Characterization of β-cyclodextrin inclusion complex with procaine hydrochloride by 1H NMR and ITC

Article abstract of DOI:10.1007/s10847-013-0350-x

The inclusion of local anesthetic drug procaine hydrochloride by β-cyclodextrin was investigated by 1D and 2D proton NMR spectroscopy and isothermal titration calorimetry (ITC) at 298 K. The stoichiometry of the complex was determinate by the method of co

Full text of DOI:10.1007/s10847-013-0350-x

J Incl Phenom Macrocycl Chem
DOI 10.1007/s10847-013-0350-x
ORIGINAL ARTICLE
Characterization of b-cyclodextrin inclusion complex
with procaine hydrochloride by H NMR and ITC
1
•
Adrian Pırnau Mihaela Mic Mircea Bogdan
•
•
ˆ
Ioan Turcu
˘
Received: 2 April 2013 / Accepted: 29 June 2013
Ó Springer Science+Business Media Dordrecht 2013
Abstract The inclusion of local anesthetic drug procaine
hydrochloride by b-cyclodextrin was investigated by 1D
and 2D proton NMR spectroscopy and isothermal titration
calorimetry (ITC) at 298 K. The stoichiometry of the
complex was determinate by the method of continuous
variation, using the chemical induced shift of both host and
guest protons. The association constant K, of the obtained
an aromatic residue. They are classified in ester types and
amide types; it depends on the group that binds to the
aromatic residue. The nature of this bond determines sev-
eral pharmacological properties for these drugs [1].
Procaine hydrochloride, also called Novocaine, is a
synthetic organic compound used in allopathic medicine as
well as in neural therapy. Generally used in a 1–10 %
saline solution, procaine hydrochloride is administered by
injection for infiltration (area flooding as in dental anes-
thesia), nerve-block, spinal, and caudal anesthesia. Unlike
cocaine, procaine is not toxic, addicting, or irritating. It has
been displaced somewhat by the chemically related drugs
lidocaine and mepivacaine, which produce prompter, more
intense anesthesia.
complex was calculated and found to be 293.17 M-1
.
Rotating frame NOE spectroscopy, was used to ascertain
the solution geometry of the host–guest complex. The
result reveals that the procaine molecule penetrates into the
b-cyclodextrin cavity with the aromatic ring. The ener-
getics of complexation process is investigated by ITC
technique. The analysis indicates that the complexation of
procaine by b-CD is an exothermic process and show that
both enthalpy and entropy contribute to the binding pro-
cess. The obtained value for the association constant is in
good agreement with that obtained from NMR.
Cyclodextrins (CDs) are cyclic oligosaccharides con-
taining six (a-CD), seven (b-CD) or eight (c-CD) a-1,
4-linked glucopyranose units with a hydrophilic hydroxyl
group on their outer surface and a hydrophobic cavity in the
center. The hydrophilic exterior of the CD molecules can
make them water soluble, but the hydrophobic cavity pro-
vides an environment for appropriate sized non-polar mole-
cules. In aqueous solution CDs are capable of forming
inclusion complex with many molecules by taking up a
whole molecule or some part or it, into the cavity. These non-
covalent complexes offer a variety of physicochemical
advantages over uncomplexed molecules including increased
water solubility and stability.
Keywords Inclusion complex ꢀ Molecular interaction ꢀ
Nuclear magnetic resonance ꢀ Isothermal titration
calorimetry
Introduction
Local anesthetics are amphiphilic molecules that have
hydrophobic and hydrophilic domains that are separated by
an intermediate alkyl chain. The hydrophilic group can be
tertiary or secondary amine, and the hydrophobic domain is
The procaine:b-CD system was also investigated by
other spectroscopic or thermodynamic methods and the
authors [2–4] obtained different values for the association
constants in the range 200–350 M-1
.
We expect that a regional administration complexed
anesthetic with b-CD improve the delivery process, leading
to a larger duration of action and an improvement of ther-
apeutic index. This inclusion complex has a strong clinical
ˆ
˘
A. Pırnau ꢀ M. Mic (&) ꢀ M. Bogdan ꢀ I. Turcu
National Institute for Research and Development of Isotopic
and Molecular Technologies, 400293 Cluj-Napoca, Romania
e-mail: mihaela.mic@itim-cj.ro
123

J Incl Phenom Macrocycl Chem
application by the possibility to use procaine in anesthesia
in vivo. Recent experimental works with several local
anesthetics highlight the interest of such and approach [5–7].
spectra were acquired in the phase sensitive mode and
residual water suppression, using Bruker standard param-
eters (pulse program roesyphpr). Each spectrum consisted
of a matrix of 8 K/4 K data points covering a spectral
width of 4,000 Hz. The spectra were obtained with a spin-
lock mixing time of 500 ms, relaxation delay 3 s and 8
scans were recorded.
Experimental
1
In this paper, we report a H NMR and ITC studies of the
The isothermal titration calorimetric experiments were
carried out on a Nano ITC2G calorimeter (TA Instruments,
New Castle, Delaware, USA) at 298 K next to the electri-
cally and chemically calibration. The volume of the titration
and reference cells was 1 ml. Small aliquots of a solution are
injected under computer control into the titration cell at pre-
defined time intervals [8, 9]. The recorded quantity is the
rate of heating as a function of time. The individual pulses
are integrated to obtain plots recording the ratio of heat to
the amount of injected molecules.
inclusion complex formed between procaine (Fig. 1a) and
b-CD (Fig. 1b), in aqueous solution. Analysis of our data
by the continuous variation method confirms that the
inclusion occurs and the complex has 1:1 stoichiometry.
The association constant was calculated by a non-linear
least squares regression analysis of the observed chemical
shift changes of the procaine and b-CD protons as a
function of b-CD concentration.
The energetics of biochemical reaction at constant
temperature was measured by isothermal titration calo-
rimetry (ITC). A single well-designed experiment can
provide complete thermodynamic characterization of a
binding reaction, including the equilibrium constant K, the
Gibbs free energy DG, the reaction enthalpy DH, the
entropic effect DS and reaction stoichiometry (n).
1H NMR measurements
In order to study the complexation process between pro-
caine and b-CD in solution by NMR spectroscopy, two
stack solutions of the procaine and b-CD in D2O, both
having 10 mM were prepared. Based on these two equi-
molar solutions, a series of nine samples (i = 1–9) con-
taining both the procaine and b-CD molecules were
prepared. This was accomplished by mixing the two solu-
tions to constant volume at varying proportions, so that a
complete range (0 \ r \ 1) of the ratio r = [X]/
([A] ? [B]) was sampled. X = A or B and [A] and [B] are
the total concentrations of the host (b-CD) and guest
(procaine), respectively. Thus the total concentration
[A] ? [B] = [C] = 10 mM was kept constant for each
solution.
Materials
b-CD (water content 8 mol/mol) was purchased from
Sigma Chimie GmbH. Germany and procaine hydrochlo-
ride from Alfa Aesar GmbH & CoKG, Germany. Both
chemicals were used without any further purification.
Deuterium oxide (99.7 % D) was obtained from Heavy
Water Plant Romag-Prod, Romania.
Apparatus
For example, taking 0.3 ml for procaine solution and
0.7 ml for b-CD solution, we obtained 1 ml solution with:
1
The H NMR experiments were performed on a Bruker
Avance III, spectrometer operating at 500.13 MHz and
equipped with a broadband observe probe. The NMR
spectra were recorded in D2O solution at 298 K and all
chemical shifts were measured relative to TMS. For each
1H NMR experiment, between 16 and 256 transients were
collected into 65 K data points over a 5,000 Hz spectral
window, using a 2 s relaxation delay. The 2D ROESY
0:3ml ꢁ 10mM ¼ 1ml ꢁ A; ½Aꢂ ¼ 3mMprocaine
0:7ml ꢁ 10mM ¼ 1ml ꢁ B; ½Bꢂ ¼ 7mM bꢃCD
and ½Aꢂ þ ½Bꢂ ¼ 10 mM
The same set of samples was used both for the
determination of stoichiometry and association constant.
Fig. 1 Chemical structure of
a procaine and b b-CD
123

J Incl Phenom Macrocycl Chem
Isothermal titration calorimetry
Results and discussions
1
Determination of the stoichiometry by H NMR
Calorimetric titration experiments were carried out at
constant temperature by injecting 90 mM procaine
hydrochloride solution into the sample cell containing
9 mM b-CD, keeping the stirring speed fixed at 250 rpm.
From a computer controlled 250 ll Hamilton syringe,
aliquots of 5 ll have been injected with a 1600 s interval
which was sufficiently long for the signal to return to the
baseline and to ensure the equilibrium for the system. The
heat associated with each injection was calculated by
integration of the peaks in the heat flow curve using the
calorimeter software. Dilution heat of the host was
measured under identical experimental conditions by
injecting the procaine into water. The blank effects were
subtracted in order to correct for dilution, mixing and
injection effects.
NMR is a technique which provides the most evidence of
inclusion of a guest molecule into the hydrophobic CD
cavity in solution. The inclusion of procaine in b-CD is
shown by the change in the chemical shift of some guest
procaine and host (b-CD) protons (in the mixture), in
comparison with the chemical shifts of the same protons in
the free components. Partial 1H NMR spectra of pure
components and procaine:b-CD mixture in a 1:1 molar
ratio is presented in Figs. 2 and 3.
The absence of new peaks that could be assigned to the
complex suggested that complexation is a dynamic process,
the included procaine being in a fast exchange between the
free and bound states.
1
Fig. 2 Partial H NMR
spectrum of a 10 mM b-CD and
b 5 mM b-CD and 5 mM
procaine
H6
H3
H2
H4
H5
(a)
H4
H2
H3
H6
Hh, Hf
H5
(b)
ppm
3.9
3.8
3.7
3.6
3.5
3.4
3.3
1
Fig. 3 Partial H NMR
spectrum of a 10 mM procaine
and b 5 mM procaine and
5 mM b-CD
(a)
Hb, Hb’
Ha, Ha’
(b)
7.9
7.8
7.7
7.6
7.5
7.4
7.3
7.2
7.1
7.0
6.9
6.8
6.7
ppm
123

J Incl Phenom Macrocycl Chem
6.8
6.7
6.6
6.5
this parameter well occur at r = n/(m ? n), where m and n
are, respectively, the proportions of procaine and b-CD in
the complex (procaine)m:(b-CD)n. The calculated quantities
Dd[bCD] are proportional to the concentration of the com-
plex [10], and can be plotted against r. The resulting con-
tinuous variation plots demonstrate that because r has a
maximum value of 0.5 and highly symmetrical shapes
(Fig. 5), the complex has 1:1 stoichiometry.
H3
H5
Ha, Ha'
3.9
3.8
3.7
3.6
3.5
The induced chemical shift variation, Dd is defined as
the difference in chemical shifts in the absence and in the
presence of the other reactant for a given ratio r.
ROESY experiments
0
2
4
6
8
10
While the 1D NMR provides unambiguous evidence on the
formation of a complex, ROESY experiments provide
information on the dynamics and the averaged relative
inter- and intramolecular proton distances. In the present
study, to gain further information on the inclusion com-
plexation mode and additional insights into the dynamic
[X] (mM)
Fig. 4 Chemical shifts variation of some representative b-CD and
procaine protons as a function of their concentration, ([b-CD] ?
[Proc] = 10 mM), where [X] = [Proc] or [b-CD]
1
The H chemical shifts variation for H3, H5 protons of
1
structure, a 2D ROESY H NMR spectrum was acquired.
the b-CD and Ha, Ha’ protons of the procaine as well as a
function of their concentration is presented in Fig. 4.
The stoichiometry of the complex must be calculated
before proceeding with any association constant calcula-
tions. Determination of the stoichiometry of the procaine-
bCD complex, by the continuous variation method was
Due to the rapid dynamics of the complexation process, the
ROESY effects were only quantitatively used and no
conclusions on intermolecular distances were extracted. An
expansion of the ROESY spectrum of the procaine-bCD
complex is reported in Fig. 6.
The 2D NMR spectrum shows several intermolecular
cross-peaks between H3, H5 and H6 protons of b-CD and
the protons of aromatic ring of procaine Ha, Ha’, Hb and
Hb’. In fact the strong correlations were found between Ha,
Ha’ from procaine and H5, H6 of b-CD. Another strong
interaction is observed between Hb, Hb’ of the procaine
and H5, H3 of the CD. These interactions indicate that the
1
based on H NMR spectra obtained for procaine and b-CD
mixtures in which the initial concentrations of the two
species were maintained constant and the ratio r varied
between 0 and 1 (see procedure). A physical parameter
directly related to the concentration of the complex (in our
case the chemical shift, d) is measured under these condi-
tions and plotted as a function of r. The maximum value for
0.6
Ha, Ha'
H5
H3
0.5
0.4
0.3
0.2
0.1
0.0
0.0
0.2
0.4
0.6
0.8
1.0
r
Fig. 5 Concentrations variation plot for: H3 and H5 protons of
b-cyclodextrin; Ha and Ha’ protons of procaine, where [X] = [Proc]
or [b-CD]
Fig. 6 Expanded region of the ROESY spectrum of procaine:b-CD
complex. [proc] = 4 mM; [b-CD] = 6 mM
123

J Incl Phenom Macrocycl Chem
The treatment of the whole set of protons studied yields
one single K value characterizing the inclusion process and
a set of calculated Ddcði;jÞ values. The software is quite
flexible since it allows observing the chemical shift varia-
tion of the host, guest or both molecules as a function of
variable guest or host concentrations.
H6
H3
Ha
H5
Hb
HO
HO
Ha’
In our case, we applied Eq. (1) for a set of protons
consisting in H3 and H5 of b-CD and Ha, Ha’ of procaine.
The association constant obtained using the above descri-
bed procedure is K = 293.17 M-1 with E = 4.28 9 10-5
and a correlation factor R = 0.9997.
H6
H5
Hb’
H3
Preliminary indications of the solution structure of the
complex can by derived from the proton chemical shift
analysis. For b-CD, the observed largest chemical shift
changes with increasing procaine concentration were those
exhibited by H3 and H5 (up-field shift) of 0.110 and
0.187 ppm respectively. Since H3 and H5 face the cavity
of b-CD, their chemical shift changes are most likely due
to the presence of the methoxybenzaldehyde moiety in the
cavity. On the other hand, the largest chemical shift vari-
ation observed for procaine protons (up-field shift) as a
function of b-CD concentration occurs at position Ha and
Ha’ of 0.134 ppm.
Fig. 7 Structure of proposed procaine:b-CD complex inclusion
geometry
procaine molecule is included with aromatic ring into the
CD cavity.
Based on these findings, the geometrical structure of the
procaine:b-CD inclusion complex, having 1:1 stoichiome-
try, can be schematically presented as shown in Fig. 7.
1
Determination of the association constant by H NMR
In order to determine the extent of the intermolecular
binding between procaine and b-CD, the association con-
stant has been evaluated. The association constant, K for a
1:1 complex can be determined according to the following
equation [11]:
Determination of the association constant by ITC
The equilibrium in guest–host reaction is described by the
following hypothetical scheme:
8
9
"
#
1=2
ꢀ
ꢁ
A þ nB $ ABn
2ꢃ4½AꢂðiÞ½BꢂðiÞ
<
=
DdðcjÞ
2½Xꢂ
1
1
Ddði;jÞ
¼
½Cꢂ þ
ꢃ
½Cꢂ þ
ð4Þ
½ABnꢂ
:
;
K
K
K ¼
n
½Aꢂ½Bꢂ
ð1Þ
where i counts the sample number and j the studied proton.
where [A], [B], and [ABn] stand for the concentrations of
procaine, b-CD and b- CD-procaine complex respectively.
In an ITC experiment the reaction heat per mole of
injected molecules after the ith injection is given by:
If the studied proton belongs to the guest or host molecule,
X = A or B respectively. DdðciÞ represents the chemical
shift difference (for a given proton) between the free
component and the pure inclusion complex. Equation (1)
involves no approximations and correlates the total
concentrations of the guest and host molecules with the
observed difference in the chemical shift:
2
3
qi
DH
1 ꢃ ri=n ꢃ ðr0 þ riÞ=nKB0
ð1 þ ri=n þ ðr0 þ riÞ=nKB0Þ ꢃ4ri=n
6
4
7
5
qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
¼
1 þ
vB0
2
2
ð5Þ
B0
A0
Bi
where r0 ¼
;
ri ¼ ¼ r0 iv , A0 and B0 are the initial
Ddði:jÞ ¼ dðfri;ejeÞ ꢃ dði;jÞ
ð2Þ
Ai
V
obs
concentrations of procaine and of b-CD respectively, DH is
the reaction enthalpy, V is the volume of reaction cell and v
is the small volume injected at each titration step [13].
The software supplied with the calorimeter was used to fit
thermodynamic parameters K and DH to the heat profiles.
The equilibrium constant obtained from ITC measure-
ment was K = 288.70 14.51 M-1 which corresponds to
a Gibbs free energy DG = -3.35 0.02 kcal/mol. The
reaction enthalpy of the host–guest inclusion complex is
DH = -2.95 0.09 kcal/mol. This value is small and
We used a computer software [12] based on an iteration
procedure following specific algorithms in order to fit the
experimental values of Dd(i,j) to the appropriate equation.
Each iteration sets up a quadratic software to determine the
direction of search and the error function:
X
until the search converges.
ðDdði;jÞ ꢃ Ddðcia;jlÞc
Þ
ð3Þ
2
E ¼
i;j
123

J Incl Phenom Macrocycl Chem
with b-CD have shown that the stoichiometry of the host–
guest complex is 1:1 (n = 0.831).
(a)
0
-5
Conclusions
-10
-15
-20
-25
The Procaine-CD system in aqueous solution has been
studied by 1H NMR. Analysis of our data by the continuous
variation method indicates that the inclusion occurs and the
complex has 1:1 stoichiometry.
The complexation-induced chemical shift of H3 and H5
protons of b-CD are those expected as a result of interac-
tion with those protons of the procaine Ha, Ha’ Hb and Hb’
which are oriented towards the b-CD cavity, thus con-
firming a procaine/b-CD interaction. The association con-
stant K for the inclusion complex was evaluated from the
observed difference in chemical shifts for procaine and
b-CD protons.
0
20000
40000
60000
80000
Time (second)
(b)
0.0
The ROESY experiment indicates that the procaine
molecule is included with aromatic ring into the CD cavity.
The microcalorimetry allowed us to study inclusion
reaction between b-CD and procaine and to determine all
the thermodynamic parameters of the CD complexation.
For this type of inclusion complex, the association
-0.5
-1.0
-1.5
-2.0
1
constant K obtained by H NMR and ITC are in good
agreement and both methods sustain a 1:1 stoichiometry.
Acknowledgments This work was financially supported by UEFI-
SCDI Romania, Project PCE-2011-3-0032.
0.0
0.5
1.0
1.5
2.0
2.5
3.0
References
Molar ratio
Fig. 8 Calorimetric titration of b-CD with procaine. a Raw data
obtained from 48 injections of 5 ll aliquots of 90 mM Proc into
9 mM b-CD at 25 °C. b The incremental heat/mol of added ligand as
a function of molar [Proc]/[b-CD] ratio. The solid line is obtained by
fitting the experimental data
1. Torres, D.R., Blanco, L.H., Vargas, E.F., Martinez, F.: Calori-
metric enthalpies of solution for lidocaine-HCl and procaine-HCl
in water at 298.15 K. J. Chem. Eng. Data 54, 786–790 (2009)
2. Merino, C., Junquera, E., Jimenez-Barbero, J., Aicart, E.: Effect
of the presence of b-cyclodextrin on the solution behaviour of
procaine hydrochloride: spectroscopic and thermodynamic stud-
ies. Langmuir 16, 1557–1565 (2000)
negative, which point out that the formation of host–guest
inclusion complexes is a weak exothermic process. These
features are usually found [2, 14] for association between
small guest molecules and an apolar cavity in water. The
entropic effect of the complexation process is DS =
1.34 0.38 cal/molꢀK and bring an additional contribution
to the negative Gibbs free energy. The negative sign of the
enthalpy change DH \ 0 combined with the opposite sign of
entropy change DS [ 0 confirm that both hydrophobic and
electrostatic interactions contribute to the binding process.
The analysis of the thermodynamic data reveals that the
driving force of the binding process is a hydrophobic
interaction with the released water molecule from the b-CD
cavity, which has positive contribution to entropy (Fig. 8).
Calorimetric measurements of the interaction of procaine
3. Iglesias, E.: Inclusion complexation of novocaine by b-cyclo-
dextrin in aqueous solutions. J. Org. Chem. 71, 4383–4392 (2006)
4. Takisawa, N., Shirahama, K., Tanaka, I.: Interactions of amphi-
philic drugs with a-, b-, and c-cyclodextrins. Colloid Polym. Sci.
271, 499–506 (1993)
5. Fraceto, L.F., Pinto, L.M.A., Franzoni, L., Braga, A.A.C., Spisni,
A., Schreier, S., de Paula, S., de Paula, E.: Spectroscopic evi-
dence for a preferential location of lidocaine inside phospholipid
bilayers. Biophys. Chem. 99, 229–243 (2002)
6. Pinto, L.M.A., Fraceto, L.F., Santana, M.H.A., Pertinhez, T.A.,
Oyama Junior, S., de Paula, E.: Physico-chemical characteriza-
tion of benzocaine-b-cyclodextrin inclusion complexes. J. Pharm.
Biomed. Anal. 39, 956–963 (2005)
´
7. Moraes, C.M., Araujo, Issa, M.G., Ferraz, H.G., Yokaichiya, F.,
Franco, M.K.K.D., Mazzaro, I., Lopes, P.S., Gonc¸alves, M.M., de
Paula, E., Fraceto, L.F.: Inclusion complex of S(-) bupivacaine
and 2-hydroxypropyl-b-cyclodextrin: study of morphology and
cytotoxicity. J Basic Appl. Pharm. Sci. 27, 207–212 (2006)
123

J Incl Phenom Macrocycl Chem
8. Pierce, M.M., Raman, C.S., Nall, B.T.: Isothermal titration calo-
rimetry of protein–protein interactions. Methods 19, 213–221 (1999)
9. Sun, D.-Z., Li, L., Qiu, X.-M., Liu, F., Yin, B.-L.: Isothermal
titration calorimetry and 1H NMR studies on host–guest inter-
action of paeonol and two of its isomers with b-cyclodextrin. Int.
J. Pharm. 316, 7–13 (2006)
10. Loukas, Y.L.: Measurement of Molecular Association in Drug:
cyclodextrin Inclusion Complexes with Improved 1H NMR
Studies. J. Pharm. Pharmacol. 49, 944–948 (1997)
12. Floare, C., Balibanu, F., Bogdan, M.: CONSTEQ—a program for
the calculation of the equilibrium constants using spectroscopic
data, Studia Univ. Babes-Bolyai Physica. L4a: 451 (2005)
13. Fini, P., Castagnolo, M.: Determination of Enthalpic Interaction
Coefficients by ITC Measurements. 2-Hydroxypropyl-b-cyclo-
dextrin in aqueous solution of NaCl. J. Therm. Anal. Calorim. 66,
91–102 (2001)
14. Junquera, E., Aicart, E.: Potentiometric Study of the Encapsula-
tion of Ketoprophen by Hydroxypropyl-b-cyclodextrin. Tem-
perature, Solvent, and Salt Effects. J. Phys. Chem. B 101,
7163–7171 (1997)
11. Bogdan, M., Caira, M.R., Farcas, S.I.: Inclusion of the niflumic
acid anion in beta-cyclodextrin: a solution NMR and X-ray
structural investigation. Supramol. Chem. 14, 427–435 (2002)
123