Cavity Supported HPLC of Cis/Trans Isomers of Proline
207
observed on the calix[nJarene phases. That means, that the chromatographic
process is only slowly going on, the interconversion of the conformers takes place
during the chromatographic run and no complete discrimination is achieved
compared to the separation on ~-CD phase. However, not to be excluded is that
hydrophobic moieties of the dipeptides interact with the cavity of calixarenes
showed by MD simulations.
3
Capillary Zone Electrophoresis (CZE)
The capillary electrophoresis as a time resolving method represents a new
method which allows the detection of conformational changes in peptides. CZE
has an advantage over HPLC methods in so far as the analytes remain in solution
and no interaction exists to a solid phase. The mobilities of peptides, however,
depend on parameters which do not vary with the conformation, such as molecular weight and the number of amino acid residues (Hilser and Freire 1995). However, Kilar and Hjerten (1993) observed changed electrophoretic mobilities for
unfolded compared to native proteins which can be attributed to changes in surface polarity and surface charges, but also to an alteration of the geometry of the
whole molecule. Referring to the fundamentals of CZE the effective mobility of a
compound depends strongly on the experimental conditions with a definitive
charge and ionic radius, but also on the frictional resistance during the migration
in an electric field. Therefore, it is possible that sterical differences such as the
hydrodynamic radius or the symmetry of the molecule can contribute to the electrophoretic discrimination of cis/trans conformers of prolyl peptides. Compared
to the presented HPLC conditions which allow the resolution of dynamically
interconverting species, the commonly prevailing conditions for the CZE of cis/
trans isomers are to be described as follows (Ma et al. 1995, 1998):
- short capillaries,
- low working temperatures,
- high voltage,
- low viscosity of the electrolyte system.
The main advantage of CZE compared to HPLC represents the possibility of
obtaining direct interrelationships to kinetic and thermodynamic parameters of
the cis/trans interconversion of the peptide bond, such as cis/trans isomer ratio or
the rate constants of the cis to trans interconversion and the corresponding back
reaction.
In recent years several studies on capillary electrophoretic separations of cis/
trans peptide bond isomers have been published (Thunecke et al. 1998, Brandsch
et al. 1998, Molle et al 1994). The rotational isomers of enalapril maleate, an
alanyl-proline derivative which is used as an angiotensin inhibitor, were resolved
by micellar CZE with SDS (Qin et al. 1992). In addition to the differences in
hydrodynamic radii in this case hydrophobic interactions with the SDS micelles
supported the separation of the peptide conformers. Meyer et al. (1994) succeeded in the electrophoretic conformer separation of proline containing thioxopeptides of the type Ala-Xaa-1\J [CS-NJ-Pro-Phe-4-nitroanilides. These com-
207
observed on the calix[nJarene phases. That means, that the chromatographic
process is only slowly going on, the interconversion of the conformers takes place
during the chromatographic run and no complete discrimination is achieved
compared to the separation on ~-CD phase. However, not to be excluded is that
hydrophobic moieties of the dipeptides interact with the cavity of calixarenes
showed by MD simulations.
3
Capillary Zone Electrophoresis (CZE)
The capillary electrophoresis as a time resolving method represents a new
method which allows the detection of conformational changes in peptides. CZE
has an advantage over HPLC methods in so far as the analytes remain in solution
and no interaction exists to a solid phase. The mobilities of peptides, however,
depend on parameters which do not vary with the conformation, such as molecular weight and the number of amino acid residues (Hilser and Freire 1995). However, Kilar and Hjerten (1993) observed changed electrophoretic mobilities for
unfolded compared to native proteins which can be attributed to changes in surface polarity and surface charges, but also to an alteration of the geometry of the
whole molecule. Referring to the fundamentals of CZE the effective mobility of a
compound depends strongly on the experimental conditions with a definitive
charge and ionic radius, but also on the frictional resistance during the migration
in an electric field. Therefore, it is possible that sterical differences such as the
hydrodynamic radius or the symmetry of the molecule can contribute to the electrophoretic discrimination of cis/trans conformers of prolyl peptides. Compared
to the presented HPLC conditions which allow the resolution of dynamically
interconverting species, the commonly prevailing conditions for the CZE of cis/
trans isomers are to be described as follows (Ma et al. 1995, 1998):
- short capillaries,
- low working temperatures,
- high voltage,
- low viscosity of the electrolyte system.
The main advantage of CZE compared to HPLC represents the possibility of
obtaining direct interrelationships to kinetic and thermodynamic parameters of
the cis/trans interconversion of the peptide bond, such as cis/trans isomer ratio or
the rate constants of the cis to trans interconversion and the corresponding back
reaction.
In recent years several studies on capillary electrophoretic separations of cis/
trans peptide bond isomers have been published (Thunecke et al. 1998, Brandsch
et al. 1998, Molle et al 1994). The rotational isomers of enalapril maleate, an
alanyl-proline derivative which is used as an angiotensin inhibitor, were resolved
by micellar CZE with SDS (Qin et al. 1992). In addition to the differences in
hydrodynamic radii in this case hydrophobic interactions with the SDS micelles
supported the separation of the peptide conformers. Meyer et al. (1994) succeeded in the electrophoretic conformer separation of proline containing thioxopeptides of the type Ala-Xaa-1\J [CS-NJ-Pro-Phe-4-nitroanilides. These com-