Enantiomeric separation of underivatized amino acids: Predictability of chiral recognition on ristocetin a chiral stationary phase

Article abstract of DOI:10.1002/chir.22291

The present work aimed to investigate the predictability of the chromatographic behavior for the separation of underivatized amino acids on ristocetin A, known as Chirobiotic R, using a DryLab high-performance liquid chromatography (HPLC) method development software, which is typically used to predict the effect of changing various chromatographic parameters on resolution in the reversed phase mode. After implementing the basic runs, and judging the predictability via the computed resolution map, it can be deduced that the chiral recognition mechanisms tend towards a hydrophilic interaction chromatography rather than the reversed phase mode, which limits the ability of DryLab software to predict separations on Chirobiotic R.

Full text of DOI:10.1002/chir.22291

CHIRALITY 26:132–135 (2014)
Short Communication
Enantiomeric Separation of Underivatized Amino Acids: Predictability
of Chiral Recognition on Ristocetin A Chiral Stationary Phase
1
1
1,2
3
*
*
HEBATALLAH A. WAGDY, RASHA S. HANAFI, RASHA M. EL-NASHAR, AND HASSAN Y. ABOUL-ENEIN
1Department of Pharmaceutical Chemistry, Faculty of Pharmacy and Biotechnology, German University in Cairo, Cairo, Egypt
2Chemistry Department, Faculty of Science, Cairo University, Giza, Egypt
3Pharmaceutical and Medicinal Chemistry, Pharmaceutical and Drug Industries Research Division, National Research Center (NRC), Cairo, Egypt
ABSTRACT
The present work aimed to investigate the predictability of the chromatographic
behavior for the separation of underivatized amino acids on ristocetin A, known as Chirobiotic R,
using a DryLab high-performance liquid chromatography (HPLC) method development
software, which is typically used to predict the effect of changing various chromatographic
parameters on resolution in the reversed phase mode. After implementing the basic runs, and
judging the predictability via the computed resolution map, it can be deduced that the chiral
recognition mechanisms tend towards a hydrophilic interaction chromatography rather than
the reversed phase mode, which limits the ability of DryLab software to predict separations on
Chirobiotic R. Chirality 26:132–135, 2014. © 2014 Wiley Periodicals, Inc.
KEY WORDS: DryLab software; ristocetin A; amino acids; chiral recognition mechanisms
INTRODUCTION
The constitutional complex structure of the macrocyclic
glycopeptides chiral selectors has been an obstacle in under-
standing their underlining chiral recognition.11 All possible
molecular interactions are responsible for retention and
enantioselectivity, as proposed by Berthod et al.,12 who found
that the interactions that may occur between the CSPs and
the analyte are: a charge–charge interaction, hydrogen bond-
ing, steric hindrances, π–π interactions, ion dipole, dipole–di-
pole, dipole-induced dipole, and Van der Waals forces.12,13
Nevertheless, the detailed recognition mechanism on a
molecular basis is not yet fully elaborated.5
The macrocyclic glycopeptides are multimodal, as they can
be coupled with different mobile phase systems: reversed,
normal, polar organic, polar ionic, or supercritical fluid
chromatography mode. The enantioselectivity of the
macrocyclic CSPs are different in each of the operating
modes, because of the different separation mechanisms that
govern in each of these modes. This work aimed to investi-
gate whether the dominating mode of Chirobiotic R is
reversed using DryLab software under reversed phase mode.
The earliest studies performed to predict the enantioselectivity
of a number of chiral compounds on derivatized β-cyclodextrin
stationary phase was based on free energy calculations of
substituents present on the stereogenic center. This study
was able to predict the possibility of enantiomeric separation
on a specific stationary phase rather than the elution order on
The macrocyclic antibiotics chiral stationary phase (CSP),
unlike other classes of chiral selectors, comprise a large
variety of structural types, including ansamycins (rifamycins),
the polypeptide antibiotic thiostrepton, and glycopeptides.
Among these, the most common and promising are the
macrocyclic glycopeptides, which have been introduced as
chiral selectors by the pioneer work of Armstrong et al.1 In
a relatively short time after their introduction to the market,
they were used successfully in most chromatographic and
electrophoretic methods of analysis. Besides the derivatized
linear or branched carbohydrates (e.g., cellulose and
amylose), macrocyclic glycopeptides appear to be the most
successful chiral selectors used to date.2,3
They represent a group of structurally diverse naturally
occurring compounds. Their molecular masses are in the
range of 1000–2100 g/mol. They all share a basket-shaped
aglycon framework, which consists of either three or four
fused macrocyclic rings but differ in size, shape, and the
geometrical arrangement of their numerous stereogenic
centers and functional groups responsible for their
enantioselective properties. The carbohydrate moieties
attached to the aglycon basket contain ionizable groups
(carboxylic and amino groups), allowing ionic interactions
involved in chiral recognitions, to take place, which are in fact
considered important players in the whole process of
enantioselectivity.4,5
Vancomycin,1 teicoplanin,6 ristocetin A,7 and the aglycone
of teicoplanin8 are commercially available and marketed
under the trade names of Chirobiotic V, T, R, and TAG,
respectively. They are prepared by covalently binding the
corresponding glycopeptides selectors to spherical silica gel
via linkage chains employing a variety of chemistries
that aim to ensure their stability without losing their chiral
recognition properties.9,10
*Correspondence to: Hassan Y. Aboul-Enein or Dr. Rasha S. Hanafi, Pharmaceu-
tical and Medicinal Chemistry, Pharmaceutical and Drug Industries Research
Division, National Research Center (NRC), Cairo 12311, Egypt, and Department
of Pharmaceutical Chemistry, Faculty of Pharmacy and Biotechnology,
German University in Cairo, Cairo, Egypt. E-mail: haboulenein@yahoo.com or
rasha.hanafi@guc.edu.eg
Received for publication 30 October 2013; Accepted 2 December 2013
DOI: 10.1002/chir.22291
Published online 22 January 2014 in Wiley Online Library
(wileyonlinelibrary.com).
© 2014 Wiley Periodicals, Inc.

ENANTIOMERIC SEPARATION OF UNDERIVATIZED AMINO ACIDS
133
this phase.14 On the other hand, ACD Lab software was able
to predict the optimum %B required for the separation of
enantiomers of eszopiclone on chiral AGP (α1-acid glycopro-
tein stationary phase).15 As well, Chromsword was used to
separate chiral drugs on the polysaccharide-based stationary
phase in normal phase mode (http://www.chromsword.
com/en/publications/).
A few reported trials aimed to predict the enantioselectivity
of chiral drugs using DryLab. The software was able to
predict the separation of N-derivatized amino acids with 2,4-
dinitrofluorobenzeneon quinine carbamate-based chiral
anion-exchanger16 and tert-butyl carbamoylated quinine17
stationary phases, where N-derivatized amino acids using
3,5-dinitrobenzylchloroformate were successfully resolved.
In both cases, the amino acids were derivatized.
DryLab software was successfully used to predict the chiral
chromatographic behavior of several compounds on
Chirobiotic V, which is expected to dramatically ease method
development of enantiomer separations of some racemic
drugs on this CSP.18 This also led to a better understanding
of the chiral recognition of the CSPs regarding the studied an-
alyte and parameters.18
Investigating the predictability of enantioseparation of
underivatized amino acids on ristocetin A chiral stationary
phase using the DryLab software will provide more informa-
tion regarding the chiral recognition mechanisms involved
in the enantioseparation of the amino acids used in this study.
column used was Chirobiotic R bonded ristocetin A-based phase (250 x
4.6 mm, 5 μm particle size) purchased from Supelco (Bellefonte, PA).
Softwares used were Chromquest 4.2 data system (UK) data acquisition
for, DryLab2000Plus and PeakMatch v. 3.60 (Molnár Institute for Applied
Chromatography, Germany).
Determination of the Predictability of the CSPs
Experimental limitations. The parameters that can be optimized by
DryLab software are: percent organic phase, temperature, pH, and flow
rate. Macrocyclic glycopeptides (Chirobiotic R) have limitations
regarding operating pressure (max 240 bar) and temperature (max 45°C),
and hence considerable variations of temperature and flow rate intervals
were not possible to consider. Accordingly, the percent organic phase was
the only investigated parameter.
Experimental preliminary runs. Preliminary runs involved injecting
both racemic amino acids (phenylalanine and valine) into two isocratic
runs at 40% and 60%B at 30 °C.
The systematic approach involved: two isocratic runs with 20% change
in organic phase.19 The isocratic runs were 40% B and 60% B. Repetitive
injections of the analytes sample solution (1 mg/mL) were performed
under each condition, until at least two consecutive reproducible
chromatograms were obtained regarding peak areas ( 10%) and retention
times ( 0.02 min). Mobile phase conditions were A = ultrapure water and
B = methanol, at a flow rate 1.0 ml/min and wavelength of detection at
225 nm. The column oven was adjusted to 30 °C.7
RESULTS AND DISCUSSION
Racemic phenylalanine and valine were used to investigate
the predictability of retention and resolution on Chirobiotic R
using DryLab and deducing the possible reversed phase
behavior as the main mode of enantiorecognition on this
phase. Temperature was not a studied variable on Chirobiotic
R (due to temperature limitations).
For phenylalanine, the preliminary run conditions were
performed with a mobile phase composed of A: water and B:
methanol at 1 mL/min and 30 °C. The two isocratic runs
involved 40% and 60% B.
MATERIALS AND METHODS
Chemicals and Reagents
Regents used were methanol (high-performance liquid chromatography
[HPLC] grade; Sigma-Aldrich, Germany) and ultrapure water (Elga lab.).
Rac–Phenylalanine and rac–valine were purchased from Merck Chemicals
(Germany).
Equipment and Software
At 40% B, phenylalanine enantiomers were separated at tR
4.452 min and 5.257 min, while at 60% B, tR were 4.524 min
HPLC (Thermo Finnigan Spectrasystem, UK) consists of: pump model
P2000, detector model UV3000, autosampler model AS3000 (UK). The
Fig. 1. Chromatograms of phenylalanine enantiomers on Chirobiotic R at different %B: (A) 40% B, (B) 60% B, (C) 80% B, and (D) 100% B.
Chirality DOI 10.1002/chir

134
WAGDY ET AL.
Fig. 2. Chromatograms of valine enantiomers on Chirobiotic R at different %B, (A) 40% B, (B) 60% B, and (C) 100% B.
and 5.374 min, for the first and the second enantiomers,
respectively (Fig. 1). The effect of an increase 20% B on
tR of phenylalanine was minimal. It was also observed that
the behavior was atypical to reversed phase mode,
because the enantiomers were slightly retarded when %B
increased. It was important to increase %B further to verify
this observation. It was confirmed that the enantiomers
were significantly retarded at higher %Bwhen it was
increased to 80% B, where tR were 5.003 min and
6.152 min. Furthermore, at 100% B, tR were 7.501 min and
11.108 min for the first and the second enantiomers,
respectively. It was also observed that Rs of
phenylalanine enantiomers was enhanced at a higher
percentage of the organic modifier.
All the chromatographic conditions performed for phenylal-
anine were repeated for the enantiomeric resolution of
racemic valine. At 40% B, valine enantiomers were separated
at tR 3.565 min and 4.031 min for the first and the second en-
antiomers, respectively. While at 60% B, tR were 3.765 min
and 4.436 min (Fig. 2). Increasing %B by 20% was found to
have small effect on retention, which was unlike the reversed
phase mode. A higher percentage of B (100%) tR were
6.058 min and 10.085 min, for the first and the second
enantiomers, respectively, and Rs improved at increasing
amounts of the organic modifier (Fig. 2).
It can be concluded that the retention mode on
Chirobiotic R, in the conditions studied in this work, is a
hydrophilic interaction chromatography rather than
reversed phase mode, where amino acids being highly
polar and charged analytes are retained predominantly by
charge–charge interactions between the amino acids
carboxylate group of the analyte and the ammonium group
on the chiral selector. The secondary interactions involved
would be π–π and H-bonding,3 with no classic reversed
phase retention mechanism involved. Hence, DryLab,
which mainly predicts reversed phase behavior, and not
hydrophilic interaction chromatography, would fail to
predict retentions on Chirobiotic R.
CONCLUSION
It can be concluded that the chiral recognition mechanism
of Chirobiotic R, in the cases under study in this work,
belongs to hydrophilic interaction chromatography rather
than reversed phase mode. This accounts for the inability of
software such as DryLab to predict chiral behavior on
Chirobiotic R.
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