LC Enantioseparation of β-Amino Acids on a Crown Ether-Based Stationary Phase

Article (PDF Available)inChromatographia 68:13-18 · October 2008with24 Reads
DOI: 10.1365/s10337-007-0498-x
Abstract
Reversed-phase high-performance liquid chromatographic methods were developed for the enantioseparation of ten unusual β-3-homo-amino acids. The underivatized analytes were separated on a chiral stationary phase containing (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid as chiral selector. The effects of organic and acidic modifiers and the mobile phase composition on the separation were investigated. The structures of the substituents in the β position substantially influenced the retention and resolution. The elution sequence was determined in some cases: the S enantiomers eluted before the R enantiomers.
LC Enantioseparation of b-Amino Acids
on a Crown Ether-Based Stationary Phase
Robert Berkecz
1
, Istva
´
n Ilisz
1
, Zolta
´
n Pataj
1
, Ferenc Fu
¨
lo
¨
p
2
, Hee Jung Choi
3
, Myung Ho Hyun
3
,
Antal Pe
´
ter
1,&
1
Department of Inorganic and Analytical Chemistry, University of Szeged, Do
´
mte
´
r 7, 6720 Szeged, Hungary;
E-Mail: apeter@chem.u-szeged.hu
2
Institute of Pharmaceutical Chemistry, University of Szeged, Eo
¨
tvo
¨
s utca 6, 6720 Szeged, Hungary
3
Department of Chemistry and Chemistry Institute for Functional Materials, Pusan National University, Busan 609-735, South Korea
Received: 31 August 2007 / Revised: 11 October 2007 / Accepted: 26 November 2007
Online publication: 5 January 2008
Abstract
Reversed-phase high-performance liquid chromatographic methods were developed for the
enantioseparation of ten unusual b-3-homo-amino acids. The underivatized analytes were
separated on a chiral stationary phase containing (+)-(18-crown-6)-2,3,11,12-tetracarb-
oxylic acid as chiral selector. The effects of organic and acidic modifiers and the mobile
phase composition on the separation were investigated. The structures of the substituents in
the b position substantially influenced the retention and resolution. The elution sequence was
determined in some cases: the S enantiomers eluted before the R enantiomers.
Keywords
Column liquid chromatography
b-3-Homo-amino acids
(+)-(18-Crown-6)-2,3,11,12-tetracarboxylic acid-based chiral stationary phase
Introduction
In recent years, b-amino acids have re-
ceived increased attention either because
of their unique pharmacological prop-
erties or in consequence of their key
components of numerous bioactive
molecules (e.g. taxane derivatives and
bestatin), developmental pharmaceuti-
cals, peptidomimetics and are important
constituents of natural products such as
alkaloids, peptides and b-lactam antibi-
otics [15]. b-Amino acids can be used as
building blocks of biologically active
peptides and b-peptides fold into com-
pact helices in solution [68].
The wide-ranging utility of these
compounds has led to much attention
being paid to their enantioselective
syntheses [911], which require analyti-
cal methods for a check on the enan-
tiopurity of the final products. For these
purposes, mainly high-performance
liquid chromatographic (LC) separation
methods are widely used. The applica-
tion of chiral crown ethers as chiral
stationary phases (CSPs) was initiated
by Cram et al. [12], and (+)-(18-crown-
6)-2,3,11,12-tetracarboxylic acid has
been successfully applied as a CSP
[1315].
The HPLC enantioseparation of
b-amino acids has been achieved both by
indirect and direct chromatographic
methods [1625]. In the last decade
D’Acquarica et al. [22] and Pe
´
ter et al.
[2632] separated different b-amino acids
on new types of CSPs, containing an
(R)-N,N-carboxymethylundecyl phenyl-
glycinol derivative, a macrocyclic glyco-
peptide antibiotic, a quinine-derived
chiral anion-exchanger or a crown ether
as chiral selector, while Hyun et al.
[3337] applied crown ether and ligand
exchange selectors.
Presented at: 7th Balaton Symposium on
High-Performance Separation Methods,
Sio
´
fok, Hungary, September 5–7, 2007.
2008, 68, S13–S18
DOI: 10.1365/s10337-007-0498-x
2008 Friedr. Vieweg & Sohn Verlag/GWV Fachverlage GmbH
Original Chromatographia Supplement Vol. 68, 2008
S13
In the present paper, direct LC
methods are described for the enantio-
separation of b-3-homo-amino acids,
with the application of a (+)-(18-crown-
6)-2,3,11,12-tetracarboxylic acid-based
CSP as chiral selector (Fig. 1). The
effects of different parameters on the
selectivity, such as the nature of the
organic and acidic modifiers, the mobile
phase composition, and the structure of
the analyte, are examined and discussed.
The separation of the stereoisomers was
optimized by variation of the chro-
matographic parameters. The elution
sequence was determined in some cases.
Experimental
Chemicals and Reagents
Racemic 3-aminobutanoic acid (1) and
3-aminopentanoic acid (2) (Table 1) were
prepared from the corresponding a,b-
unsaturated acids by benzylamine addi-
tion and subsequent debenzylation of the
products with a 20% metallic palladium
on charcoal in a hydrogen atmosphere
[38, 39]. (R)-3-Aminobutanoic acid was
prepared by the same method, but
(R)-(+)-a-methylbenzylamine was used
in the addition step instead of benzyl-
amine [40]. The other racemic b-amino
acids, 3-amino-4-methylpentanoic acid
(3), 3-amino-4,4-dimethylpentanoic acid
(4), 3-amino-4-methylhexanoic acid (5),
3-amino-4-ethylhexanoic acid (6), 3-ami-
no-3-cyclohexylpropanoic acid (9) and
3-amino-3-(3-cyclohexen-1-yl)propanoic
acid (10), were synthetized from the cor-
responding aldehydes by a modification
of the procedure of Rodionov and
Malivinskaia [41]: the aldehydes were
condensed with an equimolar amount of
malonic acid in refluxing 96% ethanol in
the presence of two equivalents of
ammonium acetate [42, 43]. Enantiome-
rically pure (R)- and (S)-3-amino-5-hex-
enoic acid (7) and (R)- and (S)-3-amino-
5-hexynoic acid (8) were from Solvay to
Peptisyntha (Brussels, Belgium).
Acetonitrile (MeCN), methanol
(MeOH) of LC grade and glacial acetic
Table 1. Chromatographic data, retention factor (k), separation factor (a) and resolution (R
S
)
for the direct separation of the stereoisomers of b-amino acids on (+)-(18-crown-6)-2,3,11,12-
tetracarboxylic acid CSP at different mobile phase compositions
Analyte Mobile phase
a,H
2
O/MeOH in 5 mM
AcOH b,H
2
O/EtOH in
5 mM AcOH c,H
2
O/MeOH/
EtOH in 5 mM AcOH
k
1
k
2
a R
S
1
COOH
NH
H
3
C
2
70/30, a 1.11 1.28 1.15 0.80
50/50, a 2.24 2.65 1.18 1.15
30/70, a 5.75 7.40 1.29 1.30
50/50, b 2.48 3.0 1.21 1.70
50/25/25, c 2.04 2.54 1.25 1.75
2
COOH
NH
2
H
3
C
30/70, a 2.37 3.25 1.37 1.80
50/50, b 1.38 1.92 1.39 2.40
50/25/25, c 1.12 1.55 1.38 2.10
3
COOH
NH
2
CH
3
H
3
C
30/70, a 1.19 1.65 1.38 1.20
50/50, b 0.75 1.02 1.36 1.30
30/70, b 1.55 2.30 1.48 1.85
50/25/25, c 0.71 0.97 1.37 1.25
4
COOH
NH
2
CH
3
H
3
C
H
3
C
30/70, a 0.40 0.63 1.57 <0.60
50/50, b 0.88 0.92 1.05 <0.40
50/25/25, c 0.43 0.52 1.21 <0.40
5
COOH
NH
2
CH
3
CH
3
30/70, a 0.90 1.11 1.23 <0.60
50/50, b 0.86 0.86 1.00 <0.40
50/25/25, c 0.71 0.83 1.17 <0.40
6
COOH
NH
2
H
3
C
H
3
C
30/70, a 0.72 1.23 1.71 1.25
50/50, b 0.73 0.98 1.34 1.35
50/25/25, c 0.86 1.38 1.60 2.05
7
COOH
CH
CH
2
NH
2
30/70, a 2.30 3.06 1.33 1.70
50/50, b 0.90 1.13 1.25 1.05
50/25/25, c 1.17 1.67 1.42 2.25
8
COOH
NH
2
C
CH
30/70, a 2.40 3.49 1.45 1.55
50/50, b 0.80 0.99 1.24 1.85
50/25/25, c 1.05 1.39 1.32 1.65
9
COOH
NH
2
30/70, a 1.13 1.69 1.50 1.40
50/50, b 0.93 1.30 1.39 1.55
50/25/25, c 0.68 1.05 1.54 2.00
10
COOH
NH
2
30/70, a 1.19 1.89 1.59 1.65
50/50, b 0.96 1.49 1.55 1.75
50/25/25, c 0.84 1.28 1.54 1.40
Column, (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid CSP; mobile phase: a,H
2
O/MeOH
(v/v) in 5 mM AcOH, b,H
2
O/EtOH ( v/v) in 5 mM AcOH, c,H
2
O/MeOH/EtOH (v/v/v)in5mM
AcOH; detection at 205 nm; flow rate, 0.5 mL min
1
; t
0
= 4.32 min
HOOC
COOH
NH
Si
O
HN
Si
O
O
O
O
O
O
O
Fig. 1. Structure of (+)-(18-crown-6)-2,3,11,
12-tetracarboxylic acid-based CSP
S14 Chromatographia Supplement Vol. 68, 2008 Original
acid (AcOH) were purchased from
Merck (Darmstadt, Germany). Ethanol
(EtOH), 1-propanol (PrOH), 2-propanol
(IPA), tert-butanol and other reagents of
analytical reagent grade were from
Sigma-Aldrich Kft (Budapest, Hungary).
Milli-Q water was further purified by
filtration on a 0.45-lm Millipore filter,
type HV (Molsheim, France).
Apparatus
The HPLC measurements were carried
out on a Waters LC system consisting of
an M-600 low-pressure gradient pump,
an M-996 photodiode-array detector
and a Millenium
32
Chromatography
Manager data system. The chromato-
graphic system was equipped with
Rheodyne Model 7125 injector (Cotati,
CA, USA) with 20-lL loops.
The (+)-(18-crown-6)-2,3,11,12-tet-
racarboxylic acid-based CSP, 5-lm par-
ticle size, 150 · 4.0 mm I.D., was
prepared via the method described in a
previous paper [13]. The columns were
thermostated in a water bath, with a
cooling-heating thermostat (MK 70,
Mechanik Pru
¨
fgera
¨
te, Medlingen, Ger-
many). The precision of the temperature
adjustment was ±0.1 C.
Results and Discussion
On the crown ether-based column, the
results of the separations of the enan-
tiomers of b-3-homo-amino acids were
evaluated by using different composi-
tions of mobile phases of H
2
O/MeOH,
H
2
O/MeCN, H
2
O/EtOH, H
2
O/IPA,
H
2
O/MeOH/EtOH or H
2
O/MeOH/IPA
containing AcOH, H
3
PO
4
or H
2
SO
4
in
different concentrations as acidic
modifier. To simplify the presentation,
mainly the results relating to partial or
baseline enantiomeric separation are
listed in Table 1. However, in a few
cases, for purposes of comparison,
examples are included where no sepa-
ration occurred. In Table 1, results
obtained with MeCN and IPA as
organic modifiers are not included. The
reason for this is that these modifiers
were much less effective in the enan-
tioseparation; only analytes 2 and 10
exhibited partial separation when they
were used.
In the process of chiral recognition
on the crown ether-based CSP the most
important interaction of analytes con-
taining a primary amino group is com-
plex formation between the protonated
primary amino group and the oxygen
atoms of the crown ether ring. Besides
this interaction, H-bonding, hydropho-
bic, dipole–dipole, steric, etc. interac-
tions should be taken into account. Lee
et al. [44] reported that the high enanti-
oselectivity of this type of CSP for
a-amino acids was due to the H-bonding
between one carboxylic acid of the CSP
and a carbonyl group oxygen of the
amino acid.
The data on analyte 1 revealed that
an increase in the content of MeOH in
the aqueous mobile phase increased the
retention, as indicated by the retention
factor k (Table 1). This behavior is
unusual in reversed-phase chromatogra-
phy. A detailed investigation of the ef-
fects of the MeOH and EtOH contents
on the enantioseparation for analytes 1,
3 and 7 is outlined in Fig. 2. The
increasing retention factor with increas-
ing alcohol content could be attributed
to the decreased polar interactions
between the less polar mobile phase and
polar b-amino acids. These trends were
observed for all of the investigated
analytes.
The a values generally slightly in-
creased or did not change significantly
with increasing alcohol content (Fig. 2).
It means that non-chiral interactions
were favored for the enantiomers both at
high or low alcohol content. As concerns
0 20 40 60 80 100
0,0
2,5
5,0
7,5
10,0
12,5
15,0
17,5
20,0
22,5
25,0
0,0
0,2
0,4
0,6
0,8
1,0
1,2
1,4
1,6
1,8
2,0
1
k
1
'
α, R
S
MeOH (%)
k
1
'
α
R
S
020406080100
0
5
10
15
20
25
30
35
40
45
50
55
0,0
0,3
0,6
0,9
1,2
1,5
1,8
2,1
2,4
2,7
3,0
3,3
1
k
1
'
α, R
S
EtOH (%)
k
1
'
α
R
S
0 20 40 60 80 100
0,0
0,5
1,0
1,5
2,0
2,5
3,0
3,5
4,0
4,5
5,0
0,0
0,3
0,6
0,9
1,2
1,5
1,8
2,1
2,4
2,7
3,0
3
k
1
'
α, R
S
MeOH (%)
k
1
'
α
R
S
020406080100
0,0
2,0
4,0
6,0
8,0
10,0
12,0
0,0
0,6
1,2
1,7
2,3
2,9
3,5
3
k
1
'
α, R
S
EtOH (%)
k
1
'
α
R
S
0 20406080100
0,0
1,0
2,0
3,0
4,0
5,0
6,0
7,0
8,0
9,0
10,0
0,0
0,2
0,4
0,6
0,8
1,0
1,2
1,4
1,6
1,8
2,0
7
k
1
'
α, R
S
MeOH (%)
k
1
'
α
R
S
0 20 40 60 80 100
0,0
2,0
4,0
6,0
8,0
10,0
12,0
14,0
16,0
18,0
20,0
22,0
0,0
0,2
0,5
0,7
1,0
1,2
1,5
1,7
2,0
2,2
2,5
2,7
7
k
1
'
α, R
S
EtOH (%)
k
1
'
α
R
S
Fig. 2. Dependence of the retention factor of the first-eluting enantiomer (k
1
), the separation
factor (a) and the resolution (R
S
) on the MeOH or EtOH content in the hydro-organic mobile
phase Column, (+)-(18-crown-6)-2,3,11,12-tetracarboxylic acid-based CSP; compounds, (1)3-
aminobutanoic acid, (3) 3-amino-4-methylpentanoic acid, (7) 3-amino-5-hexenoic acid; mobile
phase, H
2
O/MeOH or H
2
O/EtOH = 90/10, 70/30, 50/50, 30/70, 10/90 (v/v) in 5.0 mM AcOH;
flow rate, 0.5 mL min
1
; detection at 205 nm
Original Chromatographia Supplement Vol. 68, 2008 S15
the R
S
values, similarly as for the reten-
tion factors, high increases were ob-
served with increasing alcohol content
(Fig. 2). This behavior is very different
from that observed for b-amino acids
containing aromatic substituents in the b
position [32].
The nature of the alcoholic modifier
exerted a great effect on the retention
and resolution, whereas the selectivity
did not change substantially. For ana-
lyte 3, Fig. 3 shows that at constant
organic modifier and acid contents of
H
2
O/alcoholic mofifier = 30/70 (v/v)
and 5.0 mM AcOH, the retention fac-
tors and resolutions increased with
increasing chain length of the alcohol
especially in the case of alcohols with
branched and bulky side-chains such as
IPA and tert-butanol. The apolar
character of the mobile phase increased
in the sequence MeOH, EtOH, PrOH,
i.e. with increasing carbon number,
which is disadvantageous for polar
interactions between the mobile phase
and amino acids, and therefore the
retention factor increases. This behavior
is more pronounced for IPA and tert-
butanol. In these cases, the steric effect
probably contributes to the decreased
interactions between the mobile phase
and the analytes. However, the nature
of the alcoholic modifier exhibited a
small effect on the enantioselectivity, i.e.
the non-chiral interactions between the
CSP and the analytes are more favored
than the chiral ones.
The natures of the substituents on the
b-carbon atom of the analytes had sub-
stantial effects on the retention, but the
selectivity (a) changed to a much lower
extent than the k value (Table 1). At
constant organic modifier and acid con-
tents of H
2
O/MeOH = 30/70 (v/v),
H
2
O/EtOH = 50/50 (v/v) and H
2
O/
MeOH/EtOH = 50/25/25 (v/v/v) with
5.0 mM AcOH in all cases, the chro-
matographic behavior revealed larger
retention factors for analytes 1, 2, 7 and
8 than for 3–6, 9 and 10. For analytes
3–6, the longer aliphatic chain or
branching structure, especially in the
case of analyte 4, may weaken the polar
interaction with the selector or sterically
hinder the interaction with the CSP, and
therefore the retention decreased. The
increase in retention factor for analytes 7
and 8 as compared with 3–6, and for 10
as compared with 9, may be due to the
p-character of the analytes and therefore
the increased polar interaction ability of
analytes 7, 8 and 10 with the polar parts
of the selector. This increase in the
retention factor was not accompanied by
a significant increase in the separation
factor (though the resolution improved
slightly). The a values for the different
analytes at constant organic modifier
and acid contents of H
2
O/MeOH = 30/
70 (v/v) and 5.0 mM AcOH ranged
between 1.23 < a < 1.71; at H
2
O/
EtOH = 50/50 (v/v) and 5.0 mM AcOH
between 1.00 < a < 1.55; and at H
2
O/
MeOH/EtOH = 50/25/25 ( v/v/v) and
5.0 mM AcOH between 1.17 < a <
1.60. All of these data may be explained
by a separation mechanism in which,
besides complex formation inside the
crown ether cavity, there may be an
intermolecular polar interaction between
the analyte and the amino tethering
group of the CSP (another intermolecu-
lar H-bonding interaction between the
analyte and the CSP may occur between
the two protonated carboxy groups [44]).
However, for the two stereoisomers the
chiral interactions between the CSP
and the analytes did not differ substan-
tially, regardless of whether high or
small retention factors are observed,
and therefore the a factors differ only
slightly.
The effects of the nature and content
of the acidic modifier on the separation
were investigated by resolution in an
aqueous mobile phase at a constant
concentration of the organic modifier. A
comparison of the chromatographic data
obtained by using AcOH, H
2
SO
4
or
H
3
PO
4
as acidic modifier at a constant
concentration of 5.0 mM in a hydro-or-
ganic mobile phase containing MeOH
(50% v/v) supported the earlier findings
that AcOH is much more suitable than
the others in terms of retention, enanti-
oselectivity and resolution [13, 32] (data
not shown).
The effect of temperature on the
enantioseparation was investigated at a
mobile phase composition of H
2
O/
MeOH = 30/70 (v/v) and 5.0 mM
AcOH in the temperature range 10–
40 C. For analytes 1, 3, 8 and 10 higher
resolution was obtained at higher tem-
perature, indicating that the faster mass
transfer between the mobile and sta-
tionary phases compensated the loss in
separation factor at higher temperature.
The literature data demonstrate that,
with the (+)-(18-crown-6)-2,3,11,12-
tetracarboxylic acid-bonded CSP, the S
enantiomers elute before the R enantio-
mers for a-amino acids [34]. Taking the
Cahn–Ingold–Prelog rule into account, a
similar elution sequence was observed
0,0
0,5
1,0
1,5
2,0
2,5
3,0
k
1
'
α
R
S
Methanol
Organic modifiers
α
R
S
k
1
'
Ethanol 1-Propanol 2-Propanol tert-Butanol
Fig. 3. Dependence of retention factor of the first-eluting enantiomer (k
1
), the separation factor
(a) and the resolution (R
S
) on the nature of the alcoholic modifier. Column, (+)-(18-crown-6)-
2,3,11,12-tetracarboxylic acid-based CSP; alcoholic modifiers, MeOH, EtOH, PrOH, IPA, tert-
butanol; mobile phase, H
2
O/alcoholic modifier = 30/70 (v/v) in 5.0 mM AcOH; flow rate,
0.5 mL min
1
; detection at 205 nm
S16 Chromatographia Supplement Vol. 68, 2008 Original
for b-amino acids [32]. For analytes 1, 7
and 8, the elution sequence S < R was
observed.
Selected chromatograms for the
enantioseparation of the analytes are
depicted in Fig. 4.
Conclusions
The direct enantioseparation of various
b-3-homo-amino acids was carried out
on a crown ether-based CSP. The mobile
phase composition was optimized and
was found to be H
2
O/MeOH = 30/70
(v/v), H
2
O/EtOH = 50/50 (v/v), or H
2
O/
MeOH/EtOH = 50/25/25 (v/v/v), all
containing 5.0 mM AcOH. The chro-
matographic retention behavior and
resolution proved to be dependent on the
natures and concentrations of the alco-
holic and acidic modifiers, the tempera-
ture and the substituents in the b
positions. The elution sequence was
determined for some analytes, and fol-
lowed the general rule established earlier
for a- and b-amino acids.
Acknowledgments
This work was supported by OTKA
grants K 67563 and T 049407 and the
Korea Science and Engineering Founda-
tion (NCRC program: R15-2006-022-
03001-0). I. Ilisz wishes to express his
thanks for a Bolyai Ja
´
nos Postdoctoral
Scholarship to support his research work.
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0
0,001
0,002
0,003
Time (min)
A
1
0
0,001
0,002
Time (min)
A
2
0
0,0005
0,001
Time (min)
A
3
0
0,001
0,002
Time (min)
A
6
0
0,0005
0,001
0,0015
Time (min)
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7
0
0,0005
0,001
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Time (min)
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Time (min)
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0,01
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10
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15
813
7
12 17
712
712
8131823
51051015
Fig. 4. Selected chromatograms of analytes 1–3 and 6–10. Column, (+)-(18-crown-6)-2,3,11,12-
tetracarboxylic acid-based CSP; mobile phase for analytes 1, 6, 7 and 9 H
2
O/MeOH/
EtOH = 50/25/25 (v/v/v) in 5.0 mM AcOH, for 2 and 10 H
2
O/EtOH = 50/50 (v/v)in
5.0 mM AcOH, for 3,H
2
O/EtOH = 30/70 (v/v) in 5.0 mM AcOH, and for 8,H
2
O/
MeOH = 30/70 (v/v) in 5.0 mM AcOH; flow rate, 0.5 mL min
1
; detection at 205 nm
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S18 Chromatographia Supplement Vol. 68, 2008 Original
    • "Direct reversed-phase HPLC methods were developed for the separation of enantiomers of 14 aromatic substituted unnatural 3 -amino acids on a CSP containing (+)-(18-crown-6)-2,3,11,12- tetracarboxylic acid bonded to 3-aminopropyl silica gel as chiral selector [57] . The same selector was used for the enantioseparation of 13 aromatic substituted unusual 3 -amino acids and three of their ethyl esters [58] and 10 aliphatic substituted 3 -homoamino acids [59]. The effects of the mobile phase composition and the acidic modifiers on the separation were investigated. "
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