A new enzymatic strategy for the preparation of (2R,3S)-3-phenylisoserine: a key intermediate for the Taxol side chain

Article abstract of DOI:10.1016/j.tetasy.2010.04.004

Burkholderia cepacia lipase PS-IM catalysed the hydrolysis of racemic ethyl 3-amino-3-phenyl-2-hydroxypropionate with excellent enantioselectivity (E >200), when the reaction was performed with added H₂O as a nucleophile, in iPr₂O, at 50 °C. The hydrolysis of the less reactive enantiomeric ethyl 3-amino-3-phenyl-2-hydroxypropionate with 18% HCl afforded the corresponding enantiomerically pure (2R,3S)-3-amino-3-phenyl-2-hydroxypropionic acid hydrochloride, a key intermediate for the Taxol side chain.

Full text of DOI:10.1016/j.tetasy.2010.04.004

Tetrahedron: Asymmetry 21 (2010) 637–639
Contents lists available at ScienceDirect
Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
A new enzymatic strategy for the preparation of (2R,3S)-3-phenylisoserine: a
key intermediate for the Taxol side chain
*
}
Eniko Forró, Ferenc Fülöp
Institute of Pharmaceutical Chemistry, University of Szeged, H-6701 Szeged, Eötvös u 6, Hungary
a r t i c l e i n f o
a b s t r a c t
Article history:
Burkholderia cepacia lipase PS-IM catalysed the hydrolysis of racemic ethyl 3-amino-3-phenyl-2-hydroxy-
propionate with excellent enantioselectivity (E >200), when the reaction was performed with added H2O
as a nucleophile, in iPr2O, at 50 °C. The hydrolysis of the less reactive enantiomeric ethyl 3-amino-3-phe-
nyl-2-hydroxypropionate with 18% HCl afforded the corresponding enantiomerically pure (2R,3S)-3-
amino-3-phenyl-2-hydroxypropionic acid hydrochloride, a key intermediate for the Taxol side chain.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 10 March 2010
Accepted 6 April 2010
Available online 11 May 2010
Dedicated to Professor Karoly Lempert on
the occasion of his 85th birthday
1. Introduction
aration of intermediates for the taxol side chain and analogues:
optically active cis and trans
a-hydroxy-b-lactams through Baker’s
Many chiral b-amino acids have valuable pharmacological
(cispentacin1 and Icofungipen2) effects, and have wide-ranging
use in peptide,3 heterocyclic,4 combinatorial5 chemistry and in
drug research.6 Some acyclic b-amino acids have attracted much
attention due to their importance in the synthesis of potential
pharmaceutical drugs, such as TaxolÒ (paclitaxel) and its analogue
TaxotèreÒ (docetaxel), currently considered to be among the most
important drugs in cancer chemotherapy.7
yeast reduction of a racemic a-keto-b-lactam and by the hydrolysis
of a racemic
a-acetoxy b-lactam with the resting cells of Bacillus
subtilis. Very recently, we have published two very applicable
new enzymatic strategies for the synthesis of the (2R,3S)-3-ami-
no-3-phenyl-2-hydroxypropionic acid.13 Burkholderia cepacia cata-
lysed the hydrolysis of racemic cis-3-acetoxy-4-phenylazetidin-2-
one with a moderate enantioselectivity (E = 24) in an organic sol-
vent, but Candida antarctica lipase B catalysed ring cleavage of
unprotected racemic cis-3-hydroxy-4-phenylazetidin-2-one with
an excellent one (E >200). In parallel, a new type of enzymatic
two-step cascade reaction, with excellent overall enantioselectivi-
ty (E >200) has been devised and used successfully for the desired
(2R,3S)-3-amino-3-phenyl-2-hydroxypropionic acid.
Since it is now evident that a 3-phenylisoserine-derived side
chain is essential for the antitumour activity of Taxol, there is a
need for the development of efficient processes for the preparation
of (2R,3S)-3-amino-3-phenyl-2-hydroxypropionic acid (2R,3S)-2 or
its direct enantiopure sources, such as ethyl (2R,3S)-3-amino-3-
phenyl-2-hydroxypropionate (2R,3S)-1. A number of new enzy-
matic and asymmetric syntheses for the Taxol side chain have been
elaborated, and most of them have been revieved.8 As an enzy-
matic method, Rodrigues et al.9 have described a highly chemoen-
zymatic synthesis of syn-(2R,3S)-3-amino-2-hydroxy esters with
high enantiomeric excess (ee P96%) through the Saccharomyces
cerevisiae-catalysed reduction of 3-bromo-2-ketoalkanoates. Tane-
ja et al.10 recently reported the enzymatic synthesis of several 4-
substituted (phenyl, 2-furyl or 2-thienyl) N-protected (4-methoxy-
phenyl) 2-azetidinone derivatives (ee P99%) through the lipase
(Arthrobacter sp.)-catalysed enantioselective cleavage of the ester
at C3 (E >200) in phosphate buffer (pH 7.0). As another example,
Sih et al.11 described the preparation of optically active 3-hydro-
xy-4-phenyl b-lactam derivatives through lipase-catalysed asym-
metric acylations (vinyl acetate, E >100), of the secondary OH
group and hydrolysis (H2O, E >100) of the ester function at C3 in
aqueous medium. Bose et al.12 described a chemoenzymatic prep-
Earlier extensive investigations of the lipase-catalysed enantio-
selective hydrolysis of b-amino esters14 suggested us to develop
the lipase-catalysed enantioselective hydrolysis of racemic ethyl
3-amino-3-phenyl-2-hydroxypropionate [( )-1].
2. Results and discussion
Racemic cis-3-acetoxy-4-phenylazetidin-2-one was prepared
by the Staudinger reaction from the Schiff’s base from benzalde-
hyde and p-anisidine and acetoxy-acetyl chloride in the presence
of TEA (Scheme 1), according to the literature.10 Treatment of race-
mic cis-3-acetoxy-1-(4-methoxyphenyl)-2-azetidinone with CAN
followed by neutralisation with 5% NaHCO3 solution resulted in
cis-3-acetoxy-4-phenylazetidin-2-one. The hydrolysis of this lac-
tam in MeOH with saturated NaHCO3 and Na2CO3 furnished cis-
3-hydroxy-4-phenylazetidin-2-one15 and the ring opening of its
with 22% ethanolic HCl and subsequent liberation of the free base
with aqueous KOH afforded the desired ethyl 3-amino-3-phenyl-2-
hydroxypropionate ester ( )-1.14c
* Corresponding author. Tel.: +36 62 54 5564; fax: +36 62 54 5705.
E-mail address: fulop@pharm.u-szeged.hu (F. Fülöp).
0957-4166/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2010.04.004

638
E. Forró, F. Fülöp / Tetrahedron: Asymmetry 21 (2010) 637–639
AcO
O
AcO
O
AcO
O
N
NH
CH2Cl2
TEA
CAN
+
N
Cl
OMe
OMe
NaHCO3,
Na2CO3
MeOH
HO
O
HO
COOEt
NH2
HO
COOEt
22%EtOH/HCl
KOH
NH
.
NH2 HCl
( )-1
Scheme 1. Synthesis of ( )-1.
On the basis of our earlier results on the enantioselective hydro-
(50 mg) and H2O 0.025 mmol) was shaken intensely at 50 °C.
Unfortunately, practically no reaction was observed in 2 days
(conv. < 2%). In summary, although n-hexane ensured a slightly
faster reaction than that with iPr2O (entry 5 vs 4), for reasons of
substrate solubility, iPr2O was chosen as the solvent for further
preliminary experiments and gram-scale resolution.
lyses of b-amino esters by using CAL-B and lipase PS in an organic
solvent14, we started preliminary experiments relating to the
hydrolysis of ( )-1 (Scheme 2) with 0.5 equiv of H2O in t-BuOMe,
with enzyme screening, at 50 °C.
The catalytic activity of the tested lipase PS-IM in iPr2O at 50 °C
was affected by the amount of H2O added to the reaction mixture
(Table 1, entries 4 and 8–11). The shortest reaction time (the time
needed to reach 50% conversion) combined with the best E (>200)
was obtained for 0.5–2 equiv of added H2O (Table 1, entries 4 and
9). As the amount of added H2O was further increased, the reaction
became progressively faster, but the value of E decreased (Table 1,
entries 10, 11 and 7). It is noteworthy that when the reaction was
performed without the addition of H2O (entry 8), the quantity of
H2O present in the reaction medium (<0.1%) or in the enzyme
preparation (no data) was still sufficient for the hydrolysis to
proceed.
HO
Ph
COOH
NH2
HO
Ph
COOEt
NH2
2
3
HO
Ph
COOEt
NH2
2
3
H2O
+
lipase
organic solvent
50 °C
(2S,3R)-2
1
(2R,3S)-
( )-1
Δ
18% HCl,
HO
COOH
2
3
.
Ph
NH2 HCl
2.
(2R,3S)- HCl
Scheme 2. Lipase-catalysed hydrolyses of ( )-1.
An attempt was also made to improve the enzyme:substrate
mass ratio (from 5:1 to 1:1), consequently, a set of preliminary
experiments were performed with a decreased amount of en-
zyme (40, 30, 20 and 10 mg mlꢀ1) (data not shown), and similarly
good results (E >200) were obtained, but longer reaction time
was necessary (conversion = 42% after 3.5 h for 10 mg mlꢀ1
enzyme).
The lipases tested were lipase PS-IM (B. cepacia, immobilised on
diatomaceous earth), PS-SD (B. cepacia), lipase AK (Pseudomonas
fluorescens), lipase AY (Candida rugosa), CAL-A (lipase A from C. ant-
arctica) and PPL (porcine pancreatic lipase) (Table 1). Not even
traces of the substrate were detected in the presence of CAL-B (li-
pase B from C. antarctica) produced by the submerged fermenta-
tion of a genetically modified Aspergillus oryzae microorganism
and adsorbed on a macroporous resin) after 8 h, while a very minor
amount of b-amino acid, (the b-amino acid ethyl ester probably
polymerised16) was detected in racemic form (Table 1, entry 1),
and CAL-A and lipase AY did not catalyse the hydrolysis at all dur-
ing 22 h (entries 11 and 13). Lipase PS-SD (entry 2) and PPL (entry
13) catalysed the otherwise highly selective (E >200) reactions
more slowly than lipase AK (entry 15). Lipase PS-IM (entry 3)
proved to be the best enzyme (conv. 50% after only 3 h, E >200),
and was therefore chosen as the enzyme for further studies and
for the gram-scale resolution.
Several solvents were tested to study the solvent effect in the
lipase PS-IM-catalysed hydrolysis of ( )-1 at 50 °C (Table 1, entries
3–7). The reaction proceeded considerably faster in n-hexane and
in iPr2O (entries 4 and 5) than in t-BuOMe (entry 3) or in toluene
(entry 6), though all afforded high enantioselectivities (E >200).
When the solvent was H2O, the reaction was much faster, but a
dramatic drop in enantioselectivity was observed (entry 7). On
the principle that ‘the best solvent is no solvent’,17 the hydrolysis
of ( )-1 was also performed in a solvent-free system: a mixture
of thoroughly powdered ( )-1 (9 mg, 0.05 mmol), lipase PS-IM
On the basis of the preliminary results, the gram-scale resolu-
tion of ( )-1 was performed with 0.5 equiv of H2O in the presence
of lipase PS-IM (20 mg mLꢀ1), in iPr2O, at 50 °C.7
When (2R,3S)-1 was hydrolysed14b with 18% HCl, the desired
amino acid hydrochloride, (2R,3S)-2ꢁHCl was formed.
The absolute configurations were proved by comparing the
a
values with the literature data; lit19
a value for (2S,3R)-3-amino-
(2S,3R)-3-amino-2-hydroxy-3-phenylpropionic acid {a2D3 ¼ þ14:4
(c 0.5, 6 M HCl), ee = 100%} is in good accordance with the value
measured for (2S,3R)-2 under the same conditions {a2D5 ¼ þ15 (c
0.25, 6 M HCl), ee >98%}, and the
a value measured for the antipode
(2R,3S)-3-amino-2-hydroxy-3-phenylpropionic acid {a2D5 ¼ ꢀ15 (c
0.25, 6 M HCl), ee >98%}.12 Therefore, lipase PS-catalysed hydroly-
sis of ( )-1 displayed 2S selectivity.
3. Conclusion
B. cepacia lipase PS-IM catalysed the hydrolysis of ( )-1 with
excellent enantioselectivity (E >200) with added H2O (0.5 equiv)
in iPr2O at 50 °C. The advantages of the enzymatic hydrolysis of
amino ester ( )-1 in an organic solvent are the fact that the amino

E. Forró, F. Fülöp / Tetrahedron: Asymmetry 21 (2010) 637–639
639
Table 1
Conversion and enantioselectivity of hydrolysis of ( )-3a
Enzyme (50 mg mLꢀ1
)
Reaction time (h)
Solvent
Added H2O (equiv)
Conv. (%)
eeS (%)
eeP (%)
E
b
c
Entry
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
CAL-B
2 (5) (8)
t-BuOMe
t-BuOMe
t-BuOMe
iPr2O
n-Hexane
Toluene
H2O
iPr2O
iPr2O
iPr2O
iPr2O
0.5
0.5
0.5
0.5
0.5
0.5
—
—
2
10
100
0.5
0.5
0.5
0.5
59 (90) (100)e
0
30
96 (51)
83
90
52
61
77
84
80
80
0
0d
Lipase PS-SD
Lipase PS-IM
Lipase PS-IM
Lipase PS-IM
Lipase PS-IM
Lipase PS-IM
Lipase PS-IM
Lipase PS-IM
Lipase PS-IM
Lipase PS-IM
CAL-Af
22
3 (2)
2
2
2
1
2
2
2
23
50 (34)
46
48
34
64
44
46
50
52
0
>98
>98
>98
>98
>98
35
>98
97
80
74
—
>98
—
>98
>200
>200
>200
>200
>200
3.7
>200
175
21
16
—
>200
—
>200
2
22
22
22
22
t-BuOMe
t-BuOMe
t-BuOMe
t-BuOMe
PPL
13
0
42
15
0
72
Lipase AYf
Lipase AKf
a
b
c
d
e
f
0.05 M substrate, 50 °C.
According to GC.20
According to GC after double derivatisation.12
Only a very small amount of b-amino acid was detected after 8 h; the b-amino acid ethyl ester probably polymerised (polymer not characterised).
Calculated by using an internal standard (n-hexadecane).
Contains 20% (w/w) of lipase adsorbed on Celite in the presence of sucrose.
8. Borah, J. C.; Boruva, J.; Barua, N. C. Curr. Org. Synth. 2007, 4, 175–199.
9. Rodrigues, J. A. R.; Milagre, H. M. S.; Milagre, C. D. F.; Moran, P. J. S. Tetrahedron:
Asymmetry 2005, 16, 3099–3106.
group need not necessarily be protected, and the products can be
easily separated.
Present enzymatic strategy is excellently applicable for the
synthesis of (2R,3S)-3-amino-3-phenyl-2-hydroxypropionic acid
(2R,3S)-2, a key intermediate for the Taxol side chain. One advan-
tage is the use of a lipase, which is commercially available, stable,
has high enantioselectivity and can be applied on an industrial
scale.
10. Anand, N.; Kapoor, M.; Ahmad, K.; Koul, S.; Parshad, R.; Manhas, K. S.; Sharma,
R. L. S.; Qazi, G. N.; Taneja, S. C. Tetrahedron: Asymmetry 2007, 18, 1059–1069.
11. Brieva, R.; Crich, J. Z.; Sih, C. J. J. Org. Chem. 1993, 58, 1068–1075.
12. Banik, B. K.; Negi, M.; Manhas, M. S.; Bose, A. K. Mol. Med. Rep. 2010, 3, 317–
318.
13. Forró, E.; Fülöp, F. Eur. J. Org. Chem. 2010, doi:10.1002/ejoc.201000262.
14. (a) Forró, E.; Fülöp, F. Chem. Eur. J. 2007, 13, 6397–6401; (b) Forró, E.; Paál, T.;
Tasnádi, G.; Fülöp, F. Adv. Synth. Catal. 2006, 348, 917–923; (c) Tasnádi, G.;
Forró, E.; Fülöp, F. Tetrahedron: Asymmetry 2008, 19, 2072–2077; (d) Tasnádi,
G.; Forró, E.; Fülöp, F. Tetrahedron: Asymmetry 2009, 15, 1771–1777; (e)
Tasnádi, G.; Forró, E.; Fülöp, F. Org. Biomol. Chem. 2010, 8, 793–799.
15. Choong, E. S.; Sung, W. L.; Eun, J. R.; Sunggi, L.; Wonku, L. Tetrahedron:
Asymmetry 1998, 9, 983–992.
Acknowledgements
The authors acknowledge receipt of OTKA grants K 71938 and T
}
049407 and a Bolyai Fellowship for Eniko Forró. Thanks are due to
16. Ismail, H.; Lau, R. M.; van Rantwijk, F.; Sheldon, R. A. Adv. Synth. Catal. 2008,
350, 1511–1516.
Mrs. Judit Árva for her technical assistance.
17. Forró, E.; Fülöp, F. Tetrahedron: Asymmetry 2008, 19, 1005–1009.
18. Racemic 1 (1.00 g, 4.78 mmol) was dissolved in iPr2O (40 mL). Lipase PS-IM
(2 g, 50 mg mLꢀ1) and H2O (43
lL, 2.4 mmol) were added and the mixture was
References
shaken in an incubator shaker at 50 °C for 2.5 h. The reaction was stopped by
filtering off the enzyme at 50% conversion. The solvent was evaporated off, and
the residue (2R,3S)-1 crystallised out [470 mg, 47%; a2D5 ¼ ꢀ2:9 (c 0.26 in
CHCl3); mp 82–84 °C; ee >99%]. The filtered-off enzyme was washed with
distilled H2O (3 ꢂ 15 mL), and the H2O was evaporated off, yielding the
crystalline amino acid (2S,3R)-2 [389 mg, 45%; a25 ¼ þ6:7 (c 0.25, H2O),
a2D5 ¼ þ15 (c 0.25, 6 M HCl); ee >98%, {lit18 a2D3 ¼Dþ14:4 (c 0.5, 6 M HCl),
ee = 100%}; mp 251–256 °C with decomposition (lit18 255–256 °C with
decomposition)]. 1H NMR (400 MHz, CDCl3, 25 °C, TMS) data for (2R,3S)-1:
d = 1.27–1.31 (t, J = 8.2, 3H, CH2CH3), 2.37 (br s, 1H, OH), 4.21–4.31 (m, 4H
overlapping, CH2CH3 and 2 ꢂ CH), 7.30–7.42 (m, 5H, C6H5). Anal. Calcd for
1. (a) Fülöp, F. Chem. Rev. 2001, 101, 2181–2204; (b) Kuhl, A.; Hahn, M. G.; Dumic,
M.; Mittendorf, J. Amino Acids 2005, 29, 89–100; (c) Kiss, L.; Forró, E.; Fülöp, F.
In Amino Acids; Hughes, A. B., Ed.; Peptides and Proteins in Organic Chemistry;
Wiley-VCH: Weinheim, 2009; Vol. 1, pp 367–409.
2. (a) Zegarac, M.; Mestrovic, E.; Hulita, N. K.; Filic, D.; Dumic, M.; Grunenberg, A.;
Keil, B.; Ceric, H. PCT WO 2005100302 A1 20051027; (b) Hameršak, Z.; Roje, M.;
´
´
Avdagic, A.; Šunjic, V. Tetrahedron: Asymmetry 2007, 18, 635–644.
3. (a) Steer, D. L.; Lew, R. A.; Perlmutter, P.; Smith, A. I.; Aguilar, M. I. Curr. Med.
Chem. 2002, 9, 811–822; (b) Martinek, T. A.; Mándity, I. M.; Fülöp, L.; Tóth, G.
K.; Vass, E.; Hollósi, M.; Forró, E.; Fülöp, F. J. Am. Chem. Soc. 2006, 128, 13539–
13544; (c) Price, J. L.; Horne, W. S.; Gellman, S. H. J. Am. Chem. Soc. 2007, 129,
6376–6377; (d) Mándity, I. M.; Wéber, E.; Martinek, T. A.; Olajos, G.; Tóth, G. K.;
Vass, E.; Fülöp, F. Angew. Chem., Int. Ed. 2009, 48, 2171–2175.
C11H15NO3: C, 63.14; H, 7.23; N, 6.69. Found: C, 63.33; H, 7.23; N, 6.57. The 1
H
NMR (400 MHz, D2O)
d (ppm) for (2S,3R)-2: 4.35–4.37 [d, J = 5.9 Hz, 1H,
CH(OH)(COOH)], 4.59–4.60 (d, J = 5.9 Hz, 1H, CHNH2), 7.43–7.56 (m, 5H, C6H5).
Anal. Calcd for C9H11NO3: C, 59.66; H, 6.12; N, 7.73. Found: C, 59.55; H, 6.10; N,
7.873. The 1H NMR (400 MHz, D2O) d (ppm) for (2R,3S)-2ꢁHCl: 4.58–4.59 [d,
J = 6.4 Hz, 1H, CH(OH)(COOH)], 4.65–4.66 (d, J = 6.4 Hz, 1H, CHNH2), 7.44–7.51
(m, 5H, C6H5). Anal. Calcd for C9H11NO3 HCl: C, 49.67; H, 5.56; N, 6.44. Found:
C, 49.50; H, 5.44; N, 6.62.
4. (a) Carter, P. H. U.S. Patent 2005,277,666; Chem. Abstr. 2005, 144, 51452; (b)
Fülöp, F.; Miklós, F.; Forró, E. Synlett 2008, 1687–1689; (c) Kiss, L.; Nonn, M.;
Forró, E.; Sillanpää, R.; Fülöp, F. Tetrahedron Lett. 2009, 50, 2605–2608; (d) Kazi,
B.; Kiss, L.; Forró, E.; Fülöp, F. Tetrahedron Lett. 2010, 51, 82–85.
5. (a) Kiss, L.; Forró, E.; Sillanpää, R.; Fülöp, F. J. Org. Chem. 2007, 72, 8786–8790;
(b) Kanizsai, I.; Gyónfalvi, S.; Szakonyi, Z.; Sillanpää, R.; Fülöp, F. Green Chem.
2007, 9, 357–360.
19. Andrés, J. M.; Martinez, M. A.; Pedrosa, R.; Pérez-Encabo, A. Tetrahedron:
Asymmetry 2001, 12, 347–353.
20. GC method; Supelco Gamma-Dex™ 225 column (30 m) [120 °C for 7 min ?
190 °C (temperature rise 10 °C minꢀ1; 70 kPa; retention time (min), (2R,3S)-1:
23.39 (antipode: 23.58)].
6. Juaristi, E.; Soloshonok, V. A. Enantioselective Synthesis of b-Amino Acids; Wiley-
Interscience: New York, 2005; (b) Fernandez, D.; Torres, E.; Aviles, F. X.; Ortuno,
R. M.; Vendrell, J. Bioorg. Med. Chem. 2009, 17, 3824–3828.
7. (a) Miller, R. W. J. Nat. Prod. 1990, 43, 425–437; (b) Kingston, D. G. I.; Newman,
D. J. Curr. Opin. Drug Discovery Dev. 2007, 10, 130–144.