Resolution of N-protected amino acid esters using whole cells of Candida parapsilosis ATCC 7330

Article abstract of DOI:10.1016/j.tetasy.2010.02.011

Whole cells of Candida parapsilosis ATCC 7330 were used for the resolution of N-acetyl amino acid esters. Excellent enantioselectivities (E = 40 to >500) were achieved for the resolution of N-protected protein and non-protein amino acid esters giving good yields (28-50%) and high enantiomeric excesses (up to >99%) for both enantiomers.

Full text of DOI:10.1016/j.tetasy.2010.02.011

Tetrahedron: Asymmetry 21 (2010) 457–460
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Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
Resolution of N-protected amino acid esters using whole cells
of Candida parapsilosis ATCC 7330
Selvaraj Stella a, Anju Chadha a,b,
*
a Laboratory of Bioorganic Chemistry, Department of Biotechnology, Indian Institute of Technology Madras, Chennai 600 036, India
b National Center for Catalysis Research, Indian Institute of Technology Madras, Chennai 600 036, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 13 January 2010
Accepted 16 February 2010
Whole cells of Candida parapsilosis ATCC 7330 were used for the resolution of N-acetyl amino acid esters.
Excellent enantioselectivities (E = 40 to >500) were achieved for the resolution of N-protected protein and
non-protein amino acid esters giving good yields (28–50%) and high enantiomeric excesses (up to >99%)
for both enantiomers.
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
ered, but the corresponding product N-acetyl-L-amino acids were
not isolated.7 Baker’s yeast cross-linked with glutaraldehyde and
immobilized with calcium alginate was used for the enantioselec-
Enantiomerically pure natural and unnatural
a-amino acids
and their derivatives are useful intermediates in the pharmaceu-
tive hydrolysis of methyl esters of racemic N-acetyl-a-amino
tical, biotech, and chemical industries.1 Therefore, it is important
acids in reverse micellar media.8 Immobilized whole cells of Sulf-
olobus solfataricus, a thermoacidophilic archaeobacterium, trapped
in sodium alginate were reported for the hydrolysis of the methyl
to synthesize enantiomerically pure
a-amino acids in high yields
and enantiomeric excesses.2 Among the various biocatalytic
methods, the enzymatic resolution of DL-amino acids is an effi-
cient method for the preparation of amino acids as it allows the
recovery of both enantiomers. Isolated hydrolase enzymes such
as proteases and lipases have been used extensively for the reso-
lution of racemic amino acids through the enantioselective hydro-
lysis of their esters.3 Detergent (alkaline) proteases used for the
resolution of amino acid esters are mostly from bacterial origin,
and require cost-intensive technologies to obtain a microorgan-
ism-free enzyme preparation.3m Using a whole cell biocatalyst is
advantageous because the enzymes in the whole cell system are
regarded as immobilized by entrapment or attachment to the cell
wall8a and no isolation or purification of the enzyme is needed.
Industrial production of amino acids mostly involves microbial
processes because cell disruption and concentration of the en-
zyme solution are dispensable in whole cell-catalyzed reactions.4
For example, whole cells of recombinant Escherichia coli are re-
ported for the asymmetric hydrolysis of amino acid hydantoins5
and N-acyl amino acids6 to produce enantiomerically pure amino
acids. There are very few reports exploring the hydrolytic proper-
ties of whole cells for the resolution of amino acid esters. In
whole cell-mediated enzymatic resolution, baker’s yeast (Saccha-
romyces cerevisiae) was employed for the resolution of racemic
N-acetyl amino acid esters. The unreacted substrates were recov-
esters of natural racemic a
-amino acids.9 Given the advantages of
using whole cells and expanding upon the scope of Candida par-
apsilosis ATCC 7330 which is known to deracemize the racemic
a
,b-hydroxy esters and allylic alcohols, and reduce a- and b-oxo-
esters and aryl imines in an enantioselective manner,10,17 we
herein report the resolution of natural and unnatural N-acetyl
amino acid esters using this biocatalyst. Both the enantiomers
were isolated in high yields and enantiomeric excess (ee).
2. Results and discussion
In preliminary studies, the racemic N-free phenylalanine ethyl
ester 1 was subjected to hydrolysis using the whole cells of C. par-
apsilosis ATCC 7330 (Scheme 1). The natural enantiomer
alanine ethyl ester was hydrolyzed into its -acid. At 25 °C, 38%
conversion was reached in 10 min and the reaction led to rather
low enantiomeric excesses of both the remaining substrate -ester
2 (35% ee) and the -acid 3 (57% ee) with an E ratio of 5.1. A similar
L-phenyl-
L
D
L
type of hydrolysis with low E ratio (E = 1.2–4.2) was reported using
Aspergillus oryzae protease for the methyl esters of racemic N-free
amino acids11 and in this case the highest enantioselectivities were
obtained by employing esters bearing a longer alkyl chain (isobutyl
ester).
When the amino group was replaced by the acetylamino group
an increase in the enantioselectivity was observed that is, N-acetyl-
DL-phenylalanine ethyl ester was hydrolyzed with good enantiose-
* Corresponding author. Tel.: +91 44 2257 4106; fax: +91 44 2257 4102.
E-mail addresses: anjuc@iitm.ac.in, anjuchadha@hotmail.com (A. Chadha).
0957-4166/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2010.02.011

458
S. Stella, A. Chadha / Tetrahedron: Asymmetry 21 (2010) 457–460
O
O
O
Candida parapsilosis ATCC 7330
OH
O
O
+
NH2
NH2
25 ºC, water, 10 min
NH2
1
3
2
DL-phenylalanine
ethyl ester
L-acid
57%ee
D-ester
35%ee
Scheme 1. Resolution of DL-phenylalanine ethyl ester 1 using the whole cells of Candida parapsilosis ATCC 7330.
lectivity E = 105 compared to the N-free phenylalanine ethyl ester
with an increased time (2 h) of hydrolysis. This led to a further
investigation of the hydrolysis of a series of natural and unnatural
N-acetyl amino acid ethyl esters 4 using the whole cells of C. par-
apsilosis ATCC 7330 (Scheme 2). The natural enantiomer N-acet-
where the side chain is not separated by a –CH2– group was very
slow (entry h). After 96h reaction time, the substrate -ester was
isolated in 62% yield and 36% enantiomeric excess and the product
was not isolated.
D
yl-
the
L
ester was hydrolyzed into N-acetyl-
L-acid 6, while neither
3. Conclusion
D-enantiomer 5 nor the amide group was affected in both enan-
tiomers. The natural N-acetyl amino acid esters phenylalanine 4a,
tyrosine 4g, and tryptophan 4f were hydrolyzed to give good ee
(70–99%) of the products and excellent ee (95 to >99) of the sub-
strates (Table 1, entries a, f, and g). The enantioselectivities of the
N-acetyl esters of phenylalanine (E = 105) and tryptophan
(E = 40) were rather low compared to those of the other amino acid
esters. The hydrolysis of N-acetyl ethyl ester of tyrosine was slow
and 49% conversion was reached in 48 h with a high enantiomeric
ratio E >500.
This study shows that the whole cells of C. parapsilosis ATCC 7330
are efficient biocatalysts in the preparation of enantiomerically pure
natural and unnatural amino acids. The enantioselectivity of ester
hydrolysis can be increased by substituting the N-free ester with
N-acetyl amino acid esters. Significantly, both natural and unnatural
amino acids, where the R-group is separated by a –CH2– group, were
hydrolyzed with good enantioselectivity (E >500).
The unnatural N-acetyl-p-F-phenylalanine ethyl ester 4b was
hydrolyzed with an E of ratio >200. Notably, the other halogenated
phenylalanines (p-Cl and p-Br-phenylalanine 4c and 4d) were
hydrolyzed with good ee for both substrates and products with
high enantioselectivity (E >500) (Table 1, entries b–d). This is high-
er than that reported for the hydrolysis of N-protected p-haloge-
nated phenylalanine esters by porcine pancreatic lipase,12 Bacillus
subtilis13, and sulfhydryl proteases.14 Moreover, the type of halogen
atom substituted on the aromatic ring has no effect on the hydro-
lytic activity of the enzyme(s) involved and all three halogenated
amino acids were hydrolyzed within 3–4 h with high ee and
conversions close to 50%.
4. Experimental
4.1. General
C. parapsilosis ATCC 7330 was purchased from ATCC Manassas,
VA 201018, USA and maintained at 4 °C in yeast malt agar medium
that contained 5 g/l peptic digest of animal tissue, 3 g/l malt ex-
tract, 3 g/l yeast extract, 10 g/l dextrose, and 20 g/l agar. The unnat-
ural racemic amino acids were purchased from Sigma chemicals,
and the natural racemic amino acids were from SRL chemicals, In-
dia and used as received. All solvents were of analytical grade and
used as received. Thionyl chloride and acetic anhydride were dis-
tilled prior to use. HPLC analysis of the N-acetyl amino acid esters
was carried out on a Jasco PU-1580 liquid chromatograph with a
PDA detector using Daicel OJ-H, OB-H, and OD-H chiral columns,
Hexane: isopropanol mixture was used as the mobile phase with
flow rate 1 ml/min. The proportion of solvents varied for different
N-acetyl amino acid esters. HPLC analysis of the N-free phenylala-
nine ester and acid was carried out on a Crownpak CR+ column,
with HClO4 (pH 2) as the mobile phase with flow rate 0.8 ml/
min. Optical rotations were recorded on a Rudolph, Autopol IV dig-
ital polarimeter. The characterization of racemic and enantiomeri-
cally pure N-acetyl amino acid ethyl esters was carried out by 1H,
13C NMR, and mass spectrometry. 1H and 13C NMR spectra were re-
corded in CDCl3 or CD3OD solution on a Bruker AV-400 spectrom-
eter operating at 400 and 100 MHz, respectively.
Another important unnatural amino acid N-acetyl-L-homophe-
nylalanine 6e which is a chiral intermediate in the synthesis of
angiotensin converting enzyme inhibitors,15 was obtained in 98%
ee by the hydrolysis (E >200) of N-acetyl-DL-homophenylalanine
ethyl ester 4e (Table 1, entry e). Different solvent systems have
been reported for the enzymatic ester hydrolysis of N-protected
homophenylalanine; papain in 20% solution of DMF in phosphate
buffer,14 porcine pancreatic lipase in 15% solution of ionic liquid
[EtPy]+[CF3COO]ꢀ in water;3e 30% acetonitrile in phosphate buf-
fer,12c Bacillus lichenifomis alcalase in two different ionic liquid
solutions of [EMIM][BF4] and [EtPy][BF4] in water;3f and immobi-
lized
a-chymotrypsin and baker’s yeast in reverse micellar med-
ia.8b Herein, the hydrolysis was carried out in water with 7.5%
ethanol. The hydrolysis of N-acetylphenylglycine ethyl ester 4h
O
O
O
R
R
Candida parapsilosis ATCC 7330
+
R
O
OH
O
HN
HN
25 ºC, water
HN
O
5
O
6
O
4a-h
N-acetyl-D-ester
N-acetyl-L-acid
R = C6H5-CH2-, p-F-C6H4-CH2-,
36 - >99%ee
70 - 99%ee
p-Cl-C6H4-CH2-, p-Br-C6H4-CH2-, C6H5-(CH2)2 -,
C8H6 N-CH2-, p-OH-C6H4 -CH2 -, C6H5 -
40 - 62% yield
28 - 47% yield
Scheme 2. Resolution of N-acetyl-DL-amino acid ethyl esters 4a–h using the whole cells of Candida parapsilosis ATCC 7330.

S. Stella, A. Chadha / Tetrahedron: Asymmetry 21 (2010) 457–460
459
Table 1
Resolution of racemic N-acetyl amino acid esters 4a–h using the whole cells of Candida parapsilosis ATCC 7330
Entry
Time (h)
N-Acetyl-
D
-ester 5
N-Acetyl-
L
-acid 6
Cd (%) Ee
DL-N-Acetyl amino
acid ethyl esters 4
Yielda (%) ees (%) Specific rotation
Yielda (%) eep (%) Specific rotation
b
c
a 3D4
a 3D0
a 3D4
a 3D4
a 3D0
a 3D0
a 3D4
a 3D4
ꢁ
¼ ꢀ12:4 (c 1, EtOH) Lit.19a
¼ ꢀ72:5 (c 2, CHCl3) Lit.7b
¼ ꢀ16:8 (c 2, EtOH)
½
½
½
½
½
½
½
a 3D5
a 3D0
a 3D5
a 3D0
ꢁ
¼ þ28:9 (c 1, MeOH) Lit.19b
¼ þ30:8 (c 1, MeOH)
a
b
c
d
e
f
Phenylalanine
2
4
4
3
45
48
50
42
50
40
43
62
>99
99
47
47
39
38
32
28
28
nd
87
98
99
98
98
70
99
nd
53.5
50.3
50.3
50.5
49.0
58.8
49.0
—
105
>200
½
½
½
½
½
½
½
½
p-F-phenylalanine
p-Cl-phenylalanine
p-Br-phenylalanine
ꢁ
ꢁ
>99
>99
94
>500
>500
>200
40
ꢁ
ꢁ
ꢁ
¼ þ47:5 (c 1, MeOH)
ꢁ
¼ ꢀ17:5 (c 1, EtOH)
¼ þ35:5 (c 1, MeOH)
a 3D5
a 3D5
a 3D0
ꢁ
¼ þ14:6 (c 2, MeOH) Lit.20
¼ þ18:8 (c 0.5, aq NaOH) Lit.22
¼ þ44:9 (c 0.5, MeOH) Lit.23b
Homophenylalanine 24
ꢁ
¼ ꢀ1:8 (c 2, EtOH)
¼ ꢀ38:7 (c 1, CHCl3) Lit.21
¼ ꢀ16:2 (c 2, EtOH) Lit.23a
¼ ꢀ48:6 (c 2, EtOH) Lit.24
ꢁ
Tryptophan
Tyrosine
24
48
96
>99
95
ꢁ
g
h
>500
—
ꢁ
ꢁ
Phenylglycine
36
—
ꢁ
nd—not determined.
a
Isolated yield.
Determined by chiral HPLC.
b
c
Determined by chiral HPLC after derivatization into an ethyl ester.
Conversion was calculated according to ees /(ees + eep).
d
e
Enantioselectivity ratios were calculated according to E = ln[(1 ꢀ c)(1 ꢀ ees)]/ln[(1 ꢀ c)(1 + ees)],25 E values in the range 200–500 are mentioned as >200, more than 500 as
>500.
4.2. Synthesis of racemic N-free amino acid ethyl esters16a
The basic aqueous layer was then acidified with 6 M HCl to pH
2–3, extracted with ethyl acetate, and concentrated under reduced
pressure to give 6a, which was purified by column chromatogra-
phy with 1:4 methanol/dichloromethane mixture to give an iso-
lated yield of 47% (25 mg) and ee 87% {specific rotation of 6a
All racemic N-free amino acid ethyl esters were prepared in
quantitative yields by esterification with thionyl chloride and eth-
anol at reflux. The salt-free amino acid ethyl ester was obtained by
neutralizing the ester hydrochloride with saturated NaHCO3 solu-
tion, extracted with ethyl acetate, and concentrated under reduced
pressure. The residue was purified by column chromatography and
analyzed by 1H, 13C NMR and mass spectrometry.
½
a 3D5
ꢁ
¼ þ28:9 (c 1, MeOH) Lit.19b
½
a 2D2
ꢁ
¼ þ40:0 (c 1, MeOH)}. The
ee of the D-ester 5a and L-acid 6a (ethyl ester) was determined
by chiral HPLC with Chiralcel OBH column, 95:5 hexane/isopropyl
alcohol mixture with a flow rate of 1 ml/min.
4.3. Synthesis of racemic N-acetyl amino acid ethyl esters 4a–h16b
4.6. General method for the resolution of racemic N-acetyl
amino acid ethyl esters 4a–h
The N-free amino acid ethyl esters (1 mmol) were dissolved in
glacial acetic acid and acetic anhydride (1.2 mmol) was added
and stirred at rt for 1–2 h. The solvent was removed by rotary
evaporation. Further purification by column chromatography
yielded the pure N-acetylated amino acid esters.
To 6 g of wet cells of C. parapsilosis suspended in 18.5 ml water,
60 mg racemic N-acetyl amino acid ethyl ester 4 dissolved in
1.5 ml ethanol as a co-solvent was added and incubated at 25 °C,
200 rpm. The hydrolysis was monitored by determining the ee of
the unreacted substrate
D-ester 5 (ees) by chiral HPLC. The ee of
4.4. Growth conditions for C. parapsilosis ATCC 733017
the product N-acetyl- -acid 6 was determined at the maximum
L
ees. After hydrolysis, the cells were centrifuged and the superna-
tant was adjusted to pH 9 with saturated NaHCO3 solution, ex-
C. parapsilosis ATCC 7330 was grown in yeast malt broth med-
ium (50 ml) in 250 ml Erlenmeyer flasks incubated at 25 °C,
200 rpm. The cells were harvested by centrifuging the 14th h cul-
ture broth at 10,000 rpm for 10 min at 4 °C and subsequent wash-
ing with distilled water. The process was repeated twice, and the
wet cells were used for biotransformation. In total, 1 g wet cells
were obtained per 50 ml medium in 250 ml Erlenmeyer flask.
tracted with ethyl acetate to give N-acetyl-
ester. The basic aqueous layer was then acidified with 6 M HCl to
pH 2–3, and extracted with ethyl acetate to give the N-acetyl-
amino acid. The ee of the -ester 5 was determined by chiral HPLC
with Chiralcel OB-H, OJ-H, and OD-H columns.18 The
-acid 6 prod-
D-amino acid ethyl
L-
D
L
uct was esterified with SOCl2/EtOH and analyzed by chiral HPLC.
4.5. Resolution of N-acetyl-DL-phenylalanine ethyl ester 4a
Acknowledgments
To 6 g of wet cells of C. parapsilosis suspended in 18.5 ml water,
60 mg N-acetyl-DL-phenylalanine ethyl ester 4a dissolved in 1.5 ml
ethanol as a co-solvent was added and incubated at 25 °C, 200 rpm
for 2 h. The hydrolysis was monitored by determining the ee of the
One of us [S. Stella] thanks the Council of Scientific and Indus-
trial research (CSIR, India) for the fellowship. We thank the Sophis-
ticated Analytical Instrumentation Facility (SAIF) and Department
of Chemistry IITM for the NMR and Mass analysis.
unreacted substrate N-acetyl-
by chiral HPLC. The ee of the product N-acetyl-
D
-phenylalanine ethyl ester 5a (ees)
-phenylalanine
L
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