Characterization of folic acid/native cyclodextrins host-guest complexes in solution

Article abstract of DOI:10.1016/j.molstruc.2015.12.082

The complexation of folic acid (FA) with native cyclodextrins was studied and this process was used for the comparison of ¹H NMR, ITC and ESIMS for the evaluation of association constants. The stability increases in the series: α-cyclodextrin/FA < γ-cyclodextrin/FA < β-cyclodextrin/FA. ¹H NMR and ITC gave comparable results in regard to association constant values, while results obtained for MS were considerably higher due to different interactions (electrostatic instead of hydrophobic) responsible for the stabilization of the complexes. The dimerization of FA in water was also studied, as well as its impact on the process of complexation with native cyclodextrins.

Full text of DOI:10.1016/j.molstruc.2015.12.082

Journal of Molecular Structure 1109 (2016) 114e118
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: http://www.elsevier.com/locate/molstruc
Characterization of folic acid/native cyclodextrins hosteguest
complexes in solution
,
*
b
c
ꢀ b
Magdalena Ceborska a , Magdalena Zimnicka , Małgorzata Wszelaka-Rylik , Anna Troc
a Institute of Physical Chemistry, Polish Academy of Sciences, 01-224 Warsaw, Poland
b Institute of Organic Chemistry, Polish Academy of Sciences, 01-224 Warsaw, Poland
c
ꢀ
ꢀ
Faculty of Biology and Environmental Sciences, Cardinal Stefan Wyszynski University, Woycickiego 1/3, PL-01 938 Warsaw, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
The complexation of folic acid (FA) with native cyclodextrins was studied and this process was used for
the comparison of 1H NMR, ITC and ESIMS for the evaluation of association constants. The stability in-
Received 3 August 2015
Received in revised form
29 December 2015
Accepted 29 December 2015
Available online 31 December 2015
creases in the series: a-cyclodextrin/FA < g-cyclodextrin/FA < b
-cyclodextrin/FA. 1H NMR and ITC gave
comparable results in regard to association constant values, while results obtained for MS were
considerably higher due to different interactions (electrostatic instead of hydrophobic) responsible for
the stabilization of the complexes. The dimerization of FA in water was also studied, as well as its impact
on the process of complexation with native cyclodextrins.
Keywords:
Folic acid
© 2016 Elsevier B.V. All rights reserved.
Native cyclodextrins
Aggregation in water
Nuclear magnetic resonance
Isothermal Titration Calorimetry
Mass spectrometry
1. Introduction
derivatives (e.g. various folate-based prodrugs).
Native cyclodextrins (CDs) e
oligosaccharides built from 6, 7 or 8 glucose units linked by
a
,
b
and
g
(Fig. 1) e are macrocyclic
-1,4-
Synthetic folic acid (FA, Fig. 1), the fully oxidized form of tetra-
hydrofolate, belongs to the group of water-soluble vitamins B9.
Tetrahydrofolate and its derivatives take part in many biological
processes in the human body. They play important role in some
cellular pathways, where folate acts as one-carbon source e DNA
synthesis, methylation and sustenance, as well as RNA and protein
methylation. They are required for the process of biosynthesis of
proteins and nucleic acids [1]. Folate receptor is overexpressed in
many tumors, such as brain, breast, kidney and lung cancers [2].
The density of the folate receptor grows significantly in the later
stages of cancer and, in the case of classical treatment methods
failing, folate-derived therapeutics may be in use. Prodrugs are
usually conjugates of FA with various drugs, such as cytotoxic drugs,
e.g. doxorubicin [3] camptothecin [4] and taxol [5]. Due to the va-
riety of possible pharmaceutical applications it is important to
improve bioavailability and aqueous solubility of FA and its
a
glycosidic bonds. They possess hydrophobic cavity of different
radius and sizes, which is able to accommodate various molecules
and hydrophilic outer face, which makes them easily soluble in
water. They improve the solubility, stability, and bioavailability of
biologically active compounds. Due to their specific properties they
are used by cosmetic [6], food [7] and pharmaceutical industries
[8,9].
Recently, we have studied the formation of hosteguest systems
between native CDs and FA [10]. The analysis of 1H NMR and 2D
NMR spectra led to the conclusion that CDs characterized by larger
cavity volume e
b
- and
g-CDs form with FA rotaxane-like struc-
tures, while the smallest
a
-CD forms an exclusion compound. Ac-
cording to the preliminary results obtained from Mass
Spectrometry experiments the following series of complexes' sta-
bilities in the gas phase was postulated: g-CD/FA > b-CD/FA > a-CD/
FA. These results were further confirmed by Ion-Mobility Mass
Spectrometry and theoretical calculations [11]. The aim of the work
presented here is to study in detail and to compare stabilities of the
obtained hosteguest systems in solution with the previously ob-
tained results for the gas phase. Techniques involved in the study
* Corresponding author.
E-mail addresses: mceborska@ichf.edu.pl (M. Ceborska), mzimnicka@icho.edu.
pl (M. Zimnicka), m.wszelaka@uksw.edu.pl (M. Wszelaka-Rylik), annatroc@icho.
ꢀ
edu.pl (A. Troc).
http://dx.doi.org/10.1016/j.molstruc.2015.12.082
0022-2860/© 2016 Elsevier B.V. All rights reserved.

M. Ceborska et al. / Journal of Molecular Structure 1109 (2016) 114e118
115
Fig. 1. Schematic presentation of a) host molecules e
a, b and g-CDs (1e3) and b) guest e folic acid (4), numbering of cyclodextrin carbon atoms in green. (For interpretation of the
references to colour in this figure legend, the reader is referred to the web version of this article.)
include 1H NMR, Isothermal Titration Calorimetry and HPLC-MS. 1H
NMR allowed for determination of association constants of -CD/
FA, -CD/FA and -CD/FA in water, it also enabled the study of FA
dimerization in water. Isothermal Titration Calorimetry allowed for
the confirmation of the results obtained from 1H NMR studies,
giving also the answer of the association constant of the strongest
of the studied complexes in physiological buffer (HEPES). MS
method was also applied to determine the association constants of
2.2.1.1. Dimerization of folic acid sodium salt in water. D2O was
titrated in an NMR tube with the 0.22 M D2O solution of FA sodium
salt until the concentration reached c ¼ 0.038 [M]. Dimerization
constant was evaluated using HypNMR program [12,13].
a
b
g
2.2.1.2. Titration studies for evaluation of stability constants.
The ca. 0.01 M D2O solution of a receptor (a, b and g-CD, respec-
tively) was titrated in an NMR tube with the 0.25 M solution of a FA
sodium salt. The solution of the salt contained a certain amount of
the proper CD in order to keep its concentration constant during
the titration. For each titration 15 data points were recorded. The
corresponding association constants Kas were calculated using
non-linear regression algorithm on the basis of the all chemical
shift changes by the HypNMR 2008 software [12,13]. A nonlinear
curve fitting for 1:1 binding model was carried out with the
HypNMR program.
a
-CD/FA, b-CD/FA and g-CD/FA complexes. In those measurements
mass spectrometer was treated as detector for quantitative analysis
of CD/FA complexes. In contrast to the previously obtained gas-
phase stability order [10] in this approach the solution-phase
binding details such as association constants and binding stoichi-
ometry may be obtained under certain conditions. For example, it is
very important to carefully set up the experimental parameters to
avoid dissociation of the complex or change of the solution-phase
equilibria while transferring ions to the gas phase. The type of in-
teractions occurring within complex affects significantly the
agreement between solution- and gas-phase association constants,
since each type of interactions responds differently on the transi-
tion from solution to the gas phase. The stabilization from hydro-
phobic interactions which play a leading role in cyclodextrin
inclusion complexes is significantly diminished in the gas phase,
while electrostatic interactions are supposed to be strengthened in
vacuum. In this study we evaluate the MS approach to determine
the solution association constants of FA/CDs complexes on the basis
of the comparison with other methods as well as with the pure gas-
phase stabilization results.
2.2.2. Isothermal Titration Calorimetry
ITC measurements were carried out at 298.15 K on a Microcal
OMEGA ultrasensitive titration calorimeter (MicroCal Inc.).
proper cyclodextrin and FA sodium salt were dissolved in water and
degassed prior to measurements. For -CD additional experiments
A
b
in 0.1 M Hepes buffer, pH 7.40 were performed. The solutions in the
cell were stirred at 400 rpm. Equal volumes of FA sodium salt so-
lutions were injected into the sample cell containing
CD over 20 s with an interval of 170 s between injections from a
250 l injection syringe, in a series of controlled pulses. The sample
a-CD/b- CD/g-
m
cell volume was 1.3611 cm3. The integrated heat effects of each
injection were corrected by subtraction of the corresponding in-
tegrated heat effects of folic acid salt injections into the pure buffer
and water and heat effects of buffer/water injections into the CD
solutions. Standard deviation was calculated by the model of a
single set of identical sites (ITC Tutorial Guide).
2. Experimental
2.1. Materials
Folic acid was commercially available product (obtained from
2.2.3. Mass spectrometry measurements
Sigma Aldrich, Germany) and was in a form of sodium salt.
a, b and
MS measurements were carried out using a 4000 Q TRAP
(Applied Biosystems Inc, USA), equipped with an electrospray (ESI)
ion source (TurboIonSpray), the triple quadrupole/linear ion trap
mass analyzer and coupled to High-Performance Liquid Chro-
matograph Prominence LC-20 (Shimadzu). The analysis was carried
out in the negative ion mode. The ion source parameters were
tuned for the maximum response of the noncovalent complexes of
[CDs/FA ꢀ 2H]2ꢀ and were set at the following values: ion spray
voltage (IS) 4500 V, declustering potential (DP) 50 V, entrance
potential (EP) 14 V, ion source temperature 300 K. Samples were
g
cyclodextrins were obtained from Cyclolab (Hungary) and used as
received. HEPES was obtained from Roth. Distilled water was used
for aqueous solutions. D2O (99.9% D) was purchased from Cam-
bridge Isotope Laboratories.
2.2. Methods
2.2.1. Nuclear Magnetic Resonance (1H NMR)
The 1H spectra were recorded on Varian Mercury 400. All
measurements were performed in D2O. Chemical shifts are re-
ported in ppm. The splitting pattern of multiplets is described by
abbreviations (s e singlet, d e doublet, dd e doublet of doublets, m
e complex multiplicity).
infused into the mass spectrometer at injection volume of 10 ml
using HPLC system through autosampler with the flow rate of
0.2 ml/min, H2O:MeOH (1:1) of mobile phase. The spectra were
recorded in the MRM ion mode at unit mass resolution. The

116
M. Ceborska et al. / Journal of Molecular Structure 1109 (2016) 114e118
instrument was controlled and recorded data were processed using
Analyst v. 1.4.2 software package (Applied Biosystems Inc, USA).
Samples were prepared in the following manner: Stock solutions
of CDs and FA salt were prepared in water at concentrations of
0.1 mM and 1.6 mM, respectively. The calibration curve for FA salt
was prepared at a concentration range of 0.008e0.22 mM. Each
concentration was prepared in triplicate. The calibration curve was
obtained as a plot of peak areas of [FA ꢀ H] against the concentration
of FA. In the binding studies the concentration of a given cyclodex-
trin was 0.1 mM, while the concentrations of FA salt were increasing
from 0 to 0.2 mM. Unbound FA concentrations were calculated from
the calibration curve. Bound FA concentrations, equal to the con-
centrations of CD/FA complex were taken as the difference between
total and free FA concentrations. Each concentration in binding
titration studies was analyzed in triplicate and the average values
from those measurements were taken to build the Scatchard plots.
The association constants (Ka) of CD/FA complexes were determined
from the slope of the Scatchard plots [14], while the number of
ligand binding sites were estimated from their intercepts.
found in liquid crystalline phases [15] it is important to learn more
about this process as it may have an impact on the complexation
with cyclodextrins. Abu Khaled and Krumdieck [16] used 1H NMR
for studying self-association of FA, dihydrofolic acid (DHFA) and
5,6,7,8-tetrahydrofolic acid (THFA) in phosphate buffer in/without
the presence of KCl, proving the formation of the proper dimers at
low concentrations and polymeric structures at higher concentra-
tions. Association constants values were much lower upon addition
of KCl (158 0.8, 17.6 1.1 and 2.8 1.3 for all the studied com-
pounds respectively). We studied this process for the folic acid
sodium salt in D2O following subsequent changes of chemical shifts
of FA salt protons upon dilution (changes in 1H NMR signal chem-
ical shifts of protons of folic acid sodium salt upon addition of D2O
are presented in Fig. 3). The formation of dimers was observed and
dimerization constant was calculated. Its value, 6.6
0.3 differs
significantly from this obtained by Abu Khaled and Krumdieck,
which is probably caused by different experimental conditions.
Obtained value was later included in calculations of association
constants of native cyclodextrins/FA complexes.
In 1H NMR the formation of the inclusion complexes results in
the shift changes for protons of the host molecule taking part in the
complexation process, as well as for the protons of the included
guest molecule. In general, the formation of inclusion complexes of
native CDs causes upfield shifts of the H-3 and H-5 protons of
cyclodextrin, located inside the cavity. The protons located outside
the cavity exhibit negligible changes. Guest protons, especially
those taking part in the complexation, also show shift changes. 1H
3. Results and discussion
3.1. Stoichiometry of the complexes
In our previous works we established formation of 1:1 com-
plexes in solution and in the gas phase [10,11]. Previously deter-
mined stoichiometry of FA/CD complexes was further confirmed by
ITC and ESI MS experiments. The initial model, used for ITC data,
assuming one type of binding sites, demonstrated that 1.08 0.05
NMR spectra of the mixtures of FA sodium salt and a, b and g-CDs
clearly indicate the formation of the complexes between native CDs
and FA. The association constants of the formed complexes were
determined by a 1H NMR titration technique in D2O. For the cal-
culations changes of all shift changes, both for host and guest
molecules of FA were bound per
molecules of FA were bound per
b
-CD molecule and 0.96 0.07
g
-CD. The effects measured for
a-
CD were too small to observe and thus, in this case, further analysis
was impossible. ESI MS titrations (see Schatchard plots, Fig. 2)
allowed for conformation of approximately 1:1 stoichiometry in FA/
protons were taken into consideration. For
a-CD/FA the changes of
three protons belonging to -CD and four belonging to the guest
a
b
-CD and FA/
of FA/ -CD complexes (exact results are as follows: 1.0 for
for -CD, and 0.92 for -CD).
g
-CD complexes, additionally giving the stoichiometry
a
a
-CD, 1.2
b
g
3.2. Formation of the complexes and comparison of 1HNMR and ITC
for determination of association constants
Folic acid and its salts are known to form aggregates in solution.
Although they are rather weak, especially compared to aggregates
Fig. 3. a) Molecular structure of folic acid b) changes in 1H NMR signal chemical shifts
of protons of folic acid sodium salt upon addition of D2O (a e green triangle, b e plain
triangle, c e plain square, d e green square). (For interpretation of the references to
colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 2. Scatchard plots of the ESI MS titration results for binding of FA to cyclodextrins
(circle e b-CD/FA, plain circle e a-CD/FA, and plain triangle e g-CD/FA).

M. Ceborska et al. / Journal of Molecular Structure 1109 (2016) 114e118
117
Table 1
molecule were analyzed. The biggest shift changes appeared for
The values of complex association constants (Ka) as measured by 1H NMR and ITC.
cyclodextrin proton located at outer face at narrow rim e H-6 and
aromatic protons of FA. For b- and g-CD/FA complexes also three
Association constant Ka [Mꢀ1
]
1H NMR (D2O)
ITC (water)
ITC (HEPES)
protons belonging to host and five belonging to the guest molecule
were analyzed. In both complexes the biggest shift changes
appeared for inner surface cyclodextrin protons H-3 and H-5 and
for aromatic protons of the guest molecule (changes in 1H NMR
signal chemical shifts are presented in Fig. 4). The solution of the
host cyclodextrin in D2O was titrated by the solution of the FA so-
dium salt containing certain amount of the proper cyclodextrin in
order to keep its concentration constant during the titration. The
corresponding titration curves are consistent with a 1:1 binding
a-CD/FA
b-CD/FA
g-CD/FA
<5
118 11
17
e
e
162 13
56 26
136 11
e
2
model for all the experiments presented. For a-CD/FA the observed
changes were significantly smaller than for the other two, larger
cyclodextrins resulting in a very low stability constant <5. The
biggest association constant was calculated for
b-CD/FA (118 11),
with relatively smaller value for -CD/FA complex (17 2).
g
The association constants were also determined by ITC experi-
ments and later compared with values obtained by 1H NMR
(Table 1). ITC experimental data were analyzed on the basis of the
model of a single set of identical sites (ITC Tutorial Guide). The
obtained results of the ITC experiments are given in Fig. 5 for the
CD/FA inclusion complexes.
b-
It can be seen from both 1H NMR and ITC experiments (Table 1)
that complexes with
b-cyclodextrin are the strongest. Complexes
with -cyclodextrin are very weak, which may be caused by the
a
character of the obtained complex, exclusion instead of inclusion
compound, where the cavity of the host compound is empty, as it is
too small to be penetrated by the molecule of folic acid. For the
strongest system the formation of the complexes was also studied
in physiological buffer (HEPES), for which association constant did
not differ significantly from the value obtained in pure water.
3.3. Formation of the complexes and determination of association
constants studied by ESI MS
Previous MS studies were aimed at determination of the gas-
Fig. 5. ITC results for titration of a b-CD water solution against FA sodium salt aqueous
solution.
phase stability order of CDs/FA complexes [11] and their detailed
structural analysis with the use of IM-MS and theoretical calcula-
tions [12]. In this manuscript, the noncovalent interaction strength
of CDs/FA complexes has been measured for solution-phase by
using MS as a detector. The association constants (Ka) of CD/FA
complex formation, based on the titration experiments with con-
stant concentration of cyclodextrin and increasing concentration of
FA (for a detailed experimental description, please see the experi-
mental part), were evaluated and compared with that obtained by
NMR and ITC.
A calibration curve obtained for the FA was linear from 0.008 to
0.22 mM (Fig .6).
The Scatchard plots of the results from the titration binding
studies involving
approximately 1:1 stoichiometry in CD/FA complexes (1.0 for
1.2 for -CD, and 0.92 for -CD). The association constants (Ka)
a
-CD,
b
-CD or
g-CD with FA (Fig. 2) indicated
a-CD,
b
g
determined from the slopes of the Scatchard plots are presented in
Table 2. It is worth mentioning that the ESI MS value of Ka obtained
for b-CD/FA complex is higher than both reported values obtained
by capillary electrophoresis coupled with UV detection and mass
spectrometry (Table 2) [17]. However it is difficult to refer to these
earlier reported values since the measurement conditions differed
Fig. 4. Changes in chemical shifts of folic acid and
therms for titration of -CD with FA (for FA: a e blue diamond, b e blue triangle, c e
plain triangle, d e blue circle, e e blue square; for -CD: H-1 egreen triangle, H-3 e
green square, H-5 e plain square). (For interpretation of the references to colour in this
figure legend, the reader is referred to the web version of this article.)
b-CD and calculated binding iso-
b
b

118
M. Ceborska et al. / Journal of Molecular Structure 1109 (2016) 114e118
stability of the complexes in solution increases in the series:
a-CD/
FA < -CD/FA < -D/FA confirmed by all three methods used. The
g
b
obtained series differs from obtained previously from preliminary
gas phase experiments and also from theoretical calculations,
which indicated the formation of the strongest complex for the
biggest g
-CD. The 1H NMR and ITC gave comparable results in re-
gard to association constant values, while results obtained for MS
measurements were considerably higher due to different in-
teractions (electrostatic instead of hydrophobic) responsible for the
stabilization of the CD/FA complexes.
Acknowledgments
This work was financed by the Polish Ministry of Science and
Higher Education, Grant No. IP2012007472.
Fig. 6. Linear regression analysis of the calibration curve for FA.
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Table 2
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ESI MS
ACE-UV [17]
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The complexation of folic acid with native cyclodextrins was
studied in detail and was treated as a model process for the com-
parison of 1H Nuclear Magnetic Resonance, Isothermal Titration
Calorimetry and Mass Spectrometry techniques for the evaluation
of association constants of the formed complexes in water. The