Enantiomeric resolution and docking studies of chiral xanthonic derivatives on chirobiotic columns

Article abstract of DOI:10.3390/molecules23010142

A systematic study of enantioresolution of a library of xanthonic derivatives, prepared “in-house”, was successfully carried out with four commercially available macrocyclic glycopeptide-based columns, namely Chirobiotic^TM T, Chirobiotic^TM R, Chirobiotic^TM V and Chirobiotic^TM TAG. Evaluation was conducted in multimodal elution conditions: normal-phase, polar organic, polar ionic and reversed-phase. The effects of the mobile phase composition, the percentage of organic modifier, the pH of the mobile phase, the nature and concentration of different mobile phase additives on the chromatographic parameters are discussed. Chirobiotic^TM T and Chirobiotic^TM V, under normal-phase and reversed-phase modes, respectively, presented the best chromatographic parameters. Considering the importance of understanding the chiral recognition mechanisms associated with the chromatographic enantioresolution, and the scarce data available for macrocyclic glycopeptide-based columns, computational studies by molecular docking were also carried out.

Full text of DOI:10.3390/molecules23010142

molecules
Article
Enantiomeric Resolution and Docking Studies of
Chiral Xanthonic Derivatives on Chirobiotic Columns
Ye Zaw Phyo 1,2, Sara Cravo 2,3, Andreia Palmeira 2,3, Maria Elizabeth Tiritan 2,3,4
,
e‛
Anake Kijjoa 1,2 ID , Madalena M. M. Pinto 2,3,
*
and Carla Fernandes 2,3,
*
ID
1
ICBAS-Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, Rua de Jorge Viterbo Ferreira,
228, 4050-313 Porto, Portugal; chemistkophyo.ckp@gmail.com (Y.Z.P.); ankijjoa@icbas.up.pt (A.K.)
Interdisciplinary Centre of Marine and Environmental Research (CIIMAR), Edifício do Terminal de
Cruzeiros do Porto de Leixões, Av. General Norton de Matos s/n, 4050-208 Matosinhos, Portugal;
scravo@ff.up.pt (S.C.); andreiapalmeira@gmail.com (A.P.); elizabeth.tiritan@iscsn.cespu.pt (M.E.T.)
Laboratório 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
CESPU, Instituto de Investigação e Formação Avançada em Ciências e Tecnologias da Saúde (IINFACTS),
Rua Central de Gandra, 1317, 4585-116 Gandra PRD, Portugal
2
3
4
*
Correspondence: madalena@ff.up.pt (M.M.M.P.); cfernandes@ff.up.pt (C.F.);
Tel.: +35-122-042-8692 (M.M.M.P.); +35-122-042-8688 (C.F.)
Received: 29 November 2017; Accepted: 8 January 2018; Published: 11 January 2018
Abstract: A systematic study of enantioresolution of a library of xanthonic derivatives,
prepared “in-house”, was successfully carried out with four commercially available macrocyclic
glycopeptide-based columns, namely ChirobioticTM T, ChirobioticTM R, ChirobioticTM V and
ChirobioticTM TAG. Evaluation was conducted in multimodal elution conditions: normal-phase, polar
organic, polar ionic and reversed-phase. The effects of the mobile phase composition, the percentage
of organic modifier, the pH of the mobile phase, the nature and concentration of different mobile phase
additives on the chromatographic parameters are discussed. ChirobioticTM T and ChirobioticTM V,
under normal-phase and reversed-phase modes, respectively, presented the best chromatographic
parameters. Considering the importance of understanding the chiral recognition mechanisms
associated with the chromatographic enantioresolution, and the scarce data available for macrocyclic
glycopeptide-based columns, computational studies by molecular docking were also carried out.
Keywords: ChirobioticTM; docking; enantioresolution; enantioselectivity; molecular recognition;
xanthonic derivatives
1. Introduction
During the last few decades there has been widespread interest in oxygenated heterocyclic
compounds such as molecules with a xanthone scaffold [
important roles as bioactive agents and because xanthone is an attractive core for molecular
modifications [ ] and the design of new molecular entities [10 11]. Xanthonic derivatives can
be isolated from terrestrial [12 13] and marine [ ] sources, or obtained by synthesis using different
synthetic methodologies [14,15].
1–3], mainly in consideration of their
4–
9
,
,
2
Recently, some synthetic enantiomerically pure xanthone derivatives prepared “in house”
demonstrated highly interesting biological activities, namely inhibition of enzymes involved in
inflammatory processes [16], growth inhibitory effects on different tumor cell lines [17], nerve sciatic
conduction blockade [18], as well as the ability to behave as promising chiral selectors for liquid
chromatography (LC) [19].
Since the enantiomeric purity of the final product is crucial regarding both biological activities
and preparation of single enantiomer selectors for LC stationary phases, there is a great need for
Molecules 2018, 23, 142; doi:10.3390/molecules23010142
www.mdpi.com/journal/molecules

Molecules 2018, 23, 142
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suitable methods for enantioseparation and evaluation of the enantiomeric composition. One of the
most frequently applied techniques is LC with chiral columns [20]. Consequently, systematic studies of
enantioresolution using different types of LC chiral columns, i.e., macrocyclic glycopeptide-based [21],
Pirkle-type [22,23] and polysaccharide-based [24], and further determination of the enantiomeric
purity of the synthesized xanthonic compounds, were described. By comparison, of the overall
chromatographic results for the same small library of xanthonic derivatives considering the different
types of chiral columns, in general, macrocyclic glycopeptide-based showed enantioselectivity but low
resolution [25]. These results were not expected considering that this type of chiral columns are one
of the most versatile and broadly applied, allowing efficient enantioseparations of diverse classes of
chiral compounds [26–33]. Moreover, their usefulness can be increased considering the complementary
profile in enantioselectivity of the different macrocyclic glycopeptide-based selectors [34].
In order to further explore the effectiveness of this type of columns to separate the enantiomers
of this important class of compounds, herein we describe a systematic study of enantioresolution
using a larger library of chiral xanthonic derivatives and different mobile phases in normal-phase
mode (NPM), polar organic mode (POM), polar ionic mode (PIM), and reversed-phase mode (RPM).
The macrocyclic glycopeptide columns based on ristocetin, teicoplanin, teicoplanin aglycone, and
vancomycin, ChirobioticTM R, T, TAG and V, respectively, were evaluated. The effects of the mobile
phase composition, the percentage of organic modifier, the pH of the mobile phase, the nature
and concentration of different mobile phase additives on the chromatographic parameters, for each
ChirobioticTM column, are discussed. The elution order was also assessed in all cases. Finally, taking
into account the importance in understanding the chiral recognition mechanisms associated with the
chromatographic enantioresolution [35,36] and the scarce data for macrocyclic glycopeptide-based
selectors [37–40], computational studies were also carried out.
2. Results and Discussion
In our previous work, LC columns containing macrocyclic glycopeptides as chiral selectors:
ChirobioticTM R, T, TAG and V (Figure 1), were demonstrated to have enantioselectivity for six of the
seven enantiomeric pairs of the xanthonic derivatives analyzed [21]. However, poor enantioresolution
were obtained, which prompted us to pursue our investigation increasing the library of compounds
and the variables of the mobile phase to better understand the behavior of these chiral columns
towards the target analytes. In this work, a library of thirty-one chiral xanthonic analytes (Figure 2)
was used for the systematic study of enantioresolution, including the previous seven enantiomeric
pairs [21] and more twenty-four [22], applying the chiral columns based on these four macrocyclic
glycopeptide selectors.
Chiral xanthonic analytes were obtained in enantiomeric pure form (enantiomeric excess between
98% and 99%) [21,22,24], by coupling carboxyxanthones with both enantiomers of commercially
available chiral building blocks, using O-(benzotriazol-1-yl)-N,N,N0,N0-tetramethyluronium
tetrafluoroborate (TBTU) as coupling reagent (Scheme 1), according to procedures described
elsewhere [17,18].
The target library of this study, although comprising a common structural xanthone scaffold, is
structurally diverse (Figure 2). The xanthone scaffold of analytes
alkoxyamide chemical bridge for the link to the chiral moiety; analytes
at position 6 of the xanthone scaffold, and analytes 22 31 have methyl groups at positions 5 and
7. Additionally, the analytes 31 comprise an amide linkage with the chiral moiety at position 2.
1–8
has no substituents beyond an
9
–21 have a methoxyl group
–
9–
The majority of the analytes have only one stereogenic center with an alkyl or aromatic moiety, except
7, 8, 20, 21, 30 and 31 which have two stereogenic centers.

Molecules 2018, 23, 142
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Figure 1. 2D (A) and 3D (B) structures of the four macrocyclic glycopeptide-based selectors. Chiral
selectors are represented in sticks with C, O, N, and Cl atoms colored in grey, red, blue and
green, respectively.

Molecules 2018, 23, 142
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Figure 2. Chemical structures of chiral xanthonic analytes 1–31.
Scheme 1. General scheme of synthesis of chiral xanthonic analytes 1–31.
Considering that chromatographic separations are highly influenced by mobile phase components,
all chiral analytes were evaluated under four chromatographic elution modes (NPM, POM, PIM and
RPM). In NPM, n-hexane (Hex) and an organic modifier, ethanol (EtOH) or 2-propanol (2-PrOH), were

Molecules 2018, 23, 142
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evaluated. Methanol (MeOH), EtOH, 2-PrOH or acetonitrile (ACN) in different proportions were used
in POM. The mixture of MeOH with several percentages of triethylamine (TEA) and glacial acetic acid
(AcOH) were employed in PIM. Regarding RPM, different proportions of MeOH or tetrahydrofuran
(THF) and buffer (0.1 or 1% triethylammonium acetate (TEAA) or ammonium acetate (NH4OAc)) with
pH 4.0–6.0 were applied.
The experimental conditions, including the nature and proportion of the organic modifier, the
pH of the mobile phase, the buffer type and concentration, were investigated in the course of the
enantioseparation process.
2.1. Performance of Chirobiotic T Column for Enantioresolution of Chiral Xanthonic Analytes
Table 1 summarizes the best chromatographic results obtained with ChirobioticTM T column,
under multimodal elution conditions, with resolution factors
obtained with different mobile phases, but not leading to good enantioresolution, are also included for
purposes of comparison.
≥
1.00. However, in some cases, results
Table 1. Chromatographic data, retention factor of first eluted enantiomer (k1), separation factor (α),
resolution (RS), and elution order, for the chiral xanthonic analytes on ChirobioticTM T column, under
multimodal chromatographic conditions.
Elution
Mode
First Eluted
Enantiomer
Analyte
1
Mobile Phase
Hex:EtOH (80:20 v/v)
Hex:2-PrOH (70:30 v/v)
100% EtOH
k1
α
RS
NPM
NPM
POM
18.13
33.19
1.19
1.16
1.47
1.14
2.26
1.93
0.70
(R)
(R)
(R)
2
NPM
NPM
POM
Hex:EtOH (40:60 v/v)
Hex:2-PrOH (70:30 v/v)
100% EtOH
1.92
15.98
0.80
1.26
1.62
1.20
1.28
1.49
0.86
(R)
(R)
(R)
3
4
NPM
NPM
Hex:EtOH (80:20 v/v)
Hex:2-PrOH (50:50 v/v)
32.34
2.93
1.09
1.32
1.09
1.00
(R)
(R)
NPM
PIM
POM
Hex:EtOH (80:20 v/v)
MeOH:AcOH:TEA (100:0.1:1 v/v/v)
100% EtOH
19.56
1.13
1.09
1.42
1.09
1.33
2.95
0.80
1.28
(R)
(R)
(R)
7
9
NPM
Hex:EtOH (90:10 v/v)
23.91
1.29
2.83
(R)
NPM
NPM
PIM
POM
POM
Hex:EtOH (70:30 v/v)
Hex:2-PrOH (50:50 v/v)
MeOH:AcOH:TEA (100:0.1:0.1 v/v/v)
100% EtOH
9.21
7.11
1.34
1.13
1.63
1.47
1.46
1.08
1.42
1.63
2.48
1.07
0.60
1.73
1.32
(R)
(R)
(R)
(R)
(R)
100% 2-PrOH
10
11
NPM
NPM
POM
POM
Hex:EtOH (70:30 v/v)
Hex:2-PrOH (50:50 v/v)
100% EtOH
5.04
4.05
0.80
1.01
1.54
1.61
1.49
1.74
3.30
1.56
1.90
1.61
(R)
(R)
(R)
(R)
100% 2-PrOH
NPM
NPM
POM
POM
Hex:EtOH (80:20 v/v)
Hex:2-PrOH (80:20 v/v)
100% EtOH
6.93
8.35
0.63
0.69
1.29
1.33
1.25
1.41
2.43
0.84
1.00
1.14
(R)
(R)
(R)
(R)
100% 2-PrOH
12
14
15
NPM
NPM
POM
Hex:EtOH (80:20 v/v)
Hex:2-PrOH (70:30 v/v)
100% EtOH
15.31
22.72
1.03
1.32
1.38
1.25
2.31
0.50
1.00
(R)
(R)
(R)
NPM
NPM
RPM
Hex:EtOH (90:10 v/v)
Hex:2-PrOH (50:50 v/v)
MeOH:TEAA pH 4.2 (40:60 v/v)
9.78
1.53
10.54
1.29
1.15
1.19
1.55
0.78
1.53
(R)
(R)
(R)
NPM
NPM
POM
RPM
Hex:EtOH (90:10 v/v)
Hex:2-PrOH (50:50 v/v)
100% EtOH
16.67
2.33
0.67
1.26
1.23
1.15
1.33
1.97
0.89
0.70
2.46
(R)
(R)
(R)
(R)
MeOH:TEAA pH 4.2 (30:70 v/v)
20.59

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Table 1. Cont.
Elution
Mode
First Eluted
Enantiomer
Analyte
16
Mobile Phase
Hex:EtOH (90:10 v/v)
Hex:2-PrOH (50:50 v/v)
100% EtOH
k1
α
RS
NPM
NPM
POM
RPM
17.86
2.48
0.70
1.18
1.15
1.08
1.24
1.51
0.40
0.50
1.92
(R)
(R)
(R)
(R)
MeOH:TEAA pH 4.2 (30:70 v/v)
22.32
17 *
NPM
NPM
POM
POM
RPM
Hex:EtOH (80:20 v/v)
Hex:2-PrOH (80:20 v/v)
100% EtOH
100% 2-PrOH
MeOH:TEAA pH 4.2 (30:70 v/v)
2.03
3.33
0.42
0.30
21.13
2.09
2.71
1.39
1.99
1.49
7.37
3.31
1.45
1.78
3.20
(S)
(S)
(S)
(S)
(R)
18
19
20
NPM
NPM
RPM
Hex:EtOH (90:10 v/v)
Hex:2-PrOH (50:50 v/v)
MeOH:TEAA pH 4.2 (30:70 v/v)
12.05
1.67
18.89
1.16
1.13
1.18
1.43
0.30
1.37
(R)
(R)
(R)
NPM
NPM
RPM
Hex:EtOH (90:10 v/v)
Hex:2-PrOH (50:50 v/v)
MeOH:TEAA pH 4.2 (30:70 v/v)
10.68
1.59
29.61
1.13
1.10
1.17
1.28
0.30
1.30
(R)
(R)
(R)
NPM
NPM
PIM
POM
POM
RPM
Hex:EtOH (80:20 v/v)
Hex:2-PrOH (50:50 v/v)
MeOH:AcOH:TEA (100:0.1:1 v/v/v)
100% EtOH
100% 2-PrOH
MeOH:TEAA pH 4.2 (30:70 v/v)
11.20
3.45
1.10
0.85
1.09
8.55
1.31
1.30
1.11
1.30
1.46
2.26
2.24
1.11
0.74
1.32
0.99
1.66
(R)
(R)
(R)
(R)
(R)
(R)
22
NPM
NPM
POM
POM
Hex:EtOH (80:20 v/v)
Hex:2-PrOH (50:50 v/v)
100% EtOH
5.50
2.62
0.72
0.77
1.36
1.39
1.34
1.52
2.45
1.18
1.38
1.10
(R)
(R)
(R)
(R)
100% 2-PrOH
23 *
NPM
NPM
POM
POM
RPM
Hex:EtOH (50:50 v/v)
Hex:2-PrOH (50:50 v/v)
100% EtOH
100% 2-PrOH
MeOH:TEAA pH 4.2 (40:60 v/v)
1.22
1.57
0.56
0.48
4.68
1.25
1.19
1.25
1.54
2.83
1.28
0.87
1.00
1.08
6.79
(S)
(S)
(S)
(S)
(R)
24
25
26
27
28
29
30
NPM
RPM
Hex:EtOH (90:10 v/v)
MeOH:TEAA pH 4.2 (30:70 v/v)
3.57
29.11
1.11
1.23
0.79
1.55
(R)
(R)
NPM
RPM
Hex:EtOH (90:10 v/v)
MeOH:TEAA pH 4.2 (30:70 v/v)
4.09
22.38
1.09
1.16
0.92
1.08
(R)
(R)
NPM
RPM
Hex:EtOH (90:10 v/v)
MeOH:TEAA pH 4.2 (30:70 v/v)
4.53
21.58
1.12
1.22
1.16
1.49
(R)
(R)
NPM
RPM
Hex:EtOH (90:10 v/v)
MeOH:TEAA pH 4.2 (40:60 v/v)
5.91
10.87
1.16
1.26
1.40
1.97
(R)
(R)
NPM
RPM
Hex:EtOH (90:10 v/v)
MeOH:TEAA pH 4.2 (30:70 v/v)
6.41
27.13
1.11
1.26
1.13
1.87
(R)
(R)
NPM
RPM
Hex:EtOH (90:10 v/v)
MeOH:TEAA pH 4.2 (40:60 v/v)
4.67
13.04
1.07
1.16
0.89
1.18
(R)
(R)
NPM
NPM
POM
POM
Hex:EtOH (90:10 v/v)
Hex:2-PrOH (80:20 v/v)
100% EtOH
15.16
9.76
0.56
0.49
1.24
1.33
1.17
1.43
1.93
0.88
0.70
1.05
(R)
(R)
(R)
(R)
100% 2-PrOH
Chromatographic conditions: flow rate: 0.5 mL/min, UV detection at 254 nm; * Elution order changed with the
mobile phase.
ChirobioticTM T column presented good chromatographic parameters in our previous work [21],
and in the present work the same pattern was achieved, with enantioseparation of twenty-five out of
thirty-one enantiomeric mixtures of xanthonic analytes (81%) (Table 1).
In NPM, mixtures of Hex and EtOH or 2-PrOH as organic modifier, in different proportions, were
evaluated in order to achieve optimized chromatographic conditions, i.e., high enantioselectivity
and resolution in short analysis time. In general, the Hex:EtOH combinations afforded better
chromatographic results. For example, excellent enantioselectivity (α = 2.09) and resolution (RS = 7.37)

Molecules 2018, 23, 142
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were obtained for analyte 17, with a k1 value of 2.03, when Hex:EtOH (80:20, v/v) was used as mobile
phase. This was also the best chromatographic result achieved for ChirobioticTM T column. Moreover,
under NPM very high enantioselectivity and resolution were achieved for twenty-two analytes with
α
and RS values ranging from 1.11 to 2.09 and from 1.09 to 7.37, respectively. Additionally, it was found
that, when using Hex:EtOH (90:10 v/v) as mobile phase, this column also showed enantioselectivity
for analytes 24 and 29 but with slight resolution (RS < 1.00). On the other hand, POM using EtOH
or 2-PrOH as mobile phases proved to be an excellent alternative to NPM for ten xanthonic analytes
(Table 1), since good enantioselectivity and resolution were obtained (
1.25 to 1.99 and from 1.00 to 1.90, respectively), with very short retention (values ranging from 0.30
to 1.63). As an example, high enantioselectivity ( = 1.49) and resolution (RS = 1.90) were obtained
for analyte 10, when 100% EtOH was used as mobile phase, being the chromatographic run less than
α and RS values ranging from
α
10 min. The low toxicity of this solvent is also a significant advantage to be taken into account [41,42].
Following the systematic investigation, PIM were also evaluated for the ChirobioticTM T column.
In spite of the shorted retention factors obtained, absence or poor enantioselectivity was observed with
all the combination of mobile phases evaluated. Only for analytes 4, 9 and 20 α values > 1.00 were
achieved but with poor resolution (RS < 1.00).
Similar to the achieved results in our previous work [21], high retention times were observed
under RPM, however, for fourteen analytes good enantioselectivity and resolution were obtained, with
α and RS values ranging from 1.16 to 2.83 and from 1.08 to 6.79, respectively.
For example, the ChirobioticTM T column presented very high enantioseparation capabilities
for analytes 15 and 23, with
α values of 1.33 and 2.83, respectively, and RS values of 2.46 and 6.79,
respectively, using MeOH:TEAA at pH 4.2 (30:70 v/v) as mobile phase. These chromatographic results
were also the best achieved for both analytes in this column. Moreover, the proportion 30:70 v/v of
MeOH:TEAA was the optimum for the majority of the obtained enantioseparations.
Considering the overall chromatographic data, it should be noted that analyte 17 could be
baseline resolved under three different modes, namely NPM, POM and RPM. Figure 3 shows selected
chromatograms of the enantioseparation of analyte 17 using different mobile phases.
Figure 3. Chromatograms of the enantioseparation of analyte 17 on the ChirobioticTM T column using
different mobile phases.
Regarding the elution order, for the enantioseparated xanthonic analytes, the (R)-enantiomer was
the first to elute in this column. The exception was only observed for analytes 17 and 23, which elution

Molecules 2018, 23, 142
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order changed according to the elution mode: in NPM and POM the first eluted enantiomer was the
(S), while (R) enantiomer eluted first in RPM.
2.2. Performance of ChirobioticTM R Column for Enantioresolution of Chiral Xanthonic Analytes
Considering the ChirobioticTM R column, a total of seventeen chiral xanthonic analytes (55%) was
enantioseparated with resolution factors ≥1.00 (Table 2).
Table 2. Chromatographic data, retention factor of first eluted enantiomer (k1), separation factor (α),
resolution (RS), and elution order, for the chiral xanthonic analytes on ChirobioticTM R column, under
multimodal chromatographic conditions.
Elution
Mode
First Eluted
Enantiomer
Analyte
1
Mobile Phase
k1
α
RS
POM
PIM
100% MeOH
0.27
0.50
1.72
1.60
1.23
1.41
(S)
(S)
MeOH:AcOH:TEA (100:0.1:1 v/v/v)
2
4
5
9
POM
PIM
100% MeOH
MeOH:AcOH:TEA (100:0.01:0.01 v/v/v)
0.27
0.48
1.44
1.32
0.45
0.81
(R)
(R)
POM
PIM
100% MeOH
MeOH:AcOH:TEA (100:0.1:1 v/v/v)
0.23
0.48
1.88
1.52
1.00
1.37
(S)
(S)
POM
PIM
100% MeOH
MeOH:AcOH:TEA (100:0.1:1 v/v/v)
0.28
0.39
1.80
1.65
1.02
1.52
(R)
(R)
NPM
NPM
POM
PIM
Hex:EtOH (80:20 v/v)
Hex:2-PrOH (70:30 v/v)
100% 2-PrOH
MeOH:AcOH:TEA (100:0.1:0.1 v/v/v)
9.32
7.59
1.44
0.22
1.46
1.88
1.85
1.72
1.99
1.49
1.32
0.87
(R)
(R)
(R)
(R)
10 *
NPM
NPM
Hex:EtOH (90:10 v/v)
Hex:2-PrOH (80:20 v/v)
15.78
8.41
1.24
1.69
1.77
1.73
(R)
(R)
11
12
NPM
Hex:EtOH (90:10 v/v)
10.83
1.16
1.68
(R)
NPM
PIM
Hex:EtOH (90:10 v/v)
MeOH:AcOH:TEA (100:0.1:1 v/v/v)
28.07
0.67
1.16
1.26
1.37
0.84
(R)
(R)
13 *
PIM
MeOH:AcOH:TEA (100:0.01:0.01 v/v/v)
0.44
1.47
1.15
(S)
17
NPM
NPM
POM
RPM
Hex:EtOH (90:10 v/v)
Hex:2-PrOH (70:30 v/v)
100% 2-PrOH
2.32
0.39
0.29
1.09
2.91
4.65
2.05
1.35
3.95
2.05
0.91
0.70
(S)
(S)
(S)
(S)
MeOH:TEAA pH 4.2 (50:50 v/v)
19
20
RPM
MeOH:TEAA pH 4.2 (30:70 v/v)
1.90
1.21
1.00
(R)
NPM
NPM
PIM
Hex:EtOH (90:10 v/v)
Hex:2-PrOH (50:50 v/v)
MeOH:AcOH:TEA (100:0.1:1 v/v/v)
20.33
2.13
1.10
1.11
1.28
1.11
1.18
0.91
0.74
(R)
(R)
(R)
22
NPM
NPM
POM
Hex:EtOH (90:10 v/v)
Hex:2-PrOH (70:30 v/v)
100% 2-PrOH
9.69
2.06
0.61
1.58
2.21
1.97
2.81
1.83
1.00
(R)
(R)
(R)
23 *
NPM
POM
RPM
Hex:2-PrOH (80:20 v/v)
100% 2-PrOH
MeOH:TEAA pH 4.2 (40:60 v/v)
2.42
0.41
0.64
1.52
1.36
1.70
1.12
0.50
1.51
(R)
(R)
(S)
27
28
RPM
MeOH:TEAA pH 4.2 (30:70 v/v)
15.54
1.09
0.70
(S)
NPM
RPM
Hex:EtOH (90:10 v/v)
MeOH:TEAA pH 4.2 (30:70 v/v)
6.41
15.45
1.11
1.25
1.13
1.12
(S)
(S)
29
RPM
MeOH:TEAA pH 4.2 (40:60 v/v)
13.04
1.16
1.18
(S)
Chromatographic conditions: flow rate: 0.2 mL/min, UV detection at 254 nm; * Elution order changed with the
mobile phase.
Under NPM, this column showed very high enantioseparation capabilities for nine analytes, with
α
and RS values ranging from 1.11 to 4.65 and from 1.12 to 3.95, respectively. The best chromatographic
result was achieved for analyte 17, with excellent enantioselectivity (α = 4.65), high resolution

Molecules 2018, 23, 142
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(RS = 2.05), and short retention time (k1 = 0.39), when Hex:2-PrOH (70:30, v/v) was used as mobile
phase. Nevertheless, in general, mixtures of Hex:EtOH afforded better chromatographic results.
It was found that none of the analytes which xanthone core has no substituents beyond an
alkoxyamide chemical bridge for the link to the chiral moiety (
NPM. Actually, analytes exhibited large k values and poor enantioresolution (data not shown).
Consequently, under this elution mode, probably the nonspecific interactions between the
1–8) were enantioseparated, under
1–8
π-π
xanthonic and the aromatic moieties of the ristocetin selector resulted in high retention without
chiral recognition. It is noteworthy that the nature of the substituents and their position on the
xanthone core also have an important role in enantioselectivity.
Under POM, absence or poor resolution was observed for the majority of the xanthonic analytes.
However, for the analytes comprising only one aromatic moiety and an hydroxyl group next to the
stereogenic center (1, 9 and 22) good enantioselectivity and resolution was obtained, with α and RS
values ranging from 1.72 to 1.85 and from 1.00 to 1.32, respectively, using 100% MeOH or 2-PrOH as
mobile phases. Therefore, these structural molecular features of the analytes might be determining for
enantiorecognition under this elution conditions. Moreover, the nature of the alcoholic modifier also
exerted considerable effects in the retention factors. The polar nature of the mobile phase increased in
the sequence 2-PrOH, EtOH and MeOH, while at a constant percentage of alcohol modifier. Increasing
carbon number of the molecules of the organic solvent is disadvantageous for polar interactions
between the mobile phase and analytes and, consequently, the retention factor may increase [43].
This behavior was more pronounced for 2-PrOH. In this case, the steric effect probably contributes to
the decreased interactions between the mobile phase and the analytes. As an example, the retention
time of analyte
1 decreased when changing the mobile phase from 2-PrOH, EtOH and MeOH, with k1
values of 1.92, 0.56 and 0.27, respectively (data not shown).
Considering evaluation on the RPM the chromatographic results were only satisfactory for
analytes 19
RS = 1.51 for analyte 23 using MeOH:TEAA pH 4.2 (40:60 v/v) as mobile phase. When replacing water
by pure MeOH, i.e., changing RPM to PIM, interesting results were obtained for four analytes (
and 13). The presence of the protic but less polar MeOH may improve the ion-dipole interactions
, 23, 28 and 29. The best enantioselectivity and resolution achieved were α = 1.70 and
1, 4,
5
between those analytes and the ristocetin selector. Moreover, the acidic and basic additives added to
the mobile phases are important to ensure the ionization of the selector.
In order to investigate the effect of ion content on chromatographic parameters, experiments were
carried out with mobile phases containing MeOH:AcOH:TEA in the ratio of 100:1:0.1, 100:0.1:1, 100:1:1,
100:0.1:0.1 and 100:0.01:0.01 v/v/v) (Figure 4). Generally, when using MeOH:AcOH:TEA 100:0.1:1
v/v/v high retention and resolution were obtained (k1 ranging from 0.39 to 0.59 and RS ranging from
1.03 to 1.52) with good enantioselectivity (α ranging from 1.35 to 1.65). Thus, using a slightly basic
mobile phase, ion-dipole interactions between the amines of the selector and the amide or hydroxyl
groups of the analytes, in addition to other selector-analyte interactions, may be responsible for chiral
discrimination. Moreover, it was found that a higher ionic concentration resulted in lower retention.
Probably, the ions existing in the mobile phase will compete for the interaction sites with the analytes.
In ChirobioticTM R column the elution order was also determined for all the enantioseparated
xanthonic analytes. As shown in Table 2, for some analytes the first eluted enantiomer was the (R) while
for others it was the (S). Accordingly, it was found that neither the xanthonic moiety of the analytes
nor the chiral moiety determined the elution order. For example, comparing the results obtained with
the analytes 19 and 29, possessing the same chiral moiety but differing on the substituents linked to
the xanthone scaffold, it can be seen that the first eluted enantiomer was the (R), while for the latter
analyte was the (S)-enantiomer. Other situation can be observed when comparing the elution order of
analytes 9 and 17, possessing the same substituents linked to the xanthone scaffold but differing on
the chiral moiety: for analyte
9 the first eluted enantiomer was the (R) while for analyte 17 was the
(S)-enantiomer.

Molecules 2018, 23, 142
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Figure 4. Effect of the different proportion of AcOH and TEA in MeOH on chromatographic parameters
k1, α and RS for analytes 1, 4, 5 and 13.
Moreover, in the case of analytes 10
,
13 and 23 the elution order changed according to the elution
mode: in NPM and POM the first eluted enantiomer was the (R), differing for the opposite enantiomer
in RPM (some data are not shown in Table 2 since it summarizes only the best chromatographic results
obtained using ChirobioticTM R column for the tested xanthonic analytes).
2.3. Performance of ChirobioticTM V Column for Enantioresolution of Chiral Xanthonic Analytes
Considering the ChirobioticTM V column, NPM and RPM demonstrated to be the best elution
modes for the enantioresolution of xanthonic analytes. A total of seven xanthonic analytes was
successfully enantioseparated under NPM with
α and RS values ranging from 1.13 to 2.31 and from
1.16 to 7.71, respectively. Considering RPM, very high enantioselectivity and resolution were obtained
for fourteen analytes with
α and RS values ranging from 1.21 to 3.32 and from 1.18 to 4.94, respectively.
Relevant chromatographic results obtained using the ChirobioticTM V column for the target xanthonic
analytes, under multimodal elution conditions are presented in Table 3.
Table 3. Chromatographic data, retention factor of first eluted enantiomer (k1), separation factor (α),
resolution (RS), and elution order, for the chiral xanthonic analytes on the ChirobioticTM V column,
under multimodal chromatographic conditions.
Elution
Mode
First Eluted
Enantiomer
Analyte
Mobile Phase
k1
α
RS
1
2
4
6
RPM
NPM
NPM
NPM
MeOH:NH4OAc pH 6 (50:50 v/v)
Hex:EtOH (80:20 v/v)
2.32
8.72
3.37
4.69
1.12
1.09
1.08
1.09
0.92
0.88
0.66
0.89
(R)
(R)
(R)
(R)
Hex:EtOH (50:50 v/v)
Hex:EtOH (80:20 v/v)
RPM
RPM
MeOH:NH4OAc pH 6 (50:50 v/v)
MeOH:NH4OAc pH 4 (50:50 v/v)
3.21
2.68
1.39
1.39
2.29
2.67
(R)
(R)
9 *
NPM
POM
Hex:EtOH (80:20 v/v)
100% EtOH
15.72
0.72
1.19
1.17
1.16
0.90
(S)
(S)
10
NPM
POM
POM
Hex:EtOH (80:20 v/v)
100% EtOH
100% 2-PrOH
8.45
0.39
1.24
1.24
1.23
1.29
1.89
0.60
0.60
(R)
(R)
(R)
11
NPM
Hex:EtOH (80:20 v/v)
6.14
1.15
1.39
(R)

Molecules 2018, 23, 142
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Table 3. Cont.
Elution
Mode
First Eluted
Enantiomer
Analyte
12
Mobile Phase
k1
α
RS
NPM
POM
Hex:EtOH (80:20 v/v)
100% 2-PrOH
13.56
1.79
1.13
1.11
1.35
0.60
(R)
(R)
14
15
RPM
RPM
MeOH:NH4OAc pH 6 (50:50 v/v)
MeOH:NH4OAc pH 4 (50:50 v/v)
3.52
2.98
1.43
1.47
1.56
2.24
(R)
(R)
RPM
RPM
MeOH:NH4OAc pH 6 (50:50 v/v)
MeOH:NH4OAc pH 4 (50:50 v/v)
3.41
2.87
1.42
1.44
2.32
2.49
(R)
(R)
16
RPM
RPM
MeOH:NH4OAc pH 6 (50:50 v/v)
MeOH:NH4OAc pH 4 (50:50 v/v)
3.49
2.98
1.35
1.36
2.08
1.85
(R)
(R)
17 *
NPM
POM
POM
RPM
RPM
Hex:EtOH (80:20 v/v)
100% EtOH
100% 2-PrOH
MeOH:NH4OAc pH 6 (50:50 v/v)
MeOH:NH4OAc pH 4 (50:50 v/v)
1.85
0.13
0.20
3.11
2.61
2.31
2.20
2.00
1.21
1.21
7.71
1.29
1.71
1.18
1.05
(S)
(S)
(S)
(R)
(R)
18
19
20
RPM
RPM
MeOH:NH4OAc pH 6 (50:50 v/v)
MeOH:NH4OAc pH 4 (50:50 v/v)
4.26
2.66
1.04
1.37
0.90
1.89
(R)
(R)
RPM
RPM
MeOH:NH4OAc pH 6 (50:50 v/v)
MeOH:NH4OAc pH 4 (50:50 v/v)
4.09
3.03
1.53
1.55
2.79
2.47
(R)
(R)
NPM
POM
POM
Hex:EtOH (80:20 v/v)
100% EtOH
100% 2-PrOH
3.39
0.51
1.77
1.21
1.25
1.36
1.32
0.60
1.23
(R)
(R)
(R)
22
23
RPM
RPM
MeOH:NH4OAc pH 6 (50:50 v/v)
MeOH:NH4OAc pH 4 (50:50 v/v)
2.38
2.51
1.13
1.15
0.75
0.78
(R)
(R)
NPM
RPM
RPM
Hex:EtOH (80:20 v/v)
MeOH:NH4OAc pH 6 (50:50 v/v)
MeOH:NH4OAc pH 4 (50:50 v/v)
3.45
1.34
1.35
1.31
2.34
3.32
2.68
4.94
4.65
(R)
(R)
(R)
24
25
26
27
28
29
RPM
RPM
MeOH:NH4OAc pH 6 (50:50 v/v)
MeOH:NH4OAc pH 4 (50:50 v/v)
3.39
3.17
1.43
1.32
1.17
1.80
(R)
(R)
RPM
RPM
MeOH:NH4OAc pH 6 (50:50 v/v)
MeOH:NH4OAc pH 4 (50:50 v/v)
3.00
2.55
1.10
1.35
1.45
1.66
(R)
(R)
RPM
RPM
MeOH:NH4OAc pH 6 (50:50 v/v)
MeOH:NH4OAc pH 4 (50:50 v/v)
2.94
2.47
1.27
1.28
1.32
1.39
(R)
(R)
RPM
RPM
MeOH:NH4OAc pH 6 (50:50 v/v)
MeOH:NH4OAc pH 4 (50:50 v/v)
3.14
2.29
1.38
1.41
1.91
1.79
(R)
(R)
RPM
RPM
MeOH:NH4OAc pH 6 (50:50 v/v)
MeOH:NH4OAc pH 4 (50:50 v/v)
2.88
2.39
1.32
1.34
1.28
1.50
(R)
(R)
RPM
RPM
MeOH:NH4OAc pH 6 (50:50 v/v)
MeOH:NH4OAc pH 4 (50:50 v/v)
3.31
2.99
1.49
1.54
1.97
2.08
(R)
(R)
Chromatographic conditions: flow rate: 0.5 mL/min, UV detection at 254 nm; * Elution order changed with the
mobile phase.
Considering NPM, the combination of Hex and EtOH proved to be the most efficient. As an
example, when Hex:EtOH (80:20 v/v) was used as mobile phase, excellent enantioselectivity (α = 2.31)
and resolution (RS = 7.71) were obtained for analyte 17, in a good retention time (k1 = 1.85). This is also
the best chromatographic result achieved for all the chiral columns.
The ChirobioticTM V column also afforded the best chromatographic results for other analytes
(6, 14, 15, 16, 18, 19, 24, 25 and 29) but under RPM. In this elution mode, the nature and proportion of
the organic modifier, the pH of the mobile phase, the buffer type and concentration were investigated
to study their influence on the enantioseparation process as well as to optimize the chromatographic
parameters. It was found that the nature of the organic modifier exerted considerable effects on the
separation, as example when MeOH was changed to THF the chromatographic parameters were not
satisfactory (data not shown). Moreover, slight differences on enantioselectivity and resolution were
observed at different pH (ranging from 4.0 to 6.0). This can be explained taking into account the

Molecules 2018, 23, 142
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neutral character of the analytes that, consequently, do not undergo much influence to variations of pH.
The best mobile phases were the following: MeOH:NH4OAc pH 6 (50:50 v/v) and MeOH:NH4OAc
pH 4 (50:50 v/v).
It was found that all the analytes having methyl groups at positions 5 and 7 of the xanthone
scaffold and only one stereogenic center (22
Opposite situation occurred for the analytes which xanthone scaffold has no substituents beyond
an alkoxyamide chemical bridge for the link to the chiral moiety ( ) (with exception of analyte ).
Consequently, under this elution mode, the nature and position of the substituents on the xanthone
scaffold also have a relevant influence on the chiral recognition. In the case of analytes , probably
–29) were successfully enantioseparated under RPM.
1–
8
6
1–8
the nonspecific hydrophobic interactions between the xanthone scaffold and the vancomycin selector
resulted in retention without chiral recognition.
Additionally, POM was found to be successful for the enantioseparation of two xanthonic analytes
(
17 and 20). The ChirobiticTM V column provided excellent enantioselectivity (
α
= 2.00) and resolution
(RS = 1.71) and very short time of analysis (k1 = 0.20) for analyte 17 with 100% 2-PrOH as mobile phase
and, for analyte 20 the and RS values were 1.36 and 1.23, respectively. Considering evaluation on
α
PIM the chromatographic parameters were not satisfactory (data not shown).
Regarding the elution order, likewise to the ChirobioticTM T column, the (R)-enantiomer of all
the enantioseparated xanthonic analytes was the first to elute in ChirobioticTM V column, except for
analytes
9 and 17, which elution order changed according to the elution mode: in NPM and POM the
first eluted enantiomer was (S), while in RPM was the (R)-enantiomer.
Selected chromatograms for the separation of chiral xanthonic analytes on the ChirobioticTM
column are shown in Figure 5.
V
Figure 5. Chromatograms of xanthonic analytes 17, 19, 23 and 29 on the ChirobioticTM V column.
2.4. Performance of ChirobioticTM TAG Column for Enantioresolution of Chiral Xanthonic Analytes
The performance of ChirobioticTM TAG column to resolve the xanthonic analytes was also
systematically evaluated under multimodal elution conditions. However, this column showed much
lower discrimination capability compared to the others. Table 4 showed the relevant chromatographic
results obtained for the tested xanthonic analytes, under multimodal elution conditions.
As shown in Table 4, good enantioseparation was observed under NPM for five xanthonic analytes
using Hex:EtOH (70:30 v/v) as mobile phase with
α and RS values ranging from 1.37 to 1.45 and from

Molecules 2018, 23, 142
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1.02 to 3.87, respectively. Partial enantioseparation was obtained for analytes
using 100% EtOH as mobile phase.
1
and
4
, under POM,
Considering elution order of all the enantioseparated xanthonic analytes, the (R)-enantiomer
was the first to elute on the ChirobioticTM TAG column. The only exception was also observed for
analyte 17.
Table 4. Chromatographic data, retention factor of first eluted enantiomer (k1), separation factor (α),
resolution (RS), and elution order, for the chiral xanthonic analytes on the ChirobioticTM TAG column,
under multimodal chromatographic conditions.
Elution
Mode
First Eluted
Enantiomer
Analyte
Mobile Phase
100% EtOH
k1
α
RS
1
2
POM
1.26
1.24
0.60
(R)
NPM
POM
Hex:EtOH (70:30 v/v)
100% EtOH
5.01
0.73
1.42
1.39
1.45
0.80
(R)
(R)
4
NPM
POM
Hex:EtOH (70:30 v/v)
100% EtOH
10.66
1.21
1.38
1.36
1.76
0.92
(R)
(R)
5
NPM
NPM
NPM
NPM
Hex:EtOH (70:30 v/v)
Hex:EtOH (70:30 v/v)
Hex:EtOH (70:30 v/v)
Hex:EtOH (70:30 v/v)
8.29
0.80
2.01
1.57
1.37
2.77
1.45
1.27
1.02
3.87
1.16
0.50
(R)
(S)
(R)
(R)
17
30
31
Chromatographic conditions: flow rate: 0.2 mL/min, UV detection at 254 nm.
2.5. Overall Effectiveness of ChirobioticTM Columns for Enantioresolution of Chiral Xanthonic Compounds
under Multimodal Elution Conditions
After the systematic study of enantioseparation by LC under multimodal elution conditions,
ChirobioticTM T demonstrated to be the most suitable for the xanthonic analytes evaluated. In fact,
twenty-five out of thirty-one xanthonic analytes (81%) were enantioseparated with RS values
(Figure 6).
≥
1.00
Figure 6. Diagram considering the xanthonic analytes enantioseparated by ChirobioticTM T, R and
V columns, with resolution factors
1.00. ChirobioticTM TAG was not included considering its poor
enantioselectivity for the evaluted analytes.
≥
Moreover, this column was more efficient under NPM, allowing the efficient enantioseparation
of twenty-two analytes (71%), as shown in Figure 7. This result is in agreement with our previous
work [21]. Similar trend was observed by Armstrong et col. for dihydrofurocoumarins [44]. RPM
and POM were also efficient elution modes for the efficient enantioseparation of fourteen (45%) and
ten xanthonic analytes (32%), respectively, on a ChirobioticTM T column. Moreover, in some cases
these elution modes proved to be good alternatives to NPM. Additionally, it should be highlight

Molecules 2018, 23, 142
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that analytes
T column.
3
,
7
and 30 could only be enantioresolved with RS value
≥
1.00 on the ChirobioticTM
Figure 7. Comparative performance of ChirobioticTM T, R, V and TAG for baseline enantioseparation
of xanthonic analytes under multimodal elution conditions.
Interestingly, ChirobioticTM V under RPM was very efficient for the tested xanthonic analytes
(Figure 7) and, in some cases, exhibited the broadest enantioselectivity observed in all chiral columns.
It was a surprising result considering that in our previous work, this chiral column was one of the less
efficient. However, herein, for the new xanthonic analytes evaluated, particularly those having methyl
groups at positions 5 and 7 on the xanthone scaffold, this column was very effective. In fact, a total of
twenty-three analytes (74%) were baseline enantioseparated on the ChirobioticTM V column (Figure 6),
and analyte 6 could only be baseline resolved in this column.
ChirobioticTM R and TAG columns showed the lowest discrimination ability, under multimodal
elution conditions. However, under NPM nine (29%) and five (16%) analytes were baseline
enantioseparated in ChirobioticTM R and TAG, respectively (Figure 7). Additionally, ChirobioticTM
R
column was the only one useful for the efficient enantioseparation of analytes 5 and 13 (Figure 6).
Finally, it should be emphasized that the xanthonic analytes comprising two stereogenic centers
with the same configuration, i.e., analytes 21 and 31, (Figure 2) were not enantioseparated in
8,
any of the four ChirobioticTM columns. Interestingly, their diastereoisomers were enantioseparated.
These results suggest that the stereochemistry of both stereogenic centers on the xanthonic analytes
play an important role in the enantioseparation of this class of chiral compounds on macrocyclic
glycopeptide-based columns evaluated.
2.6. Computational Studies
In order to understand the binding mechanism behind the observed results of enantioresolution,
docking studies were performed, using the chiral xanthonic analytes that were enantioseparated
by LC with resolution factors
≥
1.00. Values of docking scores are presented on Table 5. The lower
the docking score, the more stable is the analyte:selector complex. Concerning ChirobioticTM T,
R, V, and TAG columns, there was a 52%, 50%, 47%, and 80% agreement between docking scores
and experimental results concerning the elution order of the enantiomers of the chiral analytes.
The differences observed may be due to the complex structures of the macrocyclic glycopeptide-based
selectors allowing different binding patterns with the molecules of the analytes. In fact, although all
the structurally related glycopeptide antibiotics can establish the same type of interactions, including
hydrogen, dipole-dipole, π-stacking, hydrophobic as well as steric repulsion [20], their morphological
differences in the aglycon macrocyclic portions as well as other structural features, are responsible for
their differences in enantioselectivity, retention times, and efficiency [45]. Moreover, computationally,
AutodockVina has a hybrid scoring function (empirical and knowledge-based function) inspired in
the X-Score function that consists of a weighted sum of steric interactions, hydrophobic interaction,
and number of active rotatable bonds with different weights [46]. Therefore, similar selectors and/or
ligands can originate very different docking scores, depending not only on the type of interactions

Molecules 2018, 23, 142
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established, but also the number of interactions, maximizing favorable and minimizing unfavorable
interactions, shape and property complementarities [47].
Table 5. Docking scores (Kcal.mol−1) of both enantiomers of xanthonic analytes on ChirobioticTM T, R,
V, TAG (I), and binding affinity difference between (R) and (S) enantiomers (II).
I
II
RS Value
Docking Score
Docking Score (R)–(S) (Delta) δ
Analytes
T
R
V
TAG
<1.00 <1.00
<1.00 <1.00 1.45
<1.00 <1.00 <1.00
T
R
V
TAG
T
R
V
TAG
(R)-1
(S)-1
−6.6
−7.1
−6.9
2.26
1.41
*
*
−0.5
−0.2
*
*
−7.1
(R)-2
(S)-2
−5.9
−5.4
−4.9
−5.1
1.49
1.09
*
*
*
*
*
−0.5
0.0
*
*
−0.2
(R)-3
(S)-3
−5.8
*
*
*
*
*
−5.8
(R)-4
(S)-4
−5.8
−5.9
−6.2
−6.0
−4.9
−4.8
2.95
1.37
1.52
<1.00
<1.00
2.67
1.76
1.02
−0.1
*
−0.2
*
−0.1
(R)-5
(S)-5
−6.3
−5.1
<1.00
*
−0.2
*
−0.1
−6.1
−5.2
(R)-6
(S)-6
−6.9
−6.6
<1.00 <1.00
2.83
<1.00 <1.00 <1.00 <1.00
<1.00
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
*
−0.3
*
*
(R,R)-7
(S,S)-7
−7.0
−6.7
<1.00 <1.00 <1.00
*
−0.3
*
*
*
(S,R)-8
(R,S)-8
*
*
*
*
*
(R)-9
(S)-9
−6.2
−6.1
−6.2
−6.7
2.48
3.30
2.43
2.31
<1.00
1.55
2.46
1.92
7.37
1.43
1.30
2.24
1.99
1.77
1.16
1.89
1.39
1.35
<1.00
<1.00
<1.00
<1.00
**
−0.1
0.0
−0.5
**
*
(R)-10
(S)-10
−5.7
−6.7
−6.6
**
**
−0.1
−0.2
−0.2
*
*
−5.7
(R)-11
(S)-11
−5.7
−5.8
−6.3
−6.1
−6.1
1.68
−0.1
−0.1
*
−0.2
*
−6.3
(R)-12
(S)-12
−5.9
−6.2
−6.7
−6.5
1.37
−0.3
*
−5.8
−6.5
(R)-13
(S)-13
1.15
<1.00 <1.00
*
**
*
*
**
*
(R)-14
(S)-14
−6.5
−6.6
−6.5
−6.6
<1.00
<1.00
<1.00
3.95
2.24
2.49
2.08
7.71
1.89
2.79
1.32
<1.00
<1.00
<1.00
3.87
−0.1
0.0
*
−0.1
−0.2
−0.3
**
*
(R)-15
(S)-15
−6.3
−7.3
*
*
*
−6.3
−7.1
(R)-16
(S)-16
−6.0
−5.9
−7.1
−7.4
*
−0.2
**
*
*
(R)-17
(S)-17
−7.3
−7.3
−4.9
−4.6
**
**
0.0
−0.3
(R)-18
(S)-18
−5.6
−5.7
−7.7
−7.9
<1.00
1.00
<1.00
<1.00
<1.00
*
*
*
*
*
*
*
*
*
*
−0.1
−0.1
0.0
*
−0.2
−0.2
−0.2
*
*
*
*
*
*
*
*
*
*
(R)-19
(S)-19
−6.5
−7.2
−7.3
−7.7
−0.1
−6.4
−7.5
(R,R)-20
(S,S)-20
−6.5
−6.5
−7.4
−7.6
−7.8
1.18
−0.3
−7.1
(S,R)-21
(R,S)-21
<1.00 <1.00 <1.00 <1.00
*
*
*
*
*
(R)-22
(S)-22
−6.5
−6.2
−7.1
−7.0
2.45
6.79
1.55
1.08
1.49
2.81
1.51
<1.00 <1.00
*
−0.3
**
−0.1
*
(R)-23
(S)-23
−6.3
−6.7
4.94
1.80
1.66
1.39
<1.00
<1.00
<1.00
<1.00
**
**
**
*
−0.4
−0.5
−0.4
−0.2
(R)-24
(S)-24
−6.7
−7.2
−8.0
<1.00
<1.00
<1.00
*
−0.5
−0.6
−0.5
−7.5
(R)-25
(S)-25
−6.1
−7.9
−8.3
*
*
−6.7
(R)-26
(S)-26
−6.5
−7.2
−7.6
*
*
−7.8

Molecules 2018, 23, 142
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Table 5. Cont.
I
II
RS Value
Docking Score
Docking Score (R)–(S) (Delta) δ
Analytes
T
R
V
TAG
T
R
V
TAG
T
R
V
TAG
(R)-27
(S)-27
−7.0
−7.5
1.97
<1.00
1.13
1.91
<1.00
*
*
−0.1
*
−0.1
*
−7.1
−7.6
(R)-28
(S)-28
−6.3
−6.8
−7.5
−7.2
−7.8
−7.5
1.87
1.18
1.93
1.50
2.08
<1.00
<1.00
1.16
*
*
−0.5
−0.5
−0.2
*
−0.3
−0.3
*
(R)-29
(S)-29
−6.6
−7.6
−7.7
1.18
0.0
*
−0.2
*
−0.1
*
−7.1
−7.6
−7.5
(R,R)-30
(S,S)-30
−6.6
−6.8
−5.4
−5.5
<1.00 <1.00
*
*
*
*
*
*
(S,R)-31
(R,S)-31
<1.00 <1.00 <1.00 <1.00
*
*
*
* not analyzed because RS < 1.00; ** not analyzed because elution order was different according to the mobile phase
used; Bold numbers: docking binding affinity in accordance with elution order.
In order to understand the binding mechanism, a visual inspection of the binding conformations
and established interactions were performed for the molecules of the xanthonic analytes. Figures 8–10
illustrate representative examples of the most stable docked conformations for enantiomers complexes
with the chiral selectors of the four chiral columns.
Figure 8. Analyte
1 (A), Analyte 14 (B), Analyte 30 (C), and Analyte 18 (D), docked on teicoplanin
selector. Chiral selectors are represented in sticks with C, O, N, and Cl atoms colored in grey, red,
blue, and green, respectively. (S) and (R) enantiomers are represented with magenta and yellow sticks,
respectively. Hydrogen interactions and
double arrow, respectively.
π-stacking interactions are represented with dashes and

Molecules 2018, 23, 142
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Figure 9. (A) Analyte 9 docked on ristocetin selector; (B) Analyte 25 docked on vancomycin selector.
Chiral selectors are represented in sticks with C, O, N, and Cl atoms colored in grey, red, blue, and green,
respectively. (S) and (R) enantiomers are represented with magenta and yellow sticks, respectively.
In (A), hydrogen interactions and π-stacking interactions are represented with dashes and double
arrow, respectively. In (B), all the interactions (apolar and polar) are represented as grey dashes.
Figure 10. (A) Analyte 2 docked on teicoplanin aglycone selector; (B) Analyte 17 docked on teicoplanin
aglycone selector. Chiral selectors are represented in sticks with C, O, N, and Cl atoms colored in grey,
red, blue, and green, respectively. (S) and (R) enantiomers are represented with magenta and yellow
sticks, respectively. Hydrogen interactions and
double arrow, respectively.
π-stacking interactions are represented with dashes and
Figure 8A show that both enantiomers of analyte
1 are folded in a “U” shape to maximize both the
π
stacking interactions between the xanthonic moiety and one of the teicoplanin aromatic rings; and the
hydrogen interactions between –OH and –NH– groups of the xanthone molecule and the teicoplanin
carbonyl groups. However, as the carbonyl group of the xanthone core of (S)-enantiomer is turned
inside of the teicoplanin molecule, an additional hydrogen interaction with a teicoplanin carbonyl
group was established. This last interaction can justify the stronger retention of (S)-enantiomer in the
ChirobioticTM T column.
However, this is not a general rule for the other enantioseparated analytes. As shown in Figure 8,
for analytes 14 (Figure 8B), 30 (Figure 8C), and 18 (Figure 8D), the docking poses of both enantiomers
of each xanthonic pair are very diverse. Consequently, different groups of both xanthonic and chiral
moieties of the analytes are involved in the interactions.
Although both enantiomers of analyte
xanthone scaffold and the aromatic rings of the ristocetin selector, only the aromatic ring of the chiral
moiety of (S)-enantiomer is in such a way that allows an additional parallel -stacking interaction
9 bind through π-stacking interactions between the
π
(Figure 9A). Moreover, the terminal –OH group of (S)-enantiomer also establishes an additional
hydrogen interaction with an –OH group of the chiral selector.
On the ChirobioticTM V column, the majority of the chiral xanthonic analytes were
enantioseparated under RPM. Concerning the enantiomers of analyte 25 docked onto the ChirobioticTM

Molecules 2018, 23, 142
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V column, both enantiomers are bound to the selector through hydrophobic interactions between
the xanthone scaffold and the aromatic rings and alkyl chain of vancomycin selector (Figure 9B).
Additionally, hydrogen interactions (three) were also established. However, one of the hydrogen
interactions on (S)-enantiomer is established with a fluorine atom [48], more electronegative than
oxygen, thus resulting on a stronger interaction.
On the ChirobioticTM TAG column, the (S)-enantiomers were always more retained,
with exception of analyte 17. Using analyte
2 as an example, both enantiomers are bound to the
ChirobioticTM TAG column by
π
stacking interactions established between the xanthone scaffold
and aromatic rings of the selector. (S)-enantiomer binds more stably to the chiral selector creating
additional hydrogen interactions with the amide groups of the chiral moiety (Figure 10A). Concerning
analyte 17, the aromatic ring and amide group of the chiral moiety on both enantiomers establish
π
-stacking and hydrogen interactions with the chiral selector, respectively. Nonetheless, only the
(R)-enantiomer establishes an extra hydrogen interaction through the xanthone carbonyl group
(Figure 10B). This different elution profile of analyte 17, comparing to the other analytes, was due to
its binding pose with the xanthone scaffold facing the outside medium, and the aromatic ring of the
chiral moiety establishing double sided
(Figure 10B); whereas for other analytes, such as
π
-stacking interactions with two aromatic rings on the selector
, the xanthone scaffold is the structural moiety that
2
is holding the analyte to the selector through parallel π-stacking interactions (Figure 10A).
3. Materials and Methods
3.1. Chemicals and Reagents
Chiral xanthonic analytes (Figure 2) were obtained “in-house” according to procedures described
elsewhere [17,18]. HPLC grade EtOH, MeOH, 2-PrOH, ACN, THF, and Hex were purchased from
Sigma-Aldrich (St. Louis, MO, USA). TEA, AcOH, NH4OAc, all p.a. grade, were also obtained from
Sigma-Aldrich. Ultrapure water was purified by a Milli-Q system (Millipore, Bedford, MA, USA).
TEAA buffers were prepared by titration of 0.1 or 1% (by volume) aqueous solutions of TEA with
AcOH to adjust to a suitable pH.
3.2. Apparatus and Chromatography
Chromatographic measurements were carried out on two chromatographic systems. One LC
system contained a model 880-PU Intelligent HPLC pump (JASCO Corporation, Tokyo, Japan),
equipped with a 7125 injector (Rheodyne LCC, Rohnert Park, CA, USA) fitted with a 20 µL loop,
a JASCO model 880-30 solvent mixer, a 875-UV intelligent UV/Vis detector A chromatography station
for Microsoft Windows 95, version 1.7 DLL, was applied. The second LC was a Dionex Ultimate
3000 (Thermo Fisher Scientific Inc., Waltham, MA, USA) system equipped with a 3000 quaternary
pump, a 3000 autosampler, and a 3000 Variable UV/Vis detector was also used. ChromeleonTM
software version 7.2 Ultimate (Thermo Fisher Scientific Inc., Waltham, MA, USA) was employed to
manage chromatographic data. Chromatographic separations were performed on four commercial
macrocyclic glycopeptide-based columns: ChirobioticTM T (150
ChirobioticTM TAG (150 m particle size), ChirobioticTM V (250
2.1 mm id, 5
particle size) and ChirobioticTM R (150
2.1 mm id, 5 m particle size) (Figure 1), from ASTEC
×
4.6 mm id, 5
µ
m particle size),
×
µ
×
4.6 mm id, 5
µm
×
µ
(Whippany, NJ, USA). Stock solutions of all chiral xanthonic analytes were prepared by dissolution
in EtOH at a concentration of 1 mg/mL. Working solutions were further prepared by dilution of the
stock solutions in the same solvent to a concentration of 10
and all the chromatographic analyses were performed in isocratic mode at 22
The dead times (t0) were considered to be equal to the peak of the solvent front, and were taken from
each particular run. The column flow rate was 0.5 mL/min (ChirobioticTM T and V) or 0.2 mL/min
(ChirobioticTM R and TAG), and the chromatograms were recorded by UV detection at a wavelength
of 254 nm. The mobile phases were prepared in a volume/volume relation, filtered using a Millipore
µ
g/mL. The injection volume was 10
µL,
±
2 ◦C, in triplicate.

Molecules 2018, 23, 142
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0.45 µm filter, and further degassed in an ultrasonic bath for 15 min before use. All the analytes
mentioned in Figure 2 were evaluated with different mobile phases under NPM, POM, PIM and RPM.
The elution order was determined for all the enantioseparated xanthonic analytes by injecting the
solutions of the enantiomeric mixtures (prepared mixing equal aliquots of each enantiomer), and then
each enantiomer separately.
3.3. Computational
The structures of the selectors were obtained from PubChem [49], and both enantiomers
of all xanthonic compounds (Figure 2) were drawn and minimized using an Austin Model 1
(AM1) semi-empirical quantum mechanics force field [50]. The calculation was finished when the
gradient between any two successive steps in the geometry search was less than 10−1 kcal/mol/Å
or the maximum steps were reached, whichever comes first. The line search used was the
Broyden-Fletcher-Golfarb-Shanno search which uses an approximate Hessian matrix to guide the
search [51]. Docking simulations between the chiral selector and the xanthonic enantiomers were
undertaken in AutoDock Vina (Molecular Graphics Lab, La Jolla, CA, USA) [46]. AutoDock Vina
considered the target conformation as a rigid unit while the ligands 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 small molecule. The lowest binding energy docking poses of
each compound were chosen. AutoDock Vina was run using an exhaustiveness of 9 and grid boxes
engulfing the selectors were built. PyMol v1.3 (Schrödinger, New York, NY, USA) [52] was used for
visual inspection of results and graphical representations.
4. Conclusions
The systematic study of enantioseparation for thirty-one chiral xanthonic analytes using four
macrocyclic glycopeptide-based chiral columns confirmed the applicability of these columns in
multimodal elution conditions for the enantioseparation of this class of compounds. It was found
that their applicability increased considering the complementary profile in enantioselectivity of the
different macrocyclic glycopeptide-based selectors. For some chiral xanthonic analytes, 100% EtOH as
mobile phase presented excellent chromatographic parameters. EtOH is considered a green solvent
due its low toxicity. As it was expected, the structural nature of the analytes and the chiral selectors as
well as the mobile phase composition proved to be fundamental factors for the molecular interactions
and consequently for chiral recognition.
The docking study showed that each macrocyclic glycopeptide-based selector allowed different
binding patterns for both enantiomers of the analytes due to their complex structures. Due to the very
diverse structural features of the thirty-one enantiomeric pairs of xanthonic analytes, with different
alkyl and aryl substituents in different positions of the xanthone scaffold, and to the differences in
spatial disposition (chirality) and tridimensional shape of the compounds, would not be suitable to
establish a generalized chiral recognition mechanism or identify which chemical characteristics are
more relevant for retention in the referred columns.
However, the results of the present study fulfilled the initial objectives since higher resolutions
were obtained for the chiral xanthonic analytes evaluated compared to our previous work, and
additional information about chiral recognition mechanisms was provided. In summary, we believe
that this work contributes to a better knowledge in enantioseparation and chiral recognition in general
and for chiral xanthonic derivatives in particular. Computational chemistry studies allowed an
improvement to understand the behavior of these important class of compounds within macrocyclic
glycopeptide-based chiral columns.
Acknowledgments: This research was partially supported by the Strategic Funding UID/Multi/04423/2013
through national funds provided by FCT—Foundation for Science and Technology and European
Regional Development Fund (ERDF), in the framework of the programme PT2020, the project
PTDC/MAR-BIO/4694/2014 (reference POCI-01-0145-FEDER-016790; Project 3599—Promover a Produção

Molecules 2018, 23, 142
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Científica e Desenvolvimento Tecnológico e a Constituição de Redes Temáticas (3599-PPCDT)) as well as by the
project INNOVMAR—Innovation and Sustainability in the Management and Exploitation of Marine Resources
(reference NORTE-01-0145-FEDER-000035, within Research Line NOVELMAR), supported by North Portugal
Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through
the European Regional Development Fund (ERDF), and Chiral_Drugs_CESPU_2017. Phyo thanks the Erasmus
Mundus Action 2 (Lotus Plus project) for a PhD’s scholarship.
e‛
Author Contributions: Ye Zaw Phyo performed both liquid chromatography enantioresolution and
docking experiments, and contributed in writing the draft of the manuscript. Sara Cravo participated in
liquid chromatography enantioresolution experiments. Andreia Palmeira participated in docking studies,
and contributed in writing the manuscript. Maria Elizabeth Tiritan gave scientific advice for the development of
liquid chromatography enantioresolution experiments, participated in discussion of the results and reviewed the
final version of the manuscript. Anake Kijjoa contributed with discussion during the preparation of the manuscript
and reviewed the final version of the manuscript. Madalena M. M. Pinto contributed with discussion during
experiments, the preparation of the manuscript and reviewed the final version. Carla Fernandes coordinated and
supervised both liquid chromatography enantioresolution and docking experiments, reviewed the data generated
and wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 1–31 are available from the authors.
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