and Eriksson,9) and Schill et al.13) also found that some amines and acidic compounds without ... 11) R, E. Feeney, G. E. Means and J. C. Bigler, J. Biol. Chem.
Vol. 35 (1987)
682
Chem. Pharm. 35( 2 ) 682-686
Bull. (1987 )_
Direct
Liquid
Chromatographic Use
Resolution
of Ovomucoid
of Racemic
as a Column
Compounds.
Ligand
TOSHINOBU MIWA,*'a MASAKI ICHIKAWA,a MASANORI TSUNO,b TEIICHI HATTORI,b TAKESHI MIYAKAWA,a MASANORI KAYANO,a and YASUO MIYAKEb Eisai Co., Ltd., Pharmaceutical Research Laboratory,a 2-1 Takehaya-machi, Kawashima-cho, Hashima-gun, Gifu 483, Japan and Eisai Tsukuba Laboratory,b 5-1-3 Tokodai, Toyosato-cho, Tsukuba-gun, Ibaragi 300-06, Japan (Received June 6, 1986)
A new chiral recognition column, with conjugated ovomucoid as the ligand, was developed. This column may be employed for the chiral resolution of acids as well as amines without derivatization. The retention time, capacity factor and resolution factor were dependent on pH, buffer strength and 2-propanol concentration of the mobile phase. For chlorpheniramine, a resolution factor of 1.5 was obtained. Keywords-chiral
resolution;
affinity
column;
HPLC;
enantiomer;
ovomucoid
Introduction Diastereomeric
derivatization
active
compounds
by
direct
resolution
many
researchers.
of
high
racemic For
poly(triphenylmethyl complex,3) In
addition,
a crown columns
to
column,
that
has
by
Hermansson,8)
a low
0.3
this
its
reported
report
variation ovomucoid
property
of
pH, as
the
a
ocracid
used
by
using
bovine
aracid
for the
ion the
glycoprotein
chiral
been
at has
serum
resolution pairing
the
been
is present
this
of in
some
Cohn
V
a
a
protein-
ability
beginning
of of
a
this
demonstrated
by
(BSA)-conjugated column.
racemic
method,
availability
HPLC.
and
albumin
of
a
developed:
recognition
started HPLC
by
gel,1)
charge-transfer
glycoprotein-conjugated
the
However,
the
the
achieved
normal-phase
column6)
compounds
a an
preferred
but
of
in
been
chiral
in
optically
recently,
silica
have
exchange
columns
employed
a
used HPLC
application
racemic
has
phase,2) been
ligand
of
but
acid-conjugated
reversed-phase
The of
who is
phases
amino
have
resolution
amines column of
can
these
fraction
The ƒ¿1
because also
be
proteins at
a
level
-
this used
is very of
only
mg/ml. we
of many
in
reported.
acids.9)
describe
ovomucoid.
by
stationary
gel4)
phase,5)
who
value,
racemic
example,
is conjugated and
pI
of
approximately In
certain
protein-conjugated
column
For
used
for
(HPLC),
stationary
resolution of
method
chromatography
silica
be
been
Bomgren,7)
resolution
limited.
may
have
and
glycoprotein
protein for
gel
usefulness
and
acid
preferred
3,5-dinitrobenzoyl
ether-conjugated
chromatographic The
Allenmark
on
stationary
silica
century.
a
the
liquid
compounds
example,
cyclodextrin-conjugated
protein
been
methacrylate)-conjugated
and
conjugated
has
performance
a new The
trypsin
nature
chiral
heat
of
inhibition11)
researchers.12) to
chiral
and recognition
resolution this
is well
Ovomucoid to
organic ligand
column
ovomucoid known.
Methods
is readily solvents. and
developed
whose has
these a method
recognition
thoroughly
for
available, For
chiral
been
its
purification
have
and
is relatively
reasons,
we
for
ligand
researched10)
chose
conjugation
been
stable
to
chicken which
is
No.
2
683
relatively
easy to perform
The resolution described.
and uses succinimide
of some racemic
compounds
and the characteristics
Materials Apparatus-
A Hitachi
monitor)
equipped
columns
of 4.6
electrode
pH
with (i.d.) •~
meter.
L-5000
system
a 655A-40 150
A
mm
hydrochloride
chlorprenaline are
in
Fig.
were
from
were by
Purification
Synthesis
different
The
overnight
in
activated
silica
procedure
time
been
buffer)
the
(pH
Tokyo
Kasei
gel . The
pH
Co.
variable-wavelength was was
used
UV
. Stainless-steel
measured
Chlorpheniramine
grade.
crystallization
with
a Toa
maleate
, chlorpremaleate and l-
d-Chlorpheniramine
methods.
The
structures
of
these
compounds
(0.1
washed was
3.0
resolution
dropwise.
6.7) were
containing calculated.
Q pH
water
water-organic to
purified
from
chicken
Gel-Aminopropyl
Unisil
M NaHCO3, with
added
Resolved by HPLC on an Ovomucoid-
was
Silica
Conditions-A
buffer and
oven
egg
white
by
the
ethanol-precipitation
Deutsch.12)
as follows:
buffer
gel had
of coupling
(k')
and
was
a coupling
Resolution phosphate
Ovomucoid
Fredericq
of Ovomucoid-Conjugated
carbonate.
30 ml
by
silica
a 655A
column
1.
of-Ovomucoid
described
are also
used.
of pharmaceutical
Fig. 1. Structures of Compounds Conjugated Column
method
with
a 655A-52
conjugated was
purchased
purified
and
ovomucoid
phenylpropanolamine
chromatograph
injector
polarimeter
was
and
hydrochloride
presented
with
digital
Chemicals ƒ¿,ƒÃ-Dibenzoyllysine naline
liquid
sample
packed
DIP-4
of the column
and Methods
(a 655A-11
automatic
were
JASCO
derivatives.
and The
NH2 6.8) then
Column
Results
at
room
with
the
gel
mixture as
the
temperature
was
activated
N,N-disuccinimidyl temperature
coupling
using
organic
used solvent
was
as
a rotary
30 ml
silica
was
the was
maintained
with
N
carbonate
buffer,
ovomucoid-conjugated
solvent 2-propanol
silica (2 g) and
gel
,N-disuccinimidyl (3 g) were reacted
evaporator
of ovomucoid was
eluent.
packed Five
used,
and
at
25 •Ž.
the
. After solution
into to
the
50 mm
change
the (2 g in
column
.
potassium of retention
and Discussion
Ovomucoid is one of the most stable proteins present in egg white. Accordingly , the ovomucoid-conjugated silica gel column prepared in this study was stable over a wide range of pH values and was stable to organic solvents. This column was also stable at room temperature for more than 3 months. Resolution of the acidic compound dibenzoyllysine was achieved with a 15cm column eluted with 20 mm potassium phosphate buffer (pH 6.0), as shown in Fig. 2. Amines (chlorpheniramine and chlorprenaline) were resolved on the same column and with the same buffer at different pH values and different 2-propanol concentrations. (Figs. 3 and 4). The pH of the mobile phase greatly affected the retention . Table I shows that higher pH values caused stronger retention of chlorprenaline and phenylpropanolamine . cx,eDibenzoyllysine was strongly retained by the ovomucoid column at lower pH values , as
Vol. 35 (1987)
684
Fig.
2.
Separation
of
the
Enantiomers
of ƒ¿,ƒÃ-
Fig.
Mobile
phase,
(pH 6.0); detection, earlier eluted peak
3. an
Dibenzoyllysine 20 mm
potassium
phosphate
220 nm; flow rate, 1.0 ml/min. is that of d-ƒ¿,ƒÃ-dibenzoyllysine.
Separation
Mobile
buffer The
Fig.
of
d,l-Chlorpheniramine
Ovomucoid-Conjugated phase,
(pH nm;
5.5) flow
the
mixture.
4.
20 mm
Separation
of
phosphate
phase,
buffer
2-propanol; detection, 220 sample amount, 2.5 ƒÊg as
d,l-Chlorprenaline
and
of l-Chlorprenaline
Ovomucoid-Conjugated Mobile
potassium
containing 6% rate, 1.2 ml/min;
Chromatogram
on
Column
on
an
Column 20 mm
potassium
(pH 6.1); detection, 210nm; flow sample amount, 1 ƒÊg as the mixture.
phosphate rate,
buffer
1.2 ml/min;
shown in Table II. These results show that the ovomucoid column exhibits a strong hydrophobic interaction with solutes. Allenmark and Bomgren7) stated, in their report on a BSA-conjugated silica gel HPLC column, that the k' values of aroyl-amino acids are influenced by hydrophobic interaction, coulombic interaction and hydrogen bonding. To eluscidate the coulombic interaction of the ovomucoid-conjugated column, the effect of the ionic strength of the mobile phase on the retention of solutes was examined (Table III). Tropic acid is strongly retained on this column at lower ionic strength. The retention of mandelic acid methyl ester, a non-ionic substance, was not influenced by mobile phase concentration. Higher k' values for chlorpheniramine were obtained at high buffer strengths. Though we have not examined these interactions in detail, it can be considered that they may be controllable by adjusting the protein and buffer conditions. Hermansson8) stated that, in an orosomucoid-conjugated column, oxazoline derivatives
No.
2
685
TABLEI. Separation of Racemic Chlorprenaline and Phenylpropanolamine by HPLC on an Ovomucoid-Conjugated Silica Gel Column
Mobile phase: 20 mm potassium
phosphate.
Column:
4.6 x 150 mm.
TABLEII. Separation of Racemic a,e-Dibenzoyllysine by HPLC on an Ovomucoid-Conjugated Silica Gel Column
Mobile
phase:
20 mm
TABLE III.
potassium
Influence
phosphate.
of Buffer
a Non-ionic
Mobile
phase:
potassium
TABLE IV.
Mobile potassium
phase: phosphate
For
phosphate.
Strength
Substance,
Column:
4.6 •~
4.6 •~
150 mm.
on the and
150
mm
Retention
of an Acid,
an Amine
.
Influence of 2-Propanol Concentration on the Resolution of Chlorprenaline and Chlorpheniramine
chlorprenaline,
(pH
Column:
6.5).
Column:
20 mm
potassium
4.6 •~ 150 mm.
phosphate
(pH
6.1);
for
chlorpheniramine,
20 mm
Vol. 35 (1987)
686
of racemic fl-blockers (e.g. propranolol), acetylated primary amines, and the ethyl ester of mandelic acid can be resolved with excellent separation factors. More recently, Hermansson and Eriksson,9) and Schill et al.13)also found that some amines and acidic compounds without derivatization could be separated on an orosomucoid-conjugated column. An ovomucoidconjugated column could resolve chlorpheniramine and chlorprenaline without derivatization. Though the two amines were resolved by this method, the k' values were strongly influenced by the pH of the mobile phase, and at high pH values (7 and above) the amines were retained on the column. Phenylpropanolamine was resolved slightly when the pH of the mobile phase was 6.4, but not at 6.1 or 6.7. The reason for this phenomenon is not yet clear. The influence of a mobile phase additive (2-propanol) on the resolution of chlorprenaline and chlorpheniramine is shown in Table IV. Addition of 2-propanol caused a decrease in the retention times of both compounds. The separation factor of chlorprenaline enantiomers decresed when the retention time was shorter. Although the resolution factor of chlorpheniramine tended to become lower as the concentration of 2-propanol was increased to 4%, a 2propanol concentration of 6% resulted in an increase of k' value to a level higher than that when no 2-propanol was present, and gave excellent resolution. Though we have not tested higher concentration of 2-propanol, an optimum concentration may exist. Conclusion Ovomucoid, a stable protein present in egg white, is useful for chiral recognition. It is readily available, and from this viewpoint, an ovomucoid column is preferable to an orosomucoid column. Though ovomucoid is stable at various pH values, a high pH will cause decomposition of the silica gel, so the use of a porous polymers as the stationary phase may allow the more extensive use of ovomucoid as a chiral stationary-phase ligand.
References
1) 2) 3) 4) 5) 6) 7) 8) 9) 10) 11) 12) 13)
and
Notes
W. H. Pirkle and M. H. Hyun, J. Chromatogr., 328, 1 (1985). H. Yuki, Y. Okainoto and I. Okamoto, J. Am. Chem. Soc., 102, 6356 (1980). F. Mikes, G. Boshart and E. Gil-av, J. Chromatogr., 122, 205 (1976). S. S. Peacock, D. M. Walba, F. C. A. Gaeta, R. C. Helgeson and D. J. Cram, J. Am. Chem. Soc., 102, 2043 (1980). D. W. Armstrong, U. S. Patent 4539399 (1985) [Chem. Abstr., No. 103226754]. V. A. Davankov, Adv. Chromatogr., 18, 139 (1980). S. Allenmark and B. Bomgren, J. Chromatogr., 264, 63 (1983). J. Hermansson, J. Chromatogr., 325, 379 (1985). J. Hermansson and M. Eriksson, J. Liq. Chromatogr., 9, 621 (1986). M. Kanamori and S. Kawabata, Nippon Nogeikagaku Kaishi, 38, 367 (1964). R, E. Feeney, G. E. Means and J. C. Bigler, J. Biol. Chem., 224, 1957 (1969). E. Fredericq and H. F. Deutsch, J. Biol. Chem., 181, 499 (1986). G. Schill, I. W. Wainer and S. A. Barkan, J. Liq. Chromatogr., 9, 641 (1986).
