New chiral stationary phases based on xanthone derivatives for liquid chromatography

Article abstract of DOI:10.1002/chir.22706

Six chiral derivatives of xanthones (CDXs) were covalently bonded to silica, yielding the corresponding xanthonic chiral stationary phases (XCSPs). The new XCSPs were packed into stainless-steel columns with 150 x 4.6 mm i.d. Moreover, the greening of the chromatographic analysis by reducing the internal diameter (150 x 2.1 mm i.d.) of the liquid chromatography (LC) columns was also investigated. The enantioselective capability of these phases was evaluated by LC using different chemical classes of chiral compounds, including several types of drugs. A library of CDXs was evaluated in order to explore the principle of reciprocity as well as the chiral self-recognition phenomenon. The separation of enantiomeric mixtures of CDXs was investigated under multimodal elution conditions. The XCSPs provided high specificity for the enantiomeric mixtures of CDXs evaluated mainly under normal-phase elution conditions. Furthermore, two XCSPs were prepared with both enantiomers of the same xanthonic selector in order to confirm the inversion order elution.

Full text of DOI:10.1002/chir.22706

Received: 6 January 2017
DOI: 10.1002/chir.22706
Revised: 27 February 2017
Accepted: 19 March 2017
R E G U L A R A R T I C L E
New chiral stationary phases based on xanthone derivatives for
liquid chromatography
Carla Fernandes1,2
| Maria Elizabeth Tiritan1,2,3
| Sara Cravo1,2 | Ye’ Zaw Phyo4 |
Anake Kijjoa2,4 | Artur M.S. Silva5 | Quezia B. Cass6 | Madalena M.M. Pinto1,2
1 Laboratório de Química Orgânica e
Abstract
Farmacêutica, Departamento de Ciências
Six chiral derivatives of xanthones (CDXs) were covalently bonded to silica,
Químicas, Faculdade de Farmácia, Porto,
Portugal
yielding the corresponding xanthonic chiral stationary phases (XCSPs). The new
XCSPs were packed into stainless‐steel columns with 150 x 4.6 mm i.d. Moreover,
the greening of the chromatographic analysis by reducing the internal diameter
(150 x 2.1 mm i.d.) of the liquid chromatography (LC) columns was also investi-
gated. The enantioselective capability of these phases was evaluated by LC using
different chemical classes of chiral compounds, including several types of drugs.
A library of CDXs was evaluated in order to explore the principle of reciprocity
as well as the chiral self‐recognition phenomenon. The separation of enantiomeric
mixtures of CDXs was investigated under multimodal elution conditions. The
XCSPs provided high specificity for the enantiomeric mixtures of CDXs evaluated
mainly under normal‐phase elution conditions. Furthermore, two XCSPs were pre-
pared with both enantiomers of the same xanthonic selector in order to confirm the
inversion order elution.
2 Interdisciplinary Centre of Marine and
Environmental Research (CIIMAR),
Matosinhos, Portugal
3 CESPU, Instituto de Investigação e
Formação Avançada em Ciências e
Tecnologias da Saúde (IINFACTS), Gandra,
PRD, Portugal
4 ICBAS‐Instituto de Ciências Biomédicas
Abel Salazar, Universidade do Porto, Porto,
Portugal
5 Departamento de Química & QOPNA,
Universidade de Aveiro, Aveiro, Portugal
6 Departamento de Química, Universidade
Federal de São Carlos, São Carlos, SP,
Brasil
Correspondence
KEYWORDS
Prof. C. Fernandes, Laboratório de Química
Orgânica e Farmacêutica, Departamento de
Ciências Químicas, Faculdade de Farmácia,
Rua de Jorge Viterbo Ferreira, 228, 4050‐
313 Porto, Portugal.
chiral derivatives of xanthones, chiral stationary phase, enantioselectivity, liquid chromatography
Email: cfernandes@ff.up.pt
Funding information
Coordenação de Aperfeiçoamento de Pessoal
de Nível Superior (CAPES/FCT), Grant/
Award Number: Project 214/08; Structured
Program of R&D&I INNOVMAR ‐ funded
by the Programme NORTE2020 under the
PORTUGAL 2020 Partnership Agreement,
through ERDF and FCT, Grant/Award
Number: NORTE‐01‐0145‐FEDER‐000035;
Research Line NOVELMAR; Research pro-
ject PTDC/MAR‐BIO/4694 /2014 funded by
FCT/MCTES, PIDDAC and ERDF through
the COMPETE (POFC) programme in the
framework of the programme PT2020
reference POCI‐01‐0145‐FEDER‐016790
3599‐PPCDT; Cooperativa de Ensino
Chirality. 2017;1–13.
wileyonlinelibrary.com/journal/chir
© 2017 Wiley Periodicals, Inc.
1

2
FERNANDES ET AL.
Superior Politécnico e Universitário
COXANT‐CESPU‐2016; Erasmus Mundus
Action 2, Grant/Award Number: PhD's
scholarship; QOPNA research project and
Portuguese NMR network, Grant/Award
Number: FCT UID/QUI/00062/2013
1 | INTRODUCTION
interactions through hydrogen bonding, dipole–dipole, π–π
interactions, and steric hindrance effects,32 similar to what
occurs with Pirkle‐type CSPs.33,34
Analytical and preparative enantiomer separations by
chromatography and related techniques play more than ever
a crucial role in the chemical industry and academic
research.1 Over the years, liquid chromatography (LC)
using chiral stationary phases (CSPs) has become a very
helpful and highly applicable method for preparative resolu-
tion of racemates,2 determination of the enantiomeric purity
in quality control,3 analysis of the stereochemistry of natural
compounds,4,5 monitoring enantiomeric reactions,6,7 and for
pharmacokinetic and environmental studies.8,9 LC can be
considered the method of choice in analytical laboratories
due to its high speed, sensitivity, and reproducibility. More-
over, a number of CSPs based on polysaccharide derivatives,
macrocyclic antibiotics, small chiral molecules, cyclodex-
trins, crown ethers, proteins, synthetic polymers, molecularly
imprinted polymers, among others, have been developed for
successful enantioseparations.10-18 However, in spite of a
large number of different types of CSPs described for enantio-
meric separation by LC, there is no universal CSP, i.e., one
CSP can only separate a limited number of chiral
compounds and, in many cases, the choice of CSP may
became a difficult task. Furthermore, even though there are
many CSPs commercially available, many chiral compounds
remain to be resolved. For this reason, the development of
new CSPs continues to be a field of great importance.
Herein we report the synthesis and evaluation of six new
CSPs, based on xanthone derivatives (Figure 1). These
xanthonic chiral stationary phases (XCSPs) are based on a
completely different type of small molecule from those
commercially available. The enantioresolution capability of
the XCSPs was evaluated by LC using different chemical
classes of chiral compounds, including drugs. A library of
enantiomeric mixtures of CDXs was also evaluated in order
to explore the principle of reciprocity,34-36 as well as the
chiral self‐recognition phenomenon.37,38 Two XCSPs
possessing enantiomers of the same CDX were prepared to
evaluate the resolving power of both enantiomers as selector
and to confirm the inversion of the elution order. The
greening choice of the chromatographic analysis, by reducing
the internal diameter of the LC columns, was also investi-
gated. To the best of our knowledge, this is the first report
of the use of CDXs as LC chiral selectors.
2 | MATERIALS AND METHODS
2.1 | Chemicals
Enantiomerically pure reagents, (S)‐(+)‐valinol, (R)‐(–)‐α‐
phenylglycinol, (S)‐(+)‐α‐phenylglycinol, (1R,2S)‐(–)‐2‐
amino‐1,2‐diphenylethanol, and (R,R)‐(+)‐2‐amino‐1,2‐
diphenylethanol, were obtained from Sigma‐Aldrich
(St. Louis, MO). O‐(Benzotriazol‐1‐yl)‐N‐N‐N'‐N'‐
Xanthone derivatives comprise an important class of
oxygenated heterocycles, many of which possess a broad
spectrum of biological/pharmacological activities.19-23 In
recent years, the synthesis of new bioactive xanthone deriva-
tives using different synthetic methodologies, including
chiral derivatives of xanthones (CDXs),24,25 has been an area
of great interest in our group.26-29
The CDXs, when anchored to a chromatographic support
by covalent linkage through a spacer, possess all the
necessary attributes to constitute chromatographic
selectors.30 The 3D quasi‐planar structure and peculiar
electronic properties of the xanthone scaffold,31 associated
with a diversity of functional groups and chiral moieties, con-
stitute structural characteristics affording the establishment of
different types of interactions, as well as contributing three‐
dimensionality factors that influence the enantioselectivity.
In fact, the xanthone‐based chiral selectors may contain
aromatic, polar, and bulky nonpolar groups, forming
FIGURE 1 Schematic representation of a stationary phase based on
chiral derivatives of xanthones

FERNANDES ET AL.
3
tetramethyluronium
tetrafluoroborate
(TBTU),
3‐
(Tokyo, Japan) model 880‐PU pump, equipped with a
Rheodyne 7125 injector fitted with a 20 μL loop, a JASCO
model 880‐30 solvent mixer, a 875‐UV Intelligent UV/Vis
Detector, and a QR‐2090 Plus Chiral Polarimeter Detector.
Data acquisition was performed using Chromatography
Station for Windows, v. 1.7 DLL. Two more HPLC systems
were employed: a system consisted of one Shimadzu (Kyoto,
Japan) LC‐10ATVP pump, equipped with a Rheodyne 7725i
injector fitted with a 100 μL loop, an SPD‐10AV UV/Vis
detector with an SCL‐10Avp interface, controlled using
Shimadzu CLASS‐VP software; and a system consisted of
two Shimadzu LC 10‐ADvp pumps, an FCV‐10AL solvent
selector valve, an automatic injector SIL10‐Advp, an
SPD‐10AV UV/Vis detector with an SCL‐10Avp interface.
A CD detector model JASCO CD 2095 Plus was also used.
Data acquisition was performed using Shimadzu LC
SOLUTIONS software. Analyses were performed at room
temperature in an isocratic mode with a multimodal
operation. The stock solutions of chiral test compounds
were prepared by dissolution in EtOH at a concentration of
1 mg/mL and further diluted in the same solvent. Working
solutions of mixtures of enantiomeric pure compounds were
prepared mixing equal aliquots of each enantiomer. The
mobile phase compositions were Hex and EtOH or 2‐PrOH
as a modifier, with or without TFA, TEA, or DEA, in
normal‐phase conditions. The evaluations in polar organic
mode were carried out using MeOH, EtOH, ACN, or mix-
tures of these solvents. MeOH and water or ACN and water,
with or without TFA or TEA, were used in reversed‐phase
conditions. The mobile phases were prepared in a volume/
volume relation and degassed in an ultrasonic bath for
15 min before use. The flow rate used was 0.5 mL/min
(columns with 150 x 4.6 mm i.d.) or 0.2 mL/min (columns
with 150 x 2.1 mm i.d.) and the chromatograms were
monitored by UV detection at a wavelength of 254 nm
and/or polarimeter detection and/or CD detection. Sample
injections (20 μL) were carried out in triplicate. The dead
time (t0) was considered to be equal to the peak of the
solvent front and was taken from each particular run. The
chromatographic parameters were established as follows:
the retention factor (k) was determined as [k = (tR–t0) / t0]
where tR is the retention time for each enantiomer; the
enantioselectivity factor (α) was calculated as [α = k2 / k1];
the resolution (Rs) was determined as [Rs = 1.18 ×
(tR2 ‐ tR1) / (W1 0.5 + W2 0.5)] where tR1, W1 0.5 and tR2,
W2 0.5 are the retention times and the peak width at half
height of the first and second eluted enantiomer, respectively.
The chiral selector loadings (μmol/m2) were determined
from the elemental analyses of carbon as follows: selector
loading = (106 × %C) / S [(100 × n × 12) – (%C × Mw)]
where %C is the elemental analyses of carbon, S is the sur-
face area, n is the number of carbon atoms in the modified
chain, and Mw is the relative molecular mass.
(triethoxysilyl)propylisocyanate, and other reagents and sol-
vents were commercially available materials as pro analysis,
from Sigma‐Aldrich or Merck (Darmstadt, Germany), and
were used without further purification. Porous spherical
silica gel Nucleosil‐100Å‐5μm from Macherey‐Nagel
(Dϋren, Germany) was used as the support material of the
six XCSPs. Ethanol (EtOH), 2‐propanol (2‐PrOH), n‐hexane
(Hex), methanol (MeOH), and acetonitrile (ACN) for high‐
performance liquid chromatography (HPLC) were purchased
from Sigma‐Aldrich. Triethylamine (TEA), diethylamine
(DEA), and trifluoroacetic acid (TFA) (all p.a. grade) were
also obtained from Sigma‐Aldrich. Ultrapure water was pro-
duced by a Millipore Milli‐Q system (Millipore, Bedford,
MA). Commercial chiral test compounds used to evaluate
the XCSPs were obtained from Sigma‐Aldrich or from
Merck. Kielcorins and CDXs were synthesized previously
in our laboratory according to procedures described
elsewhere.24,25,39
2.2 | Instrumentation and chromatography
Data melting points are uncorrected and were obtained with
a Köfler microscope. Optical rotation measurements were
carried out on a Polartronic Universal polarimeter. Infrared
spectra were recorded in a KBr microplate (cm‐1) in an FTIR
spectrometer Nicolet iS10 from Thermo Scientific
(Waltham, MA) with Smart OMNI‐Transmission accessory
(Software OMNIC 8.3). 1H and 13C NMR spectra were
taken in DMSO‐d6 at room temperature, on Bruker Avance
300 and 500 instruments (Billerica, MA; 300.13 and
1
500.13 MHz for H and 75.47 and 125.77 MHz for 13C).
Chemical shifts are expressed in δ (ppm) values relative to
tetramethylsilane (TMS) as an internal reference. Coupling
constants are reported in Hz. 13C nuclear magnetic reso-
nance (NMR) assignments were made by 2D HSQC and
HMBC experiments (long‐range C, H coupling constants
were optimized to 7 Hz). HRMS mass spectra were mea-
sured on a Bruker Daltonics micrOTOF Mass Spectrometer,
recorded as ESI (electrospray) mode. Elemental analyses
were conducted on an Elemental Carlo Erba 1108 apparatus.
Flash chromatography was carried out using Grace Resolv,
silica gel 5 g/25 mL cartridges. Thin‐layer chromatography
(TLC) was performed using Merck silica gel 60 (GF254
)
plates, with appropriate mobile phases and detection at 254
and/or 365 nm. The column packing system was an SSI
Lab Alliance – Pack in a Box, portable HPLC column pack-
ing system model CP constant pressure pump, nonflush,
0.1–24 mL/min, 10.000 psig, with transducer and RS‐232
control, SS, 110/220V column packer assembly (50 mL
reservoir, column adapter 5/16” 4.6 mm) with Quick‐Set
pump control software. A Shandon column packing pump
was also used. The HPLC system used consisted of a JASCO

4
FERNANDES ET AL.
1
(7.58 mg/mL, dichloromethane); H NMR, 13C NMR, IR,
2.3 | Development of XCSPs 1‐6
XCSPs 1–6 were prepared following similar synthetic proce-
and HRMS data are shown in the Supplementary Material.
dures according to Schemes 1 and 2.
2.5.2 | (R)‐2‐(6‐methoxyxanthone‐2‐
carboxamido)‐2‐phenylethyl [3‐(triethoxysilyl)
propyl] carbamate (13)
2.4 | General procedure for the synthesis of
CDXs 7–11 and 18
Compound 13 was obtained as white crystals (yield: 87%);
m.p.: 96–98°C (ethyl acetate); [α]D20 = +44.3° (7.5 mg/mL,
CDXs 7–11 and 18 were synthesized and characterized
according to the described procedure.24,25
1
ethyl acetate); H NMR, 13C NMR, IR, and HRMS data are
shown in the Supplementary Material.
2.5 | General procedure for the synthesis of
silylated derivative of CDXs 12–16 and 19
2.5.3 | (S)‐2‐(6‐methoxy‐9‐oxo‐9H‐xanthene‐2‐
carboxamido)‐2‐phenylethyl [3‐(triethoxy‐silyl)
propyl] carbamate (14)
The appropriate CDX (2.12 mmol) was dissolved in
anhydrous toluene (60 mL), and triethylamine (303 μL,
2.18 mmol) and 3‐(triethoxysilyl)propylisocyanate (550 μL,
2.22 mmol) were added. The mixture was refluxed for 76 h.
The solvent was evaporated under reduced pressure and the
crude product was purified by flash chromatography (Grace
Resolv, silica gel 5 g / 25 mL, n‐hexane/ethyl acetate in
gradient) and by crystallization from ethyl acetate / n‐hexane
affording the silylated derivative.
Compound 14 was obtained as white crystals (yield: 74%);
20
m.p.: 96–98°C (ethyl acetate / n‐hexane); [α]D = –44.3°
1
(7.5 mg/mL, ethyl acetate); H NMR, 13C NMR, IR, and
HRMS data are shown in the Supplementary Material.
2.5.4 | (1S,2R)‐2‐(6‐methoxy‐9‐oxo‐9H‐xan-
thene‐2‐carboxamido)‐1,2‐diphenylethyl [3‐
(triethoxysilyl)propyl] carbamate (15)
2.5.1 | (S)‐2‐(6‐methoxyxanthone‐2‐
carboxamido)‐3‐methylbutyl [3‐(triethoxysilyl)
propyl] carbamate (12)
Compound 15 was obtained as white crystals (yield: 63%);
20
m.p.: 219–220°C (ethyl acetate / n‐hexane); [α]D
=
–150.0° (0.20 mg/mL, dichloromethane); 1H NMR, 13C
NMR, IR, and HRMS data are shown in the
Supplementary Material.
Compound 12 was obtained as white crystals (yield: 91%); m.
20
p.: 144–146°C (ethyl acetate / n‐hexane); [α]D = +15.8°
SCHEME 1 Synthesis of XCSPs 1–5

FERNANDES ET AL.
5
SCHEME 2 Synthesis of XCSP 6
dichloromethane, and n‐hexane. The bonded phase was dried
in a desiccator under vacuum for 24 h, yielding the XCSP.
Each new CSP (2.30 g) was mixed with Hex:2‐PrOH
(50:50 v/v) (50 mL), and then sonicated for 3 min. The sus-
pension was poured into the chamber of a column packer
and was packed into an empty HPLC column (150 x 4.6
mm i.d.) with Hex:2‐PrOH (90:10 v/v) as packing solvent,
under a pressure no more than 7500 psi.
2.5.5 | (1R,2R)‐2‐(6‐methoxy‐9‐oxo‐9H‐xan-
thene‐2‐carboxamido)‐1,2‐diphenylethyl [3‐
(triethoxysilyl)propyl] carbamate (16)
Compound 16 was obtained as white crystals (yield: 51%);
20
m.p.: 155–158°C (ethyl acetate / n‐hexane); [α]D
=
+22.0° (0.5 mg/mL, chloroform); H NMR, 13C NMR, IR,
1
and HRMS data are shown in the Supplementary Material.
2.5.6 | (R)‐2‐[2‐(3‐oxyxanthone)acetamide]‐2‐
phenylethyl [3‐(triethoxysilyl)propyl] carbamate
(19)
3 | RESULTS AND DISCUSSION
Compound 19 was obtained as light pink crystals (yield:
Six new CSPs, based on xanthone derivatives (XCSP 1–6),
were developed according to Schemes 1 and 2. XCSPs 1–5
were prepared using the xanthone derivative 6‐methoxy‐9‐
oxo‐9H‐xanthene‐2‐carboxylic acid (1) (Scheme 1), synthe-
sized via Ullmann reaction with the formation of diaryl
ether intermediate according to the previously described
procedure,25 as a chemical substrate. For XCSP 6, the
xanthone derivative 2‐[(9‐oxo‐9H‐xanthen‐3‐yl)oxy]acetic
acid (17), synthesized via benzophenone intermediate,24
was used as a substrate (Scheme 2). The CDXs were syn-
thesized by coupling these two suitable functionalized
xanthone derivatives with commercially available
enantiomerically pure building blocks.24,25 The activation
of the carboxylic acid group of the xanthone scaffold was
carried out with TBTU. Accordingly, xanthone 1 was
coupled with the enantiomerically pure amino alcohols
(S)‐(+)‐valinol (2), (R)‐(–)‐α‐phenylglycinol (3), (S)‐(+)‐α‐
20
76%); m.p.: 108–110°C (ethyl acetate / n‐hexane); [α]D
=
1
–19.2° (3.1 mg/mL, ethyl acetate); H NMR, 13C NMR, IR,
and HRMS data are shown in the Supplementary Material.
2.6 | General procedure of covalent linkage of
silylated derivative of CDXs onto the silica gel
and HPLC column packing
Anhydrous toluene (30 mL) was added to silica gel Nucleosil
(2890 mg, 100 Å‐5μm, Macherey‐Nagel), previously dried in
a desiccator under vacuum and with phosphorus pentoxide
for 24 h. Following this, the appropriate silylated derivative
of CDX (1.42 mmol) dissolved in the same solvent (30 mL)
was added. The mixture was gently stirred at reflux for
76 h. The solid was then filtered and washed successively
with (100 mL) toluene, methanol, acetone, ethyl acetate,

6
FERNANDES ET AL.
phenylglycinol (4), (1R,2S)‐(–)‐2‐amino‐1,2‐diphenylethanol
(5), and (1R,2R)‐(+)‐2‐amino‐1,2‐diphenylethanol (6), to
afford CDXs (7–11), respectively. CDX 18 was synthesized
by coupling xanthone 17 with the amino alcohol (R)‐(–)‐α‐
phenylglycinol (3). Our selection included enantiomerically
pure building blocks with no tendency towards racemization
or enantiomeric interconversion, having a primary amine as
a reactive group for the coupling reaction with the
carboxyxanthone derivative and a hydroxyl group to further
linkage to the spacer.
The amino alcohols valinol and phenylglycinol were
chosen to obtain XCSPs, similar to valine‐derived40 and
phenylglycine‐derived41 CSPs developed by Pirkle et al. In
fact, these amino acid‐derived CSPs were shown to be useful
to separate a broad spectrum of racemates on both analytical
and preparative scales.33,34 Recognizing the crucial role of
π–π interactions between each enantiomer and this type of
CSPs for chiral recognition, the amino alcohol 2‐amino‐1,2‐
diphenylethanol was also selected in order to increase these
interaction types, as well as to obtain a more rigid structure
(cleft type selector), similar to the most employed and
successful Pirkle‐type CSP, i.e., Whelk‐O1 CSP.42
The strategy used for binding the CDXs (chiral
selectors) to the chromatographic support was through
the synthesis of derivatives that allowed the covalent
linkage to the silica, specifically silylated derivatives.43,44
Consequently, all CDXs reacted with 3‐(triethoxysilyl)
propylisocyanate giving the corresponding silylated deriva-
tives. Then, the silylated derivatives of all the CDXs were
anchored to silica gel (Nucleosil‐100‐5) giving the corre-
spondent XCSPs 1–6 (Schemes 1 and 2). This synthetic
methodology involves only a few steps and the reactions
provided good yields.
that incorporates the enantiomeric precursors to XCSPs 1–3
and XCSP 5 and analogs.
The enantioseparation by XCSPs 1–6 was evaluated
under normal, polar organic, and reversed‐phase elution
conditions. Table 2 shows the overall best results.
The results in Table
2
show that the best
enantioselectivity was achieved mainly under normal‐phase
elution conditions using EtOH as a modifier.
It was found that XCSP 1 showed enantioselective
capabilities only for enantiomeric mixtures of CDXs.
Concerning, for example, the enantiomeric mixtures of
CDXs 23, 24, and 25, good enantioselectivity (α =
1.14, 1.15, and 1.15) and resolution (RS = 2.16, 2.24,
and 2.18) were respectively obtained, with Hex:EtOH
(90:10 v/v) as mobile phase.
XCSP 2 was found to have more versatility, showing
enantioselectivity not only for the enantiomeric mixtures of
CDXs (α ranging from 1.04 to 1.32) but also for the chiral
compounds 1‐(9‐anthryl)‐2,2,2‐trifluoroethanol (19) (α =
1.07), N‐[1‐(1‐naphthyl)ethyl]‐3,5‐dinitrobenzamide (20)
(α = 1.31), and bi‐naphthol (22) (α = 1.08). Unsurprisingly,
in general, XCSP 2 showed the best performance and versa-
tility, when taking into account that: 1) XCSP 2 is similar to
the successfully commercially available Phenylglycine
CSP41; 2) the selector of this CSP has another aromatic group
in addition to the xanthone scaffold, both of which were
essential for chiral recognition by this type of CSPs through
π–π interactions between enantiomers and the CSP; and 3)
an excellent enantioselectivity and resolution were achieved
for the enantiomeric mixture of chiral selector of XCSP 2
using macrocyclic antibiotics,7 (S,S)‐Whelk‐O1,32 and
polysaccharide‐based CSPs.6
As expected, XCSP
3 proved to have similar
The chiral selector loadings for all the XCSPs were calcu-
lated from the elemental analyses of carbon (Table 1).
Finally, all the XCSPs were packed in a column with
150 x 4.6 mm i.d. using Hex:2‐PrOH (90:10 v/v). XCSP 2
and XCSP 5 were also packed in a column with 150 x 2.1
mm i.d.
enantioselectivity capabilities to XCSP 2. Actually, these
two XCSPs have the same chiral selector but with opposite
configurations. However, the XCSP 3 generally presented
higher retention for the enantiomers, which can be explained
by its slightly higher selector loading (1.31 μmol/m2) when
compared with XCSP 2 (0.90 μmol/m2).
The LC enantioselective resolution capabilities of XCSPs
1‐6 were evaluated with several enantiomeric mixtures of
structurally different chiral compounds, including some types
of drugs. Furthermore, a library of CDXs was also chosen
Regarding XCSP 4, the additional aromatic groups of the
chiral selector of this CSP did not contribute to its potential
enantiorecognition. In fact, the steric hindrance of the bulky
and rigid structural elements may be responsible for non‐
TABLE 1 Elemental analysis and chiral selector loadings for XCSPs 1–6
Elemental analysis
XCSP 1
XCSP 2
XCSP 3
XCSP 4
XCSP 5
XCSP 6
% C
7.09
1.01
2.60
0.80
8.66
0.82
1.84
0.90
11.87
1.14
1.65
1.31
8.36
1.10
1.59
0.70
10.11
0.85
0.97
0.85
9.87
0.89
1.76
1.04
% N
% H
Selector loading (based on C) μmol/m2

FERNANDES ET AL.
7
TABLE 2 Chromatographic resolution of enantiomeric mixtures on XCSPs 1‐6
Enantiomeric mixture
XCSP
Mobile phase (v/v)
Hex/2‐PrOH: 90/10
k1
α
Rs
XCSP 2
1.81
1.07
< 1.00
XCSP 2
Hex/EtOH: 80/20
Hex/2‐PrOH: 70/30
Hex/EtOH: 80/20
Hex/EtOH: 80/20
2.90
2.33
3.07
3.35
1.17
1.31
1.17
1.22
1.95
1.93
1.95
2.96
XCSP 3
XCSP 6
XCSP 4
XCSP 2
ACN/H2O: 10/90
MeOH/H2O: 15/85
EtOH/H2O: 15/85
10.29
8.97
4.61
1.07
1.06
1.07
< 1.00
< 1.00
< 1.00
Hex/EtOH: 90/10
with 0.01% TFA
3.96
1.08
< 1.00
XCSP 1
XCSP 2
Hex/EtOH: 90/10
Hex/EtOH: 80/20
Hex/2‐PrOH: 70/30
Hex/EtOH: 80/20
Hex/EtOH: 85/15
Hex/2‐PrOH: 70/30
Hex/EtOH: 80/20
Hex/EtOH: 80/20
9.76
5.27
2.36
6.12
9.59
3.48
6.72
6.01
1.14
1.13
1.18
1.12
1.13
1.15
1.11
1.06
2.16
1.49
< 1.00
1.33
1.57
< 1.00
1.30
XCSP 3
XCSP 6
< 1.00
XCSP 1
XCSP 2
Hex/EtOH: 90/10
Hex/EtOH: 80/20
Hex/2‐PrOH: 70/30
Hex/EtOH: 80/20
Hex/EtOH: 85/15
Hex/2‐PrOH: 70/30
EtOH
ACN/MeOH: 50/50
ACN/MeOH: 5:95
Hex/EtOH: 80/20
Hex/EtOH: 80/20
Hex/EtOH: 80/20
18.79
7.53
4.35
10.99
17.89
6.82
0.96
0.57
0.96
1.15
1.13
1.16
1.15
1.15
1.32
1.10
1.10
1.08
1.12
1.05
1.10
2.24
1.68
< 1.00
1.98
1.60
< 1.00
< 1.00
< 1.00
< 1.00
1.95
XCSP 3
XCSP 6
XCSP 5
10.67
12.35
4.45
< 1.00
< 1.00
XCSP 1
Hex/EtOH: 90/10
Hex/EtOH: 80/20
16.63
6.94
1.05
1.04
< 1.00
< 1.00
(Continues)

8
FERNANDES ET AL.
TABLE 2 (Continued)
Enantiomeric mixture
XCSP
Mobile phase (v/v)
k1
α
Rs
XCSP 1
Hex/EtOH: 90/10
Hex/EtOH: 80/20
Hex/2‐PrOH: 70/30
Hex/EtOH: 80/20
Hex/EtOH: 85/15
Hex/2‐PrOH: 70/30
Hex/EtOH: 80/20
Hex/EtOH: 80/20
7.59
3.55
1.86
4.85
6.69
2.63
6.42
4.79
1.15
1.13
1.18
1.11
1.11
1.16
1.11
1.08
2.18
1.38
< 1.00
1.21
1.40
< 1.00
1.10
XCSP 2
XCSP 3
XCSP 6
1.06
XCSP 1
XCSP 2
Hex/EtOH: 90/10
Hex/EtOH: 80/20
Hex/2‐PrOH: 70/30
Hex/EtOH: 80/20
Hex/EtOH: 85/15
Hex/2‐PrOH: 70/30
EtOH
5.88
3.66
1.82
4.92
7.07
2.92
0.78
6.72
4.99
1.07
1.06
1.08
1.09
1.10
1.12
1.08
1.10
1.08
< 1.00
< 1.00
< 1.00
1.14
1.38
< 1.00
< 1.00
1.15
XCSP 3
XCSP 6
Hex/EtOH: 80/20
Hex/EtOH: 80/20
1.11
XCSP 1
XCSP 2
XCSP 3
Hex/EtOH: 90/10
Hex/EtOH: 80/20
Hex/EtOH: 80/20
Hex/EtOH: 85/15
Hex/EtOH: 80/20
18.08
7.06
9.59
13.52
12.54
1.06
1.05
1.04
1.04
1.03
< 1.00
< 1.00
< 1.00
< 1.00
< 1.00
XCSP 1
XCSP 3
Hex/EtOH: 90/10
Hex/EtOH: 80/20
10.07
7.53
1.05
1.05
< 1.00
< 1.00
XCSP 1
Hex/EtOH: 90/10
Hex/EtOH: 80/20
Hex/EtOH: 80/20
Hex/EtOH: 80/20
8.40
3.43
4.82
6.24
1.08
1.06
1.07
1.07
< 1.00
< 1.00
< 1.00
< 1.00
XCSP 2
XCSP 3
XCSP 1
XCSP 3
Hex/EtOH: 90/10
Hex/EtOH: 80/20
18.44
12.35
1.04
1.02
< 1.00
< 1.00
(Continues)

FERNANDES ET AL.
9
TABLE 2 (Continued)
Enantiomeric mixture
XCSP
Mobile phase (v/v)
k1
α
Rs
XCSP 1
XCSP 2
XCSP 3
XCSP 6
Hex/EtOH: 90/10
Hex/EtOH: 80/20
Hex/EtOH: 80/20
Hex/EtOH: 80/20
5.89
4.66
6.33
4.79
1.04
1.06
1.07
1.04
< 1.00
< 1.00
< 1.00
< 1.00
enantioselective interactions with the chiral compounds.
Interestingly, it was found that XCSP 4 presented some
enantioselectivity (α ~1.07) only for the chiral compound
albendazole sulfoxide (21) under reversed‐phase mode. This
result indicates that the chiral recognition mechanism of
XCSP 4 is different from other XCSPs.
Interestingly, although the chiral selector of XCSP 5
differs only in the configuration of one stereogenic center
compared with that of XCSP 4, the chromatographic
behavior of XCSP 5 was similar to that of XCSPs 1–3.
However, XCSP 5 revealed poor enantioresolution capabili-
ties for the tested compounds.
On the other hand, the best chromatographic value was
achieved with XCSP 6, with α = 1.22 and RS = 2.96 for
N‐[1‐(1‐naphthyl)ethyl]‐3,5‐dinitrobenzamide (7) with Hex:
EtOH (80:20 v/v) as a mobile phase. The selector of
XCSP 6 comprises the same chiral moiety as that of
XCSP 2, but with different substituents on the xanthone
scaffold. Nevertheless, in general, less discrimination
ability was observed compared to XCSP 1 and XCSP 2
(Table 2).
It is important to highlight that the enantioselectivity
for the enantiomeric mixtures of CDXs, tested on XCSPs,
was mainly achieved using a low percentage of the modi-
fier (5–30%). However, with those elution conditions high
retention factors were observed (k1 ranging from 1.82 to
18.79). To overcome this situation, the polarity of the
mobile phase was increased to consequently decrease the
retention time, but concomitantly the enantioselectivity also
decreased. Considering evaluation on the polar organic and
reversed‐phase elution conditions, the chromatographic
results were not satisfactory. In general, short retention
times were observed with absence or very poor resolutions.
Characteristic chromatograms obtained during the evalu-
ation of the enantioselective resolution capabilities of the
XCSPs are shown in Figure 2.
on XCSP 1 and 9 of them (75%) on XCSP‐2. This
specificity for the CDXs is very important, since previous
studies, using commercial CSPs, namely, (S,S)‐Whelk‐O1
and L‐Phenylglycine CSPs, demonstrated less versatility for
this important class of chiral compounds.32 In fact, the
enantiomeric mixture of CDX 27 was the only pair resolved
on L‐Phenylglycine CSP, while (S,S)‐Whelk‐O1 CSP showed
enantioselectivity only for CDXs possessing an aromatic
moiety bonded to the stereogenic center.
The chromatographic results also suggested that XCSPs
can be developed by applying the concept of reciprocity.35,36
As an example, the best resolution achieved on XCSP 1 was
with the enantiomeric mixture of CDX 24, with RS = 2.24
using Hex:EtOH (90:10 v/v) as a mobile phase. Therefore,
the immobilized enantiomers of CDX 24 (XCSP 2 and
XCSP 3), using similar interactions, were able to distinguish
between the enantiomeric precursors of XCSP 1, i.e.,
CDX 23 (Table 2). For example, good enantioselectivity
(α = 1.13) and resolution (RS = 1.57) were obtained on
XCSP‐2 with Hex:EtOH (85:15 v/v).
It was also found that the chiral self‐recognition phenom-
enon occurs with the XCSPs. As, for example, XCSP 1 and
XCSP 2 can effectively separate the enantiomeric mixtures
of their precursors, namely, CDX 23 and CDX 24, with RS
= 2.16 and 1.98, respectively. In both cases, the enantiomer
that forms the homochiral diastereisomeric complex with
the CSP was more retained. Similar results were described
in the literature using Pirkle‐type CSPs.33,37,38
In order to confirm the inversion of elution order of
XCSPs, the two enantiomers of CDX 24 (compounds 8 and
9) were also eluted separately on XCSP 2, and on the
opposite enantiomeric XCSP (XCSP 3). The chromatograms
are shown in Figure 3.
As shown in Figure 3, with these new CSPs, the elution
order of the enantiomers of CDX 24 is inverted by using
the same type of CSP. This is an important advantage
for enantiomeric purity determinations or preparative
separations, giving the possibility to elute before the trace
enantiomer or the desired enantiomer, respectively.
Considering the overall chromatographic results, it was
also found that all the XCSPs (except for XCSP 4) provided
enantioselectivity for the enantiomeric mixtures of CDXs.
Accordingly, for example, from the 12 enantiomeric mixtures
of CDXs tested, 10 of them (83%) showed enantioselectivity
Additionally, concerning ecological, practical and
economic reasons, the greening of the chromatographic

10
FERNANDES ET AL.
FIGURE 2 Chromatograms of enantiomeric mixtures of compounds 20, 23, 24, 26, and 27 on XCSPs. Flow rate, 0.5 mL/min; detection, 254 nm
analysis45,46 was investigated by reducing the internal
diameter of the LC columns from 4.6 to 2.1 mm i.d.
Accordingly, the best obtained XCSPs, XCSP 2, and
XCSP 5 were packed in a column with 150 x 2.1 mm
i.d. and their enantiomeric performance was evaluated
using a flow rate of 0.2 mL/min. The major advantage

FERNANDES ET AL.
11
under the project PTDC/MAR‐BIO/4694/2014 (reference
POCI‐01‐0145‐FEDER‐016790) supported 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) programme and the
Project 3599
Desenvolvimento Tecnológico e a Constituição de Redes
Temáticas (3599‐PPCDT), COXANT–CESPU‐2016,
– Promover a Produção Científica e
QOPNA research project (FCT UID/QUI/00062/2013) and
Portuguese NMR network. Y.Z. Phyo thanks the Erasmus
Mundus Action 2 (Lotus Plus project) for a PhD's scholar-
ship. We thank also Coordenação de Aperfeiçoamento
Pessoal de Nível Superior (CAPES) and FCT for the joint
international Project 214/08.
FIGURE 3 Schematic chromatograms of enantiomeric mixture of
CDX 24, (S)‐CDX 9, and (R)‐CDX 8 on XCSP 2 and XCSP 3. Mobile
phase, Hex:EtOH (80:20 v/v); flow rate, 0.5 mL/min; detection, 254 nm
REFERENCES
was the decrease of the solvent consumption without
compromising the enantioseparation.
1. Ward TJ, Ward KD. Chiral separations: A review of current topics
and trends. Anal Chem. 2012;84(2):626‐635.
2. Sousa ME, Tiritan ME, Belaz KRA, et al. Multimilligram
enantioresolution of low‐solubility xanthonolignoids on polysac-
charide chiral stationary phases using a solid‐phase injection
system. J Chromatogr A. 2006;1120(1‐2):75‐81.
4 | CONCLUSION
Six new xanthonic chiral stationary phases (XCSPs) were
developed using a well‐established synthetic methodology
that involves only a few steps of reactions with good
yields. The XCSPs were covalently bonded to fully
porous silica, which guarantee long‐lasting columns, and
show high stability, versatility in the selection of the
mobile phase composition, and reproducibility. It was
found that, in general, they show enantioselectivity for
CDXs and other chiral compounds, such as 1‐(9‐
anthryl)‐2,2,2‐trifluoroethanol, N‐[1‐(1‐naphthyl)ethyl]‐3,5‐
dinitrobenzamide, binaphthol, and albendazole sulfoxide.
Moreover, this type of chiral stationary phase gives the
possibility to invert the elution order (by using the oppo-
site enantiomer as chiral selector). Therefore, this work
represents a breakthrough that is extremely interesting
not only in the field of chiral LC but also as new chem-
ical tools for further studies of chiral molecular
recognition.
3. Silva B, Fernandes C, Tiritan ME, et al. Chiral enantioresolution of
cathinone derivatives present in “legal highs”, and enantioselectivity
evaluation on cytotoxicity of 3,4‐methylenedioxypyrovalerone
(MDPV). Forensic Toxicol. 2016;1‐14.
4. Prompanya C, Fernandes C, Cravo S, et al. A new cyclic
hexapeptide and a new isocoumarin derivative from the marine
sponge‐associated fungus Aspergillus similanensis KUFA 0013.
Mar Drugs. 2015;13(3):1432‐1450.
5. Zin WWM, Buttachon S, Dethoup T, et al. New cyclotetrapeptides
and a new diketopiperzine derivative from the marine sponge‐
associated fungus Neosartorya glabra KUFA 0702. Mar Drugs.
2016;14(7):
6. Fernandes C, Brandão P, Santos A, et al. Resolution and determina-
tion of enantiomeric purity of new chiral derivatives of xanthones
using polysaccharide‐based stationary phases. J Chromatogr A.
2012;1269:143‐153.
7. Fernandes C, Tiritan ME, Cass Q, Kairys V, Fernandes MX, Pinto
M. Enantioseparation and chiral recognition mechanism of new
chiral derivatives of xanthones on macrocyclic antibiotic stationary
phases. J Chromatogr A. 2012;1241:60‐68.
ACKNOWLEDGMENTS
This research was partially supported by the Structured
Program of R&D&I INNOVMAR – Innovation and
Sustainability in the Management and Exploitation of
Marine Resources (reference NORTE‐01‐0145‐FEDER‐
000035, Research Line NOVELMAR), funded by the
Northern Regional Operational Programme (NORTE2020)
through the European Regional Development Fund (ERDF)
and by Foundation for Science and Technology (FCT),
8. Ribeiro AR, Gonçalves VMF, Maia AS, Ribeiro C, Castro PML,
Tiritan ME. Dispersive liquid–liquid microextraction and HPLC to
analyse fluoxetine and metoprolol enantiomers in wastewaters. Envi-
ron Chem Lett. 2015;13(2):203‐210.
9. Ribeiro AR, Maia AS, Cass QB, Tiritan ME. Enantioseparation of
chiral pharmaceuticals in biomedical and environmental analyses
by liquid chromatography: An overview. J Chromatogr B Analyt
Technol Biomed Life Sci. 2014;968:8‐21.

12
FERNANDES ET AL.
10. Cavazzini A, Pasti L, Massi A, Marchetti N, Dondi F. Recent appli-
cations in chiral high performance liquid chromatography: A review.
Anal Chim Acta. 2011;706(2):205‐222.
of dual anticoagulant/antiplatelet agents.
2011;54(15):5373‐5384.
J
Med Chem.
29. Sousa E, Paiva A, Nazareth N, et al. Bromoalkoxyxanthones as prom-
ising antitumor agents: Synthesis, crystal structure and effect on
human tumor cell lines. Eur J Med Chem. 2009;44(9):3830‐3835.
11. Kalíková K, Riesová M, Tesařová E. Recent chiral selectors for
separation in HPLC and CE. Cent Eur J Chem. 2011;1‐22.
12. Lämmerhofer M. Chiral recognition by enantioselective liquid
chromatography: Mechanisms and modern chiral stationary phases.
J Chromatogr A. 2010;1217(6):814‐856.
30. Pinto M, Tiritan ME, Fernandes C, Cass Q. In: Industrial BdP, ed.
Fases Estacionárias Quirais baseadas em Derivados Xantónicos.
Porto, Portugal: In; 2011.
13. Chankvetadze B. Recent developments on polysaccharide‐based
chiral stationary phases for liquid‐phase separation of enantiomers.
J Chromatogr A. 2012;1269:26‐51.
31. Gales L, Damas AM. Xanthones — A structural perspective. Curr
Med Chem. 2005;12(21):2499‐2515.
32. Fernandes C, Palmeira A, Santos A, Tiritan ME, Afonso C, Pinto
MM. Enantioresolution of chiral derivatives of xanthones on (S,S)‐
Whelk‐O1 and l ‐phenylglycine stationary phases and chiral
recognition mechanism by docking approach for (S,S)‐Whelk‐O1.
Chirality. 2013;25(2):89‐100.
14. Tang M, Zhang J, Zhuang S, Liu W. Development of chiral station-
ary phases for high‐performance liquid chromatographic separation.
Trends Anal Chem. 2012;39:180‐194.
15. Carreira EM, Yamamoto H. Comprehensive Chirality. In: Volume 8:
Separations and Analysis. Amsterdam: Elsevier; 2012:96‐310.
33. Welch CJ. Evolution of chiral stationary phase design in the Pirkle
laboratories. J Chromatogr A. 1994;666(1–2):3‐26.
16. Bocian S, Skoczylas M, Buszewski B. Amino acids, peptides,
and proteins as chemically bonded stationary phases — A review.
J Sep Sci. 2016;39(1):83‐92.
34. Fernandes C, Tiritan ME, Pinto M. Small molecules as chromato-
graphic tools for HPLC enantiomeric resolution: Pirkle‐type chiral
stationary phases evolution. Chromatographia. 2013;76(15‐16):
871‐897.
17. Zhou J, Tang J, Tang W. Recent development of cationic cyclodex-
trins for chiral separation. Trends Anal Chem. 2015;65:22‐29.
35. Taylor DR, Maher K. Chiral separations by high‐performance liquid
chromatography. J Chromatogr Sci. 1992;30(3):67
18. Shen J, Okamoto Y. Efficient separation of enantiomers using
stereoregular chiral polymers. Chem Rev. 2016;116(3):1094‐1138.
36. Pirkle WH, Däppen R. Reciprocity in chiral recognition. Compari-
19. Pinto MMM, Sousa ME, Nascimento MSJ. Xanthone derivatives:
New insights in biological activities. Curr Med Chem.
2005;12(21):2517‐2538.
son of several chiral stationary phases.
J Chromatogr A.
1987;404(C):107‐115.
37. Pirkle WH, Pochapsky TC. Considerations of chiral recognition
relevant to the liquid chromatographic separation of enantiomers.
Chem Rev. 1989;89(2):347‐362.
20. Masters KS, Bräse S. Xanthones from fungi, lichens, and bacteria:
The natural products and their synthesis. Chem Rev.
2012;112(7):3717‐3776.
38. Lee W, Snyder SE, Volkers PI, et al. Assessing chiral
self‐recognition using chiral stationary phases. Tetrahedron.
2011;67(37):7143‐7147.
21. Wezeman T, Bräse S, Masters KS. Xanthone dimers: A compound
family which is both common and privileged. Nat Prod Rep.
2015;32(1):6‐28.
39. Sousa EP, Silva AMS, Pinto MMM, Pedro MM, Cerqueira FAM,
Nascimento MSJ. Isomeric kielcorins and dihydroxyxanthones:
Synthesis, structure elucidation, and inhibitory activities of growth
of human cancer cell lines and on the proliferation of human
lymphocytes in vitro. Helv Chim Acta. 2002;85(9):2862‐2876.
22. Pinto MMM, Castanheiro RAP, Kijjoa A. Xanthones from
marine‐derived microorganisms: isolation, structure elucidation,
and biological activities. In: Encyclopedia of Analytical Chemistry.
Vol.27 Hoboken, NJ: John Wiley & Sons L; 2014:1‐21.
23. Shagufta AI. Recent insight into the biological activities of synthetic
40. Pirkle WH, Finn JM. Chiral high‐pressure liquid chromatographic
stationary phases. 3. General resolution of arylalkylcarbinols.
J Org Chem. 1981;46(14):2935‐2938.
xanthone derivatives. Eur J Med Chem. 2016;116:267‐280.
24. Fernandes C, Masawang K, Tiritan ME, et al. New chiral derivatives
of xanthones: Synthesis and investigation of enantioselectivity as
inhibitors of growth of human tumor cell lines. Bioorg Med Chem.
2014;22(3):1049‐1062.
41. Pirkle WH, House DW, Finn JM. Broad spectrum resolution of
optical isomers using chiral high‐performance liquid chromato-
graphic bonded phases. J Chromatogr. 1980;192(1):143‐158.
25. Fernandes C, Oliveira L, Tiritan ME, et al. Synthesis of new chiral
xanthone derivatives acting as nerve conduction blockers in the rat
sciatic nerve. Eur J Med Chem. 2012;55:1‐11.
42. Pirkle WH, Welch CJ. An improved chiral stationary phase for the
chromatographic separation of underivatized naproxen enantiomers.
J Liq Chromatogr. 1992;15(11):1947‐1955.
26. Azevedo CMG, Afonso CMM, Sousa D, et al. Multidimensional
optimization of promising antitumor xanthone derivatives. Bioorg
Med Chem. 2013;21(11):2941‐2959.
43. Welch CJ, Perrin SR. Improved chiral stationary phase for ß‐blocker
enantioseparations. J Chromatogr A. 1995;690(2):218‐225.
44. Ihara T, Sugimoto Y, Asada M, Nakagama T, Hobo T. Influence
of the method of preparation of chiral stationary phases on
enantiomer separations in high‐performance liquid chromatography.
J Chromatogr A. 1995;694(1):49‐56.
27. Castanheiro RAP, Pinto MMM, Cravo SMM, Pinto DCGA, Silva
AMS, Kijjoa A. Improved methodologies for synthesis of prenylated
xanthones by microwave irradiation and combination of heteroge-
neous catalysis (K10 clay) with microwave irradiation.
Tetrahedron. 2009;65(19):3848‐3857.
45. Sandra P, Vanhoenacker G, David F, Sandra K. Pereira A. Green
chromatography (Part 1): Introduction and liquid chromatography.
LC‐GC Eur. 2010;23(5)
28. Correia‐Da‐Silva M, Sousa E, Duarte B, et al. Polysulfated
xanthones: Multipathway development of
a new generation

FERNANDES ET AL.
13
46. Majors RE, Raynie D. The greening of the chromatography
laboratory. LC‐GC N Am. 2011;29(2):118‐134.
How to cite this article: Fernandes C, Tiritan ME,
Cravo S, et al. New chiral stationary phases based on
xanthone derivatives for liquid chromatography.
Chirality. 2017;1‐13. https://doi.org/10.1002/
chir.22706
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