720 Szeged, Hungary. Tel.: +36 62 545564;
fax: +36 62 545705.
E-mail addresses: forro.eniko@pharm.u-szeged.hu (E. Forró),
fulop@pharm.u-szeged.hu (F. Fülöp).
http://dx.doi.org/10.1016/j.molcatb.2015.03.015
1381-1177/© 2015 Elsevier B.V. All rights reserved.

102
E. Forró et al. / Journal of Molecular Catalysis B: Enzymatic 116 (2015) 101–105
Scheme 1. Enzymatic O-acylation of (3S*,4R*)-1.
Scheme 2. Enzymatic O-acetylation of (3S*,4R*)-1.
In view of the earlier results on enzymatic acylations of pri-
mary alcohols with a remote stereocentre [2,4,26,27], our aim
in the present research work was to develop an enantioselective
strategy for the preparation of (2R,3S)-3-phenylisoserine, through
the enzymatic O-acylation of racemic N-hydroxymethylated cis-3-
acetoxy-4-phenylazetidin-2-one [(3S*,4R*)-1] (Scheme 1).
2. Results and discussion
Racemic cis-3-acetoxy-4-phenylazetidin-2-one was pre-
pared according to the literature [19], and then transformed
with paraformaldehyde under sonication [28] into the N-
hydroxymethylated lactam (3S*,4R*)-( )-1. The hydrolysis of
(3S*,4R*)-( )-1 in MeOH with saturated NaHCO3 and Na2CO3
[29] furnished racemic N-hydroxymethylated cis-3-hydroxy-4-
phenylazetidin-2-one [(3S*,4R*)-( )-4].
Scheme 3. The intramolecular acyl migration.
AY) were also tested for the acetylation of (3S*,4R*)-1 under the
same conditions (0.015 M substrate, 30 mg mL−1 enzyme, 6 equiv.
of VA, iPr2O, 50 ◦C). CAL-B catalyzed the ring cleavage of ˇ-lactam,
in good accordance with our earlier results [25,35–37], while all
the other enzymes tested catalyzed only the acetylation reactions
(Table 1), but enantiomerically enriched 4 (due to the acyl migra-
tion) appeared in all of the reaction mixtures. Thus, lipase PS-IM
was selected for further preliminary acylations.
In an attempt to suppress the acyl migration, several solvents,
such as toluene, t-BuOMe and 2-methyltetrahydrofuran (2-Me-
THF), were tested in the lipase PS-IM (30 mg mL−1)-catalyzed
acetylation of (3S*,4R*)-1 with 6 equiv. of VA at 25 ◦C (Table 2).
Unfortunately, no significant beneficial effect was observed (2–4%
mole fractions of 4 were also detected).
To avoid the inaccuracy in the determination of ee for (3R,4S)-2
as a result of enzymatic acetylation (due to the intramolecular acyl
migration), two other acyl donors, vinyl butyrate (VB) and 2,2,2-
trifluoroethyl butyrate (2,2,2-TFB) (6 equiv.), were also tested for
the acylation of racemic (3S*,4R*)-1 in the presence of lipase PS-IM
(30 mg mL−1) in iPr2O at 50 ◦C (Scheme 4).
On the basis of earlier results on the enzymatic acy-
lations of cyclic [28] and acyclic [30] N-hydroxymethylated
ˇ-lactams, we first carried out the enzymatic acylation of
racemic N-hydroxymethylated cis-3-acetoxy-4-phenylazetidin-2-
one [(3S*,4R*)-1] with vinyl acetate (VA) (1.2 equiv.) as acyl donor
in the presence of PS-IM (30 mg mL−1) in iPr2O at 50 ◦C (Scheme 2).
Due to an unexpected acyl migration, allowed by the closeness
of the OH and O-acyl groups and any H2O present in the reaction
medium, a considerable amount of the undesirable diol 4 (∼4%
mole fraction, ee = 46%) was formed besides to the desired diac-
etate (3R,4S)-2 (6% mole fraction, apparent ee = 45%) and unreacted
(3S,4R)-1 (81% mole fraction, ee = 10%) after 24 h. The mole frac-
tions were determined with n-heptadecane as internal standard,
by using GC on an l-Val column (Supporting information, S1).
When the concentration of (3S*,4R*)-1 (in iPr2O at 50 ◦C) was
decreased from 0.015 to 0.007 M, and then increased to 0.03 M,
practically no change was observed in the mole fractions of 1, 2,
4 and 5 (Scheme 3) after 20 min. The intramolecular character of
the acyl migration was therefore assumed. The acyl migration in
the presence of enzyme [31–33] (PS-IM in iPr2O at 50 ◦C) was also
investigated, when 95% 1, 3% 2, 2% 4 and traces (<1%) of 5 were
determined in the reaction mixture after 1 h.
Excellent E values were observed (>200) at around 50% con-
version, but racemic 2 (∼4% mole fraction) and enantiomerically
In view of our recent results on the enzyme-catalyzed acylation
of ˇ-hydroxy esters with regard to the competition of acylation and
hydrolysis [34], the acetylation of (3S*,4R*)-1 was next performed
with 6 equiv. of VA, when only traces of enantiomerically enriched
4 were detected after 10 min, at a conversion of 53% (ee1 = 91%
and apparent ee2 = 80%, apparent E = 28). When Et3N was added
to the reaction mixture under the same conditions, practically
no changes were observed (conv. = 54%, ee1 = 95% and apparent
ee2 = 80%, apparent E = 33).
Table 1
Enzyme screening for the acetylation of (3S*,4R*)-1.a
b
b,c
b
Enzyme
1 (mol%) ee1 (%) 2 (mol%) ee2
(%) 4 (mol%) ee4 (%)
(30 mg mL−1
)
CAL-A
Lipase AK
Lipase AY
79
46
91
16
91
1
14
48
7
40
80
17
7
6
2
42
67
15
a
0.015 M substrate, 6 equiv. of VA, iPr2O, 50 ◦C, after 30 min.
According to GC (Section 4).
Apparent values.
b
c
Besides PS-IM, the enzymes CAL-B, C. antarctica lipase A (CAL-
A), Pseudomonas fluorescens (lipase AK) and Candida rugosa (lipase

E. Forró et al. / Journal of Molecular Catalysis B: Enzymatic 116 (2015) 101–105
103
Scheme 4. Enzymatic O-butyrylation of (3S*,4R*)-1.
Table 2
Table 5
Solvent screening for the acetylation of (3S*,4R*)-1.a
Effects of temperature on the conversion and enantioselectivity of butyrylation of
(3S*,4R*)-1.a
c
c,d
Solvent
Conv.b (%)
ee1 (%)
ee2
(%)
Apparent E
Temperature (◦C) Reaction time
(min)
Conv.b (%) ee1 (%) ee6 (%)
E
c
c
Toluene
t-BuOMe
2-Me-THF
53
56
40
93
90
50
81
70
76
32
17
12
50
25
4
5
10
30
49
46
41
92
86
70
99
99
99
>200
>200
>200
a
0.015 M substrate, lipase PS-IM (30 mg mL−1), 6 equiv. of VA, in the solvent
tested, 50 ◦C, after 1 h.
b
a
Conversion values calcd. from ee1 (unreacted substrate) and ee2 (product) values
0.015 M substrate, lipase PS-IM (30 mg mL−1), 6 equiv. of VB, iPr2O.
Conversion values calcd. from ee1 (unreacted substrate) and ee6 (product) val-
b
through the equation c = ee1/(ee1 + ee2).
c
According to GC (Section 4).
Apparent values.
ues.
d
c
According to GC (Section 4).
Table 3
Conversion and enantioselectivity of butyrylation of (3S*,4R*)-1.a
In view of the best combination of enantioselectivity and reaction
rate, iPr2O was chosen as reaction medium for the preparative-scale
acylation.
c
c
Acyl donor (6 equiv.)
Conv.b (%)
ee1 (%)
ee6 (%)
E
VB
2,2,2-TFB
48
26
92
34
99
99
>200
>200
When the PS-IM-catalyzed butyrylation of (3S*,4R*)-1 with VB
was carried out at different temperatures, on lowering of the
reaction temperature from 50 to 25 ◦C and then to 4 ◦C, a con-
siderable decrease in reaction rate was observed, without a drop
in E (Table 5). Racemic 2 and enantiomerically enriched 4 were
also present in the reaction mixtures (≤7% mole fractions). Finally,
25 ◦C was chosen as reaction temperature for the preparative-scale
reaction.
a
0.015 M substrate, 30 mg mL−1 PS-IM, iPr2O, 50 ◦C, after 5 min.
Conversion values calcd. from ee1 (unreacted substrate) and ee6 (product) val-
b
ues.
c
According to GC (Section 4).
Table 4
Solvent screening for the butyrylation of (3S*,4R*)-1.a
Since the small-scale butyrylation of (3S*,4R*)-1 at 25 ◦C slowed
considerably at around 47% conversion, and E also decreased
slightly (ee1 = 92%, ee6 = 94%, conv. = 49%, after 40 min), increased
amounts of acyl donor (10 and 20 vs. 6 equiv.) were tested
(10 equiv.: ee1 = 95%, ee6 = 98%, conv. = 49%, E > 200, after 30 min;
20 equiv.: ee1 = 97%, ee6 = 97%, conv. = 50%, E > 200, after 20 min).
Finally, 10 equiv. of VB was selected for the preparative-scale
butyrylation.
On the basis of the preliminary results, the preparative-scale
resolutions of (3S*,4R*)-1 were performed under the optimized
conditions (see footnote to Table 6); and the results are reported in
Table 6 and the Section 4.
Acidic hydrolysis of enantiomeric (3R,4S)-6 (ee = 99%) and
(3S,4R)-1 (ee = 98%) (Scheme 5) resulted in the desired amino
acid hydrochlorides [(2R,3S)-3·HCl and (2S,3R)-3·HCl], [(2R,3S)-
3-phenylisoserine, the key intermediate for the taxol side-chain
[(2R,3S)-3·HCl, ee = 99%], being one of them.
c
c
Solvent
Conv.b (%)
ee1 (%)
ee6 (%)
E
Toluene
t-BuOMe
2-Me-THF
26
48
23
34
87
28
99
93
96
>200
78
64
0.015 M substrate, lipase PS-IM (30 mg mL−1), 6 equiv. of VB, in the solvent
a
tested, 50 ◦C, after 5 min.
b
Conversion values calcd. from ee1 (unreacted substrate) and ee6 (product) val-
ues.
c
According to GC (Section 4).
enriched 4 (ee ≤ 54%) (∼3% mole fraction) were also detected in the
reaction mixtures besides to the desired products [(3R,4S)-6 and
(3S,4R)-1] (Table 3 and Supporting information, S2).
In order to prolong the reaction (the time needed to reach 50%
conversion), solvent screening (toluene, t-BuOMe and 2-Me-THF)
was performed (Table 4). Racemic 2 and enantiomerically enriched
4 were also detected in the reaction mixtures (≤6% mole fractions).
Table 6
Lipase PS-IM-catalyzed preparative-scale acylations of (3S*,4R*)-( )-1.
Acyl donor Reaction time (min) Conv.a (%)
E
Product enantiomer
Yield (%) Isomer
22
(3R,4S)-2 99f
Unreacted enantiomer
25
25
eeb (%) [␣]D EtOH Yield (%) Isomer
eeb (%) [␣]D (EtOH)
(3S*,4R*)-( )-1c VA
(3S*,4R*)-( )-1i VB
30
20
48(54d)
50
nde
>200 46
+27g
+35j
38
46
(3S,4R)-1 91
(3S,4R)-1 98
−36h
−46k
(3R,4S)-6 99
a
Values calcd. from ee1 (unreacted substrate) and ee2 or ee6 (product) values.
b
c
d
e
f
Determined by GC (Section 4 and Supporting information, S1 and S2).
6 equiv. of VA; lipase PS-IM (10 mg mL−1) in iPr2O at 25 ◦C.
Overall conversion.
Not determined.
Recrystallized three times from Et2O.
c 0.30.
g
h
i
c 0.26.
10 equiv. of VB; lipase PS-IM (30 mg mL−1) in iPr2O at 25 ◦C.
j
c 0.30.
c 1.325.
k

104
E. Forró et al. / Journal of Molecular Catalysis B: Enzymatic 116 (2015) 101–105
Optical rotations were measured with a Perkin-Elmer 341
polarimeter. 1H NMR spectra were recorded on a Bruker Avance
DRX 400 spectrometer (Supporting information, Figs. S4–S7). Melt-
ing points (m.p.s) were determined on a Kofler apparatus.
4.2. Gram-scale acetylation of racemic N-hydroxymethylated
cis-3-acetoxy-4-phenylazetidin-2-one [(3S*,4R*)-1]
(3S*,4R*)-1 (200 mg, 0.85 mmol) was dissolved in iPr2O (40 mL).
Lipase PS-IM (400 mg, 10 mg mL−1) and VA (487 ␮L, 5.10 mmol)
were added and the mixture was shaken in an incubator shaker
at 25 ◦C for 30 min. The reaction was stopped by filtering off the
enzyme at 54% overall conversion. The solvent was then evapo-
rated off, and the residue was subjected to column chromatography
(EtOAc:n-hexane 1:1). The resulting diacetylated product (3R,4S)-2
was crystallized out and then recrystallized three times from Et2O
[49 mg, 22%; [˛]D25 = +27 (c 0.30, EtOH); ee = 99%; m.p.: 75–77 ◦C].
The unreacted (3S,4R)-1 was similarly crystallized out from Et2O
[75 mg, 38%; [˛]D25 = −36 (c 0.26, EtOH); ee = 91%; m.p.: 61–63 ◦C].
The 1H NMR (400 MHz, CDCl3, 25 ◦C, TMS) data for (3S,4R)-1
were similar to those for (3S*,4R*)-1: ı = 1.67 (s, 3H, CH3), 4.44–4.47
(d, J = 11.5 Hz, 1H, CHAH), 5.06–5.13 (d, J = 11.5 Hz, 1H, CHBH),
5.13–5.18 (d, J = 4.8 Hz, 1H, CH2OH) 5.82–5.87 (d, J = 4.8 Hz, 1H,
CH2OH) 7.27–7.40 (m, 5H, C6H5). Analysis: calcd. for C12H13NO4: C,
61.27; H, 5.57; N, 5.95; found: C, 61.34; H, 5.54; N, 5.81.
Scheme 5. Preparation of (2R,3S)-3·HCl and (2S,3R)-3·HCl.
The absolute configurations were proved by comparing the ˛
values with the literature data; the lit [25] ˛ value for (2R,3S)-
3-phenylisoserine {[˛]D25 = −15 (c 0.25, 6N HCl), ee > 98%} is in
good accordance with the value measured for enantiomeric 3, pre-
pared from diacetate 6 (Scheme 5) under the same conditions
{[˛]D25 = −15 (c 0.25, 6 N HCl), ee = 99%}. The lipase PS-IM catalyzed
acylation of (3S*,4R*)-1 therefore displayed 4S selectivity.
3. Conclusions
A
new enzyme-catalyzed strategy has been devised for
the synthesis of enantiomeric (ee = 99%) 3-amino-3-phenyl-2-
hydroxypropionic acid [(2R,3S)-3], a key intermediate for the taxol
side-chain. B. cepacia lipase PS-IM catalyzed the S-selective acy-
lation of (3S*,4R*)-1 with VB (10 equiv.) in iPr2O at 25 ◦C with
excellent enantioselectivity (E > 200).
It should be highlighted that the possibility of intramolecular
acyl migration during an enzymatic transformation of substrates
containing both OCOR and OH functions needs to be carefully inves-
tigated.
The 1H NMR (400 MHz, CDCl3, 25 ◦C, TMS) data for (3R,4S)-
2: ı = 1.68 (s, 3H, CH2CH3), 1.99 (s, 3H, CHCH3), 4.94–4.99 (d,
J = 11.36 Hz, 1H, CHAH), 5.05–5.09 (d, J = 4.76 Hz, 1H, CH2OH),
5.38–5.43 (d, J = 10.60 Hz, 1H, CHBH), 5.84–5.88 (d, J = 4.88 Hz, 1H,
CH2OH), 7.26–7.40 (m, 5H, C6H5). Analysis: calcd. for C14H15NO5:
C, 60.64; H, 5.45; N, 5.05; found: C, 60.66; H, 5.44; N, 5.08.
4.3. Gram-scale butyrylation of racemic N-hydroxymethylated
cis-3-acetoxy-4-phenylazetidin-2-one
4. Experimental
Via the procedure described above, the reaction of racemic
(3S*,4R*)-1 (100 mg, 0.43 mmol) and VB (545 ␮L, 4.30 mmol) in
4.1. Materials and methods
iPr2O (10 mL) in the presence of Lipase PS-IM (300 mg, 30 mg mL−1
)
Lipase PS-IM (immobilized on diatomaceous earth) was from
Amano Enzyme Europe Ltd. CAL-B (lipase B from C. antarctica), pro-
duced by the submerged fermentation of a genetically modified
Aspergillus oryzae microorganism and adsorbed on a macroporous
resin (Catalogue No. L4777), was from Sigma-Aldrich. CAL-A (lipase
A from C. antarctica) was purchased from Roche Diagnostics Corpo-
ration. Lipase AK (P. fluorescens) was from Amano Pharmaceuticals,
and lipase AY (C. rugosa) was from Fluka. The solvents were of the
highest analytical grade.
In a typical small-scale experiment, racemic substrate (0.015 M
solution) in an organic solvent (1 mL) was added to the lipase tested
(30 mg mL−1). The acyl donor (1.2, 6, 10 or 20 equiv.) was next
added. The mixture was shaken at 4, 25 or 50 ◦C. The progress
of the reaction was followed by taking samples from the reac-
tion mixture at intervals and analysing them by GC on an l-Val
column (25 m) [80 ◦C for 5 min → 160 ◦C for 40 min (temperature
rise 10 ◦C min−1) → 190 ◦C (temperature rise 20 ◦C min−1); 70 kPa;
retention times (min), (3S,4R)-1: 40.73 (antipode: 39.07); (3R,4S)-
2: 30.84 (antipode: 29.88); (3S,4R)-4: 61.04 (antipode: 60.11);
(3R*,4S*)-5: 35.25 (antipode: 35.93); (3R,4S)-6: 50.00 (antipode:
47.58)]. The ee values for the ˇ-amino acids prepared were deter-
mined by a GC method [38] on a Chrompack Chirasil-Dex CB
column after a simple and rapid double derivatization with (i)
CH2N2 (caution! derivatization with diazomethane should be per-
formed under a well-working hood); (ii) Ac2O in the presence of
4-dimethylaminopyridine and pyridine [140 ◦C for 7 min → 190 ◦C
(temperature rise 10 ◦C min−1; 100 kPa; retention times (min),
(2R,3S)-3: 19.01 (antipode: 18.70)] (Supporting information, S3).
at 25 ◦C afforded the diacylated product (3R,4S)-6 [59 mg, 46%;
[˛]D25 = +35 (c 0.30, EtOH); ee = 99%; m.p.: 61.5–63.5 ◦C (crystallized
out from Et2O)] and unreacted (3S,4R)-1, [46 mg, 46%; [˛]D25 = −46
(c 1.325, EtOH); ee = 99%; m.p.: 61–63 ◦C (crystallized out from
Et2O)] after 20 min.
The 1H NMR (400 MHz, CDCl3, 25 ◦C, TMS) data for (3S,4R)-1 in
the butyrylation reaction were similar to those for (3S,4R)-1 in the
acetylation reaction.
The 1H NMR (400 MHz, CDCl3, 25 ◦C, TMS) data for (3R,4S)-
6: ı = 0.86–0.93 (t, J = 7.4 Hz, 3H, CH2CH2CH3), 1.50–1.63 (m, 2H,
CH2CH2CH3), 1.67 (s, 3H, OCOCH3), 2.15–2.29 (m, 2H, CH2CH2CH3),
4.94–5.01 (d, J = 11.2 Hz, 1H, CHAH), 5.04–5.08 (d, J = 4.9 Hz, 1H,
CH2OCO), 5.37–5.43 (d, J = 11.24 Hz, 1H, CHBH), 5.83–5.87 (d,
J = 4.89 Hz, 1H, CH2OCO), 7.27–7.40 (m, 5H, C6H5). Analysis: calcd.
for C16H19NO5: C, 62.94; H, 6.27; N, 4.59; found: C, 62.90; H, 6.30;
N, 4.61.
4.4. Preparation of (2R,3S)- and (2S,3R)-3-phenylisoserine
hydrochlorides [(2R,3S)-3·HCl and (2S,3R)-3·HCl]
Enantiomeric (3R,4S)-6 (49 mg, 0.16 mmol) or (3S,4R)-1 (53 mg,
0.23 mmol) was dissolved in 18% HCl (20 mL) and refluxed for
5 h. The solvent was then evaporated off, and the product was
recrystallized from EtOH and Et2O, which afforded white crystals
of (2R,3S)-3·HCl [33 mg, 95%, [˛]D25 = −15 (c 0.25, 6 N HCl), m.p.
215–217 ◦C, ee = 99%] or (2S,3R)-3·HCl [48 mg, 95%, [˛]D25 = +14.6
(c 0.33, 6 N HCl), m.p. 215–217 ◦C, ee = 99%].

E. Forró et al. / Journal of Molecular Catalysis B: Enzymatic 116 (2015) 101–105
105
The 1H NMR (400 MHz, D2O, 25 ◦C, TMS) data for (2R,3S)-3·HCl
were similar to those for (2S,3R)-3·HCl: ı = 4.36–4.37 [d, J = 5.9 Hz,
1H, CH(OH)(COOH)], 4.59–4.61 (d, J = 5.9 Hz, 1H, CHNH2), 7.43–7.58
(m, 5H, C6H5). Analysis: calcd. for C9H12ClNO3: C, 49.67; H, 5.56; N,
6.44; Analysis: found for (2R,3S)-3·HCl: C, 49.48; H, 5.61; N, 6.38.
Analysis: found for (2S,3R)-3·HCl: C, 49.50; H, 5.59; N, 6.44.
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Supplementary data associated with this article can be
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