Enantioseparation using cyclosophoraoses as a novel chiral additive in capillary electrophoresis

Article abstract of DOI:10.1016/S0008-6215(03)00083-1

Cyclosophoraoses, cyclic β-(1→2)-D-glucans produced by Rhizobium meliloti 2011, were used as a novel chiral additive for the separation of terbutaline, amethopterin, thyroxine and N-acetylphenylalanine enantiomers in aqueous capillary electrophoresis (CE). Enantioseparation took place in the normal- or reversed-polarity mode when a high concentration of neutral (60 mM) or anionic (40 mM) cyclosophoraoses was added to the background electrolyte (BGE).

Full text of DOI:10.1016/S0008-6215(03)00083-1

Carbohydrate Research 338 (2003) 1143–1146
www.elsevier.com/locate/carres
Note
Enantioseparation using cyclosophoraoses as a novel chiral
additive in capillary electrophoresis
Sanghoo Lee, Seunho Jung*
Department of Microbial Engineering and Bio/Molecular Informatics Center, Konkuk Uni6ersity, 1 Hwayang-dong, Gwangjin-gu, Seoul 143-701,
South Korea
Received 17 October 2002; accepted 17 February 2003
Abstract
Cyclosophoraoses, cyclic b-(1“2)-D-glucans produced by Rhizobium meliloti 2011, were used as a novel chiral additive for the
separation of terbutaline, amethopterin, thyroxine and N-acetylphenylalanine enantiomers in aqueous capillary electrophoresis
(CE). Enantioseparation took place in the normal- or reversed-polarity mode when a high concentration of neutral (60 mM) or
anionic (40 mM) cyclosophoraoses was added to the background electrolyte (BGE). © 2003 Elsevier Science Ltd. All rights
reserved.
Keywords: Cyclosophoraoses; R. meliloti 2011; Enantiomeric separation; Capillary electrophoresis; Chiral additive
bones. The glucopyranose unit of neutral and anionic
cyclosophoraoses composed of glycosidic b-(1“2) link-
age is shown in Fig. 1. Recently, we reported that these
macrocyclic molecules function as a chiral NMR sol-
vating agent.6 To the best of our knowledge, there have
been no reports on the separation of enantiomers using
cyclosophoraoses as a chiral additive in CE.
Herein, we describe for the first time that a family of
cyclosophoraoses functions as a chiral selector in CE
for enantiomeric separations of the antiasthmatic drug,
terbutaline, the antineoplastic agent, amethopterin, thy-
roxine hormone, and an a-amino acid derivative, N-
acetylphenylalanine.
Chirality remains an important consideration for
many compounds such as pharmaceuticals, food addi-
tives and agrochemicals. Enantiomeric separation by
capillary electrophoresis (CE) has rapidly attracted at-
tention as a promising technique because of its high
efficiency, low cost, wide range of separation mecha-
nisms, simplicity and flexibility, and very low consump-
tion of buffer and samples. Recent capillary
electrophoretic literature focuses on the utilization of
chiral selectors in the background electrolyte (BGE) for
the resolution of enantiomers.1
Cyclosophoraoses are a family of unbranched cyclic
b-(1“2)-D-glucans produced as intra- or extraoligosac-
charides by many strains of Rhizobium and Agrobac-
terium as a mixture of molecules with various sizes in a
neutral or anionic form.2 Recent reports have shown
that cyclosophoraoses have potential as a complexation
host for the solubility enhancement of poorly soluble
guest molecules.3b–d Though the exact three-dimen-
sional structure of cyclosophoraose is not known, re-
cent nuclear magnetic resonance (NMR) studies5 and
molecular dynamics simulations5,6,15,17 have provided
molecular models with flexible glycosidic linkage back-
* Corresponding author. Tel.: +82-2-4503520; fax: +82-
2-4523611
E-mail address: shjung@konkuk.ac.kr (S. Jung).
Fig. 1. The glucopyranose unit of neutral and anionic cy-
closophoraoses.
0008-6215/03/$ - see front matter © 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0008-6215(03)00083-1

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S. Lee, S. Jung / Carbohydrate Research 338 (2003) 1143–1146
Table 1
buffer (separation factor (h)=1.07, Table 1) in the
normal polarity mode. It has been reported that the
chiral additives such as CD derivatives containing ion-
izable sulfate groups,10 nonionizable dimethyl19 and
hydroxypropyl groups11 as well as native b-CD19
provide moderate enantiomeric resolutions for racemic
terbutaline in CE. In the present study, the racemic
terbutalines were separated into each enantiomer by
adding neutral cyclosophoraoses to the BGE.
Racemic amethopterin was known to be moderately
resolved by macrocyclic antibiotics as chiral additives in
CE.12,13 In the present study, chiral separation occurred
with much higher enantioselectivity (h=1.48, Rs=
11.2, Fig. 2A) in the normal polarity mode when the 60
mM neutral cyclosophoraoses were added as a chiral
additive to the BGE. The (+)-enantiomer of
amethopterin migrated before the (−)-enantiomer.
Retention time (tR), separation factor (h) and resolution (Rs)
of enantiomers resolved by adding neutral a or anionic b
cyclosophoraoses
Enantiomers
Migration time (min)
tR
h
Rs
c
Terbutaline
Amethopterin
Thyroxine
5.06 a
1.64 a
9.91 a
28.85 b
1.07 3.9
1.48 11.2
1.02 13.6
1.13 18.3
Thyroxine
N-Acetylphenylalan 5.95 b
1.14
8.4
ine
a 60 mM neutral cyclosophoraoses were added to the BGE.
b 40 mM anionic cyclosophoraoses were added to the BGE.
c Migration time of the first enantiomers eluted from capil-
lary column.
Structural analyses of the isolated neutral and an-
ionic cyclosophoraoses were carried out as described
previously.3b–f The ring sizes of the neutral3b or
anionic3e cyclosophoraoses were confirmed through ma-
trix-assisted laser desorption ionization-mass spec-
trometry
(MALDI-MS)
and
electrospray
ionization-mass spectrometry (ESI-MS) analyses, rang-
ing from DP 17 to 27, respectively. Based on the
MALDI-MS and ESI-MS analyses, the number-aver-
age molecular weight (Mn) of the neutral and anionic
cyclosophoraoses was determined as 3568.63d and
3940.2,3e respectively. Based on the previous results,3e,18
we confirmed that the anionic cyclosophoraoses used in
the present study contained the phosphoglycerol
molecules as the substituent at C-6 of the glucose
residue as shown in Fig. 1.
As pointed out by Wren and Rowe,7 weakly interact-
ing analytes with a chiral additive typically require
higher concentrations of the chiral additive than
strongly interacting ones. However, the longer migra-
tion time accompanying higher concentrations of a
chiral additive may also limit the utility of the chiral
additive. To overcome this problem, negatively charged
chiral addtives have been predominantly employed to
the effective separations in reversed polarity mode.8–10
As the representative chiral additives, various cyclomal-
tooligosaccharides (cyclodextrins, CDs), which contain
a-(1“4)-glycosidically linked glucopyranose units, and
their derivatives have been employed.12
Herein, we performed effective separations of the
enantiomers with cyclosophoraoses used as a novel
chiral additive in the normal/reversed polarity mode
(Table 1 and Fig. 2). Intrinsic high solubility3a of
cyclosophoraoses in aqueous solution gave benefit to
the CE experiment.
In the case of racemic terbutaline, a resolution (Rs)
value of 3.9 was obtained with 60 mM neutral cy-
closophoraoses added to the acidic (pH 3.0) running
Fig. 2. Electropherograms showing enantiomeric separations
induced by neutral or anionic cyclosophoraoses used as a
novel chiral additive in CE. (A) Enantiomeric separation of a
nonracemic mixture of amethopterin ((+):(−)=3:2)) in-
duced by 60 mM neutral cyclosophoraoses in the normal
polarity mode at 10 kV. (B) Enantiomeric separation of
nonracemic mixture of thyroxine (D:L=1:2) induced by 40
mM anionic cyclosophoraoses in the reversed polarity mode,
pH 3.0 at −10 kV.

S. Lee, S. Jung / Carbohydrate Research 338 (2003) 1143–1146
1145
The enantiomers of thyroxine were known to be
resolved by using anionic CDs as chiral additives in
CE.14 Likewise, thyroxine enantiomers were separated
with a very high resolution (h=1.13, Rs=18.3) by
adding 40 mM anionic cyclosophoraoses to the BGE in
the reversed polarity mode (Table 1 and Fig. 2B). This
enantiomers were also fully resolved (h=1.02, Rs=
13.6) in the normal polarity mode with 60 mM neutral
cyclosophoraoses added as a chiral additive (Table 1).
closophoraoses were achieved as our previous
reports.3b–f
1.3. Structural analyses of neutral and anionic
cyclosophoraoses
The structure and molecular weight of neutral and
anionic cyclosophoraoses was confirmed through NMR
spectroscopy,3c–e ESI-MS3e and MALDI-MS3b analyses
as our previous reports.
The
L enantiomer of thyroxine migrated before the D
enantiomer by adding either neutral or anionic
cyclosophoraoses.
1.4. Capillary electrophoretic conditions
In the previous study, we showed that the N-
acetylphenylalanine enantiomers were discriminated
throughout the NMR analysis by neutral cy-
closophoraoses in aqueous solution.4 A nonracemic
mixture (D:L=2:3) of this a-amino acid derivative was
also resolved in a CE experiment at the reversed polar-
ity mode (h=1.14, Rs=8.4, Table 1).The N-acetyl-D-
All CE experiments were performed using a Agilent 3D
CE System (Wilmington, DE, USA) equipped with a
diode array detector. Separations were carried out with
uncoated fused-silica capillary (50 mm i.d., 33 cm total
length, effective length 24.5 cm). The polyimide coating
of the capillary was stripped to create a 0.5-cm detec-
tion window. The BGE consisted of an aqueous solu-
tion of 50 mM KH2PO4, adjusted to pH 3.0 with
phosphoric acid. The sample solutions were prepared in
the run buffer to give final concentrations of 0.02 mg
mL−1 for terbutaline, 1.36 mg mL−1 for amethopterin,
2.33 mg mL−1 for thyroxine and 1.0 mg mL−1 for
N-acetylphenylalanine, respectively. The analytes were
monitored at 211 nm for terbutaline, at 201 nm for
amethopterin, at 220 nm for thyroxine, and at 205 nm
for N-acetylphenylalanine, respectively. The neutral or
anionic cyclosophoraoses were used as chiral additives
for enantioseparation of terbutaline, amethopterin, thy-
roxine and N-acetylphenylalanine in the normal/re-
versed polarity mode at 910 kV. The samples were
injected by a pressure of 5 kPa for 1–3 s. Before each
analysis, the capillary was rinsed for 2 min with water
and 3 min with the BGE solution. The capillary was
thermostated at 20 °C.
phenylalanine enantiomer migrated before the
L
enantiomer. The migration order of each enantiomer in
CE could explain the extent of the interaction of cy-
closophoraoses with each enantiomer as shown in the
previous NMR data.4
Throughout the present investigation, we showed
that a family of neutral or anionic cyclosophoraoses
was successfully utilized as novel chiral additives for
enantioseparation in CE. Cyclosophoraoses seemed to
provide the capacity for the required difference in both
the binding of the enantiomers and the appropriate
mobility. They shortened the analysis time as well as
greatly increased the efficiency of enantioseparation of
the analytes in the normal or reversed polarity mode.
Although the exact molecular mechanism of the chiral
separation by cyclosophoraoses remains to be eluci-
dated, we postulate that the chiral recognitions are
likely to be induced by the portions associated with the
b-glycosidic linkages of cyclosophoraoses as we de-
scribed previously.6
Acknowledgements
1. Experimental
We thank Professor D.S. Chung in Seoul National
University for his helpful discussion. We also thank CE
group in national instrumentation center for environ-
mental management (NICEM). This work was sup-
ported by grants from Bioproducts and Biotechnology
Research Group (01-J-BP-01-B-59) in Ministry of Sci-
ence and Technology in South Korea. SDG.
1.1. Chemicals and reagents
All chemicals, including enantiomers or racemates of
terbutaline,
amethopterin,
thyroxine
and
N-
acetylphenylalanine used in this study were purchased
from Sigma Chemical Co. (St. Louis, MO, USA).
1.2. Preparation of neutral and anionic
cyclosophoraoses
References
1. Chankvetadze, B.; Blaschke, G. J. Chromatogr. A 2001,
R. meliloti 2011 was cultured in a 5-L jar fermenter
containing GMS medium as previous report.16 Isolation
and purification of neutral and anionic cy-
906, 309–363.
2. Breedveld, M. W.; Miller, K. J. Microbiol. Re6. 1994, 58,
145–161.

1146
S. Lee, S. Jung / Carbohydrate Research 338 (2003) 1143–1146
8. Gahm, K.; Stalcup, A. M. Chirality 1996, 8, 316–324.
3. (a) Koizumi, K.; Okada, Y.; Horiyama, S.; Utamura, T.;
9. Gahm, K.; Chang, L. W.; Armstrong, D. W. J. Chro-
matogr., A 1997, 759, 149–155.
10. Gratz, S. R.; Stalcup, A. M. Anal. Chem. 1998, 70,
5166–5171.
11. Kim, K. H.; Kim, H. J.; Jeun, E. Y.; Seo, S. H.; Hong, S.
P.; Kang, J. S.; Youm, J. R.; Lee, S. C. Arch. Pharm. Res.
2001, 24, 281–285.
12. Fanali, S. J. Chromatogr. A 2000, 875, 89–122.
13. Gasper, M. P.; Berthod, A.; Nair, U. B.; Armstrong, D.
W. Anal. Chem. 1996, 68, 2501–2514.
14. Fillet, M.; Hubert, P.; Crommen, J. Electrophoresis 1998,
19, 2834–2840.
15. Palleschi, A.; Crescenzi, V. Gazz. Chim. Ital. 1985, 115,
243–245.
16. Breedveld, M. W.; Zevenhuizen, L. P. T. M.; Zehnder, A.
J. B. Appl. En6iron. Microbiol. 1990, 56, 2080–2086.
17. York, W.; Thomsen, J.; Meyer, B. Carbohydr. Res. 1993,
248, 55–80.
18. Miller, K. J.; Gore, R. S.; Benesi, A. J. J. Bacteriol. 1988,
170, 4569–4575.
Higashiura, T.; Ikeda, M. J. Incl. Phenom. 1984, 2,
891–899;
(b) Kwon, C.; Choi, Y.; Kim, N.; Yoo, J.; Yang, C.;
Kim, H.; Jung, S. J. Incl. Phenom. 2000, 36, 55–65;
(c) Lee, S.; Kwon, C.; Choi, Y.; Seo, D.; Kim, H.; Jung,
S. J. Microbiol. Biotechnol. 2001, 11, 463–468;
(d) Lee, S.; Seo, D.; Kim, H.; Jung, S. Carbohydr. Res.
2001, 334, 119–126;
(e) Seo, D.; Lee, S.; Park, H.; Yi, D.; Ji, E.; Shin, D.;
Jung, S. Bull. Korean Chem. Soc. 2002, 23, 899–902;
(f) Seo, D.; Lee, S.; Park, H.; Kwon, T.; Jung, S. J.
Microbiol. Biotechnol. 2002, 12, 522–525.
4. Lee, S.; Jung, S. Carbohydr. Res. 2002, 337, 1785–1789.
5. Mimura, M.; Kitamura, S.; Gotoh, S.; Takeo, K.;
Urakawa, H.; Kajiwara, K. Carbohydr. Res. 1996, 289,
25–37.
6. Chio, Y.; Yang, C.; Kim, H.; Jung, S. Carbohydr. Res.
2000, 326, 227–234.
7. Wren, S. A. C.; Rowe, R. C. J. Chromatogr. 1992, 603,
235–241.
19. Fanali, S. J Chromatogr. A 1991, 545, 437–444.