Enantioselective hydrolysis of 3,4-disubstituted β-lactams. An efficient enzymatic method for the preparation of a key Taxol side-chain intermediate

Article abstract of DOI:10.1016/j.molcatb.2015.11.011

3,4-Disubstituted β-lactams 3-benzyloxy-4-(4-chlorophenyl)azetidin-2-one [(3S?,4R?)-(±)-1], 3-benzyloxy-4-phenylazetidin-2-one [(3S?,4R?)-(±)-2] and 4-(4-chlorophenyl)-3-phenoxyazetidin-2-one [(3S?,4R?)-(±)-3] were resolved through immobilized CAL-B-catalysed ring-cleavage reactions. Excellent enantioselectivities (E > 200) were obtained for (3S?,4R?)-(±)-1 and (3S?,4R?)-(±)-2 when the reactions were performed with added H₂O as nucleophile in tert-butyl methyl ether at 70 °C, whereas only moderate E (12) was achieved for (3S?,4R?)-(±)-3 under the same conditions but in diisopropyl ether. The resulting ring-opened β-amino acids [(2R,3S)-4 (ee > 98%), (2R,3S)-5 (ee > 98%) and (2R,3S)-6 (ee = 50%)] and the unreacted β-lactams [(3S,4R)-1-3] (ee > 98%) could be easily separated.

Full text of DOI:10.1016/j.molcatb.2015.11.011

Accepted Manuscript
Title: Enantioselective hydrolysis of 3,4-disubstituted
␤-lactams. An efficient enzymatic method for the preparation
of a key Taxol side-chain intermediate
˝
´
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Author: Zsolt Galla Ferenc Beke Eniko Forro Ferenc Fulo¨p
PII:
S1381-1177(15)30104-1
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.molcatb.2015.11.011
MOLCAB 3275
To appear in:
Journal of Molecular Catalysis B: Enzymatic
Received date:
Revised date:
Accepted date:
2-9-2015
6-11-2015
6-11-2015
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Please cite this article as: Zsolt Galla, Ferenc Beke, Eniko Forro, Ferenc Fulddotop,
Enantioselective hydrolysis of 3,4-disubstituted rmbeta-lactams.An efficient enzymatic
method for the preparation of a key Taxol side-chain intermediate, Journal of Molecular
Catalysis B: Enzymatic http://dx.doi.org/10.1016/j.molcatb.2015.11.011
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Enantioselective hydrolysis of 3,4-disubstituted β-lactams. An efficient
enzymatic method for the preparation of a key Taxol side-chain
intermediate
Zsolt Galla1, Ferenc Beke1, Enikő Forró1*, Ferenc Fülöp1,2*
1Institute of Pharmaceutical Chemistry
2Stereochemistry Research Group of the Hungarian Academy of Sciences University of
Szeged, H-6720 Szeged, Eötvös u. 6, Hungary
1

GRAPHICAL ABSTRACT
2

Highlights
 CAL-B-catalysed ring-cleavage of 3,4-disubstituted β-lactams (E > 200).
 A suitable method for the preparation of Taxol® side-chain was developed.
 Debenzylation was performed in a continuous flow system by using 10% Pd/C.
Abstract
3,4-Disubstituted β-lactams 3-benzyloxy-4-(4-chlorophenyl)azetidin-2-one [(3S*,4R*)-(±)-1],
3-benzyloxy-4-phenylazetidin-2-one and 4-(4-chlorophenyl)-3-
[(3S*,4R*)-(±)-2]
phenoxyazetidin-2-one [(3S*,4R*)-(±)-3] were resolved through immobilized CAL-B-
catalysed ring-cleavage reactions. Excellent enantioselectivities (E > 200) were obtained for
(3S*,4R*)-(±)-1 and (3S*,4R*)-(±)-2 when the reactions were performed with added H2O as
nucleophile in tert-butyl methyl ether at 70 ºC, whereas only moderate E (12) was achieved
for (3S*,4R*)-(±)-3 under the same conditions but in diisopropyl ether. The resulting ring-
opened β-amino acids [(2R,3S)-4 (ee > 98%), (2R,3S)-5 (ee > 98%) and (2R,3S)-6 (ee =
50%)] and the unreacted β-lactams [(3S,4R)-1–3] (ee > 98%) could be easily separated.
Keywords: Candida antarctica lipase B; ring-cleavage; enzyme catalysis; Taxol; ß-amino acid
3

1. Introduction
A large number of recent published articles and reviews have stressed the biological and
chemical importance of β-lactams and β-amino acids [1]. Molecules containing a 2-
azetidinone ring may possess antibacterial activity, e.g. carumonam is a β-lactamase-resistant
monobactam antibiotic [2], while others containing a cis 3,4-disubstituted β-lactam ring may
display PPAR α/γ agonist [3], vasopressin VIa agonist [4] or anticancer [5,6] activity. β-
Amino acids and some of their derivatives are widely used in combinatorial, peptide, organic
and medicinal chemistry [7-9]. Numerous non-proteinogenic amino acids are available can
serve as relevant components of fibrinogen receptor antagonists [10]. Taxol®, one of the most
efficient anticancer agents of the past decade [11,12], contains (2R,3S)-3-amino-3-phenyl-2-
hydroxypropanoic acid [(2R,3S)-7] in its side-chain. Since the total synthesis of Taxol is a
very lengthy and expensive process [13,14], chemists are continuously working on the
development of semi-synthetic methods which involve coupling of the C(13)-O of baccatin III
derivatives [15] to the corresponding side-chain.
Earlier enzymatic studies on the ring opening of a set of cyclic and acyclic β-lactams [16-19]
were continued with successful enzymatic syntheses of a Taxol side-chain key intermediate
through the enantioselective ring opening of racemic cis-3-hydroxy-4-phenylazetidin-2-one
(0.5 equiv. of H2O in t-BuOMe at 60 °C, with immobilized CAL-B) and sequential kinetic
resolution of racemic cis-3-acetoxy-4-phenylazetidin-2-one (1 equiv. of H2O in iPr2O at 60
°C, with immobilized CAL-B) [20]. To extend the substrate scope, and also to analyse how
different-sized substituents on C3 or C4 influence the ring cleavage of β-lactams, in the
present work we set out to develop immobilized CAL-B-catalysed methods for the enzymatic
ring opening of racemic 3,4-disubstituted β-lactams, such as 3-benzyloxy-4-(4-
chlorophenyl)azetidin-2-one, 3-benzyloxy-4-phenylazetidin-2-one and 4-(4-chlorophenyl)-3-
phenoxyazetidin-2-one [(3S*,4R*)-(±)-1–3] (Scheme 1), and then to synthetize (2R,3S)-3-
phenylisoserine (2R,3S)-7, the key intermediate of the Taxol side-chain, from the
corresponding enantiomeric compound.
4

2. Results and discussion
2.1. Synthesis of (3S*,4R*)-(±)-1–3
Racemic β-lactams (3S*,4R*)-(±)-1–3 were synthesized according to a literature method [21].
A mixture of p-ethoxyaniline and the appropriate aldehyde furnished the Schiff bases (Z)-N-
(4-chlorobenzylidene)-4-ethoxybenzenamine
(10)
and
(Z)-N-benzylidene-4-
ethoxybenzenamine (11), which, through cycloadditions in the presence of the appropriate
acyl chlorides, 2-phenoxyacetyl chloride (8) or 2-benzyloxyacetyl chloride (9), resulted in the
N-protected β-lactams 12–14. CAN-mediated oxidative removal of the 4-ethoxyphenyl groups
gave the desired β-lactams 1–3 (Scheme 2).
2.2. Immobilized CAL-B-catalysed ring-opening of (3S*,4R*)-(±)-1–3
In earlier studies, immobilized CAL-B proved to be applicable for the enantioselective (E >
200) ring opening of both 4-aryl-substituted [17] and carbocyclic β-lactams [22], and we
therefore carried out the ring opening of model compound (3S*,4R*)-(±)-1 with 1 equiv. of
H2O in iPr2O at 60 °C, with immobilized CAL-B as catalyst (Table 1, entry 1).
In order to find the optimum conditions for the gram-scale resolution of (3S*,4R*)-(±)-1,
solvent screening (Table 1, entries 1–6) was first performed in order to determine the effects
on E and the reaction rate. Practically, no reaction was detected during 65 h when the
reactions were performed in THF (entry 4) or 2-Me-THF (entry 5). The reactions proceeded
enantioselectively (E > 200), but slowly in t-BuOMe and iPr2O (conv. = 5–8% after 65 h)
(entries 1 and 6) and with somewhat higher conversions in toluene (conv. = 15% after 65 h, E
= 32) (entry 2) or n-hexane (conv. = 17% after 65 h, E = 39) (entry 3). In view of the results,
t-BuOMe was chosen for further preliminary experiments.
H2O, as a nucleophile, is essential for the ring-opening reaction, through its quantity in the
reaction medium can affect the enzymatic activity [18,22]. Experiments were therefore also
performed with different quantities of added H2O (Table 1, entries 7–10 and 12-15). On
increase of the amount of H2O up to 50 equiv., the reactions became faster without a drop in E
(entries 8–10), but a further increase of the H2O content resulted in considerably decreases in
5

both reaction rate and E (entries 12–15). It is noteworthy that, in accordance with our earlier
observation that a hydrolytic reaction proceeded even without added H2O in the reaction
mixture (due to the H2O present in the reaction medium) [22], the quantity of H2O present in
the reaction medium (< 0.1%) or at the surface of the immobilized CAL-B (2–5%) was
sufficient for the ring cleavage of (±)-1 (entry 7). Finally, 25 equiv. of H2O was chosen as the
optimum quantity.
On increase of the temperature of the ring-opening reaction from 60 °C (Table 1, entry 10) to
70 °C, the reaction rate increased without any decrease in enantioselectivity (Table 1, entry
11). Accordingly, 70 °C was chosen as the reaction temperature.
The above-optimized reaction conditions (25 equiv. of H2O, t-BuOMe, 70 °C) were next
applied for the ring cleavage of (±)-2 and (±)-3. Excellent results were observed for (±)-2 (E >
200), but a very poor E (5) for (±)-3 (Table 2, entry 1). We therefore continued the
optimizations for (±)-3 with a new solvent screening, changing the amount of added H2O and
also the temperature of the reaction (Table 2).
The reactions in toluene and n-hexane proceeded relatively slowly, with low E (entries 2 and
3) while in MeCN and THF the enzyme did not display activity during 65 h (entries 6 and 7).
A slightly increased E (8) was noted in iPr2O vs. t-BuOMe (E = 2) (entries 4 and 5). Variation
of the quantity of water (from 2 to 100 equiv., entries 8–11) and temperature (50 and 70 °C,
entries 12 and 13) led to the same results as observed earlier for (±)-1. In summary, E was
increased slightly (E = 14, entry 13) when the reaction was carried out with 25 equiv. of water
in iPr2O at 70 °C.
On the basis of the preliminary results, the immobilized CAL-B-catalysed preparative-scale
ring-opening reactions of (±)-1 and (±)-2 were performed with 25 equiv. of H2O in t-BuOMe
at 70 °C, while the preparative-scale resolution of (±)-3 was performed with 25 equiv. of H2O
in iPr2O at 70 °C. In order to obtain (2R,3S)-6 with a good ee value, the reaction was overrun
to 66% conversion. The results are reported in Table 3 and in Section 3 (Experimental part).
6

2.3. Synthesis of Taxol side-chain intermediate
To prepare (2R,3S)-3-amino-3-phenyl-2-hydroxypropanoic acid [(2R,3S)-7], the key
intermediate of Taxol, the debenzylation of (2R,3S)-5 (ee = 99%) was performed in a
continuous flow system (H-CUBE®) by using a CatCart® filled with 10% Pd/C, operating at a
flow rate of 0.1 mL/min, 50 bar, 40 °C (Scheme 4). Thus, (2R,3S)-7 was obtained with good
ee (99%) and in nearly quantitative yield (93%) after four cycles. The absolute configuration
for the enantiomeric 7 obtained was proved by comparing the literature [20] [α] value for
25
(2R,3S)-3-amino-3-phenyl-2-hydroxypropanoic acid {[α]D = -7.2 (c = 0.34, H2O), ee >
25
99%} with the [α] value measured for enantiomeric 7 {[α]D = -7.2 (c = 0.34, H2O), ee =
99%}. Thus, immobilized CAL-B catalysed the ring opening of (3S*,4R*)-(±)-2 with (2R,3S)
selectivity, while for (3S*,4R*)-(±)-1 and (3S*,4R*)-(±)-3 the analysed chromatograms
indicated the same enantiopreference for immobilized CAL-B.
2.4. Conclusions
An efficient enzymatic method was developed for the ring opening of 3,4-disubstituted β-
lactams (3S*,4R*)-(±)-1–3. High enantioselectivities (E > 200) were obtained for the ring-
opening reactions of (3S*,4R*)-(±)-1 and (3S*,4R*)-(±)-2 when immobilized CAL-B was used
as catalyst, with 25 equiv. of H2O as nucleophile, in t-BuOMe at 70 °C, while a relatively
modest E (12) was obtained for immobilized CAL-B-catalysed ring opening of (3S*,4R*)-(±)-
3 in iPr2O with 25 equiv. of H2O at 70 ºC. The great differences in E for (±)-1 and (±)-2 vs.
(±)-3 are presumably consequences of the very different steric hindrance of BzO vs. PhO,
which influences the accommodation for the enantiomers in the active site of immobilized
CAL-B. The products could be easily separated. The present enzymatic method proved
suitable for the preparation of (2R,3S)-3-amino-3-phenyl-2-hydroxypropanoic acid [(2R,3S)-
7], a key intermediate for the Taxol® side-chain.
7

3. Experimental section
3.1. Materials and methods
Immobilized CAL-B (lipase B from Candida antarctica) immobilized on acrylic resin
(L4777) was purchased from Sigma. All solvents were of the highest analytical grade. In a
typical small-scale experiment, immobilized CAL-B (30 mg), then H2O (1, 2, 10, 25, 50, 100
or 1850 equiv.) were added to the racemic substrate (0.015 M solution) in an organic solvent
(1 mL). The mixture was shaken (167 rpm) at 50, 60 or 70 °C. The progress of the reactions
was followed by taking samples from the reaction mixtures and analysing them by HPLC
with a chiral column. The ee values for the unreacted β-lactams (3S,4R)-1 and (3S,4R)-3 and
the product β-amino acid (2R,3S)-6 [after pre-column derivatization [23] with CH2N2
(Caution! derivatization with CH2N2 should be performed under a well-working hood)] were
determined on a Chiralpak IA column (4.6 × 250 mm); detection at 228 nm; eluent: n-
hexane/Et2N/iPA (90/0.1/10); flow rate: 0.5 mL min-1; retention times (min) for (3S,4R)-1:
27.86 (antipode: 25.51), (3S,4R)-3: 25.33 (antipode: 22.55), (2R,3S)-6: 32.43 (antipode:
27.08). (2R,3S)-4 and (2R,3S)-5 [after pre-column derivatization with CH2N2]; Chiralpak IA
column (4.6 × 250 mm); detection at 228 nm; eluent: n-hexane/Et2N/iPA (50/0.1/50); flow
rate: 0.5 mL min-1; retention times (min) for (2R,3S)-4: 13.80 (antipode: 11.50), (2R,3S)-5:
12.36 (antipode: 10.61). (3S,4R)-2: Chiralpak IA column (4.6 × 250 mm); detection at 228
nm; eluent: n-hexane/Et2N/iPA (50/0.1/50); flow rate: 0.5 mL min-1; retention times (min) for
(3S,4R)-2: 9.09 (antipode: 9.79).The ee value for the Taxol key intermediate (2R,3S)-7
prepared was determined by a GC method on a Chrompack Chirasil-Dex CB column after
double derivatization [23] with (i) CH2N2; (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)-7: 19.01 (antipode: 18.70)] (Supporting
Information S1–S7).
1
All melting points were measured on an X-4 melting-point apparatus with a microscope. H
NMR spectra were recorded on a Bruker Avance DRX 400 spectrometer in CDCl3 D2O and
CD3OD. 10% Pd/C CatCart® was from ThalesNano (03378 and the product ID: THS 01111).
Optical rotations [α] were measured with a Perkin-Elmer 341 polarimeter.
8

3.2. Synthesis of 3-benzyloxy-4-(4-chlorophenyl)azetidin-2-one [(±)-1]
A solution of benzyloxyacetyl chloride (8, 0.23 mL, 1.5 mmol) in dry CH2Cl2 was slowly
added to a solution of 4-chlorobenzylidene-4-ethoxyphenylamine (11, 0.26 g, 1.0 mmol) and
Et3N (0.42 mL, 3.0 mmol) in CH2Cl2 (20 mL) at -10 °C. The reaction was then allowed to
warm up to room heat, stirred for 12 h, washed with NaHCO3 solution (20 mL) and brine (20
mL), then dried (Na2SO4) and evaporated. The product 3-benzyloxy-4-(4-chlorophenyl)-1-(4-
ethoxyphenyl)azetidin-2-one (12) was recrystallized from EtOAc [265 mg, 65%; m.p. 166–
168 °C]. A solution of CAN (0.75 g, 1.4 mmol) in H2O (15 mL) was added dropwise to the β-
lactam solution (12, 0.2 g, 0.5 mmol) in MeCN (15 mL) at 0 °C. The reaction was stirred at 0
°C for 30 min, 15 mL H2O was then added and the mixture was extracted with EtOAc (3 × 20
mL) and washed with 10% aqueous NaHCO3 (20 mL). The organic layer was combined and
washed with 10% Na2SO3 (2 × 15 mL), 10% NaHCO3 (10 mL), and brine (20 mL), and dried
with Na2SO4. After filtration, the solvent was evaporated off, and the product 3-benzyloxy-4-
(4-chlorophenyl)azetidin-2-one (1) was recrystallized from EtOAc [76 mg, 53%; m.p. 199–
1
201 °C]. This product was described in 1998, but no H NMR data and m.p. were then
reported [24].
1H NMR (400 MHz, DMSO, TMS) δ (ppm) for (±)-1: 4.11–4.17 (d, J = 11.64 Hz, 1H, C3H);
4.29–4.35 (d, J = 11.16); 4.86–4.90 (d, J = 4.64 Hz, 1H, CH2); 4.93–4.98 (d, J = 4.2 Hz, 1H,
CH2); 6.88–6.95 (m, 2H, Ar); 4.19–4.26 (m, 3H, Ar); 7.35–7.48 (dd, J = 8.46 Hz, 4H, Ar);
8.63–8.69 (bs, 1H, NH). Analysis: calcd. For C16H14ClNO2: C, 66.79; H, 4.90; N, 4.87;
Analysis: found for (3S*,4R*)-(±)-1: C, 66.81; H, 4.87; N, 4.89.
3.3. Synthesis of 3-benzyloxy-4-phenylazetidin-2-one [(±)-2]
Compound 13 was prepared from benzyloxyacetyl chloride (8, 0.23 mL, 1.5 mmol) and
benzylidene-4-ethoxybenzenzamine (10, 0.23 g, 1.0 mmol) according to the procedure
described in Section 3.2. [254 mg, 68%; m.p. 145–147 °C]. Removal of the 4-ethoxyphenyl
group gave the desired β-lactam (±)-2 [73 mg, 58%; m.p. 202–204 °C {lit [25]: m.p. = 188–
189 ºC}].
9

1H NMR (400 MHz, DMSO, TMS) δ (ppm) for (±)-2: 4.09–4.14 (d, J = 11,06 Hz, 1H, C3H);
4.25–4.30 (d, J = 11.44 Hz, 1H, C4H); 4.86–4.89 (d, J = 4.44 Hz, 1H, CH2); 4.93–4.96 (m,
1H, CH2); 6.84–6.89 (m, 2H, Ar); 6.85–6.89 (m, 3H, Ar); 7.33–7.41 (m, 5H, Ar); 8.61–8.67
(bs, 1H, NH). Analysis: calcd. For C16H15NO2: C, 75.87; H, 5.97; N, 5.53; Analysis: found for
(3S*,4R*)-(±)-2: C, 75.89; H, 5.95; N, 5.55.
3.4. Synthesis of 4-(4-chlorophenyl)-3-phenoxyazetidin-2-one [(±)-3]
Compound 14 was prepared from phenoxyacetyl chloride (9, 2.07 mL, 15 mmol) and 4-
chlorobenzylidene-4-ethoxyphenylamine (11, 2.6 g, 10 mmol) according to the procedure
described in Section 3.2. [2.88 g, 80%; m.p. 170–172 °C]. Removal of the 4-ethoxyphenyl
group gave the desired β-lactam (±)-3 [434 mg, 53%; m.p. 192–193 °C {lit [21]: m.p. = 188–
190 ºC}].
1H NMR (400 MHz, DMSO, TMS) δ (ppm) for (±)-3: 5.10–5.14 (d, J = 4.6 Hz, 1H, C3H);
5.59–5.65 (dd, J = 2.12 & 4.54, 1H, C4H); 6.76–6.83 (d, J = 8 Hz, 2H, Ar); 6.86–6.93 (m,
1H, Ar); 7.12–7.22 (m, 2H Ar); 7.29–7.37 (m, 4H, Ar); 8.83–8.91 (bs, 1H, NH). Analysis:
calcd. For C15H12ClNO2: C, 65.82; H, 4.42; N, 5.12; Analysis: found for (3S*,4R*)-(±)-3: C,
65.83; H, 4.40; N, 5.15.
3.5. Preparative-scale resolution of racemic 3-benzyloxy-4-(4-chlorophenyl)azetidin-2-
one [(±)-1]
Racemic 1 (300 mg, 1.05 mmol) was dissolved in t-BuOMe (40 mL), immobilized CAL-B
(2.0 g, 30 mg mL-1) and H2O (375 µL, 20.83 mmol) were added, and the mixture was stirred
at 70 °C for 144 h. The reaction was stopped by filtering off the enzyme at 50% conversion.
The solvent was evaporated off, affording the unreacted β-lactam (3S,4R)-1 {105 mg, 35%,
25
0.37 mmol, ee = 98%; [α]D = -20 (c 0.3; CHCl3); m.p. = 188–190 °C}. The filtered
immobilized CAL-B was washed with distilled H2O (3 × 15 mL), and the H2O was
25
evaporated off. The crystalline β-amino acid was (2R,3S)-4 {96 mg, 30%; ee = 99%; [α]D
+38 (c 0.1; MeOH); m.p. = 238–240 °C}.
=
The 1H NMR (400 MHz, DMSO, TMS) δ (ppm) data for (3S,4R)-1 were the same as those for
(±)-1.
10

1H NMR (400 MHz, CD3OD, TMS) δ (ppm) for (2R,3S)-4: 3.96–4.01 (d, J = 5.08 Hz, 1H,
C2H); 4.41–4.46 (d, J = 11.6 Hz, 1H, C3H); 4.49–4.62 (d, J = 4.88 Hz, 1H, CH2) overlapping
with 4.52–4.55 (bs, 2H, NH2); 4.77–4.79 (s, 1H, CH2); 7.25–7.33 (m, 4H, Ar); 7.37–7.46 (m,
5H, Ar). Analysis: calcd. For C16H16ClNO3: C, 62.85; H, 5.27; N, 4.58; Analysis: found for
(2R,3S)-4: C, 62.87; H, 5.29; N, 4.55.
3.6. Preparative-scale resolution of racemic 3-benzyloxy-4-phenylazetidin-2-one [(±)-2]
Racemic 2 (200 mg, 0.79 mmol) was dissolved in t-BuOMe (30 mL), immobilized CAL-B
(1.5 g, 30 mg/mL) and H2O (356 µL, 19.78 mmol) were added, and the mixture was stirred at
70 °C for 24 h. The reaction was stopped by filtering off the enzyme at 50% conversion. The
solvent was evaporated off, affording the unreacted β-lactam (3S,4R)-2 {96 mg, 48%; 0.37
25
mmol, ee = 98%; [α]D = -15 (c 0.21; CHCl3); m.p. = 192–193 °C} . The filtered
immobilized CAL-B was washed with distilled H2O (3 × 15 mL), and the H2O was
25
evaporated off. The crystalline β-amino acid was (2R,3S)-5 {101 mg, 47%; ee = 99%; [α]D
+70 (c 0.3; EtOH); m.p. = 218–222 °C}.
=
The 1H NMR (400 MHz, DMSO, TMS) δ (ppm) data for (3S,4R)-2 were the same as those for
(±)-2.
1H NMR (400 MHz, CD3OD, TMS) δ (ppm) for (2R,3S)-5: 4.02–4.05 (d, J = 5.2 Hz, 1H,
C2H); 4.41–4.46 (d, J=11.52 Hz, 1H, C3H); 4.49–4.52 (d, J = 8.0 Hz, 1H, CH2) overlapping
with 4.51–4.54 (bs, 2H, NH2); 4.76–4.78 (s, 1H, CH2); 7.24–7.32 (m, 5H, Ar); 7.39–7.49 (m,
5H, Ar). Analysis: calcd. For C16H17NO3: C, 70.83; H, 6.32; N, 5.16; Analysis: found for
(2R,3S)-5: C, 70.81; H, 6.32; N, 5.14.
3.7. Preparative-scale resolution of racemic 4-(4-chlorophenyl)-3-phenoxyazetidin-2-one
[(±)-3]
Racemic 3 (200 mg, 0.73 mmol) was dissolved in iPr2O (30 mL), immobilized CAL-B (1.5 g,
30 mg/mL) and H2O (328.5 µL, 18.25 mmol) were added and the mixture was stirred at 70 °C
for 336 h. The reaction was stopped by filtering off the enzyme at 66% conversion. The
solvent was evaporated off, affording the unreacted β-lactam (3S,4R)-3 {32 mg, 16%; 0.12
25
mmol, ee = 98%; [α]D = +45 (c 0.21; CHCl3); m.p. = 194–195 °C} . The filtered
11

immobilized CAL-B was washed with distilled H2O (3 × 15 mL), and the H2O was
25
evaporated off. The crystalline β-amino acid was (2R,3S)-6 {100 mg, 47%; ee = 50%; [α]D
+11 (c 0.2; MeOH); m.p. = 250–258 °C}.
=
The 1H NMR (400 MHz, DMSO, TMS) δ (ppm) data for (3S,4R)-3 were the same as those for
(±)-3.
1H NMR (400 MHz, CD3OD, TMS) δ (ppm) for (2R,3S)-6: 4.51–4.56 (s, 2H, NH2); 4.65–
4.70 (m, 2H, C3H, C4H); 6.92–7.05 (m, 3H, Ar); 7.21–7.30 (m, 2H, Ar); 7.41–7.55 (m, 4H,
Ar). Analysis: calcd. For C15H14ClNO3: C, 61.76; H, 4.84; N, 4.80; Analysis: found for
(2R,3S)-6: C, 61.76; H, 4.86; N, 4.82.
3.8. Debenzylation of (2R, 3S)-5
The debenzylation was carried out in a continuous flow system. (2R,3S)-5 (17 mg) was
dissolved in MeOH (20 mL), and the solution was pumped through the compressed and
heated 10% Pd/C cartridge at a flow rate of 0.1 mL min-1. The pressure was 50 bar, the
temperature 40 °C and the H-CUBE system was in ‘Hydrogen’ mode. After four cycles, the
25
solvent was evaporated off. (2R,3S)-7{11 mg, 97%; ee = 99%; [α]D = -7.1 (c 0.34; H2O)}
these data being approximately equivalent to the literature [20] [α] data for (3S,4R)-5 {ee =
99%; [α]D25 = -7.2 (c 0.34; H2O)}.
1H NMR (400 MHz, D2O, TMS) δ (ppm) for (2R,3S)-7: 4.31–4.37 [d, J = 5.9 Hz, 1H,
CH(OH)(COOH)], 4.55–4.60 (d, J = 5.9 Hz, 1H, CHNH2), 7.40–7.55 (m, 5H, C6H5).
Analysis: calcd. For C9H11NO3: C, 59.66; H, 6.12; N, 7.73; Analysis: found for (2R,3S)-7: C,
59.69; H, 6.10; N, 7.73.
Acknowledgements
The authors acknowledge the receipt of OTKA Grants K-108943, K-108943 and TÁMOP-
4.1.1.C-13/1/KONV-2014-0001 for financial support.
12

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L. N. Gentile, J. H. Liu, J. Am. Chem. Soc.116 (1994) 1597-1598.
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13

[19] E. Forró, F. Fülöp, Tetrahedron: Asymmetry 15 (2004) 2875-2880.
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4375-4386.
14

R1
R2
O
R1
R2
CAL-B
H2O
R1
R2
COOH
NH2
O
+
solvent
NH
HN
(3S*,4R*)-(±)-1-3
(3S,4R)-1-3
(2R,3S)-4-6
1, 4: R1 = BnO, R2 = p-ClPh
2, 5: R1 = BnO, R2 = Ph
3, 6: R1 = PhO, R2 = p-ClPh
Scheme 1. Immobilized CAL-B-catalysed hydrolysis of (±)-1–3.
R1
O
R2
R1
R2
O
R1
R2
O
TEA
CAN
+
CH2Cl2
-10°C
MeCN
0°C
N
NH
N
R3
R3
10,11
Cl
8,9
(3S*,4R*)-(±)-1-3
(3S*,4R*)-(±)-12-14
12: R1 = BnO, R2 = p-ClPh, R3 = p-EtOPh
13: R1 = BnO, R2 = Ph, R3 = p-EtOPh
14: R1 = PhO, R2 = p-ClPh, R3 = p-EtOPh
1: R1 = BnO, R2 = p-ClPh
2: R1 = BnO, R2 = Ph
3: R1 = PhO, R2 = p-ClPh
8: R1 = BnO
9: R1 = PhO
10: R2 = Ph, R3 = p-EtOPh
11 R2 = p-ClPh, R3 = p-EtOPh
Scheme 2. Synthesis of (±)-1–3.
15

CAL-B
H2O
O
O
O
O
O
COOH
NH2
+
Solvent
NH
HN
Cl
Cl
Cl
(3S*,4R*)-(±)-1
(3S,4R)-1
(2R,3S)-4
Scheme 3. Immobilized CAL-B-catalysed ring opening of (±)-1.
H-CUBE
10% Pd/C, H2
0.1 mL min-1
50 bar, 40 °C
HO
COOH
NH2
O
COOH
NH2
(2R,3S)-7
(2R,3S)-5
Scheme 4. Debenzylation of (2R,3S)-5.
16

Table 1. Effects of solvents and the quantities of H2O on the immobilized CAL-B-catalysed
ring cleavage of (±)-1a
c
H2O
Temperature eesb
eep
(%)
99
Conv.
(%)
5
Entry
Solvent
E
(equiv.)
(°C)
60
(%)
1
2
3
4
iPr2O
1
1
1
1
1
5
> 200
32
toluene
n-hexane
THF
60
16
93
15
60
20
94
17
39
60
no reaction
2-Me-
THF
60
5
no reaction
6
t-BuOMe
t-BuOMe
t-BuOMe
1
0
2
60
60
60
60
60
70
60
60
60
60
9
99
99
99
99
99
99
96
96
95
95
8
> 200
> 200
> 200
> 200
> 200
> 200
133
7
5
5
8
10
21
36
54
67
41
45
35
9
9
t-BuOMe 10
t-BuOMe 25
t-BuOMe 25
t-BuOMe 50
t-BuOMe 100
t-BuOMe 1850
18
27
35
41
30
32
27
10
11
12
13
14
15
73
61
H2O
-
55
a 0.015 M substrate, H2O, 30 mg mL-1 immobilized CAL-B, after 65 h.
b According to HPLC (Experimental part).
c According to HPLC after derivatization (Experimental part).
17

Table 2. Immobilized CAL-B-catalysed ring-opening of (±)-3a
Reaction
c
H2O
eesb
eep
Conv.
(%)
Temperature
(°C)
time
Entry
Solvent
E
(equiv.) (%)
(%)
(h)
1
t-BuOMe 120
toluene 65
n-hexane 65
iPr2O 65
t-BuOMe 65
70
60
60
60
60
60
60
60
60
60
60
50
70
25
1
42
7
55
38
53
75
37
43
15
10
10
12
5
2
3
8
2
2
3
1
6
4
1
8
5
1
5
6
MeCN
THF
65
65
1
no reaction
no reaction
7
1
8
t-BuOMe 65
t-BuOMe 65
t-BuOMe 65
t-BuOMe 65
t-BuOMe 65
t-BuOMe 65
2
8
75
10
34
39
76
15
54
8
9
10
25
100
25
25
38
46
70
14
81
73
72
21
78
70
9
10
11
12
13
10
3
9
14
a 0.015 M substrate, H2O, 30 mg mL-1 immobilized CAL-B.
b According to HPLC (Experimental part).
c According to HPLC after derivatization (Experimental part).
18

Table 3. Preparative-scale resolution of (±)-1a, (±)-2aand (±)-3b
β-Lactam
β-Amino acid
Conv.
Time
eec
eed
Substrate
E
Yield
(%)
Yield
Isomer
(%)
(h)
25
25
(%)
Isomer
[α]D
[α]D
(%)
(%)
3S,4R-
2R,3S-
(±)-1
(±)-2
(±)-3
144
24
50
50
66
> 200
> 200
12
35
48
16
98
98
98
-20e
-15g
30
99
99
50
+38f
+70h
+11j
1
4
3S,4R-
2R,3S-
47
2
5
3S,4R-
2R,3S-
336
+45i
61
3
6
a 0.015 M substrate, 25 equiv. of H2O, 30 mg mL-1 immobilized CAL-B in t-BuOMe at 70 ºC.
b 0.015 M substrate, 25 equiv. of H2O, 30 mg mL-1 immobilized CAL-B, in iPr2O at 70 ºC.
c According to HPLC (Experimental part).
d According to HPLC after derivatization (Experimental part).
f
g
h
i
j
ec 0.30; CHCl3; c 0.10; MeOH; c 0.21; CHCl3; c 0.30; EtOH; c 0.21; CHCl3; c 0.20;
MeOH.
19