New chiral stationary phases for liquid chromatography based on small molecules: Development, enantioresolution evaluation and chiral recognition mechanisms

Article abstract of DOI:10.1002/chir.23142

Recently, we reported the development of new chiral stationary phases (CSPs) for liquid chromatography (LC) based on chiral derivatives of xanthones (CDXs). Based on the most promising CDX selectors, 12 new CSPs were successfully prepared starting from suitable functionalized small molecules including xanthone and benzophenone derivatives. The chiral selectors comprising one, two, three, or four chiral moieties were covalently bonded to a chromatographic support and further packed into LC stainless-steel columns (150?×?2.1?mm I.D.). The enantioselective performance of the new CSPs was evaluated by LC using different classes of chiral compounds. Specificity for enantioseparation of some CDXs was observed in the evaluation of the new CSPs. Besides, assessment of chiral recognition mechanisms was performed by computational studies using molecular docking approach, which are in accordance with the chromatographic parameters. X-Ray analysis was used to establish a chiral selector 3D structure.

Full text of DOI:10.1002/chir.23142

Received: 10 May 2019
DOI: 10.1002/chir.23142
Revised: 15 September 2019
Accepted: 6 October 2019
R E G U L A R A R T I C L E
New chiral stationary phases for liquid chromatography
based on small molecules: Development, enantioresolution
evaluation and chiral recognition mechanisms
Ye' Zaw Phyo1,2 | Joana Teixeira3 | Maria Elizabeth Tiritan2,3,4
| Sara Cravo2,3
|
Andreia Palmeira2,3 | Luís Gales1,5,6 | Artur M.S. Silva7 | Madalena M.M. Pinto2,3
Anake Kijjoa1,2 | Carla Fernandes2,3
|
1 ICBAS‐Instituto de Ciências Biomédicas Abel Salazar, Universidade do Porto, Porto, Portugal
2 Interdisciplinary Centre of Marine and Environmental Research, CIIMAR, Matosinhos, Portugal
3 Faculdade de Farmácia, Departamento de Ciências Químicas, Laboratório de Química Orgânica e Farmacêutica, Porto, Portugal
4 CESPU, Instituto de Investigação e Formação Avançada em Ciências e Tecnologias da Saúde (IINFACTS), Gandra, PRD, Portugal
5 Instituto de Biologia Molecular e Celular (IBMC), Universidade do Porto, Porto, Portugal
6 i3S – Instituto de Investigação e Inovação em Saúde, Porto, Portugal
7 QOPNA & LAQV‐REQUIMTE, Departamento de Química, Universidade de Aveiro, Aveiro, Portugal
Correspondence
Carla Fernandes, Faculdade de Farmácia,
Abstract
Recently, we reported the development of new chiral stationary phases (CSPs)
for liquid chromatography (LC) based on chiral derivatives of xanthones
(CDXs). Based on the most promising CDX selectors, 12 new CSPs were suc-
cessfully prepared starting from suitable functionalized small molecules
including xanthone and benzophenone derivatives. The chiral selectors com-
prising one, two, three, or four chiral moieties were covalently bonded to a
chromatographic support and further packed into LC stainless‐steel columns
(150 × 2.1 mm I.D.). The enantioselective performance of the new CSPs was
evaluated by LC using different classes of chiral compounds. Specificity for
enantioseparation of some CDXs was observed in the evaluation of the new
CSPs. Besides, assessment of chiral recognition mechanisms was performed
by computational studies using molecular docking approach, which are in
accordance with the chromatographic parameters. X‐Ray analysis was used
to establish a chiral selector 3D structure.
Departamento de Ciências Químicas,
Laboratório de Química Orgânica e
Farmacêutica, Rua de Jorge Viterbo
Ferreira, 228, 4050‐313 Porto, Portugal.
Email: cfernandes@ff.up.pt
Funding information
European Regional Development Fund,
Grant/Award Number: PTDC/MAR‐BIO/
4694/2014; Fundação para a Ciência e a
Tecnologia, Grant/Award Number: FCT
UID/QUI/00062/2019 Strategic Funding
UID/Multi/04423/2019; IINFACTS,
Grant/Award Number:
CHIRALBIOACTIVE‐PI‐3RL‐IINFACTS‐
2019
KEYWORDS
benzophenone, chiral derivative of xanthone, chiral recognition, chiral stationary phase, docking,
enantioselectivity, liquid chromatography
1 | INTRODUCTION
industry,1 for both analytical2-4 and preparative pur-
poses.5-7 The development of CSPs for LC brought a
Liquid chromatography (LC) enantioresolution using chi-
ral stationary phases (CSPs) is one of the most significant
separation approaches in academic research and
new breath to enantioseparation processes, and nowa-
days, several types are available including Pirkle‐type,
ion‐exchange‐type, molecularly‐imprinted, and based on
Chirality. 2019;1–17.
wileyonlinelibrary.com/journal/chir
© 2019 Wiley Periodicals, Inc.
1

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PHYO ET AL.
macrocyclic antibiotics, proteins, polysaccharides, cyclo-
dextrins, crown ethers, cyclofructans, and synthetic poly-
mers.8,9 Among them, more than a hundred are currently
commercially available.10 Nevertheless, although many
different types are described, the development of new
CSPs continues to be a field of research with great impor-
tance to follow the constant challenges on different areas
as well as the advances in chromatographic instrumenta-
tion.11 Of note are the Pirkle‐type CSPs that have evolved
over the years, showing more progress reported, mainly
due to the possibility of using a wide variety of small mol-
ecules as chiral selectors.12,13
Recently, we described the development of new CSPs
based on chiral derivatives of xanthones (CDXs).14 This
class of small molecules is proved to be structurally prom-
ising chiral selectors for LC,14 in addition to their broad
spectrum of bioactivities.15,16 The best enantioselectivity
University of Vigo, Spain. HRMS mass spectra were mea-
sured on a Bruker Daltonics micrOTOF Mass Spectrome-
ter, recorded as ESI (electrospray) mode. Melting points
were obtained in a Kofler microscope and are uncor-
rected. The optical rotations were recorded on a
Polartronic Universal polarimeter (ADP 410 polarimeter).
IR spectra were recorded on a FTIR spectrometer Nicolet
iS10 from Thermo Scientific with Smart OMNI‐
Transmission accessory (Software 188 OMNIC 8.3) in
KBr. The column packing was performed in an SSI Lab
Alliance–Pack in a Box, portable HPLC column packing
system model CP constant pressure pump, non‐flush,
0.1 to 24 mL min−1, 10.000 psi, with transducer and RS‐
232 control, SS, 110/220 V column packer assembly (50‐
mL reservoir, column adapter 5/16″ 2.1 mm) Quick‐Set
pump control software. The empty stainless‐steel
columns were purchased from SSI Lab Alliance. The
chiral selector loadings (μmol m−2) were determined
from the elemental analyses of carbon and nitrogen
results
were
achieved
for
xanthone‐(R)‐(−)‐α‐
phenylglycinol‐derived CSPs.14 In the last years, other
amino alcohol‐derived CSPs can be found in literature,
and among them, (R)‐phenylglycinol‐derived CSPs
showed good results for the LC enantioseparation of
many chiral analytes.17-20
as follows: selector loading
(106
[(100 12) (%C × Mw)] and selector
=
×
%C)/
×
n
×
−
loading = (106×%N)/S[(100 × n × 14) − (%N × Mw)],
where %C and %N are the elemental analyses of carbon
and nitrogen, respectively, S is the surface area, n the
number of carbon or nitrogen atoms in the modified
chain and Mw is the relative molecular mass.
In this study, 12 new CSPs were successfully prepared
by multistep pathways starting from suitable functional-
ized small molecules (xanthone and benzophenone deriv-
atives) based on the most promising selectors (CDXs)
previously reported.14 Herein, the majority of the
synthetized chiral selectors was (R)‐phenylglycinol deriv-
atives comprising one, two, three, or four chiral moieties
and was covalently bonded through one or more sites to a
chromatographic support.
Computational modeling studies by molecular docking
were performed to gain the insight of structural features
associated with chiral recognition mechanisms,21,22
which not only allow the understanding the chromato-
graphic parameters at a molecular level23-25 but also to
improve the design of new selectors.26 These studies
allowed a closer look towards the mechanisms involved
in the enantiorecognition ability of this type of CSPs.
2.2 | Chemicals and reagents
All reagents used in this study were purchased from Sigma‐
Aldrich Co (St Louis, Missouri) or Merck (Darmstadt, Ger-
many). The organic solvents were obtained from Sigma‐
Aldrich Co (St Louis, Missouri) and were used without fur-
ther purification. Silica gel 60 GF254 (Merck, Darmstadt,
Germany) precoated plates were used for TLC. Silica gel
60 (0.04‐0.063 mm, Macherey‐Nagel, Dϋren, Germany)
was used for flash column chromatography. Porous spher-
ical silica gel Nucleosil (100 Å‐5 μm, Macherey‐Nagel) was
used as the support material of the CSPs.
2 | MATERIALS AND METHODS
2.1 | General methods
2.3 | Preparation Of CSPs 1‐12
CSPs 1‐12 (Figures 1 and 2) were prepared following sim-
1H and 13C NMR spectra were taken in DMSO‐d6 or
acetone‐d6 at room temperature on a DRX‐300 spectrom-
eter. Chemical shifts are expressed in δ (ppm) values rel-
ative to TMS. 13C NMR assignments were made by HSQC
and HMBC experiments (long‐range JC‐H was optimized
to 7 Hz). Elemental analyses were conducted on an
Elemental Carlo Erba 1108 apparatus in C.A.C.T.I.–
ilar synthetic procedures according to Schemes S1 to S4.
2.3.1 | Synthesis of hydroxyxanthones 1‐7
The following hydroxyxanthones were synthesized
according to previously described procedures.27-33

PHYO ET AL.
3
FIGURE 1 The chemical structures of CSPs based on xanthone derivatives, CSPs 1‐10
1
1‐Hydroxy‐9H‐xanthen‐9‐one (1): Yield (51%). 1H
NMR and 13C NMR data are shown in Supporting
Information.
1,3‐Dihydroxy‐9H‐xanthen‐9‐one (2): Yield (51%). H
NMR and 13C NMR data are shown in Supporting
Information.

4
PHYO ET AL.
FIGURE 2 The chemical structures of
CSPs based on benzophenone derivatives,
CSPs 11‐12
Starting from a poly‐hydroxylated compound
1,3,6‐Trihydroxy‐9H‐xanthen‐9‐one (3): Yield (69%).
1H NMR and 13C NMR data are shown in Supporting
Information.
Compounds 11‐18 were prepared from suitable hydroxyl-
ated compounds (2‐9) using the same method as
described for 10, increasing the amount of K2CO3 and
BrCH2COOCH3 according to the number of hydroxyl
groups of the chemical substrate (2‐9) and the stoichiom-
etry of the reaction.
1,3,8‐Trihydroxy‐9H‐xanthen‐9‐one (4): Yield (26%).
1H NMR and 13C NMR data are shown in Supporting
Information.
1,3,6,8‐Tetrahydroxy‐9H‐xanthen‐9‐one (5): Yield
(24%). 1H NMR and 13C NMR data are shown in
Supporting Information.
Dimethyl 2,2′‐[(9‐oxo‐9H‐xanthene‐1,3‐diyl)bis(oxy)]
diacetate (11): Yield (98%, white solid). m.p. 157°C‐159°
1
C (MeOH); H NMR, 13C NMR, and IR data are shown
1
3,6‐Dihydroxy‐9H‐xanthen‐9‐one (6): Yield (99%). H
NMR and 13C NMR data are shown in Supporting
Information.
in Supporting Information.
Trimethyl 2,2′,2″‐[(9‐oxo‐9H‐xanthene‐1,3,6‐triyl)tris
(oxy)]triacetate (12): Yield (91%, white solid). m.p. 142°
1
3,4‐Dihydroxy‐9H‐xanthen‐9‐one (7): Yield (70%). H
1
C‐144°C (MeOH); H NMR, 13C NMR, and IR data are
NMR and 13C NMR data are shown in Supporting
Information.
shown in Supporting Information.
Trimethyl 2,2′,2″‐[(9‐oxo‐9H‐xanthene‐1,3,8‐triyl)tris
(oxy)]triacetate (13): Yield (93%, white solid). m.p. 151°
2.3.2 | Synthesis of acetate compounds 10‐
18
1
C‐153°C (MeOH); H NMR, 13C NMR, and IR data are
shown in Supporting Information.
Tetramethy
2,2′,2″,2‴‐[(9‐oxo‐9H‐xanthene‐
Starting from a mono‐hydroxylated compound
1,3,6,8 tetrayl)tetrakis(oxy)]tetraacetate (14): Yield (95%,
white solid). m.p. >310°C (MeOH); 1H NMR, 13C
NMR, and IR data are shown in Supporting
Information.
2,2′‐[(9‐Oxo‐9H‐xanthene‐3,6‐diyl)bis(oxy)]diacetate
(15): Yield (88%, white solid). m.p. 228°C‐230°C (MeOH);
1H NMR, 13C NMR, and IR data are shown in Supporting
Information.
The hydroxylated compound (1) (0.30 g, 1.40 mmol) was
dissolved in anhydrous acetone (40 mL), and K2CO3
(0.58 g, 1.40 mmol) and BrCH2COOCH3 (0.2 mL,
1.70 mmol) were added. The mixture was kept under reflux
and magnetic stirring for 24 hours. Afterwards, the mixture
was concentrated under reduced pressure and extracted
with dichloromethane (2
× 100 mL) and water
(2 × 100 mL). The organic layer was evaporated under
reduced pressure, and the crude product was crystallized
from methanol (MeOH), to afford 10 as a white solid.
Methyl 2‐[(9‐oxo‐9H‐xanthen‐1‐yl)oxy]acetate (10):
Dimethyl 2,2′‐[(9‐oxo‐9H‐xanthene‐3,4‐diyl)bis(oxy)]
diacetate (16): Yield (95%, white solid). m.p. >310°C
1
(MeOH); H NMR, 13C NMR, and IR data are shown in
1
Supporting Information.
Dimethyl 2,2′‐[(carbonylbis(4,1‐phenylene))bis(oxy)]
diacetate (17): Yield (94%, white solid). m.p. 175°C‐177°
Yield (95%, white solid). m.p. 156°C‐158°C (MeOH); H
NMR, 13C NMR, and IR data are shown in Supporting
Information.

PHYO ET AL.
5
C (MeOH); 1H NMR, 13C NMR, and IR data are shown in
Supporting Information.
2,2′,2″,2‴‐[(Carbonylbis(benzene‐4,1,3‐triyl))tetrakis
(oxy)]tetraacetic acid (27): Yield (80%, white solid). m.p.
1
Tetramethyl
2,2′,2″,2‴‐[(carbonylbis(benzene‐4,1,3‐
232°C‐235°C (MeOH); H NMR, 13C NMR, and IR data
triyl))tetrakis(oxy)]tetraacetate (18): Yield (93%, white
are shown in Supporting Information.
1
solid). m.p. 128°C‐130°C (MeOH); H NMR, 13C NMR,
and IR data are shown in Supporting Information.
2.3.4 | Synthesis of chiral selectors 28‐39
Starting from a monocarboxylated compound
The carboxylated compound 19 (0.25 g, 0.93 mmol) was
dissolved in tetrahydrofuran (THF, 50 mL), and
triethylamine (TEA, 380 μL, 2.66 mmol) was added. Then
O‐(benzotriazol‐1‐yl)‐N,N,N′,N′‐tetramethyluronium tet-
rafluoroborate (TBTU, 0.37 g, 1.11 mmol) and (R)‐(−)‐α‐
phenylglycinol (0.16 g, 1.11 mmol) were added. The mix-
ture was stirred at room temperature for 5 hours. The sol-
vent was evaporated under reduced pressure, and the
crude product was dissolved in CHCl3 (100 mL). This
solution was washed with 1M HCl (2 × 50 mL), saturated
solution of NaHCO3 (2 × 50 mL), and water (3 × 50 mL).
The organic layer was dried with anhydrous sodium sul-
phate and filtered, and the solvent was evaporated under
reduced pressure. The crude product was recrystallized
from CHCl3/MeOH to afford 28.
2.3.3 | Synthesis of carboxylated com-
pounds 19‐27
A suitable acetate compound (1.40 mmol) was dissolved in
CH2Cl2/MeOH (1:1 v/v, 100 mL) and 8M NaOH (15‐30 mL)
was added. The mixture was kept at room temperature and
magnetic stirring for 24 hours. Afterwards, the organic sol-
vents were evaporated under reduced pressure, and water
(30 mL) was added. The aqueous phase was acidified with
5M HCl solution resulting in the formation of a precipitate
that was collected by filtration under reduced pressure,
washed with water, and crystallized from MeOH, to pro-
vide a carboxylated compound (20‐27).
2‐[(9‐Oxo‐9H‐xanthen‐1‐yl)oxy]acetic acid (19): Yield
1
(93%, white solid). m.p. 139°C‐141°C (MeOH); H NMR,
13C NMR, and IR data are shown in Supporting
Information.
(R)‐N‐(2‐Hydroxy‐1‐phenylethyl)‐2‐[(9‐oxo‐9H‐
xanthen‐1‐yl)oxy]acetamide (28): Yield (86%, white solid).
25°C
2,2′‐[(9‐Oxo‐9H‐xanthene‐1,3‐diyl)bis(oxy)]diacetic
acid (20): Yield (94%, white solid). m.p. 212°C‐214°C
m.p. 168°C‐170°C (CHCl3:MeOH); [α]D
−219.0
(c = 0.32 × 10−3 g mL−1 in CHCl3); H NMR, 13C NMR,
IR, and HRMS data are shown in Supporting Informa-
tion; e.r. >99% (LC; Column: (S,S)‐Whelk‐O1
(25 cm × 4.6 mm I.D., 5‐μm particle size), mobile phase:
MeOH/CH3CN (50:50 v:v), 1.0 mL min−1, λmax 254 nm,
k1: 0.63, k2: 0.85). The structure of compound 28 was
determined by X‐ray crystallography (Section 2.4).
1
1
(MeOH); H NMR, 13C NMR, and IR data are shown in
Supporting Information.
2,2′,2″‐[(9‐Oxo‐9H‐xanthene‐1,3,6‐triyl)tris(oxy)]
triacetic acid (21): Yield (89%, white solid). m.p. 225°C‐
226°C (MeOH); 1H NMR, 13C NMR, and IR data are
shown in Supporting Information.
2,2′,2″‐[(9‐Oxo‐9H‐xanthene‐1,3,8‐triyl)tris(oxy)]
triacetic acid (22): Yield (95%, white solid). m.p. 219°C‐
221°C (MeOH); 1H NMR, 13C NMR, and IR data are
shown in Supporting Information.
Starting from polycarboxylated compounds
Compounds 29‐39 were prepared from suitable carboxyl-
ated compounds (20‐27) using the same method as
described for 19, increasing the amount of TEA, TBTU,
and appropriate chiral reagent according to the number
of carboxylic groups of the chemical substrates (20‐27)
and the stoichiometry of the reaction.
2,2′,2″,2‴‐[(9‐Oxo‐9H‐xanthene‐1,3,6,8‐tetrayl)tetrakis
(oxy)]tetraacetic acid (23): Yield (88%, white solid). m.p.
1
>310°C (MeOH); H NMR, 13C NMR, and IR data are
shown in Supporting Information.
2,2′‐[(9‐Oxo‐9H‐xanthene‐3,6‐diyl)bis(oxy)]diacetic
acid (24): Yield (88%, yellow solid). m.p. 226°C‐228°C
2,2′‐{[9‐Oxo‐9H‐xanthene‐1,3‐diyl)bis(oxy)]bis[N‐((R)‐
2‐hydroxy‐1 phenylethyl]acetamide]} (29): Yield (85%,
1
25°C
(MeOH); H NMR, 13C NMR, and IR data are shown in
white solid). m.p.: 154°C‐156°C (CHCl3:MeOH); [α]D
1
Supporting Information.
−300.0 (c = 0.1 × 10−3 g mL−1 in CHCl3); H NMR, 13C
2,2′‐[(9‐Oxo‐9H‐xanthene‐3,4‐diyl)bis(oxy)]diacetic
acid (25): Yield (81%, white solid). m.p. >310°C (MeOH);
1H NMR, 13C NMR, and IR data are shown in Supporting
Information.
NMR, IR, and HRMS data are shown in Supporting Infor-
mation; e.r. >99% (LC; Column: (S,S)‐Whelk‐O1
(25 cm × 4.6 mm I.D., 5‐μm particle size), mobile phase:
MeOH/CH3CN (50:50 v:v), 1.0 mL min−1, λmax 254 nm,
k1: 0.61, k2: 1.33).
2,2′,2″‐{[9‐Oxo‐9H‐xanthene‐1,3,6‐triyl)tris(oxy)]tris
[N‐(R)‐2‐hydroxy‐1‐phenylethyl]acetamide} (30): Yield
(88%, white solid). m.p.: 163°C‐165°C (CHCl3:MeOH);
2,2′‐[(Carbonylbis(4,1‐phenylene))bis(oxy)]diacetic
acid (26): Yield (88%, white solid). m.p. 234°C‐236°C
1
(MeOH); H NMR, 13C NMR, and IR data are shown in
Supporting Information.

6
PHYO ET AL.
25°C
−285.0 (c = 0.21 × 10−3 g mL−1 in CHCl3); H
white solid). m.p. 177°C‐179°C (CHCl3:MeOH); [α]D
25°C
1
[α]D
NMR, 13C NMR, IR, and HRMS data are shown in
Supporting Information; e.r. >99% (LC; Column: (S,S)‐
Whelk‐O1 (25 cm × 4.6 mm I.D., 5‐μm particle size),
−455.0 (c = 0.088 × 10−3 g mL−1 in CHCl3); H NMR,
1
13C NMR, IR, and HRMS data are shown in Supporting
Information; e.r. >99% (LC; Column: (S,S)‐Whelk‐O1
(25 cm × 4.6 mm I.D., 5‐μm particle size), mobile phase:
MeOH/CH3CN (50:50 v:v), 1.0 mL min−1, λmax 254 nm,
k1: 0.72, k2: 1.77).
mobile phase: MeOH/CH3CN (50:50 v:v), 1.0 mL min−1
λmax 254 nm, k1: 0.63, k2: 1.90).
,
2,2′,2″‐{[9‐Oxo‐9H‐xanthene‐1,3,8‐triyl)tris(oxy)]tris
[N‐(R)‐2‐hydroxy‐1‐phenylethyl]acetamide} (31): Yield
(90%, white solid). m.p. 133°C‐136°C (CHCl3:MeOH);
2,2′‐{[(9‐Oxo‐9H‐xanthene‐3,4‐diyl)bis(oxy)]bis[N‐(R)‐
2‐hydroxy‐1‐phenylethyl]acetamide} (37): Yield (57%,
−300.0 (c = 0.1 × 10−3 g mL−1 in CHCl3); H
white solid). m.p. 202°C‐204°C (CHCl3:MeOH); [α]D
25°C
1
25°C
[α]D
NMR, 13C NMR, IR, and HRMS data are shown in
Supporting Information; e.r. >99% (LC; Column: (S,S)‐
Whelk‐O1 (25 cm × 4.6 mm I.D., 5‐μm particle size),
−300.0 (c = 0.1 × 10−3 g mL−1 in CHCl3); H NMR, 13C
1
NMR, IR, and HRMS data are shown in Supporting Infor-
mation; e.r. >99% (LC; Column: (S,S)‐Whelk‐O1
(25 cm × 4.6 mm I.D., 5‐μm particle size), mobile phase:
MeOH/CH3CN (50:50 v:v), 1.0 mL min−1, λmax 254 nm,
k1: 0.53, k2: 1.21).
mobile phase: MeOH/CH3CN (50:50 v:v), 1.0 mL min−1
λmax 254 nm, k1: 0.60, k2: 2.49).
,
2,2′,2″,2‴‐{[(9‐Oxo‐9H‐xanthene‐1,3,6,8‐tetrayl)
tetrakis(oxy)]tetrakis[N‐(R)‐2‐hydroxy‐1‐phenylethyl]
2,2′‐{[(Carbonylbis(4,1‐phenylene)bis(oxy)]bis[N‐(R)‐
2‐hydroxy‐1‐phenylethyl]acetamide} (38): Yield (86%,
acetamide} (32): Yield (78%, white solid). m.p. >350°
C (CHCl3:MeOH); [α]D25°C −308.0 (c = 0.13 × 10−3 g mL−1
white solid). m.p. 173°C‐175°C (CHCl3:MeOH); [α]D
25°C
1
1
in CHCl3); H NMR, 13C NMR, IR, and HRMS data are
−238.0 (c = 0.21 × 10−3 g mL−1 in CHCl3); H NMR,
shown in Supporting Information; e.r. > 99% (LC; Col-
umn: (S,S)‐Whelk‐O1 (25 cm × 4.6 mm I.D., 5‐μm par-
ticle size), mobile phase: MeOH/CH3CN (50:50 v:v),
1.0 mL min−1, λmax 254 nm, k1: 0.42, k2: 0.62).
13C NMR, IR, and HRMS data are shown in Supporting
Information; e.r. >99% (LC; Column: (S,S)‐Whelk‐O1
(25 cm × 4.6 mm I.D., 5‐μm particle size), mobile phase:
MeOH/CH3CN (50:50 v:v), 1.0 mL min−1, λmax 254 nm,
k1: 0.43, k2: 0.77).
2,2′,2″,2‴‐{[(Carbonylbis (benzene‐4,1,3‐triyl)tetrakis
(oxy)]tetrakis[N‐(R)‐2‐hydroxy‐1‐phenylethyl]acetamide}
(39): Yield (79%, white solid). m.p. >350°C (CHCl3:
2,2′‐{[(9‐Oxo‐9H‐xanthene‐3,6‐diyl)bis(oxy)]bis[N‐(S)‐
2‐hydroxy‐1‐phenylethyl]acetamide} (33): Yield (80%,
25°
white solid). m.p. 178°C‐180°C (CHCl3:MeOH); [α]D
+455.0 (c = 0.088 × 10−3 g mL−1 in CHCl3); H NMR,
C
1
13C NMR, IR, and HRMS data are shown in Supporting
Information; e.r. >99% (LC; Column: (S,S)‐Whelk‐O1
(25 cm × 4.6 mm I.D., 5‐μm particle size), mobile phase:
MeOH/CH3CN (50:50 v:v), 1.0 mL min−1, λmax 254 nm,
k1: 0.72, k2: 1.77).
MeOH); [α]D
−333.0 (c = 0.12 × 10−3 g mL−1 in
25°C
CHCl3); 1H NMR, 13C NMR, IR, and HRMS data are
shown in Supporting Information; e.r. >99% (LC; Col-
umn: (S,S)‐Whelk‐O1 (25 cm × 4.6 mm I.D., 5‐μm parti-
cle size), mobile phase: MeOH/CH3CN (50:50 v:v),
1.0 mL min−1, λmax 254 nm, k1: 0.39, k2: 0.62).
2,2′‐{[(9‐Oxo‐9H‐xanthene‐3,6‐diyl)bis(oxy)]bis[N‐(S)‐
1‐hydroxy‐4‐methylpentan‐2‐yl]acetamide} (34): Yield
(80%, white solid). m.p. 228°C‐230°C (CHCl3:MeOH);
2.3.5 | Synthesis and covalent linkage of
silylated selectors onto the silica gel
25°C
1
[α]D
+400.0 (c = 0.12 × 10−3 g mL−1 in CH3CN); H
NMR, 13C NMR, IR, and HRMS data are shown in
Supporting Information; e.r. >99% (LC; Column: Lux
celullose‐2 (15 cm × 4.6 mm I.D., 3‐μm particle size),
mobile phase: 100% EtOH, 0.5 mL min−1, λmax 254 nm,
k1: 1.34, k2: 2.90).
Starting from a selector with one chiral unit
The chiral compound 28 (0.51 mmol) was dissolved in
anhydrous toluene (20 mL), and TEA (108 μL, 1.51 mmol)
and
3‐(triethoxysilyl)propylisocyanate
(200
μL,
2,2′‐{[(9‐Oxo‐9H‐xanthene‐3,6‐diyl)bis(oxy)]bis[N‐(S)‐
1‐hydroxy‐3‐methylbutan‐2‐yl]acetamide} (35): Yield
(80%, white solid). m.p. 236°C‐238°C (CHCl3:MeOH); [α]
1.51 mmol) were added. The mixture was refluxed for
72 hours. The solvent was evaporated under reduced
pressure, and the crude product was dissolved in anhy-
drous toluene (30 mL). Silica gel Nucleosil (800 mg,
100 Å‐5 μm, Macherey‐Nagel) previously dried, in a des-
iccator under vacuum with phosphorus pentoxide for
24 hours, was suspended in the same solvent (30 mL).
Following, the crude product solution was added. The
reaction mixture was gently stirred at reflux for 72 hours.
The modified silica was filtered and washed successively
with toluene, MeOH, acetone, ethyl acetate,
25°C
1
+417.0 (c = 0.12 × 10−3 g mL−1 in CH3CN); H
D
NMR, 13C NMR, IR, and HRMS data are shown in
Supporting Information; e.r. >99% (LC; Column: Lux
celullose‐2 (15 cm × 4.6 mm I.D., 3‐μm particle size),
mobile phase: 100% EtOH, 0.5 mL min−1, λmax 254 nm,
k1: 1.55, k2: 2.89).
2,2′‐{[(9‐Oxo‐9H‐xanthene‐3,6‐diyl)bis(oxy)]bis[N‐(R)‐
2‐hydroxy‐1‐phenylethyl]acetamide} (36): Yield (85%,

PHYO ET AL.
7
TABLE 1 Elemental analysis and chiral selector loadings for
CSPs 1‐12
dichloromethane, and n‐hexane (100 mL). The bonded
phase was dried in a desiccator under vacuum for
24 hours, affording the CSP.
Elemental analysis
% selector loading % selector loading
Starting from a selector with more than one chiral
unit
(based on C), μ
%C %N %H mol m−2
(based on N), μ
mol m−2
The silylation reaction of selectors with more than one
chiral unit (29‐39) was performed using the same condi-
tions as described for selector 28, increasing the amount
of TEA and 3‐(triethoxysilyl)propylisocyanate, according
to the number of chiral units of the selector (29‐39) and
the stoichiometry of the reaction. In some cases, anhy-
drous CH3CN (34‐36, 39) or anhydrous CH3CN with
few drops of DMSO (29, 33) or anhydrous toluene with
few drops of THF and DMSO (30) were used due to the
low solubility of selectors. The covalent linkage of
silylated selectors onto the silica gel was performed using
the same method as described for 28. Elemental analyses
were performed to compare the extent of covalent bind-
ing of each chiral selector to silica. The chiral selector
loadings (μmol/m2) were determined from the elemental
analyses of carbon and nitrogen (Table 1).
CSP 18.21 2.91 1.99 2.32
1
6.94
1.75
1.77
0.59
6.72
7.59
2.21
18.7
0.36
0.82
1.71
0.35
CSP 13.49 2.21 1.99 1.03
2
CSP 13.66 2.26 2.13 1.22
3
CSP 7.58 0.98 1.29 0.61
4
CSP 18.84 4.21 2.91 1.67
5
CSP 21.63 4.37 2.44 2.09
6
CSP 13.11 2.55 2.02 1.00
7
CSP 23.66 5.95 3.91 1.87
8
CSP 6.15 0.91 1.21 0.30
9
2.3.6 | LC column packing
CSP 14.06 2.19 3.06 0.67
10
Each packing material (approximately 1.0 g) was sieved,
slurried in Hex: 2‐PrOH (50:50 v/v) (50 mL), and soni-
cated for 3 minutes. Then the suspension was poured into
the chamber of a column packer and packed into a
stainless‐steel column (150 × 2.1 mm I.D.) using Hex: 2‐
PrOH (90:10 v/v) as a packing solvent, under a pressure
no more than 6000 psi.
CSP 14.81 2.19 2.19 1.18
11
CSP 9.13 1.15 1.33 0.38
12
with isotropic displacement parameters. The flack x
parameter was refined with SHELXL‐97 by means of
TWIN and BASF to yield 0.097(3). The refinement con-
2.4 | X‐ray crystallography
A single crystal of (R)‐N‐(2‐hydroxy‐1‐phenylethyl)‐2‐[(9‐
oxo‐9H‐xanthen‐1‐yl)oxy]acetamide (28) was mounted
on a cryoloop using paratone. X‐ray diffraction data were
collected at 290 K with a Gemini PX Ultra equipped with
CuKα radiation (λ = 1.54184 Å). The structure was solved
by direct methods using SHELXS‐97 and refined with
SHELXL‐97.34 Crystal was orthorhombic, space group
P212121, cell volume 3820.8(3) Å3, and unit cell
dimensions a = 8.3606(3) Å, b = 8.9346(3) Å, and
c = 51.149(3) Å (uncertainties in parentheses). There
were two molecules in the asymmetric unit, one of them
with an oxygen atom with alternate conformation. Non‐
hydrogen atoms were refined anisotropically. Hydrogen
atoms were either placed at their idealized positions using
appropriate HFIX instructions in SHELXL and included
in subsequent refinement cycles or were directly found
from difference Fourier maps and were refined freely
verged to
R (all data) = 8.44% and wR2 (all
data) = 12.14%.
Full details of the data collection and refinement and
tables of atomic coordinates, bond lengths and angles,
and torsion angles have been deposited with the Cam-
bridge Crystallographic Data Centre (CCDC 1908277).
2.5 | Chromatography
HPLC grade ethanol (EtOH), 2‐propanol (2‐PrOH),
MeOH, CH3CN, and n‐hexane (Hex) were purchased
from Sigma‐Aldrich Co (St Louis, Missouri). Ultrapure
water was produced by a Millipore Milli‐Q system
(Millipore, Bedford, Massachusetts). Commercial chiral
analytes used to evaluate the CSPs, trifluoroacetic acid

8
PHYO ET AL.
(TFA), and TEA were obtained from Sigma‐Aldrich (St
Louis, Missouri) or Merck (Darmstadt, Germany). The
“in‐house” chiral analytes were synthesized previously
by our group.35,36 Working solutions of all chiral analytes
were prepared using HPLC grade EtOH at a concentra-
tion of 10 μg mL−1, and the injection volume was
10 μL. The HPLC system comprised a Jasco model 880‐
PU Intelligent HPLC pump (JASCO Corporation, Tokyo,
Japan), equipped with a 7125 injector (Rheodyne LCC,
Rohnert Park, California) fitted with a 20‐μL loop, a
JASCO model 880‐30 solvent mixer involving an 875‐UV
intelligent UV‐vis detector. The data were handled on
ChromNAC Chromatography Data System (version
1.19.1) from JASCO Corporation (Tokyo, Japan). The
mobile phases were prepared in a volume/volume ratio
and degassed in an ultrasonic bath for 15 minutes before
use. The chromatographic analyses were carried out in
3 | RESULTS AND DISCUSSION
Our previous results of enantioselectivity obtained with
recent CSPs based on CDXs,14 the structural attributes
of those small molecules for suitability as chiral selectors,
and the high enantioselectivity observed for a series of
CDXs in diverse CSPs2,3,23-25 were crucial to pursue this
approach and explore other strategies with CDXs as chi-
ral selectors. Therefore, we report herein the preparation
and evaluation of the LC enantioresolution behavior of
twelve new CSPs (CSPs 1‐12) (Figures 1 and 2).
The synthetic routes for preparation of the new CSPs
are shown in Schemes S1 to S4. Nine different hydroxyl-
ated small molecules (1‐9) were selected as a basis for
molecular modifications. The hydroxy‐9H‐xanthen‐9‐
ones 1‐5 were obtained through the condensation of
appropriate building blocks in the presence of Lewis acid
ZnCl2 (1)31 or P2O5/MeSO3H (Eaton's reagent) (2‐5)27-
isocratic mode at 22
2°C, in triplicate, at a flow rate
29,32
of 0.2 mL min−1. The UV detection was performed at a
wavelength of 254 nm. The column void time was consid-
ered to be equal to the peak of the solvent front and was
taken from each particular run.
(Scheme S1). A dehydrative cyclization of 2,2′,4,4′‐
tetrahydroxybenzophenone (9) in a furnace at 200°C
afforded the 3,6‐dihydroxy‐9H‐xanthen‐9‐one (6),30 in a
very high yield (99%) (Scheme S2). The 3,4‐dihydroxy‐
9H‐xanthen‐9‐one (7) was synthetized by a classical intra-
molecular acylation of a benzophenone intermediate and
further demethylation31 (Scheme S3). The commercially
available 2,2′,4,4′‐tetrahydroxybenzophenone (9) and
4,4′‐dihydroxybenzophenone (8) were also used as build-
ing blocks for preparation of new CSPs (Scheme S4). The
hydroxylated small molecules (1‐9) were further oxidized
to afford acetate intermediates (10‐18) and then carboxyl-
ated derivatives (19‐27). The synthetic procedure to
obtain the acetate intermediates (10‐18) involved a
Williamson ether synthesis using an alkyl halide contain-
ing an ester group (BrCH2COOCH3). Then, resorting to
an alkaline hydrolysis of the respective esters, the carbox-
ylated derivatives (19‐27) were obtained. Compounds
comprising a carboxylic acid, such as carboxyxanthones,
have already proved to be suitable molecular scaffolds
for synthesis of analogues and derivatives, including
chiral compounds.41 The chiral selectors (28‐39) were
synthetized by coupling the carboxylated derivatives (19‐
27) with commercially available enantiomerically pure
amino alcohols at room temperature. The synthetic strat-
egy was based on previous works using TBTU as a cou-
pling reagent, in the presence of a catalytic amount of
TEA in THF as solvent,35,36 providing good yields. The
structures of the chiral selectors 28‐39 and all intermedi-
ates were successfully established, and for hydroxy‐9H‐
xanthen‐9‐ones (1‐7), all the data were in accordance
with the literature.28,30,42-45 The enantiomeric ratio (e.r.)
of all chiral selectors was determined by LC46 using two
commercial chiral columns, (S,S)‐Whelk‐O1 and Lux
Celullose‐2, and was higher than 99%. The next step
2.6 | Computational
The structures of the synthesized selectors were drawn
in Chemdraw and minimized in Chem3D 16.0, and
both enantiomers of analytes were drawn and mini-
mized using an Austin Model 1 (AM1) semi‐empirical
quantum mechanics force field.37 The calculation was
finished when the gradient between any two successive
steps in the geometry search was less than 10−-
1
kcal mol−1 Å−2 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.38
Docking simulations between the synthesized chiral
selectors and the analytes were undertaken in
AutoDock Vina (Molecular Graphics Lab, La Jolla, Cal-
ifornia).39 AutoDock Vina considered the target confor-
mation 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 8, and grid boxes with
dimensions 16.0, 15.0, and 13.0 engulfing the selectors
were built. PyMol v1.3 (Schrödinger, New York, New
York)40 was used to visualize the conformations and
interactions as well as the graphical representations.

PHYO ET AL.
9
was the synthesis of silylated derivatives, by reaction with
3‐(triethoxysilyl)propylisocyanate,47,48 to allow the fur-
ther covalent linkage of the chiral selectors to a chro-
matographic support (silica gel, Nucleosil‐100‐5),
affording CSPs 1‐12 (Figures 1 and 2). Therefore, after
the synthesis of suitable chemical substrates, the carbox-
ylated derivatives 19‐27, all CSPs were prepared through
simple three steps: amidation, silylation, and bonding to
silica gel.
The majority of the new CSPs was covalently bonded
to silica gel within appropriate levels (0.61‐2.32 μmol m−2
based on carbon), as shown in Table 1, when compared
with previous CSPs.14 It was found that for some CSPs
(1, 5‐8) the percent of selector loading based on nitrogen
analysis was higher than the percent based on carbon.
This may be because often the ethoxy groups of the silox-
anes are not fully hydrolyzed. The silica gels in CSPs 9
and 12 contain about 0.30 μmol m−2. It is assumed that
the lower amount of loaded chiral selectors in those CSPs
was caused by the low solubility of their silylated chiral
selectors in toluene. Even though we conducted addi-
tional experiments by testing other solvents such as
THF, CH3CN, and DMSO, the solubility remained low.
The columns capacity for enantioresolution is highly
dependent on coverage density of CSP; thus, the low
chiral selector loading for CSPs 9 and 12 will certainly
influence their chromatographic performance.
All the CSPs were packed in LC columns with
150 × 2.1 mm I.D. This internal diameter was chosen
because, previously, we demonstrated that columns con-
taining the same chiral selector based on a CDX and only
differing in the internal diameters, specifically 4.6 and
2.1 mm, showed similar enantiomeric performance.14 In
fact, by reducing the internal diameter of the LC col-
umns, two main advantages can be pointed out such as
a significant reduction of the material to be synthetized
and packed and a considerable decrease of the solvent
consumption during the packing and chromatographic
analysis. The ecological, practical, and economic benefits
are undeniable.49,50
Initially, several commercial and “in house” chiral
analytes (A1‐A38) were selected (Figure S1) to evaluate
the LC enantioselective performance of CSPs 1‐12. Addi-
tionally, enantiomeric mixtures of the chiral selectors
(28‐39) used for its preparation were included. The chro-
matographic analyses were performed under elution in
the normal phase mode, using different proportions of
Hex and EtOH or 2‐PrOH as mobile phases. Baseline
enantioseparation was only obtained for analytes A24
and A30. For the other tested analytes, short retention
factors (analysis time generally lower than 3 min) and
the absence or poor resolutions were found for all the
combinations of mobile phases evaluated. Table 2 shows
the overall best results. For the enantioseparated
analytes, the elution order was determined by injecting
each enantiomer separately.
Taking into account that in our previous work, the
best enantioselectivity was achieved with two
xanthone‐(R)‐(−)‐α‐phenylglycinol‐derived CSPs,14 we
first developed the CSP 1 comprising a chiral selector
based on a xanthone derivative with a chiral moiety
derived from (R)‐(−)‐α‐phenylglycinol (52) in position 1
of the xanthone scaffold (28) (Figure 1). The strategy
was to use the same chiral moiety but changing its posi-
tion in the xanthone scaffold. It was found that CSP 1
showed good enantioselectivity, α values of 2.02 and
2.04, and resolution, Rs values of 2.44 and 2.13, for A24
and A30, respectively, using Hex: EtOH (90:10 v/v) as
mobile phase. Moreover, in this mobile phase, the theo-
retical plate numbers (N) for the first and second eluted
enantiomers were 242/349 and 206/509 for A24 and
A30, respectively.
The next step was the introduction of an extra chiral
moiety affording CSP 2 (Figure 1). The aim was to
attempt more versatility and better enantioresolution
by increasing the number of chiral moieties for
enantiorecognition. Nevertheless, CSP 2 comprising two
chiral moieties in positions 3 and 6 of the xanthone scaf-
fold (symmetrical structure) presented similar chromato-
graphic performance to CSP 1, showing only a slight
increase of retention as well as enantioselectivity and res-
olution for A24 and A30 (Table 2). Nevertheless, an
increasing of the efficiency was observed, being the N
values for the first and second eluted enantiomers of
A24 and A30 744/832 and 636/788, respectively, in Hex:
EtOH (80:20 v/v).
For comparison purposes, in addition to the (R)‐(−)‐α‐
phenylglycinol (52), the amino alcohols (S)‐(+)‐leucinol
(53) and (S)‐(+)‐valinol (54) were chosen to obtain CSPs
3 and 4, respectively (Figure 1). Surprisingly, both CSPs
showed similar enantioselectivity and resolution to those
of CSP 2 for the enantioseparated analytes (A24 and A30),
as shown in Table 2. For CSP 3, the N values for the first
and second eluted enantiomers were 371/295 and
266/270 for A24 and A30, respectively, using Hex: EtOH
(80:20 v/v) as mobile phase; while for CSP 4, the N values
were 291/344 and 528/655.
An advantage of CSPs comprising small molecules as
selectors is the possibility of inversion of elution order
by preparation of two different CSPs switching the config-
uration of the chiral selector.11 Previously, we have con-
firmed the inversion of elution order for a selector based
on a xanthone derivative comprising one chiral aromatic
moiety.14 To verify if with selectors comprising two chiral
aromatic moieties the inversion of elution order would
occur, CSP 5 was prepared. The selector of this CSP is

10
PHYO ET AL.
TABLE 2 Chromatographic enantioseparation of analytes A24 and A30 on CSPs 1‐12
A24
A30
k1
Mobile Phase (v/v)
k1
α
Rs
2.44
1.15
1.66
α
Rs
CSP 1
CSP 2
Hex/EtOH: 90/10
Hex/EtOH: 80/20
Hex/2‐PrOH: 80/20
1.50
0.61
0.64
2.02
1.69
2.26
1.84
0.93
0.79
2.04
1.73
2.21
2.13
1.21
1.52
Hex/EtOH: 80/20
Hex/EtOH: 70/30
Hex/2‐PrOH: 80/20
Hex/2‐PrOH: 70/30
2.40
1.63
3.22
2.03
1.92
1.74
2.51
2.19
3.50
2.61
3.38
2.56
2.39
1.53
3.00
1.75
2.13
2.03
3.02
2.83
3.81
3.11
3.71
3.13
CSP 3
CSP 4
Hex/EtOH: 90/10
Hex/EtOH: 80/20
Hex/EtOH: 70/30
Hex/2‐PrOH: 80/20
Hex/2‐PrOH: 70/30
2.36
1.58
0.99
1.91
1.36
2.00
1.86
1.59
2.31
1.49
1.95
1.88
<1.00
2.26
3.26
1.83
0.99
2.05
1.21
1.74
1.63
1.62
1.93
1.87
1.67
1.36
1.06
1.83
1.29
1.15
Hex/EtOH: 90/10
Hex/EtOH: 80/20
Hex/EtOH: 70/30
Hex/2‐PrOH: 80/20
Hex/2‐PrOH: 70/30
2.24
1.02
0.86
0.95
0.61
2.16
1.89
1.46
2.00
1.80
3.20
1.66
1.17
1.95
1.46
2.59
1.68
0.71
1.16
0.91
2.22
2.09
1.93
1.91
1.52
3.73
2.12
2.08
1.70
1.00
CSP 5
CSP 6
Hex/EtOH: 80/20
Hex/EtOH: 70/30
Hex/2‐PrOH: 80/20
Hex/2‐PrOH: 70/30
2.09
1.44
1.82
1.73
2.29
2.16
2.29
2.08
1.33
1.11
1.09
1.08
1.75
1.06
3.28
1.10
3.33
3.48
2.24
3.01
1.74
1.64
1.76
1.59
Hex/EtOH: 90/10
Hex/EtOH: 80/20
Hex/2‐PrOH: 80/20
0.55
0.34
0.22
2.25
1.27
2.48
<1.00
<1.00
<1.00
0.99
0.39
0.26
1.00
1.00
1.00
ND
ND
ND
CSP 7
CSP 8
CSP 9
Hex/EtOH: 80/20
Hex/2‐PrOH: 80/20
2.13
2.01
1.61
1.81
1.61
1.48
2.08
1.65
2.37
2.98
2.91
2.54
Hex/EtOH: 90/10
Hex/EtOH: 80/20
2.63
1.43
2.43
2.05
<1.00
<1.00
2.96
1.46
2.57
1.00
<1.00
<1.00
Hex/EtOH: 90/10
Hex/EtOH: 80/20
Hex/EtOH: 70/30
1.62
0.75
0.32
2.28
1.86
1.56
1.68
1.32
<1.00
2.25
0.96
0.38
1.00
1.00
1.00
ND
ND
ND
CSP 10
CSP 11
Hex/EtOH: 90/10
Hex/EtOH: 80/20
Hex/2‐PrOH: 80/20
2.55
1.43
1.10
2.41
2.02
2.30
2.02
1.36
1.31
3.15
1.29
1.08
2.05
2.12
2.55
1.59
1.22
1.56
Hex/EtOH: 90/10
Hex/EtOH: 80/20
Hex/EtOH: 70/30
Hex/2‐PrOH: 80/20
Hex/2‐PrOH: 70/30
1.68
1.14
0.69
1.21
0.75
2.07
1.84
1.67
2.57
1.77
2.15
1.56
<1.00
1.69
1.68
0.99
0.59
0.78
0.55
2.13
2.17
1.98
2.28
1.25
2.24
1.88
1.24
1.27
1.38
1.11
CSP 12
Hex/EtOH: 90/10
Hex/EtOH: 80/20
Hex/EtOH: 70/30
Hex/2‐PrOH: 80/20
Hex/2‐PrOH: 70/30
3.00
1.32
0.70
1.17
1.02
2.20
1.87
1.71
2.07
2.13
3.56
2.24
1.30
1.57
1.75
3.70
1.41
0.70
1.16
0.89
1.96
1.68
1.56
2.00
2.09
3.04
1.69
1.15
1.95
1.72
Note. ND, not determined. Detection at 254 nm; flow rate, 0.2 mL min−1. Size of all columns: 150 mm length, 2.1 mm I.D.
the enantiomeric pair of CSP 2 selector. In this study,
with more structurally complex xanthone selectors, it
was found that the inversion of elution order did not
occur for the enantioseparated analytes (A24 and A30).
The results obtained in the subsequent docking studies
justify why.
Two more CSPs comprising two chiral moieties
derived from (R)‐(−)‐α‐phenylglycinol in different

PHYO ET AL.
11
positions in the xanthone scaffold, specifically positions 1
and 3 (CSP 6) and positions 3 and 4 (CSP 7) (Figure 1)
results for both analytes A24 and A30, as shown in
Table 2.
were developed. It was found that CSP
7
In order to understand the role of the rigidity of the
quasi‐planar structure of the xanthone scaffold in the
enantioresolution properties of these CSPs and also
aiming to improve the enantioselectivity by increasing
the flexibility of the central aromatic core, CSPs 11 and
12 were prepared (Figure 2). These CSPs are derived from
benzophenone derivatives, one of the most common
intermediates for the synthesis of xanthones.51,52 It was
found that CSPs 11 and 12 afforded similar chromato-
graphic results to their xanthone‐based analogue CSPs,
specifically CSPs 5 and 10 (Table 2).
Regarding the influence of the organic modifier in
the mobile phase, it was found that, despite a slight
increase of enantioselectivity observed for Hex/2‐PrOH
combinations, shorter retention factors and higher reso-
lution were obtained using mixtures of Hex and EtOH.
For these reasons, the use of EtOH was a better choice.
Regarding the elution order, for the enantioseparated
analytes, the (R)‐enantiomer was the more retained in
all CSPs. In Figures 3 and 4, a set of selected
enantioseparated the same two analytes with equivalent
enantioselectivity and resolution, compared with CSP 2.
As example, for analyte A30, using Hex:EtOH (80:20 v/
v) as a mobile phase, the α and Rs values obtained on
CSP 2 were 2.13 and 3.81, while for CSP 7, those values
were 2.37 and 2.91, respectively. However, CSP 7 showed
lower efficiency, being the N values for the first and sec-
ond eluted enantiomers of A24 and A30 360/349 and
334/337, respectively, in Hex: EtOH (80:20 v/v). CSP 6
showed some enantioselectivity only for analyte A24,
however, with poor resolution (Rs < 1.00 in all tested
mobile phases).
The next step was to work with an increase of the
number of chiral moieties to three (CSPs 8 and 9) and
four (CSP 10) (Figure 1). It was found that both CSPs
comprising three chiral moieties presented less discrim-
ination ability. Nevertheless, CSP 10 comprising chiral
units in positions 1, 3, 6, and 8 of xanthone scaffold
(symmetrical structure) showed good chromatographic
FIGURE 3 Chromatograms for the enantioseparation of analyte A24 on different CSPs, at optimized chromatographic conditions

12
PHYO ET AL.
chromatograms for analytes A24 and A30 in different
CSPs are presented.
In order to understand the binding mechanism behind
the observed results of enantioresolution, docking studies
were performed using the selectors of CSPs 1‐12. The
structure of the chiral selector of CSP 1 (compound 28)
was determined by single crystal X‐Ray diffraction, and
its ortep view is shown in Figure 5.
The central pyranoid ring has a partial aromatic char-
acter, and as usual, the xanthone skeleton adopts a kind
of flattened boat conformation.53 The absolute configura-
tion of the chiral center of the compound was confirmed
to be (R) by X‐Ray analysis.
Values of docking scores are presented on Table 3.
The lower the docking score, the more stable the
analyte‐selector complex is. Docking scores between
−3.4 and − 6.5 kcal mol−1 and energy differences from
−0.1 to −0.9 kcalmol−1 were obtained for A24; and
docking scores between −4.0 and − 6.1 kcal mol−1
and energy differences from 0 to −0.4 kcal mol−1 were
obtained for A30.
FIGURE 5 Ortep view of the crystal structure of chiral selector
28
and 10‐12, indicating that they have different affinities
for these selectors, which is in accordance with the chro-
matographic data (separations were achieved, α = 1.25 to
3.02). In contrast, the binding energies were equal for
The binding energies were different for the enantio-
mers of A24 in CSPs 1‐12 and A30 in CSPs 1‐5, 7, 8,
FIGURE 4 Chromatograms for the enantioseparation of analyte A30 on different CSPs, in optimized chromatographic conditions

PHYO ET AL.
13
TABLE 3 Docking scores and elution order of both enantiomers of analytes A24 and A30 on CSPs 1‐12
A24
A30
Docking Score,
kcal mol−1
Energy Difference,
kcal mol−1
Docking Score,
kcal mol−1
Energy Difference,
kcal mol−1
CSP 1 (X‐ray structure)
CSP 2
(S): −4.5
(R): −4.8
−0.3
−0.1
−0.1
−0.2
−0.5
−0.2
−0.1
−0.9
−0.2
−0.1
−0.1
−0.1
(S): −4.5
(R): −4.7
−0.2
(S): −5.4
(R): −5.5
(S): −5.2
(R): −5.4
−0.2
−0.3
−0.3
−0.2
0.0
CSP 3
(S): −5.0
(R): −5.1
(S): −4.6
(R): −4.9
CSP 4
(S): −5.5
(R): −5.7
(S): −5.1
(R): −5.4
CSP 5
(S): −5.1
(R): −5.6
(S): −5.0
(R): −5.3
CSP 6
(S): −5.5
(R): −5.7
(S): −5.2
(R): −5.2
CSP 7
(S): −3.4
(R): −3.5
(S): −4.0
(R): −4.4
−0.4
−0.1
0.0
CSP 8
(S): −5.0
(R): −5.9
(S): −4.1
(R): −4.2
CSP 9
(S): −6.3
(R): −6.5
(S): −6.1
(R): −6.1
CSP 10
CSP 11
CSP 12
(S): −6.0
(R): −6.1
(S): −5.6
(R): −5.7
−0.1
−0.2
−0.1
(S): −5.3
(R): −5.4
(S): −5.0
(R): −5.2
(S): −6.0
(R): −6.1
(S): −5.6
(R): −5.7
both enantiomers of A30 in CSP 6 (−5.2 kcal mol−1) and
CSP 9 (−6.1 kcal mol−1), indicating that they have identi-
cal affinity for these selectors, which is in agreement with
the experimental results (no separation was achieved,
α = 1.00). Docking analytes into selectors of CSPs 1‐12
revealed that the (R)‐enantiomers had higher affinity to
the target (lower docking scores) than the corresponding
(S)‐enantiomers, although the average docking score dif-
ference was small in some cases. Consequently, there
was 100% agreement between docking scores and experi-
mental results for enantioselectivity and elution order of
the enantiomers of the chiral analytes in all CSPs.
In order to understand the binding mechanism and
the chromatographic results, a visual inspection of the
binding conformations and established interactions were
performed.
Figure 6A illustrates a representative example of the
most stable docked conformations for enantiomers of
FIGURE 6 (A) Crystallographic
selector of CSP 1 and docked A30
enantiomers (top‐view of the docking
poses is represented on the bottom‐left
corner of the image); (B) A17 docking pose
of A17 enantiomers on crystallographic
selector of CSP 1. Selector of CSP 1 is
represented as grey sticks; (R) and (S)
enantiomers are represented as yellow and
pink sticks, respectively. Hydrogen
interactions and π‐stacking interactions
are represented in yellow broken lines and
yellow double arrow, respectively

14
PHYO ET AL.
analyte A30 complexed with the chiral selector of CSP 1.
The (R)‐enantiomer of A30 was more retained in the CSP
and, accordingly, presented lower docking score than the
(S)‐enantiomer. In fact, only the (R)‐enantiomer binds in
a conformation that allows the establishment of a hydro-
gen interaction with the ether group of the xanthone tri-
cyclic system of the selector. Furthermore, the tricyclic
ring system of (R)‐enantiomer establishes parallel π‐
stacking interactions with the xanthone scaffold of the
selector, while its antipoda establishes parallel‐displaced
π‐stacking interactions (Figure 6A). On the other hand,
regarding for example analyte A17, which have the same
chiral moiety as analyte A30 only differing in the nature,
number, and positions of the substituents on the xan-
thone scaffold (Figure S1), both enantiomers docked on
the identical docking pose with the tricyclic ring systems
superimposed and an RMSD of only 1.5 Å (Figure 6B). It
was found that A17 enantiomers, which were not sepa-
rated on CSP 1, only established π‐stacking interactions
with the selector of CSP 1.
It is also important to highlight that, through docking
studies, it was confirmed that the aromatic ring bonded to
the stereogenic center of the side chain of the selector is
not involved in interactions with analytes, as shown in
Figure 7A,B. The same occurs when the group is an alkyl
chain. This justifies the reason why CSPs 2 and 5 (derived
from phenylglycinol), CSP 3 (derived from leucinol), and
CSP 4 (derived from valinol) have similar chromato-
graphic behavior. It was found that with selectors of CSPs
comprising three chiral units (CSPs 8 and 9), the interac-
tions responsible for the enantioselectivity did not occur
successfully due to steric hindrance of the three bulky
chiral moieties preventing the establishment of parallel
π‐stacking interactions between the xanthone scaffolds
of analytes and selectors. Moreover, the available space
for the interactions between these two CSPs and the
enantiomers is smaller. For selectors of CSPs comprising
four bulky chiral moieties (CSPs 10 and 12), they are in
positions of the xanthone or benzophenone scaffold
allowing to establish the interactions essential for enan-
tiomeric discrimination (Figure 7C,D). Regarding CSP
10, for example, enantiomers of A30 docks on the same
side of the selector, but with the carbonyl group of the
xanthone scaffold pointing in opposite directions. There-
fore, the (R)‐enantiomer is able to form two hydrogen
interactions through the xanthone carbonyl and side
chain hydroxyl groups. The (S)‐enantiomer establishes
only one hydrogen interaction (Figure 7C). As far as
CSP 12 is concerned, the enantiomers of A30 also dock
on the same side of the selector. The (S)‐enantiomer of
A30 establishes three hydrogen interactions with the ben-
zophenone carbonyl group, amide, and hydroxyl groups
of the side chains of the selector. The (R)‐enantiomer of
A30 only establishes one hydrogen interaction by means
of the side chain hydroxyl group, but it establishes addi-
tional π‐stacking interactions (parallel and T‐shaped)
(Figure 7D).
Regardless the nature of the central aromatic core of
the selector, xanthone for CSP 10 or benzophenone for
CSP 12, both CSPs were able to establish parallel π‐
stacking interactions with (R)‐enantiomer of analyte
A30 that, in addition to other interactions, are
FIGURE 7 (A) Selector of CSP 2 and docked A30 enantiomers; (B) selector of CSP 5 and docked A30 enantiomers; (C) selector of CSP 10
and docked A30 enantiomers; (D) selector of CSP 12 and docked A30 enantiomers. Selectors of CSPs 2, 5, 10, and 12 are represented as grey
sticks; (R) and (S) enantiomers are represented as yellow and pink sticks, respectively. Hydrogen interactions and π‐stacking interactions are
represented in yellow broken lines and yellow double arrow, respectively

PHYO ET AL.
15
responsible for enantioselectivity that explains the similar
enantioresolution properties of these CSPs.
chromatographic performance of the new CSPs for
enantioseparation of a large library of chiral amines
should be further investigated.
To enlarge the number of enantiomeric separations, 24
more chiral analytes were selected, including structurally
diverse commercial compounds from different chemical
nature (neutral, acid, and basic). Moreover, the chromato-
graphic analysis was performed using several mobile
phases in the three elution modes (normal phase, polar
organic, and reversed‐phase elution conditions) for all
the tested analytes (including the enantiomeric mixtures
of the chiral selectors (28‐39) and analytes A1‐A38. Consid-
ering that the majority of the new CSPs presented similar
enantioselective performance, as they enantioseparated
the same two analytes (A24 and A30), CSP 2 was selected
to carried out further analysis since it showed the best effi-
ciency in the previous chromatographic evaluation. The
overall best results were summarized in Table 4.
Excellent enantioselectivity, α value of 5.08, and reso-
lution, Rs value of 3.13, were obtained for 4‐methoxy‐α‐
methylbenzylamine, in reversed‐phase mode, using
CH3CN/H2O/TEA: 80/20/0.01 as mobile phase. More-
over, enantioselectivity was also obtained for other
analytes, including the commercial amines N‐benzyl‐
1‐(1‐naphthyl)ethylamine and alanine‐2‐naphthylamide,
although with low resolution (Table 4). Therefore, the
4 | CONCLUSION
Twelve new CSPs were successfully prepared aiming to
obtain versatile and efficient CSPs as well as to explore
the role of some structural characteristics that could be
crucial for enantiorecognition. It was found that the num-
ber and position of the chiral units positioned in the xan-
thone scaffold of the chiral selectors can interfere with
the chiral recognition mechanism by steric hindrance.
Moreover, for the CSPs comprising equal numbers of chi-
ral units in the same positions, the nature of the substitu-
ent next to the stereogenic center was shown not to be
pivotal because either the CSPs having an aromatic ring
or an alkyl chain showed similar results. The central
aromatic core of the selector, either xanthone or benzo-
phenone scaffold, aligned with the xanthone tricyclic ring
of the analytes to maximize π‐stacking interactions, and
the hydrogen bonding interactions between the donor
and acceptor polar groups of analytes and selectors,
demonstrated to be essential for enantioselective recogni-
tion. The docking studies proved to be a meaningful
tool for the structural requirements necessary for the
effective elucidation of the intermolecular CSP‐analyte
interactions.
TABLE 4 Chromatographic data obtained on CSP 2 (not include
analytes A24 and A30)
Analyte
Mobile Phase (v/v) k1
α
Rs
The development of new CSPs for LC is a continuous
and challenging issue. This work is an important
contribution to improve the design of new selectors and
further preparation of more efficient CSPs based on xan-
thone and benzophenone derivatives possessing wider
applicability.
4‐Methoxy‐α‐
methylbenzylamine
CH3CN/H2O/TEA: 0.23 5.08 3.13
80/20/0.01
N‐Benzyl‐1‐(1‐
naphthyl)ethylamine
CH3CN/H2O/TEA: 0.82 1.44 <1.00
80/20/0.01
Alanine‐2‐
naphthylamide
Hex/EtOH/TEA: 90/ 3.28 1.36 <1.00
10/0.01
Enantiomeric mixture
of 28
CH3CN: 100
0.95 1.42 <1.00
ACKNOWLEDGMENTS
Enantiomeric mixture
of 35
CH3CN/MeOH: 80/ 0.40 1.33 <1.00
20
This work was supported by the Strategic Funding
UID/Multi/04423/2019 through national funds provided
by FCT—Foundation for Science and Technology and
European Regional Development Fund (ERDF), through
the COMPETE – Programa Operacional Factores de
Competitividade (POFC) program in the framework of
the program PT2020; the project PTDC/MAR‐BIO/4694/
2014 (reference POCI‐01‐0145‐FEDER‐016790 and 3599‐
PPCDT), co‐financed by COMPETE 2020, under
the PORTUGAL 2020 Partnership Agreement,
through the European Regional Development Fund
(ERDF), CHIRALBIOACTIVE‐PI‐3RL‐IINFACTS‐2019
financed by IINFACTS and QOPNA research project
(FCT UID/QUI/00062/2019), and Portuguese NMR
A5a
Hex/EtOH: 80/20
CH3CN: 100
3.40 1.28 1.00
1.38 1.78 <1.00
A17a
A19a
CH3CN/MeOH: 80/ 0.41 1.17 <1.00
20
A20a
A27a
A28a
CH3CN/MeOH: 80/ 0.42 1.10 <1.00
20
CH3CN/EtOH: 80/
20
0.38 1.39 <1.00
CH3CN/EtOH: 80/
20
0.37 1.39 <1.00
Note. Detection at 254 nm; flow rate, 0.2 mL min−1
aChemical structures shown in Figure S1.
.

16
PHYO ET AL.
12. Fernandes C, Phyo YZ, Silva AS, Tiritan ME, Kijjoa A, Pinto
MMM. Chiral stationary phases based on small molecules: an
update of the last 17 years. Sep Pur Rev. 2018;47(2):89‐123.
network. Y.Z. Phyo thanks the Erasmus Mundus Action 2
(Lotus Plus project) for a PhD scholarship.
13. Fernandes C, Tiritan ME, Pinto M. Small molecules as chro-
matographic tools for HPLC enantiomeric resolution: Pirkle‐
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ORCID
Maria Elizabeth Tiritan https://orcid.org/0000-0003-3320-
730X
Carla Fernandes https://orcid.org/0000-0003-0940-9163
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