Enantioresolution of chiral derivatives of xanthones on (S,S)-Whelk-O1 and l -phenylglycine stationary phases and chiral recognition mechanism by docking approach for (S,S)-Whelk-O1

Article abstract of DOI:10.1002/chir.22112

The resolution of seven enantiomeric pairs of chiral derivatives of xanthones (CDXs) on (S,S)-Whelk-O1 and l-phenylglycine chiral stationary phases (CSPs) was systematically investigated using multimodal elution conditions (normal-phase, polar-organic, and reversed-phase). The (S,S)-Whelk-O1 CSP, under polar-organic conditions, demonstrated a very good power of resolution for the CDXs possessing an aromatic moiety linked to the stereogenic center with separation factor and resolution factor ranging from 1.91 to 7.55 and from 6.71 to 24.16, respectively. The chiral recognition mechanisms were also investigated for (S,S)-Whelk-O1 CSP by molecular docking technique. Data regarding the CSP-CDX molecular conformations and interactions were retrieved. These results were in accordance with the experimental chromatographic parameters regarding enantioselectivity and enantiomer elution order. The results of the present study fulfilled the initial objectives of enantioselective studies of CDXs and elucidation of intermolecular CSP-CDX interactions. Copyright

Full text of DOI:10.1002/chir.22112

CHIRALITY 25:89–100 (2013)
Enantioresolution of Chiral Derivatives of Xanthones on (S,S)-Whelk-O1
and L-Phenylglycine Stationary Phases and Chiral Recognition
Mechanism by Docking Approach for (S,S)-Whelk-O1
CARLA FERNANDES,1,2 ANDREIA PALMEIRA,1,2 ALEXANDRE SANTOS,1,2 MARIA ELIZABETH TIRITAN,1,3 CARLOS AFONSO,1,2 AND
MADALENA M. PINTO1,2
*
1Centro de Química Medicinal da, Universidade do Porto (CEQUIMED-UP), Rua de Jorge Viterbo Ferreira 228, 4050-313, Porto, Portugal
2Laboratório de Química Orgânica e Farmacêutica, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Rua de
Jorge Viterbo Ferreira 228, 4050-313, Porto, Portugal
3Centro de Investigação em Ciências da Saúde, Instituto Superior de Ciências da Saúde-Norte, (CICS-ISCS-N), Rua Central de Gandra 1317, 4585-116,
Gandra PRD, Portugal
ABSTRACT
The resolution of seven enantiomeric pairs of chiral derivatives of xanthones
(CDXs) on (S,S)-Whelk-O1 and L-phenylglycine chiral stationary phases (CSPs) was systematically
investigated using multimodal elution conditions (normal-phase, polar-organic, and reversed-phase).
The (S,S)-Whelk-O1 CSP, under polar-organic conditions, demonstrated a very good power of reso-
lution for the CDXs possessing an aromatic moiety linked to the stereogenic center with separation
factor and resolution factor ranging from 1.91 to 7.55 and from 6.71 to 24.16, respectively. The chiral
recognition mechanisms were also investigated for (S,S)-Whelk-O1 CSP by molecular docking tech-
nique. Data regarding the CSP–CDX molecular conformations and interactions were retrieved.
These results were in accordance with the experimental chromatographic parameters regarding
enantioselectivity and enantiomer elution order. The results of the present study fulfilled the initial
objectives of enantioselective studies of CDXs and elucidation of intermolecular CSP–CDX interac-
tions. Chirality 25:89–100, 2013.
© 2012 Wiley Periodicals, Inc.
KEY WORDS: Pirkle-type chiral stationary phases; chiral derivatives of xanthones; enantioselectivity;
chiral recognition; intermolecular interactions; docking
INTRODUCTION
H bonding, face-to-face, and edge-to-face p–p interactions with the
enantiomers of naproxen. Moreover, the Whelk-O1 CSP has also
been effective for separating many others racemates16–20
including drugs.15,16,21,22
Enantiomers are often readily distinguished by biological
systems, presenting different pharmacokinetic and pharmaco-
dynamic properties. Thus, chiral separation has been a major
focus for analytical and organic chemistry during the last three
decades being nowadays crucial for drug development
process.1 Over the years, high-performance liquid chroma-
tography (HPLC) has emerged as one of the most useful
methods for analyses and preparation of enantiomerically
pure bioactive compounds.2 The remarkable development
and applications of chiral stationary phases (CSPs) have
revolutionized the field of chiral separation providing both
enantiomers in high enantiomeric purity, which is one of
the conditions required for biological, pharmacological, or
toxicological evaluations of chiral bioactive compounds.3
Among the most useful CSPs described in the literature is
the brush-type or Pirkle-type CSPs. The selectors of these CSPs
are chiral small molecules chemically bonded to silica or silica
derivatives. Pirkle et al.4 were the pioneers on the development
of those type of CSPs, with almost a hundred CSPs reported
being many of them commercially available.5 The principle of
reciprocity6 and the chiral recognition phenomena, based on
chromatographic7,8 and spectroscopic studies,9,10 were the
centerpieces of the evolution of Pirkle-type CSPs. Pirkle et
col. inspired other groups to develop numerous Pirkle-type
CSPs with chiral low-molecular mass selectors,3,11–13 including
our group.14 Among the great number of Pirkle-type CSPs
described in the literature, Whelk-O1 CSP (Fig. 1a) is one of
the most employed with the broadest application in industrial
and academic laboratories.5 Applying an immobilized guest
strategy, this CSP has been designed in the 1990s15 to undergo
© 2012 Wiley Periodicals, Inc.
The p-acceptor amino acid-derived CSP phenylglycine
(Fig. 1b) was one of the first CSP developed by Pirkle et al.23
Although its enantioselectivity power is lower than Whelk-O1
CSP, it was demonstrated to be useful for separation of different
classes of enantiomers, such as non-steroidal anti-inflammatory
drugs24 and benzodiazepinones,25 among others.
In recent years, the computational study of chromatographic
separations has become a very important tool in understanding
the chiral recognition mechanisms for diverse CSPs,5 particu-
larly Whelk-O1 CSP which has been investigated in detail by
molecular modeling,26,27 chemoinformatics,28,29 and molecular
simulation30–32 studies. The elucidation of chiral recognition
mechanisms is essential to clarify the kind of intermolecular
interactions between each enantiomer and the CSP and may
provide valuable information to estimate the magnitude of the
enantioselectivity, anticipate the elution order, predict that
other classes of racemates can be separated, and establish
Contract grant sponsor: This work was funded by FCT – Fundação para a Ciência
e a Tecnologia under the project CEQUIMED-PEst-OE/SAU/UI4040/2011 and
also by FEDER funds through the COMPETE program under the project
FCOMP-01-0124-FEDER-011057.
*Correspondence to: Madalena M. Pinto, Centro de Química Medicinal da
Universidade do Porto (CEQUIMED-UP), Laboratório de Química Orgânica e
Farmacêutica, Departamento de Ciências Químicas, Universidade do Porto, Rua
de Jorge Viterbo Ferreira 228, 4050–313 Porto, Portugal. E-mail: madalena@ff.up.
pt; madalenakijjoa@gmail.com
Received for publication 14 April 2012; Accepted 04 August 2012
DOI: 10.1002/chir.22112
Published online 10 December 2012 in Wiley Online Library
(wileyonlinelibrary.com).

90
FERNANDES ET AL.
Fig. 1. The chemical structures of (a) (S,S)-Whelk-O1 and (b) L-phenylglycine CSPs.
the more suitable chromatographic conditions. Moreover, it is
also useful to guide structural modifications of the molecules
of the CSPs in order to obtain a higher enantiomeric selectivity
for a specific class of enantiomers.
The importance of xanthone derivatives is well recognized in
medicinal chemistry concerning their broad spectrum of biolog-
ical and pharmacological activities.33–36 Our group has been
active in synthesizing compounds based on the xanthonic
scaffold for biological activity evaluation,34–40 including chiral
derivatives.14,40 In the literature, chiral derivatives of xanthones
(CDXs) have been reported to reveal antitumoral,40 antifungal,
and antibacterial,41 antiepileptic and anticonvulsant42–45 antiar-
rhythmic,46 and local anesthetic45 activities, among others.
Herein, we describe an investigation of the resolution of
seven enantiomeric pairs of CDXs (Fig. 2) on (S,S)-Whelk-O1
and L-phenylglycine CSPs. Besides that, this work explores
the influence of different mobile phases on enantiomeric
separation. So, multimodal elution conditions: normal-phase,
polar-organic, and reversed-phase modes were explored on
both CSPs.
Fig. 2. The chemical structures of enantiomeric pairs of CDXs: (R)-(+) and (S)-(ꢀ)-CDX-1 (N-(2-hydroxy-1-phenylethyl)-2-((9-oxo-9H-xanthen-3-yl)oxy)acetamide);
(R)-(+) and (S)-(ꢀ)-CDX-2 (N-(2-hydroxy-1-phenylethyl)-6-methoxy-9-oxo-9H-xanthene-2-carboxamide); (R)-(ꢀ) and (S)-(+)-CDX-3 (6-methoxy-9-oxo-N-(1-(p-tolyl)
ethyl)-9H-xanthene-2-carboxamide); (R)-(+) and (S)-(ꢀ)-CDX-4 (N-(1-hydroxy-4-methylpentan-2-yl)-6-methoxy-9-oxo-9H-xanthene-2-carboxamide); (R)-(ꢀ) and
(S)-(+)-CDX-5 (N-(1-hydroxypropan-2-yl)-6-methoxy-9-oxo-9H-xanthene-2-carboxamide); (R)-(+) and (S)-(ꢀ)-CDX-6 (N-(1-hydroxy-3-methylbutan-2-yl)-6-methoxy-9-oxo-
9H-xanthene-2-carboxamide); and (R)-(ꢀ) and (S)-(+)-CDX-7 (N-(2-hydroxypropyl)-6-methoxy-9-oxo-9H-xanthene-2-carboxamide).
Chirality DOI 10.1002/chir

ENANTIORESOLUTION OF CHIRAL DERIVATIVES OF XANTHONES ON (S,S)-WHELK-O1 AND L-PHENYLGLYCINE AND DOCKING APPROACH 91
X-ray crystallographic studies.48,49 The docking simulations were done
considering only the structural features which are assumed to be essential
for enantioselectivity. For this study, the support (silica) and the spacer were
Based on chromatographic data obtained on (S,S)-Whelk-O1
CSP, docking studies were performed in order to better
understand the chromatographic behavior at a molecular
level, as well as the structural features associated with the
chiral recognition mechanism.
The enantiomeric resolution of the same library of enantio-
replaced by a methyl group. The CDXs were subject to energy minimization
by using HyperChem version 8.0.50 The semi-empirical Austin Model 151
method with the Polak–Ribière algorithm was employed for molecular
minimization. Docking simulations between the CDXs and the
(S,S)-Whelk-O1 selector were undertaken in AutoDock Vina52 embedded
in PyRx–Virtual Screening Tool software, release 0.8 for Windows. AutoDock
Vina considered the target conformation (selector) as a rigid unit, whereas
the ligands (CDXs) were allowed to be flexible and adaptable to the target.
Vina searched for the lowest binding affinity conformations and returned
nine different conformations for each CDX. AutoDock Vina was run using
an exhaustiveness of eight and a grid box with the dimensions 13.0, 13.0,
and 10.0, engulfing the entire selector. Conformations and interactions were
visualized using PyMOL version 1.3.53
To position explicit solvent (MeOH) in the binding site, AutoDock
Vina52 was used to obtain the top-scoring poses using the previously
described protocol. The selector plus MeOH was used as “receptor” in
a new docking study using CDXs 1–7 as potential ligands.
AutoDock 4 implemented in AutoDockTools was used to dock CDXs 1–7
into flexible (S,S)-Whelk-O1. Amide bond between dinitrophenyl group
and 1,2,3,4-tetrahydrophenanthrene group was defined as flexible. A
three-dimensional grid box of 13.0, 13.0, and 10.0 (x,y,z) that encompassed
the selector cleft was defined. AutoDock 4 was used to dock CDXs 1–7 to
the selector, and each structure was scored and ranked.
meric pairs of CDXs using four macrocyclic antibiotic CSPs
under multimodal elution conditions as well as the chiral
recognition mechanisms by docking studies was recently
demonstrated.47 Regarding the enantiomeric pairs of CDXs
and comparing the enantioselectivity on both type of CSPs,
this work confirms that (S,S)-Whelk-O1, L-phenylglycine,
and macrocyclic antibiotic CSPs reveal a distinct pattern of
enantioselectivity.
To our best knowledge, this is the first report of the use of
(S,S)-Whelk-O1 and L-phenylglycine CSPs for the enantiose-
paration of this important class of compounds.
MATERIALS AND METHODS
Chemicals
The procedures to synthesize the CDXs (Fig. 2) were described else-
where.14 Briefly, carboxyxanthone derivatives were coupled with both
enantiomers of commercially available chiral building blocks using
O-(benzotriazol-1-yl)-N,N,N0,N0-tetramethyluronium tetrafluoroborate
as coupling reagent. Ethanol (EtOH), 2-propanol (2-prOH), n-hexane
(Hex), methanol (MeOH), and acetonitrile (ACN) HPLC grade were
purchased from Sigma-Aldrich Co (St. Louis, MO, USA). Ultrapure
water was produced by a Millipore Milli-Q system (Millipore, Bedford,
MA, USA). The stock solutions of the CDXs were prepared by dissolu-
tion in EtOH at the concentration of 0.5 mg/ml. Working solutions of
enantiomeric pairs of CDXs were prepared by mixing equal aliquots of
each enantiomer.
RESULTS AND DISCUSSION
The Whelk-O1 CSP has been designed mainly to operate
under normal-phase HPLC conditions. However, the Whelk-O1
CSP has also been reported to be useful in polar-organic
and reversed-phase elution conditions, within a wide range
of mobile phases.16,22,54,55 Indeed, recent publications are
devoted to the adsorption of naproxen on Whelk-O1 CSP
under reversed-phase conditions,56,57 concerning the effect
of mobile phase58 and buffer composition.59 As a general
rule, enantiomers that exhibit an H-bond acceptor and an
aromatic moiety close to the stereogenic center tend to be
well resolved on Whelk-O1 CSP.16 L-Phenylglycine CSP has
also been proven to be capable to operate not only under
normal-phase elution conditions but also reversed-phase condi-
tions.24 Hence, the enantioresolution of seven enantiomeric
pairs of CDXs (1–7) (Fig. 2) was evaluated on (S,S)-Whelk-O1
and L-phenylglycine CSPs (Fig. 1), under normal-phase,
polar-organic, and reversed-phase elution conditions.
Chromatography
The HPLC system was a JASCO model 880-PU pump, a Rheodyne model
7125 injector fitted with a 20-ml sample loop, a JASCO model 880-30 solvent
mixer, and a JASCO model 875-UV detector (Tokyo, Japan). A DataApex
CSW17
- chromatography station for Microsoft Windows 95 was
employed. The chiral columns (S,S)-Whelk-O1 and L-phenylglycine
(25 cm ꢁ 4.6 mm i.d., 5-mm particle size, 100-Å pore size) were commer-
cially available from Regis Technologies, Inc. (Morton Grove, IL, USA).
Analyses were performed at room temperature in isocratic mode using
multimodal conditions. The mobile phase compositions were Hex and EtOH
or 2-prOH as a modifier, in normal-phase condition; MeOH and water were
used in reversed-phase condition. MeOH, EtOH, ACN, or mixtures with dif-
ferent proportions of these solvents were used in polar-organic elution condi-
tions. The mobile phases were prepared in a volume/volume relation and
degassed in an ultrasonic bath for 15 min before use. The flow rate used
was 1.0 ml/min, and the chromatograms were monitored by ultraviolet
detection at a wavelength of 254 nm. The measurements were carried out
at the laboratory temperature (22 ꢂ 2ꢃC). The sample injections (20 ml) were
carried out in triplicate. The dead time (t0) was considered to be equal to the
peak of the solvent front and was taken from each particular run. The
retention factor (k) was calculated using the equation (k = [tR ꢀ t0]/t0).
The separation factor (a) was calculated as (a = k2/k1). The resolution
factor (Rs) was calculated using the equation (Rs = 1.18 [tR2 ꢀ tR1]/[W1
0.5 + W2 0.5]) where tR1 and tR2 are the retention times of the first and
Performance of (S,S)-Whelk-O1 CSP for Resolution of
CDXs
Three out of seven enantiomeric pairs of CDXs (1–3)
were enantioseparated with excellent enantioselectivity on
(S,S)-Whelk-O1 CSP, with a ranging from 1.91 to 7.55 and
resolutions ranging from 6.71 to 24.16. The overall best
results are shown on Table 1.
Briefly, under the normal-phase elution conditions using
EtOH as the modifier, very high retention factors were
observed with low percentage of the modifier, being the
chromatographic run up to 120 min. In order to overcome
this situation, the polarity of the mobile phase was increased
to consequently decrease the retention time of the enantio-
mers. Thus, the EtOH : Hex mobile phase was evaluated
systematically, changing the content of EtOH from 10% to
90% (by volume) in increments of 10% each time (data not
shown). Meanwhile, with EtOH : Hex (90:10 v/v), excellent
enantioselectivity and resolution were achieved for the
enantiomeric pairs of CDXs 1–3, with a = 2.99, 4.25, and
Chirality DOI 10.1002/chir
second enantiomers, respectively, and W1
and W2
are the
0.5
0.5
corresponding peak width measured on half height. The elution
order was determined for the enantiomers of CDXs 1–3 on
(S,S)-Whelk-O1 CSP by injecting the solutions of the enantiomeric
mixtures and then each enantiomer separately, using ACN : MeOH
(50:50 v/v) and with only MeOH as mobile phases.
Computational
The (S,S)-Whelk-O1 selector was retrieved from the Cambridge Crystallo-
graphic Data Centre (deposition number 273851), and it is based on previous

92
FERNANDES ET AL.
TABLE 1. Separation performance of (S,S)-Whelk-O1 CSP, under multimodal chromatographic conditions, for the enantiomeric
pairs of CDXs (1–3)
CDX-1
CDX-2
CDX-3
Mobile phase
k1
k2
a
Rs
k1
k2
a
Rs
k1
k2
a
Rs
EtOH/Hex: 90/10
EtOH
MeOH
2.51
2.30
1.36
1.03
0.64
9.09
7.50
6.73
3.08
2.11
1.14
22.44
2.99
2.93
2.27
2.06
1.91
2.47
10.78
10.05
10.63
8.16
6.71
13.14
1.94
1.84
1.28
1.21
0.60
7.04
8.24
7.57
3.55
3.22
1.41
7.04
4.25
4.11
2.78
2.66
2.47
1.00
13.17
12.14
12.77
10.29
8.85
–
2.59
2.58
1.98
1.24
0.91
16.21
19.58
18.58
9.95
6.82
3.61
7.55
7.21
5.02
5.50
4.08
1.00
17.58
16.15
22.04
24.16
19.46
–
ACN
ACN/MeOH: 50/50
MeOH/H2O: 80/20
16.21
CSP, chiral stationary phase; CDX, chiral derivative of xanthone; EtOH, ethanol; Hex , n-hexane; MeOH, methanol; ACN, acetonitrile.
7.55 and RS = 10.78, 13.17, and 17.58, respectively (Table 1).
However, with this mobile phase, the retention factors of the
second eluted enantiomer were still very high (k2 ranging
from 7.50 to 19.58), leading to a high analysis time. There-
fore, to overcome this situation, the strategy was using only
EtOH as mobile phase (polar-organic elution conditions).
Nevertheless, when EtOH was used as mobile phase, the
results were similar as those described for EtOH : Hex
(90:10 v/v). So, the uses of other polar-organic solvents
were attempted by switching to MeOH and ACN as mobile
phases. Lower retention factors were observed when
changing from EtOH to MeOH or ACN (Fig. 3), whereas
the enantioselectivity and resolution are at a standstill
excellent (Fig. 4). Moreover, the mixtures of two polar-organic
solvents as ACN and MeOH (50:50v/v) allowed shortened
retention factors of both enantiomers of CDXs 1–3, when
compared to 100% MeOH or ACN as mobile phase (Fig. 3).
Actually, when a mixture of ACN : MeOH (50:50 v/v) was
used, the enantioselectivity and resolutions decreased only
slightly (Fig. 4); however, the overall chromatographic
parameters were excellent. Figure 5 compares characteristic
chromatograms with different mobile phases, with ACN :
MeOH (50:50 v/v) presenting the best performance.
Finally, on the reversed-phase elution conditions, very high
retention factors were achieved for the enantiomeric pairs of
CDXs 1–3 (Table 1). When MeOH : H2O (80:20 v/v) was
used as mobile phase, only the enantiomeric pair of CDX-1
was baseline separated.
Considering enantiomeric pairs of CDXs 4–7, the results
were not satisfactory on (S,S)-Whelk-O1 CSP (data not shown).
However, using acetonitrile as mobile phase, the enantiomeric
pair of CDX-4 was slightly separated (a = 1.10), although with
poor resolution (RS = 0.61). Similarly, the enantiomeric pair
of CDX-5 was only partially resolved with MeOH : H2O
Fig. 3. Comparison of retention factors of the (a) first enantiomer (k1) and (b) the second enantiomer (k2) of (◆) CDX-1, (■) CDX-2, and (▲) CDX-3 on
(S,S)-Whelk-O1 CSP under normal-phase and polar-organic elution conditions; flow rate 1.0 ml/min; detection wavelength 254 nm.
Fig. 4. Comparison of (a) enantioselectivity (a) and (b) resolution (RS) of (◆) CDX-1, (■) CDX-2, and (▲) CDX-3 on (S,S)-Whelk-O1 CSP under normal-phase
and polar-organic elution conditions; flow rate 1.0 ml/min; detection wavelength 254 nm.
Chirality DOI 10.1002/chir

ENANTIORESOLUTION OF CHIRAL DERIVATIVES OF XANTHONES ON (S,S)-WHELK-O1 AND L-PHENYLGLYCINE AND DOCKING APPROACH 93
TABLE 2. Separation performance of L-phenylglycine CSP,
under multimodal chromatographic conditions, for the
enantiomeric pair of CDX-3
CDX-3
Mobile phase
k1
a
Rs
EtOH/Hex: 25/75
EtOH/Hex: 40/60
EtOH/Hex: 50/50
2-PrOH/Hex: 50/50
EtOH
MeOH
ACN
5.26
2.80
2.26
4.87
0.92
0.64
0.51
0.22
4.54
1.24
1.23
1.23
1.35
1.21
1.16
1.21
1.00
1.19
2.51
2.08
1.90
1.60
1.24
1.13
0.66
–
ACN/MeOH: 50/50
MeOH/H2O: 80/20
1.71
Fig. 5. Chromatograms of enantiomeric pair of CDX-3 on (S,S)-Whelk-O1
CSP in the mobile phases (a) EtOH : Hex (90:10 v/v), (b) EtOH, (c) MeOH,
(d) ACN, and (e) ACN : MeOH (50:50 v/v); flow rate 1.0 ml/min; detection
wavelength 254 nm.
CSP, chiral stationary phase; CDX, chiral derivative of xanthone; EtOH, ethanol;
Hex, n-hexane; 2-PrOH, 2-propanol; MeOH, methanol; ACN, acetonitrile.
most successful elution condition on (S,S)-Whelk-O1 CSP
(Table 1), on L-phenylglycine CSP, the poorest results were
obtained with only polar-organic solvents, with a 1.21 and
RS 1.24 (Table 2). Finally, good enantioselectivity (a = 1.19)
and resolution (RS = 1.71) were obtained on reversed-phase
elution condition, using MeOH : H2O (80:20 v/v) as mobile
phase, although with high retention (k1 = 4.54). Charac-
teristic chromatograms showing the separation performance
of L-phenylglycine CSP, under normal-phase and polar-
organic elution conditions, for the enantiomeric pair of
CDX-3 are depicted in Figure 6.
The enantiomeric pairs of CDXs 1–2 and 4–7 were not
separated, under all the chromatographic elution conditions
tested, and in general, high retentions were observed (data
not shown).
Comparing the differences among the structures of the
CDXs, it can be seen that CDX-3 is the only one that has no
primary alcoholic group linked to the stereogenic center.
Concerning all these features, it can be inferred that this group
might be responsible for strong and not enantioselective inter-
actions for enantiomeric pairs of CDXs 1–2 and 4–7.
(80:20v/v), also with poor enantioselectivity (a = 1.06) and
resolution (RS = 0.76). The enantiomeric pairs of CDX-6 and
CDX-7 were not separated, under all the chromatographic
elution conditions evaluated.
Thus, as predicted for (S,S)-Whelk-O1,16 the chromatographic
results demonstrated that the best resolved enantiomeric pairs of
CDXs were the ones that, in addition to an H-bond acceptor, have
also an aromatic moiety next to the stereogenic center, i.e.,
CDXs 1–3 (Fig. 2). In spite of the fact that, for these xanthonic
enantiomeric pairs, the best enantioselectivity and resolution
were achieved using EtOH : Hex (90:10 v/v) as mobile phase
(Table 1), polar-organic conditions proved to be a best
alternative to the normal-phase conditions, since lower reten-
tion factors with high enantioselectivity and resolution were
obtained when ACN : MeOH (50:50 v/v) was used as mobile
phase, indicating that this might be preferable for faster
analytical separations.
The elution order for the enantiomers of CDXs 1–3 using
either MeOH or ACN : MeOH (50:50 v/v) as mobile phase
demonstrated that the (S)-enantiomer of pairs of CDXs 1–2
was the first to elute, whereas for CDX-3, the (S)-enantiomer
was the more retained.
Docking Studies
Comparing the major structural differences between the
two Pirkle-type CSPs evaluated, the (S,S)-Whelk-O1 CSP
has a semi-rigid framework (cleft type) formed by an
Performance of L-Phenylglycine CSP for Resolution of CDXs
The capability of L-phenylglycine CSP to resolve the CDXs
series was also systematically evaluated under multimodal
elution conditions. However, the L-phenylglycine CSP showed
much lower discrimination capability for the enantiomeric
pairs of CDXs evaluated compared to (S,S)-Whelk-O1 CSP. In
fact, enantiomeric pair of CDX-3 was the only pair resolved
on this CSP. Indeed, under normal elution conditions, the
L-phenylglycine CSP was found to be very effective for the
enantioseparation of enantiomeric pair of CDX-3 using EtOH :
Hex (25:75 v/v) as mobile phase with enantioselectivity and
resolution of a = 1.24 and RS = 2.51, respectively (Table 2). When
EtOH was used in a 40% or 50% proportion in Hex, enantioreso-
lution was decreased, with RS = 2.08 and 1.90, respectively.
In an attempt to optimize the mobile phase composition,
2-PrOH was evaluated as an organic modifier. The enantios-
electivity (a = 1.35) was improved, but the resolution was
decreased (RS = 1.60), when 2-PrOH : Hex (50:50 v/v) was
used. In spite of the fact that polar-organic mode was the
Fig. 6. Chromatograms of enantiomeric pair of CDX-3 on L-phenylglycine CSP
in the mobile phases (a) EtOH : Hex (25:75 v/v), (b) EtOH : Hex (40:60 v/v), (c)
EtOH : Hex (50:50 v/v), (d) EtOH, (e) MeOH, and (f) ACN; flow rate 1.0 ml/min;
detection wavelength 254 nm.
Chirality DOI 10.1002/chir

94
FERNANDES ET AL.
electron-deficient 3,5-dinitrophenyl group, spatially oriented
in a perpendicular way to an electron-rich phenanthryl group,
as well as an amide H in the cleft formed by the two aromatic
systems.16 Accordingly, it should be noted that the semi-rigid
selector of the (S,S)-Whelk-O1 CSP may be crucial for the
enantioresolution of this important class of compounds as
an artificial “active site” for chiral recognition.
Considering the structural features of the (S,S)-Whelk-O1
selector, three simultaneous interactions were proposed by
Pirkle et al.: an H-bonding interaction between the amide hy-
drogen of the selector and an H acceptor in the enantiomer; a
face-to-face p–p interaction between the 3,5-dinitrophenyl
group of the selector and an aromatic moiety in the enantio-
mer; and an edge-to-face p–p interaction between the phenan-
thryl group of the selector and an aromatic moiety in the en-
antiomer.16 However, several studies demonstrated that the
details of interactions between enantiomers and Whelk-O1
CSP can be more diverse and complex than this simple
three-point model.27,31
The CDXs used in this study share a common structural
xanthonic scaffold, with a methoxyl group at position 6,
linked by an amide bond as a chemical bridge to a chiral
moiety. An exception is CDX-1 in which the xanthonic
scaffold has no methoxyl group, and the link to the chiral
moiety is through an ether chemical bridge (Fig. 2). The
major structural differences for these molecules are in the
nature of the chiral moiety, namely, a phenyl ring linked to
the stereogenic center, in CDXs 1–3. Thus, it is expectable
that these molecular features might be determining for
enantiorecognition on (S,S)-Whelk-O1 CSP, specifically
the phenyl ring next to the stereogenic center, since the
intermolecular interactions related to this group are crucial
for the chiral recognition mechanism on this Pirkle-type
CSP.16 Moreover, several molecular interactions might
occur with the CSP such as p–p and H bonding with the
xanthonic scaffold, H bonding with the polar sites of the
chiral moiety, and as well as p–p interactions with the phenyl
ring (CDXs 1–3). Besides, not only the structures of the
CDXs and the CSP suggest that p–p and H-bonding interac-
tions play an important role but also the chromatographic
results achieved under normal-phase and polar-organic
conditions contribute to this conclusion.
In order to elucidate the chiral recognition mechanisms of
CDXs on (S,S)-Whelk-O1 CSP, all the enantiomeric pairs
were used to perform computational simulations using
AutoDock Vina.52,60 The docking simulations produced nine
docked conformations for each CDX. The values of binding
affinity for the best scoring, i.e., the lowest binding affinity
conformation, for each CDX are displayed in Table 3. Energy
difference values were calculated as the difference in energy
of the lowest binding affinity conformation of (R)-CDX-CSP
complex versus (S)-CDX-CSP complex.
The PyRx/AutoDock Vina calculations lead to a successful
reproduction of the enantioselectivity for all enantiomeric
pairs of CDXs, whereas only an 86% of accordance was
obtained concerning elution order. Accordingly, regarding
enantiomeric pair of CDX-2, the more retained enantiomer,
(R)-(+)-CDX-2, binds to the (S,S)-Whelk-O1 selector with
higher affinity, i.e., lower binding energy (ꢀ6.2 kcal/mol),
compared to the first eluted enantiomer (ꢀ5.3 kcal/mol).
Concerning enantiomeric pair of CDX-3, there is also an
energetic difference between each enantiomer–CSP complex:
the (S)-enantiomer binds to the chiral selector with higher affin-
ity (ꢀ6.1 kcal/mol) than the (R)-enantiomer (ꢀ4.7 kcal/mol).
The computational calculations confirmed the experimental
chromatographic data, since (S)-(+)-CDX-3 was the more
retained enantiomer.
Furthermore, the calculated binding affinities differences
between the enantiomers of the same enantiomeric pair of
CDXs are also directly related with the magnitude of the
experimental separation factors (except enantiomeric pair of
CDX-1). For example, the enantiomeric pair of CDX-3 has
the highest energy difference (1.4 kcal/mol) and also the
highest enantioselectivity (a = 4.08). Finally, considering
enantiomeric pairs of CDXs 4–7, low separation factors
(1.00 to 1.10) are in agreement with the low docking energy
difference (0 to ꢀ0.5). For example, the binding energies
are equal for both enantiomers of CDX-7 (ꢀ5.1 kcal/mol)
indicating that they have identical affinity for the (S,S)-
Whelk-O1 selector, which is in accordance with the
chromatographic results at any elution conditions attempted
(no separation was achieved, a = 1.00).
Interestingly, the enantiomeric pair of CDX-1 was the only
pair which the order of elution obtained by docking studies
TABLE 3. Calculated binding energy values of the lowest binding affinity conformation for each enantiomer of CDXs 1–7
CDX
Binding affinity (kcal/mol)
Energy difference (kcal/mol)
1.4
Elution order
a
(R)-(+)-CDX-1
(S)-(ꢀ)-CDX-1
(R)-(+)-CDX-2
(S)-(ꢀ)-CDX-2
(R)-(ꢀ)-CDX-3
(S)-(+)-CDX-3
(R)-(+)-CDX-4
(S)-(ꢀ)-CDX-4
(R)-(ꢀ)-CDX-5
(S)-(+)-CDX-5
(R)-(+)-CDX-6
(S)-(ꢀ)-CDX-6
(R)-(ꢀ)-CDX-7
(S)-(+)-CDX-7
ꢀ4.5
ꢀ5.7
ꢀ6.2
ꢀ5.3
ꢀ4.7
ꢀ6.1
ꢀ5.5
ꢀ5.0
ꢀ5.4
ꢀ5.2
ꢀ5.2
ꢀ5.0
ꢀ5.1
ꢀ5.1
Second
First
Second
First
First
Second
1.91
ꢀ0.9
1.4
2.47
4.08
1.10
1.06
1.00
1.00
ꢀ0.5
ꢀ0.21
ꢀ0.21
0.0
–
–
–
–
–
–
–
–
CDX, chiral derivative of xanthone.
1Energy differences not considered significant.
Chirality DOI 10.1002/chir

ENANTIORESOLUTION OF CHIRAL DERIVATIVES OF XANTHONES ON (S,S)-WHELK-O1 AND L-PHENYLGLYCINE AND DOCKING APPROACH 95
was found to be the opposite of the experimental data.
Comparing the differences among the structures of the
CDXs, it can be seen that the substituent and its position
on the xanthonic scaffold were different on CDX-1. These
structural features and the limitations associated with the
stochastic docking algorithms may justify this incorrect
docking calculation.
The docked conformations with the lowest binding energy
of each enantiomer of CDXs 2–3 and 7 are demonstrated
in Figures 7–9, respectively. The selection includes two
enantiomeric pairs of CDXs which were resolved with very
high Rs values, CDX-2 and CDX-3 (Table 1), and one
enantiomeric pair without any degree of resolution under
any elution condition, CDX-7.
Figure 7 demonstrates that both enantiomers of CDX-2
interact inside the cleft of the (S,S)-Whelk-O1 selector.
However, the superimposition of (R) and (S) enantiomers
(Fig. 7a) shows that their positions are not similar. Contrary
to (S)-enantiomer, it must be noted that the (R)-(+)-CDX-2
adopts a conformation where the xanthonic scaffold lies
parallel to the phenanthryl group of the selector as well as
the phenyl ring relatively to the dinitrophenyl group of the
selector. So, the p–p interactions between these aromatic
rings may be stronger for this enantiomer (Fig. 7d), com-
pared to (S)-enantiomer (Fig. 7e). Another difference is the
position of the NH amide of the chiral moiety, which is inside
the cleft for (R)-enantiomer and outside for (S)-enantiomer.
So, the (R)-(+)-CDX-2 can establish an H-bonding interaction
with a nitro oxygen of a dinitrophenyl group of the selector
(Fig. 7b). However, this H-acceptor group of the selector also
establishes an H-bonding interaction with the S-enantiomer,
but with the H-donor hydroxyl group of the chiral moiety
(Fig. 7c). Additionally, both enantiomer–CSP complexes
display H-bonding interactions between the xanthonic carbonyl
group and NH amide of the selector.
Concerning the enantiomeric pair of CDX-3, it is important
to highlight that each enantiomer interacts in a very different
way with the (S,S)-Whelk-O1 selector: whereas the less
retained enantiomer, (R)-(ꢀ)-CDX-3, interacts with the selec-
tor in a more outward position in the cleft, the more retained
enantiomer, (S)-(+)-CDX-3, docks on the opposite side of the
dinitrophenyl group, in a more inward position (Fig. 8a). Thus,
these differences are decisive for the chiral recognition mecha-
nism. Figure 8 demonstrated that both enantiomers of CDX-3
establish one H bonding and two p–p interactions with the
chiral selector. However, the functional groups implicated on
the interactions are not the same. Accordingly, the NH amide
of the selector forms an H-bonding interaction with the oxygen
of the xanthonic heterocycle ring of the (S)-(+)-CDX-3
(Fig. 8b), whereas the H-bond acceptor of (R)-(ꢀ)-CDX-3
is the xanthonic carbonyl group (Fig. 8c). Additionally, for
(S)-(+)-CDX-3, two p–p interactions are established: one
with the xanthonic scaffold and the dinitrophenyl group of
the selector, and another with the phenyl ring, bonded to
the stereogenic center, and the phenanthryl group of the
selector (Fig. 8d). Contrary to (S)-enantiomer, the xanthonic
scaffold of (R)-(ꢀ)-CDX-3 establishes a p–p interaction with
the phenanthryl group of the selector, and the phenyl ring
establishes a p–p interaction with dinitrophenyl group of
the selector (Fig. 8e).
Concerning enantiomeric pair of CDX-7, both enantio-
mers adopt similar conformations inside the cleft of the
(S,S)-Whelk-O1 selector (Fig. 9a). They also establish
similar intermolecular interactions, namely, H bonding
Fig. 7. The most stable docked conformations of both enantiomers of CDX-2 complexed to (S,S)-Whelk-O1 selector (gray): (a) superimposed CDX–CSP com-
plexes, H-bonding interactions of (b) (R)-(+)-CDX-2 and (c) (S)-(ꢀ)-CDX-2, and p–p interactions of (d) (R)-(+)-CDX-2 and (e) (S)-(ꢀ)-CDX-2.The non-aromatic
carbon atoms of (R)-(+)-CDX-2 and of (S)-(ꢀ)-CDX-2 are represented in green and purple, respectively; red dashes represent H-bonding interactions; yellow
dashes represent all the other interactions; p–p interactions are highlighted with a red double edge arrow.
Chirality DOI 10.1002/chir

96
FERNANDES ET AL.
Fig. 8. The most stable docked conformations of both enantiomers of CDX-3 complexed to (S,S)-Whelk-O1 selector (gray): (a) superimposed CDX–CSP com-
plexes, H-bonding interactions of (b) (S)-(+)-CDX-3 and (c) (R)-(ꢀ)-CDX-3, and p–p interactions of (d) (S)-(+)-CDX-3 and (e) (R)-(ꢀ)-CDX-3.The non-aromatic
carbon atoms of (S)-(+)-CDX-3 and of (R)-(ꢀ)-CDX-3 are represented in pink and yellow, respectively; red dashes represent H-bonding interactions; yellow dashes
represent all the other interactions; p–p interactions are highlighted with a red double edge arrow.
Fig. 9. The most stable docked conformations of both enantiomers of CDX-7 complexed to (S,S)-Whelk-O1 selector (gray): (a) Superimposed CDX–CSP com-
plexes, H-bonding interactions of (b) (R)-(ꢀ)-CDX-7 and (c) (S)-(+)-CDX-7, and p–p interactions of (d) (R)-(ꢀ)-CDX-7 and (e) (S)-(+)-CDX-7.The non-aromatic
carbon atoms of (R)-(ꢀ)-CDX-7 and of (S)-(+)-CDX-7 are represented in orange and cyan, respectively; red dashes represent H-bonding interactions; yellow dashes
represent all the other interactions; p–p interactions are highlighted with a red double edge arrow.
between the xanthonic carbonyl oxygen and the NH
amide of the selector. H bonding between the NH amide
of CDXs and a nitro group of the selector (Fig. 9b and
c), and p–p interaction between the xanthonic scaffold
and the (S,S)-Whelk-O1 phenanthryl group (Fig. 9d and
e). As anticipated by the absence of chromatographic
enantioselectivity, none of these interactions is stereoche-
mically dependent.
Chirality DOI 10.1002/chir

ENANTIORESOLUTION OF CHIRAL DERIVATIVES OF XANTHONES ON (S,S)-WHELK-O1 AND L-PHENYLGLYCINE AND DOCKING APPROACH 97
TABLE 4. H bonding and p–p interactions statistics for the (S,S)-Whelk-O1 selector
H-bonding interaction with
p–p interaction with
Dinitrophenyl
Analyte
Amide hydrogen1
Nitro oxygen2
Phenanthryl
(R)-(+)-CDX-1
(S)-(ꢀ)-CDX-1
(R)-(+)-CDX-2
(S)-(ꢀ)-CDX-2
(R)-(ꢀ)-CDX-3
(S)-(+)-CDX-3
(R)-(+)-CDX-4
(S)-(ꢀ)-CDX-4
(R)-(ꢀ)-CDX-5
(S)-(+)-CDX-5
(R)-(+)-CDX-6
(S)-(ꢀ)-CDX-6
(R)-(ꢀ)-CDX-7
(S)-(+)-CDX-7
Total
4
2
6
6
5
2
5
4
5
4
6
5
5
4
63
1
4
3
2
0
0
2
2
1
2
3
2
1
1
24
7
8
5
5
6
5
7
4
7
6
8
9
7
6
90
0
4
5
4
4
8
0
1
0
0
1
0
1
3
31
CDX, chiral derivative of xanthone.
1The amide oxygen did not form any H-bonding interaction.
2H-bonding interactions only formed with the nitro oxygen facing the interior of the cleft.
TABLE 5. Calculated binding energy values of the lowest binding affinity conformation for each enantiomer of CDXs 1–7 on solvated1
(S,S)-Whelk-O1
CDX
Binding affinity (kcal/mol)
Energy difference (kcal/mol)
Elution order
a
(R)-(+)-CDX-1
(S)-(ꢀ)-CDX-1
(R)-(+)-CDX-2
(S)-(ꢀ)-CDX-2
(R)-(ꢀ)-CDX-3
(S)-(+)-CDX-3
(R)-(+)-CDX-4
(S)-(ꢀ)-CDX-4
(R)-(ꢀ)-CDX-5
(S)-(+)-CDX-5
(R)-(+)-CDX-6
(S)-(ꢀ)-CDX-6
(R)-(ꢀ)-CDX-7
(S)-(+)-CDX-7
ꢀ5.3
ꢀ5.0
ꢀ6.0
ꢀ5.2
ꢀ4.9
ꢀ5.8
ꢀ4.7
ꢀ4.7
ꢀ4.6
ꢀ4.5
ꢀ4.6
ꢀ4.5
ꢀ4.6
ꢀ4.7
ꢀ0.3
Second
First
Second
First
First
Second
2.27
ꢀ0.8
0.9
2.78
5.02
1.00
1.00
1.00
1.00
0.0
–
–
–
–
–
–
–
–
ꢀ0.12
ꢀ0.12
ꢀ0.12
CDX, chiral derivative of xanthone.
1MeOH inside the selector cleft.
2Energy differences not considered significant.
Compared to the enantioseparated CDXs (1–3), an alkyl
group bonded to the stereogenic center exists on CDX-7 instead
of the phenyl ring. Thus, the simultaneous p–p interactions that
may be established by the phenyl ring at the stereogenic center
as well as the xanthonic scaffold with the selector, as observed
for CDXs 1–3, are crucial for enantioresolution.
Bearing in mind the importance of H bonding and p–p
interactions for the mechanism of chiral recognition for
(S,S)-Whelk-O1, additional research was done in order to
evaluate the frequency of these interactions among the total
calculated conformations (Table 4). Given that AutoDock
Vina retrieved nine conformations for each enantiomer of
CDXs 1–7, in a total of 126 conformations, it was found that
the most frequent H-bonding interaction was with the amide
H of the selector, whereas the most frequent p–p interac-
tions were with the phenanthryl group (Table 4).
The interactions established by the xanthonic scaffold of all
enantiomeric pairs of CDXs (1–7) were also examined, with
the carbonyl group participating in more H-bonding interac-
tions than the oxygen of the heterocyclic ring. Also, the
xanthonic scaffold established more frequently p–p interac-
tions with phenanthryl group than with 3,5-dinitrophenyl
group of the selector.
An additional aspect of this study was the analysis of
the influence of the presence of solvent molecules on the
ligand binding affinity and on the ligand binding orien-
tation. Docking results described in the literature have
already shown that when solvent molecules were explicitly
included in the docking calculations as a part of the recep-
tor61 or when solvent molecules were added by a Monte
Carlo-based solvated docking approach,62,63 the docking
results are more in accordance with the experimental data.
Chirality DOI 10.1002/chir

98
FERNANDES ET AL.
TABLE 6. Calculated binding energy values of the lowest binding affinity conformation for each enantiomer of CDXs 1–7 on flexible
(S,S)-Whelk-O1
CDX
Binding affinity (kcal.mol-1)
Energy difference (kcal.mol-1)
Elution order
a
(R)-(+)-CDX-1
(S)-(ꢀ)-CDX-1
(R)-(+)-CDX-2
(S)-(ꢀ)-CDX-2
(R)-(ꢀ)-CDX-3
(S)-(+)-CDX-3
(R)-(+)-CDX-4
(S)-(ꢀ)-CDX-4
(R)-(ꢀ)-CDX-5
(S)-(+)-CDX-5
(R)-(+)-CDX-6
(S)-(ꢀ)-CDX-6
(R)-(ꢀ)-CDX-7
(S)-(+)-CDX-7
ꢀ5.5
ꢀ5.2
ꢀ5.6
ꢀ4.8
ꢀ5.2
ꢀ5.4
ꢀ5.1
ꢀ5.1
ꢀ5.1
ꢀ5.1
ꢀ5.2
ꢀ5.2
ꢀ5.1
ꢀ5.1
ꢀ0.3
Second
First
Second
First
First
Second
1.91
ꢀ0.8
0.2
2.47
4.08
1.10
1.06
1.00
1.00
0.0
–
–
–
–
–
–
–
–
0.0
0.0
0.0
CDX, chiral derivative of xanthone.
To obtain a complete description of (S,S)-Whelk-O1-ligand
interactions, the study of potential solvent-mediated inter-
actions was made. For this purpose, MeOH was used as
an example of the influence of solvent molecules in the
docking poses and scores of enantiomeric pairs of CDXs
1–7 (Table 5).
The docking calculations lead to a 100% successful repro-
duction of the enantioselectivity and elution order. Thus, it
is shown that the accurate placement of explicit solvent into
the systems can potentially improve docking results.
A final study revealed that the introduction of flexibility in
the selector amide chain did not improve the overall docking
scores results (Table 6).
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The (S,S)-Whelk-O1 CSP showed the highest enantios-
electivity and resolution for the enantiomeric pairs of
CDXs evaluated, whereas the L-phenylglycine CSP
showed the lowest discrimination ability. Actually, under
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