A convenient method for the enantiomeric separation of α-amino acid esters as benzophenone imine schiff base derivatives

Article abstract of DOI:10.1007/s12272-012-0609-6

A convenient liquid chromatographic method for the separation of α-amino acid esters as benzophenone Schiff base derivatives on coated chiral stationary phases (CSPs) (Chiralcel OD-H, Chiralcel OD, Chiralpak AD-H, Chiralpak AD, and Chiralpak AS) or covalently immobilized CSPs (Chiralpak IA, Chiralpak IB, and Chiralpak IC) derived from polysaccharide derivatives is described. Benzophenone imine derivatives of α-amino acid esters were readily prepared by stirring benzophenone imine and the hydrochloride salts of α-amino acid esters in 2-propanol. The chromatographic separations were conducted at a flow rate 1.0 mL/min and a detection wavelength of 254 nm; 0.5% 2-propanol/hexane (v/v) was used on CSPs. In general, the resolution of Chiralpak IC was superior to those of the other CSPs. In addition, the resolutions of other arylimine derivatives of α-amino acid esters and the effects of different mobile phases on the enantiomeric separation of α-amino acid esters as benzophenone imine derivatives on Chiralpak IC were investigated.

Full text of DOI:10.1007/s12272-012-0609-6

Arch Pharm Res Vol 35, No 6, 1015-1019, 2012
DOI 10.1007/s12272-012-0609-6
A Convenient Method for the Enantiomeric Separation of α-Amino
Acid Esters as Benzophenone Imine Schiff Base Derivatives
Hu Huang1, Wen Jun Xu1, Jing-Yu Jin1, Joon Hee Hong1, Hyun-Jae Shin2, and Wonjae Lee1
2
1College of Pharmacy, Chosun University, Gwangju 501-759, Korea and Department of Chemical and Biochemical Engi-
neering, Chosun University, Gwangju 501-759, Korea
(Received January 28, 2012/Revised February 14, 2012/Accepted February 20, 2012)
A convenient liquid chromatographic method for the separation of α-amino acid esters as ben-
zophenone Schiff base derivatives on coated chiral stationary phases (CSPs) (Chiralcel OD-H,
Chiralcel OD, Chiralpak AD-H, Chiralpak AD, and Chiralpak AS) or covalently immobilized
CSPs (Chiralpak IA, Chiralpak IB, and Chiralpak IC) derived from polysaccharide derivatives
is described. Benzophenone imine derivatives of
α
-amino acid esters were readily prepared by
-amino acid esters in 2-propanol.
stirring benzophenone imine and the hydrochloride salts of
α
The chromatographic separations were conducted at a flow rate 1.0 mL/min and a detection
wavelength of 254 nm; 0.5% 2-propanol/hexane (v/v) was used on CSPs. In general, the reso-
lution of Chiralpak IC was superior to those of the other CSPs. In addition, the resolutions of
other arylimine derivatives of
the enantiomeric separation of
Chiralpak IC were investigated.
α
-amino acid esters and the effects of different mobile phases on
-amino acid esters as benzophenone imine derivatives on
α
Key words: Chiral stationary phase, Benzophenone imine derivative, Enantiomer separation
imines for enantiomer separation, and this work resulted
in the selection of the benzophenone imine moiety used
INTRODUCTION
Several methods have been developed to determine as an amine protecting group (O’Donnell and Polt,
the enantiomeric purities and/or configurations of 1982; Greene and Wuts, 1999). The benzophenone imine
α
-
amino acids in the pharmaceutical field (Subramanian, group is expected to function as an aromatic auxiliary
2001; Francotte and Lindner, 2006). Of these techniques, group for enantiomer resolution by the chiral selectors
liquid chromatographic enantiomer separation on of CSPs (Duchateau et al., 1994). Benzophenone Schiff
chiral stationary phase (CSPs) has been known to be base derivatives have been previously used for the en-
one of the most convenient and versatile methods. In antioselective synthesis of
a previous study, a liquid chromatographic method phase-transfer alkylation (O’Donnell and Polt, 1982).
was described for the separation of enantiomers of Especially, the benzophenone imine derivatives of α-
α-amino acid esters by
α
-
amino acid esters and chiral amines as 9-anthraldimine amino acid esters are readily prepared by simply stir-
Schiff base derivatives on polysaccharide-derived CSPs ring benzophenone imine and the hydrochloride salts
(Huang et al., 2011). Of the CSPs studied, Chiralcel OD of
(OD-H) provided greatest resolution of the enantiomers ture (Fig. 1) (O’Donnell and Polt, 1982; Greene and Wuts,
of several -amino acid esters and chiral amines as 9- 1999). In this study, we describe a convenient chroma-
α-amino acid esters in 2-propanol at room tempera-
α
anthraldimine derivatives. Subsequently, we attempted tographic separation of the enantiomers of
to develop a convenient method for the derivatization
α-amino
of
α-amino acid esters using aromatic Schiff base
Correspondence to: Wonjae Lee, College of Pharmacy, Chosun
University, Gwangju 501-759, Korea
Tel: 82-62-230-6376, Fax: 82-62-222-5414
E-mail: wlee@chosun.ac.kr
Fig. 1. Preparation of the benzophenone imine derivatives
of -amino acid esters.
α
1015

1016
H. Huang et al.
acid esters as their benzophenone imine derivatives OD, Chiralpak AD and Chiralpak AS (250 mm L × 4.6
on coated and on covalently bonded polysaccharide- mm I.D., 10 m), and Chiralcel OD-H, Chiralpak AD-
H, Chiralpak IA, Chiralpak IB, and Chiralpak IC (250
mm L × 4.6 mm I.D., 5 m) were purchased from the
µ
derived CSPs.
µ
Daicel Chemical Company. Chromatography was per-
formed using a flow rate 1 mL/min, a detection wave-
length of UV 254 nm, and 0.5% 2-propanol/hexane (v/v)
MATERIALS AND METHODS
Instruments and reagents
Chromatography was performed at room tempera- as mobile phase for all CSPs. In addition, other solvents,
ture using an HPLC Breeze system (Waters) equipped such as, tetrahydrofuran, ethyl acetate, and dichloro-
with a Waters model 1525 binary pump, an autosam- methane in hexane were added when the covalently
pler, and a dual absorbance detector (Waters 2487 de- immobilized CSP, Chiralpak IC was examined.
tector). HPLC-grade hexane and 2-propanol were ob-
tained from J. T. Baker. Benzophenone imine and all
-amino acid esters were obtained from Aldrich or
RESULTS AND DISCUSSION
α
Sigma or Advanced ChemTech. The racemic and D- or
L-analytes used were prepared, as shown in Fig. 1, by
Enantiomeric separation of -amino acid esters
as benzophenone imine Schiff base derivatives
α
stirring 0.5 mmol of
α
-amino acid ester hydrochloride
Tables I and II summarize chromatographic data for
and an equimolar amount of benzophenone imine in the separation of the enantiomers of several α-amino
10 mL of 2-propanol at room temperature for 12 h. acid esters as benzophenone imine Schiff base derivatives
Reaction mixtures were filtered to remove ammonium on coated polysaccharide-derived CSPs (Chiralcel OD-
chloride and resulting solutions were directly injected H, Chiralcel OD, Chiralpak AD-H, Chiralpak AD and
into the chromatograph.
Chiralpak AS) and on covalently bonded polysaccharide-
derived CSPs (Chiralpak IA, Chiralpak IB, and Chiral-
pak IC) using 0.5% 2-propanol/hexane (v/v) as mobile
Chromatographic conditions
All polysaccharide-derived CSPs, that is, Chiralcel phase. Of the commonly used coated type CSPs (Chiralcel
Table I. Separation of the enantiomers of
OD-H, Chiralcel OD, Chiralpak AD-H, Chiralpak AD, and Chiralpak AS
α-amino acid methyl esters as benzophenone imine derivatives on Chiralcel
Chiralcel OD-H
Chiralcel OD
k'1b Rsc Conf.d αa
Chiralpak AD-H
Chiralpak AD
k'1b Rsc Conf.d αa
Chiralpak AS
k'1b Rsc Conf.d
Analyte
αa
k'1b Rsc Conf.d αa
k'1b Rsc Conf.d αa
alanine
asparagine
aspartic acid 1.47 11.31 6.40
leucine 1.00 1.42
phenylalanine 3.79 3.32 19.29
phenylglycine 1.38 3.24 4.78
1.07 2.45 0.87
D
1.00 2.98
1.00 14.50
1.37 10.60 3.61
1.00 1.43
3.65 3.96 9.70
1.42 3.09 2.77
1.00 1.18
-
-
1.12 2.33 1.08
1.00 9.59
1.31 8.30 5.78
1.00 1.32
1.23 3.16 1.25
1.35 4.71 2.23
D
1.00 1.53
1.00 6.83
2.27 3.05 5.24
1.00 1.16
1.00 2.25
1.16 3.27 0.91
1.00 1.14
-
-
1.00 2.69 -
1.00 16.38
-
-
1.00 4.71
1.00 5.50
-
-
L
L
D
D
D
-
-
-
-
-
1.87 0.78 0.96
1.83 2.14 1.63
1.39 2.79 1.25
1.79 0.75 0.97
D
D
D
D
L
L
L
L
D
D
valine
1.00 1.25
-
-
1.00 1.51
-
-
Mobile phase: 0.5% 2-propanol/hexane (v/v); Flow rate = 1.0 mL/min; Detection UV 254 nm. aSeparation factor; bCapacity factor for
the first enantiomer eluted; cResolution factor; dAbsolute configuration of the second enantiomer eluted.
Table II. Separation of the enantiomers of
IA, Chiralpak IB, and Chiralpak IC
α
-amino acid methyl esters as benzophenone imine derivatives on Chiralpak
Chiralpak IB Chiralpak IC
Chiralpak IA
Analyte
αa
k'1b
Rsc
Conf.d
αa
k'1b
Rsc
Conf.d
αa
k'1b
Rsc
Conf.d
alanine
1.00
1.16
1.12
1.05
1.36
5.95
4.99
1.30
2.05
3.32
1.14
-
1.00
1.55
1.49
1.00
2.62
1.17
1.00
1.26
4.40
4.23
0.61
1.58
1.63
0.56
-
1.48
1.00
1.00
1.58
1.12
1.75
1.98
4.92
5.87
5.88
2.26
8.48
5.50
2.41
8.77
-
-
7.03
2.08
8.46
12.94
L
asparagine
aspartic acid
leucine
phenylalanine 1.00
phenylglycine
3.01
2.20
1.08
-
2.16
0.17
D
D
D
6.19
5.70
-
8.39
2.04
-
L
L
L
L
D
L
L
L
1.23
1.06
D
L
valine
Mobile phase: 0.5% 2-propanol/hexane (v/v); Flow rate = 1.0 mL/min; Detection UV 254 nm. aSeparation factor; bCapacity
factor for the first enantiomer eluted; cResolution factor; dAbsolute configuration of the second enantiomer eluted.

Separation of the Enantiomers of
α-Amino Acid Esters
1017
OD-H, Chiralcel OD, Chiralpak AD-H, Chiralpak AD, acid esters is much more straightforward. Table III
and Chiralpak AS) listed in Table I, Chiralcel OD-H contains the chromatographic data of different aro-
showed greatest enantioselectivity, and Chiralpak AD matic imine derivatives of several α-amino acid esters
the least. Also, it is natural that the separation factors on Chiralpak IC. Benzophenone imine derivatives were
and resolution factors on Chiralcel OD-H and Chiral- better enantioseparated than benzaldimine, 1-naph-
pak AD-H with the silica particle size 5
higher than those on Chiralcel OD and Chiralpak AD the analyte is sterically hindered, the enantioselectiv-
with the silica particle size 10 m, respectively. Of the ity increases (entries 1-3 and 8-10). The resolution of
µm showed thaldimine, and 9-anthraldimine derivatives. Also, as
µ
CSPs examined (Tables I and II), Chiralpak IC pro- benzophenone imine derivatives of amino acid methyl
vided best enantioseparation, although it did not esters was greater than that of the corresponding
resolve the benzophenone imine derivatives of two amino acid ethyl esters. In addition, the elution orders
polar analytes (asparagine and aspartic acid). It was of all benzaldimine and 1-naphthaldimine derivatives
interesting to find that the performances of Chiralpak were consistent with those of the corresponding benzo-
IB and Chiralpak IC were complementary, for whereas phenone imine derivatives. However, the observed re-
Chiralpak IC failed to separate these two polar analytes, versal of the elution orders of π-basic 9-anthraldimine
they were well separated by Chiralpak IB. Orders of derivative implies the different chiral recognition pro-
enantioselectivity were OD-H > AD-H > AS~OD > AD cesses (entries 7 and 14).
for the coated CSPs and IC > IB~IA for the covalently
bonded CSPs. In a previous study on the enantiomer
separation of 9-anthraldehyde Schiff base derivatives
Effects of mobile phase on the separation of α-
amino acid esters as benzophenone imine deriv-
atives and the application to measure the en-
antiomeric purity
of
α-amino acid esters, it was found that among the
CSPs investigated, Chiralcel OD (or OD-H) had greatest
enantioselectivity, and Chiralpak IC least (Huang et
In normal phase, coated type CSPs are not compat-
al., 2011). The degree of enantiomer separation of ben- ible with all solvents and for example, the use of ethyl
zophenone imine derivatives on Chiralpak IC in this acetate, tetrahydrofuran, and halogenated solvents as
study was lower than that of their 9-anthraldimine mobile phases or solvents for analytes is prohibited
derivatives on Chiralcel OD (or OD-H) (Huang et al., (Jin et al., 2006, 2009; Thunberg et al., 2008; Zhang et
2011). However, the derivatization process required to al., 2008). On the other hand, the solvent versatility of
produce benzophenone imine derivatives of amino covalently bonded Chiralpak IC for the separation of
Table III. Separation of the enantiomers of α-amino acid esters as arylimine derivatives on Chiralpak IC
Entry
R1
R2
R3
R4
αa
k'1b
Rsc
Conf.d
1
2
3
4
5
6
7
Ph
Ph
Ph
Ph
Ph
Ph
H
H
H
Me
i-Bu
i-Pr
i-Pr
i-Pr
i-Pr
i-Pr
Me
Me
Me
Me
Me
Me
Me
1.32
1.40
1.47
1.37
1.17
1.10
1.23
3.03
1.55
1.56
2.61
5.40
3.28
4.07
3.67
4.18
4.80
6.12
3.32
1.51
2.89
L
L
L
L
L
L
D
Ph
4-MeO-Ph
1-Naphthyl
9-Anthryl
H
8
9
Ph
Ph
Ph
Ph
Ph
Ph
H
H
H
Me
i-Bu
i-Pr
i-Pr
i-Pr
i-Pr
i-Pr
Et
Et
Et
Et
Et
Et
Et
1.29
1.38
1.43
1.29
1.15
1.05
1.22
3.01
1.45
1.52
2.25
5.34
3.05
3.40
1.75
2.13
4.20
3.45
1.81
0.44
3.09
L
L
L
L
L
L
D
10
11
12
13
14
Ph
4-MeO-Ph
1-Naphthyl
9-Anthryl
H
Mobile phase = 1% 2-propanol/hexane (v/v); Flow rate = 1.0 mL/min; Detection UV 254 nm. aSeparation factor; bCapacity
factor for the first enantiomer eluted; cResolution factor; dAbsolute configuration of the second enantiomer eluted.

1018
H. Huang et al.
Table IV. Effect of mobile phase on the enantiomeric separation of the benzophenone imine derivatives of α-amino acid
methyl esters on Chiralpak IC
1% tetrahydrofuran/
hexane
30% dichloromethane/
hexane
Mobile phase 0.5% 2-propanol/hexane
αa k'1b Rsc Conf.d αa
1.48 4.92 8.77
2% ethyl acetate/hexane
Analyte
alanine
k'1b
Rsc Conf.d αa
k'1b
Rsc Conf.d αa
k'1b
Rsc Conf.d
L
1.40 4.60 2.73
L
1.27 4.70 4.89
L
1.25 2.17 2.68
L
asparagine
aspartic acid
leucine
phenylalanine 1.12 8.48 2.08
phenylglycine 1.75 5.50 8.46
1.00 5.87
1.00 5.88
1.58 2.26 7.03
-
-
1.00 8.52
1.00 8.76
1.50 2.42 5.92
1.11 16.25 1.33
1.64 6.64 5.01
1.68 2.56 7.36
-
-
1.00 5.60
1.00 5.63
1.44 3.11 2.93
1.10 10.12 0.88
1.51 7.35 3.71
1.51 3.32 6.10
-
-
1.00 2.39
1.00 2.41
1.33 1.58 2.91
1.10 3.51 1.20
1.04 2.14 0.47
1.19 1.60 3.07
-
-
L
L
D
L
L
D
L
L
D
L
L
D
D
L
valine
1.98 2.41 12.94
L
L
Chromatographic conditions; Flow rate = 1.0 mL/min; Detection UV 254 nm. aSeparation factor; bCapacity factor for the
first enantiomer eluted; cResolution factor; dAbsolute configuration of the second enantiomer eluted.
Table V. Optical purity results of the stability test for the
α
-amino acid methyl esters as benzophenone imine
benzophenone imine derivative of L-aspartic acid dimethyl
derivatives was examined (Table IV). Enantioselectiv-
ities and resolutions were found to be greatly influenced
by the nature of the mobile phase (Jin et al., 2006, 2009).
Of the mobile phases investigated, 0.5% 2-propanol/
hexane (v/v) best resolved the benzophenone imine
ester (Aldrich) stored at 4oC
Storage period
L:D ratioa
RSDb
1 Day
3 Days
5 Days
8 Days
9 Days
15 Days
16 Days
22 Days
30 Days
99.91:0.09
99.91:0.09
99.89:0.11
99.89:0.11
99.88:0.12
99.85:0.15
99.84:0.16:
99.82:0.18
99.81:0.19
1.2%
0.9%
3.1%
2.1%
1.0%
0.9%
1.0%
3.1%
3.0%
derivatives of α-amino acid methyl esters, whereas the
resolution provided by 30% dichloromethane/hexane (v/
v) was poorest. Also, optical purity results of the sta-
bility test for the benzophenone imine derivative of L-
aspartic acid dimethyl ester (Aldrich) stored at 4oC
after derivatization with benzophenone imine in 2-pro-
panol are shown in Table V. After 30 days of storage at
4oC, its optical purity was almost unaffected. Further-
more, stabilities of benzophenone imine derivatives of
amino acid esters were found to be considerably more
stable than those of the corresponding 9-anthraldehyde
aAverages of three determinations; Relative standard de-
viation.
b
derivatives (Huang et al., 2011). We also used our de- chromatograms of the benzophenone imine derivative
veloped chromatographic methods to determine the of commercially available L-phenylglycine methyl ester
enantiomeric purities of several commercially available on Chiralpak IC are presented in Fig. 2.
D- and L-amino acid methyl esters. As shown in Table
VI, enantiomeric impurity levels of <0.01-0.56% were method was developed for the resolution
found after benzophenone imine derivatization. Typical esters as their benzophenone imine Schiff base deriva-
In conclusion, a convenient liquid chromatographic
-amino acid
α
Table VI. Determination of the enantiomeric purities of the benzophenone imine derivatives of commercially available
α
-amino acid methyl esters
Entry
Analyte
Company
Sigma
Aldrich
Aldrich
Aldrich
Advanced ChemTech
Advanced ChemTech
Aldrich
D:L ratioa
RSDb
1
2
3
4
5
6
7
8
D-alanine methyl ester
L-alanine methyl ester
99.99:0.01
< 0.01: > 99.99
0.09:99.91
0.05:99.95
99.99:0.01
0.02:99.98
99.44:0.56
0.11:99.89
1.0%
0.9%
2.0%
0.8%
1.0%
0.8%
3.6%
L-aspartic acid dimethyl ester
L-leucine methyl ester
D-phenylalanine methyl ester
L-phenylalanine methyl ester
D-phenylglycine methyl ester
L-phenylglycine methyl ester
Aldrich
2.4%
a
b
Refer to the experimental section for chromatographic conditions. Average value of three determinations; Relative
standard deviation.

Separation of the Enantiomers of
α-Amino Acid Esters
1019
Fig. 2. Chromatograms of the enantiomeric resolution of the benzophenone imine derivative of racemic phenylglycine
methyl ester ( ) and L-phenylglycine methyl ester (Aldrich reagent) ( ) (D:L = 0.11:99.89) on Chiralpak IC. Mobile phase:
1% 2-propanol/hexane(v/v); flow rate = 1.0 mL/min; detection wavelength: UV 254 nm; injection amount: 5 g.
A
B
µ
tives using several coated and covalently bonded poly-
saccharide-derived CSPs. Of the CSPs studied, in gen-
eral, the covalently immobilized CSP, Chiralpak IC,
provided excellent resolution of the benzophenone
imine derivatives of α-amino acid esters, and greater
solvent choice. The developed methods were also used
to measure the enantiomeric purities of several com-
Jin, J. Y., Bae, S. K., and Lee, W., Comparative studies between
covalently immobilized and coated chiral stationary phases
based on polysaccharide derivatives for enantiomer sep-
aration of N-protected
α-amino acids and their ester
derivatives. Chirality, 21, 871-877 (2009).
Jin, J. Y., Lee, W., Park, J. H., and Ryoo, J. J., Covalently
bonded and coated chiral stationary phases derived from
poltsaccharide derivatives for enantiomer separation on
N-fluorenylmethoxycarbonyl α-amino acids with fluores-
cence detection. J. Liq. Chromatogr. Relat. Technol., 29,
1793-1801 (2006).
Jin, J. Y., Lee, W., Park, J. H., and Ryoo, J. J., Liquid chro-
matographic enantiomer separation of N-phthaloyl protected
mercially available
expected that the described liquid chromatographic
α-amino acid methyl esters. It is
method will provide a useful means of resolving
amino acid esters.
α-
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