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ROMANIAN ACADEMY
„Ilie Murgulescu” Institute of Physical Chemistry
THESIS ABSTRACT
THE STUDY OF THERMODYNAMIC STABILITY OF LIGAND –
CYCLODEXTRIN
MOLECULAR STRUCTURES. STRUCTURAL AND MORPHOLOGICAL ASPECTS
CORRELATED WITH THERMODYNAMIC DATA.
Scientific Advisor,
CS I Dr. Tănăsescu Speranţa Valeria
PhD Student,
Neacșu Dana-Andreea
BUCUREŞTI
2018
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Summary
1
I. Current state of research in the field 6
1. Introduction 7
2. Theoretical aspects related to phase equilibria
thermodynamics in "receptor-ligand"
systems
11
2.1 The phase rule 11
2.2 Thermodynamic functions for phase transformations in the
guest – cyclodextrin
molecular systems
11
2.3 Equilibrium conditions between phases 12
2.4 Thermodynamic theory involving First-Order Phase Transitions
13
2.5 Phase equilibria thermodynamics in one-component systems
14
2.6 Phase equilibria thermodynamics in multicomponent systems
15
3. Current state of research in the thermodynamics field of
cyclodextrine inclusion
compounds
18
3.1 Aspects involved in the cyclodextrine characterisation
18
3.1.1 General considerations, structure and properties of
cyclodextrins 18
3.2 Correlations between the structure and thermodynamics of
compound complexation 22
3.2.1 Basic interactions involved in the stability of complexes
23
3.2.2 Steric factors affecting the inclusion phenomena 24
3.2.2.1 Effect of ligand structure 24
3.2.2.2 Effect of cyclodextrin cavity 25
3.2.2.3 Effect of cyclodextrin cavity functionalization 25
3.2.3 Enthalpic and entropic contributions 26
3.2.3.1 Effect of enthalpy-entropy compensation 26
3.2.4 Influence of the reaction medium on the complexity
thermodynamics 27
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3.3 Methods of preparation widely used in the preparation of
inclusion compounds 28
3.4 Applications. Advantages of cyclodextrins and
cyclodextrin-ligand complexes 30
II. Experimental methodes 33
4. Techniques used for the characterisation of inclusion
complexes 34
4.1 Differential scanning calorimetry (DSC) and thermogravimetry
(TG) 34
4.1.1 The relative degree of crystallinity (RDC) 38
4.1.2 Determination of the purity of a substance 38
4.2 Isothermal titration calorimetry (ITC) 39
4.3 Spectral and morphological analysis 41
4.3.1 Ultraviolet–visible absorption spectroscopy (UV-VIS)
41
4.3.1.1 Stoichiometry determination 41
4.3.1.2 Binding constant determination 41
4.3.1.3 The obtaining of thermodynamic parameters from UV-VIS
spectroscopy
data using van’t Hoff analysis
42
4.3.2 Fourier transform infrared spectroscopy (FT-IR) 43
4.3.3 Scanning electron microscopy (SEM) 44
4.4 Other methodes involved in sample characterisation 44
4.4.1 Quantum chemistry calculations (QC) 44
4.4.2 pH measurements 44
III. Original contributions to the characterization of
ligand-cyclodextrin type systems 45
5. Thermodynamic characterization of cyclodextrins used in the
synthesis of complexes 46
5.1 Thermodynamic study of αCD, βCD, HPαCD, HPβCD, γCD 46
5.2 Conclusions 58
6. Comparative study of the inclusion compounds of uracil and
5-fluorouracil with
cyclodextrins
60
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6.1 General considerations. Study motivation 60
6.2 Experimental details and results from UV-Vis measurements
61
6.2.1 Materials and synthesis of inclusion compounds 61
6.2.2 Determination of stoichiometry of cyclodextrin-uracil and
cyclodextrin-5-
fluorouracil inclusion compounds by UV-Vis spectroscopy
measurements
62
6.3 The experimental study of inclusion compounds of the
cyclodextrin-uracil and
cyclodextrin-5-fluorouracil, in solid state
64
6.3.1 The thermodynamic study of inclusion compounds between
uracil/5-
fluorouracil and cyclodextrins
64
6.3.1.1 Experimental results obtained by TG/DTG 64
6.3.1.2 Experimental results obtained by DSC 68
6.3.2 Structural and morphological characterization of pure
compounds and inclusion
complexes
73
6.3.2.1 Analysis of the results obtained by FTIR 73
6.3.2.2 Morphological characterization by SEM 76
6.4 Theoretical study of inclusion compounds of the
cyclodextrin-uracil and
cyclodextrin-5-fluorouracil
78
6.4.1 Evaluation of inclusion compounds based on QC 78
6.5 Conclusions 88
7. Cyclodextrin inclusion compounds with L- and D- alpha-amino
acids stereoisomers 90
7.1 General considerations. Study motivation 90
7.2 Materials and synthesis of inclusion compounds 92
7.3 The thermodynamic study of pure amino acids and inclusion
compounds between the
optical isomers of arginine, histidine, tryptophan with
cyclodextrins
94
7.3.1 TG/DTG analysis of pure amino acids used in the synthesis
of complexes
formed with cyclodextrins
94
7.3.2 TG/DTG results for the inclusion compounds between the
optical isomers of
arginine and cyclodextrins
96
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7.3.3 TG/DTG results for the inclusion compounds between the
optical isomers of
histidine and cyclodextrins
99
7.3.4 TG/DTG results for the inclusion compounds between the
optical isomers of
tryptophan and cyclodextrins
102
7.3.5 DSC results for the inclusion compounds between the
optical isomers of
arginine and cyclodextrins
104
7.3.6 DSC results for the inclusion compounds between the
optical isomers of
histidine and cyclodextrins
108
7.3.7 DSC results for the inclusion compounds between the
optical isomers of
tryptophan and cyclodextrins
111
7.4 Structural and morphological characterization of the
inclusion compounds between
the optical isomers of arginine, histidine, tryptophan and
cyclodextrins
114
7.4.1 FT-IR spectra of the inclusion compounds between the
optical isomers of
arginine, histidine, tryptophan and cyclodextrins
114
7.4.2 SEM images of the inclusion compounds between the optical
isomers of
arginine, histidine, tryptophan and cyclodextrins
119
7.5 Correlations between thermodynamics, structure and
morphology of inclusion
compounds
122
7.6 Conclusions 124
8. The study of interaction between cichonine and cyclodextrins
125
8.1 General considerations. Study motivation. 125
8.2 Sample synthesis 127
8.3 Thermodynamic study of inclusion compounds between
cinchonine and cyclodextrins 128
8.3.1 TG/DSC results for pure cinchonine 129
8.3.2 TG/DTG results for the inclusion compounds of cinchonine
and beta-
cyclodextrin, at various molar ratios
130
8.3.3 TG/DTG results for the inclusion compounds of cinchonine
and 2-
hydroxypropyl-beta-cyclodextrin, at various molar ratios
132
8.3.4 TG/DTG results for the inclusion compounds of cinchonine
and gamma- 135
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cyclodextrin, at various molar ratios
8.3.5 DSC results for the inclusion compounds of cinchonine and
beta-cyclodextrin,
at various molar ratios
138
8.3.6 DSC results for the inclusion compounds of cinchonine and
2-hydroxypropyl-
beta-cyclodextrin, at various molar ratios
142
8.3.7 DSC for the inclusion compounds of cinchonine and
gamma-cyclodextrin, at
various molar ratios
141
8.4 Structural and morphological characterization of inclusion
compounds between
cinchonine and cyclodextrins
147
8.4.1 FT-IR spectra for the inclusion compounds of cinchonine
and cyclodextrins, at
various molar ratios
147
8.4.2 SEM imagines for the inclusion compounds of cinchonine and
2-
hydroxypropyl-beta-cyclodextrin, at 1:1 and 1:2 molar ratios
151
8.5 Correlations between thermodynamics, structure and
morphology of inclusion
compounds
152
8.6 Conclusions 156
9. Study of interaction between doxorubicin hydrochloride (Dox)
and γ-cyclodextrin 158
9.1 General considerations. Study motivation. 158
9.2 Sample synthesis 159
9.3 Experimental study of the complexity between Dox and γ-CD,
in liquid state 160
9.3.1 Dox/γCD analysis by UV-Vis spectroscopy 160
9.3.1.1 Experimental details about UV-Vis measurements 160
9.3.1.2 Analysis of UV-Vis spectra and determination of
stoichiometry 160
9.3.1.3 Determination of association constants and thermodynamic
parameters
for the Dox / γCD system
162
9.3.2 pH measurements and distribution of Dox microspecies
165
9.3.3 Dox / γCD system analysis by isothermal calorimetric
titration (ITC) 167
9.3.3.1 Experimental details related to ITC measurements 167
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9.3.3.2 Analysis of thermodynamic parameters obtained from ITC
168
9.3.4 Correlations between the thermodynamic parameters and the
driving
forces involved in the structure of the complex
170
9.4 Experimental study of the complexity between Dox and γCD, in
solid state 172
9.4. Thermodynamic study of inclusion compounds between Dox and
γCD 172
9.4.1.1 TG/DTG results 172
9.4.1.2 DSC results 173
9.4.2 Structural and morphological characterization of the Dox /
γCD system 175
9.4.2.1 FT-IR spectra 175
9.4.2.2 SEM images 177
9.5 Conclusions 178
10. General Conclusions. Original contributions 179
11. Perspectives of expanding and continuing research 182
Bibliography 183
List of scientific articles and communications 204
KEYWORDS: thermodynamics, stability, morphology, cyclodextrin,
cinchonine, uracil
1. Introduction
“Cyclodextrins are universal molecular recipients for: organic,
inorganic and organometallic
compounds, neutral, cationic, anionic or even organic radicals"
[1]. The main property of
cyclodextrins is to form complexes with almost any type of guest
molecule. Due to its remarkable
molecular architecture, cyclodextrins play an important role in
medicine, biology, pharmaceuticals,
food and consumer goods, with particular importance being
reflected in the number of papers and
patents dedicated to cyclodextrin and its inclusion
compounds.
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In the thesis were made original contributions regarding the
thermoanalytic study of some
complexes of organic molecules with cyclodextrins and their
thermal behavior was correlated with
the structural and morphological aspects involved in their
formation. In order to support the
information derived from the thermodynamic data, quantum
chemistry calculations were used to
obtain the energy parameters of the complexation interaction and
new structural and morphological
data were presented in relation to the studied complexes.
The motivation of the study is also related to other aspects,
such as:
- the need of new information on the structure, morphology and
quantum chemistry
calculations for ligand / cyclodextrin systems with biochemical
importance;
- the role of energy parameters in highlighting the correlation
between the ligand
structure and the properties of the complex obtained with
cyclodextrin;
- the importance of the studied systems, which can be models
within a series of
complexes obtained between cyclodextrins and ditopic
molecules.
The study in the frame of this thesis is based on the analysis
of the results obtained by
correlating several experimental methods, namely:
- Thermal analysis and calorimetry methods (Differential
Scanning Calorimetry - DSC,
thermogravimetric analysis - TG, isothermal titration
calorimetry - ITC).
- Structural and morphological characterization methods (Fourier
transformed infrared
spectroscopy measurements - FT-IR, scanning electron microscopy
- SEM, ultraviolet –
visible absorption spectroscopy - UV-Vis).
- Determination of pH.
- Quantum chemistry (QC) calculations.
In this study, a series of well-known amino acids and active
biochemicals have been selected
as guest molecules for which inclusion complexes with different
cyclodextrins [alpha-cyclodextrin
(αCD), beta-cyclodextrin (βCD),
2-hydroxypropyl-alpha-cyclodextrin (HPαCD), 2-
hydroxypropyl-beta-cyclodextrin (HPβCD), gamma-cyclodextrin
(γCD)] have been synthesized.
In the table below are listed the studied systems and the
methods of investigation used within this
thesis.
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Cyclodextrin type /
Ligand type
αCD βCD 2HPαCD 2HPβCD γCD Analysis methods
L-Arginine ● ● ● DSC, TG/DTG, FTIR, SEM,
D-Arginine ● ● ● DSC, TG/DTG, FTIR, SEM,
L-Histidine ● ● ● DSC, TG/DTG, FTIR, SEM,
D-Histidine ● ● ● DSC, TG/DTG, FTIR, SEM,
L-Tryptophan ● ● ● DSC, TG/DTG, FTIR, SEM,
D- Tryptophan ● ● ● DSC,TG/DTG, FTIR, SEM,
Uracil ● ● ● ● DSC, TG/DTG, FTIR, SEM, QC,
UV-Vis
5-Fluorouracil ● ● ● ● DSC, TG/DTG, FTIR, SEM, QC,
UV-Vis
Doxorubicin ● DSC, TG/DTG, ITC, FTIR, SEM,
UV-Vis, pH
Cinchonine ● ● ● DSC,TG/DTG, FTIR, SEM, UV-
Vis
Taking into consideration the approached field of study, this
thesis aimed to achieve the
following specific objectives:
1) Synthesis of inclusion complexes starting from the ligand and
the corresponding cyclodextrin,
using inexpensive and simple preparation methods.
2) Thermochemical characterization of pure substances from which
the synthesis of complexes was
started and determination of the corresponding thermochemical
parameters.
3) Thermodynamic and thermochemical characterization, stability
and essential thermal
characteristics of the synthesized complexes using appropriate
thermal analysis and calorimetry
methods.
4) Spectral analysis in order to check the formation of
inclusion complexes.
5) Morphological characterization of the complexes
6) Use of UV-Vis, QC analysis methods for the obtaining of
energetic parameters and
stoichiometry of complexes.
7) To highlight the influence of the reaction medium and of the
component structure on the process
of formation of inclusion complexes.
The PhD thesis is structured in 11 Chapters, included in three
main parts:
Ist part (chapters 1, 2 3) is devoted to general considerations
of phase equilibria
thermodynamics in receptor-ligand systems as well as to the
state of the art of cyclodextrin-
inclusion compounds thermodynamics.
IInd
part (chapter 4) is dedicated to the experimental methods and
techniques involved in
sample characterization.
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IIIrd
part (chapters 5, 6, 7, 8, 9, 10) of the thesis is devoted to
the original contributions to the
ligand-cyclodextrin systems.
At the end of the thesis are presented the general conclusions
and perspectives related to this
study, followed by the bibliographic references and the list of
scientific articles and communications
related to the thesis theme.
Chapter 1 presents a brief introduction that discloses
information on the importance of cyclodextrins
and inclusion complexes formed with cyclodextrins. The goal/aim,
the specific objectives and the
structure of the PhD thesis are presented.
Chapter 2 contains essential information related to the
thermodynamics of first-order phase
transitions. In this chapter are presented fundamental aspects
related to the thermodynamic functions
of the phase transformations involved in the guest-molecule –
cyclodextrin type systems. It is also
described based on thermodynamic equations approach, the theory
applied in determining the purity
of substances.
Chapter 3 contains generalities on the structure and properties
of cyclodextrins, as well as the
description of the most commonly methods in preparing inclusion
compounds. This chapter
provides the main information about driving forces and steric
factors that influence the structure and
thermodynamics of the complexation process.
Chapter 4 presents the equipments and analysis methods involved
in this thesis to study pure
components and synthesized complexes.
Chapter 5 contains the thermal analysis data obtained for pure
cyclodextrins (αCD, βCD, HPαCD,
HPβCD, γCD). The characterization of pure cyclodextrins is
imperiously needed in order to evaluate
the properties of CDs before preparing the complexes, as well as
to analyze their formation.
Chapter 6 presents the original contributions resulted from the
investigation of inclusion complexes
between the uracil or 5-fluorouracil guest molecules and
cyclodextrines. There are 16 systems
synthesized that were thermodynamically investigated using DSC /
TG. The complexation process
was validated by the FTIR and SEM methods. The stoichiometry of
the complexes was determined
before obtaining the solid powders, and quantum chemistry
calculations were performed to provide
the energetic parameters characteristic for the complexation
interaction.
Chapter 7 includes experimental data on the solid state study of
complexes formed between the
optical isomers of amino acids: arginine, histidine and
tryptophan with cyclodextrins.
Thermodynamic parameters of pure amino acids and complexes were
established by thermal
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analysis. Comparative discussions on the complexes stability
were performed. FTIR and
morphology data for all studied complexes and for the
corresponding pure amino acids were
provided. Taking into account the obtained data, the influence
of the various factors that determined
the thermodynamics of the complexation and the structure of the
complexes was discussed.
Chapter 8 presents the study of inclusion complexes between
cinchonine and cyclodextrins for
molar ratios 1: 1, 2: 1 and 1: 2 (ligand-cyclodextrin). Based on
thermal analysis data the
thermograms and the parameters corresponding to the thermal
effects were provided and the
stability of the complexes was discussed, the thermochemical
data being supported by the structural
and morphological analysis. Influence of structural factors and
of the reaction medium on the
complexity behavior were correlated.
Chapter 9 contains the original contributions resulting from the
analysis of the complex formed
between doxorubicin and γ-cyclodextrin, in solid and liquid
state. The stoichiometry of the solution
was investigated and the thermodynamic parameters were
determined by isothermal calorimetric
titration and UV-Vis spectroscopy data. The solid powder of the
complex was investigated by
thermal analysis and the corresponding thermograms and
thermochemical parameters were
presented. Based on the FTIR spectroscopy data, the complexation
porcess was evaluated. The
morphological analysis was also presented.
Chapter 10 and 11 outline the final conclusions and prospects
for expanding and continuing
research.
Original contributions to the characterization of
ligand-cyclodextrin type systems
2. Comparative study of the inclusion compounds of uracil and
5-fluorouracil with
cyclodextrins
The preparation of solid state inclusion compounds (U/αCD,
5FU/αCD, U/βCD, 5FU/βCD,
U/HPαCD, 5FU/HPαCD, U/HPβCD, 5FU/HPβCD) was done by “melting in
solution” method,
using 1:1 molar ratios of ligand-CD. The system stoichiometry
was determined by the continuous
variation method. Each system was equilibrated 24h, after that
the absorbance variation was
observed at 258nm for U systems and at 265nm for 5FU formed
systems respectively. In this
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experiment, stoichiometry was determined for 10-5
M aqueous solution and for each of the studied
systems, the stoichiometric ratio was 1:1.
These determinations were necessary to verify if the
stoichiometry of the diluted aqueous
solution correspond to the molar ratio of the systems
DSC thermograms of complexes formed between 5FU or U with
cyclodextrins show no
presence of thermal process attributed to the melting of the
ligand in presence of CD, thus for all the
studied complexes the existence of strong interactions between
the guest molecule and the CD
cavity took places, as indicated in Fig. 2.1.
50 100 150 200 250 300 350 400 450
Flu
x c
alo
ric
(m
W)
En
do
Temperatura (C)
CD
5FU/CD
5FU
U/CD
U
50 100 150 200 250 300 350 400 450
Flu
x c
alo
ric
(m
W)
En
do
Temperatura (C)
HPaCD
5FU/HPCD
5FU
U/HPCD
U
50 100 150 200 250 300 350 400 450
Flu
x c
alo
ric
(m
W)
En
do
Temperatura (C)
CD
5FU/CD
5FU
U/CD
U
50 100 150 200 250 300 350 400 450
Flu
x c
alo
ric
(m
W)
En
do
Temperatura (C)
HPCD
5FU/HPCD
5FU
U/HPCD
U
Fig. 2.1 DSC thermograms for U, 5FU, αCD, βCD, HPαCD, HPβCD and
synthesized inclusion
compounds.
Formation of complexes with functionalised cyclodextrins was
more effective than
formation of complexes with native cyclodextrins, and this
aspect could be highlighted by following
the decomposition temperature of the complexes, as shown in
Table 2 - 1.
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Tabelul 2 - 1
The order of decreasing the decomposition temperature of the
inclusion complexes formed between
U/5FU and cyclodextrins
Fig. 2.2 TG curves (continuous line) and DTG (dotted line) for
U, 5FU and for complexes formed
between U, 5FU and cyclodextrins.
The formation of inclusion complexes for all studied systems
took places and the analysis of
TG/DTG data (Figure 2.2) confirms the results obtained by DSC
[2].
Fig. 2.3 shows FTIR-ATR spectra for U, 5FU, cyclodextrins and
inclusion compounds. For
pure cyclodextrins, broadband present in the spectrum with the
maximum absorption at 3288 cm-1
for αCD, 3314 cm-1
for βCD, 3302 cm-1
for HPαCD and 3330 cm-1
for HPβCD is characteristic.
Complex
5FU/HPβCD>5FU/HPαCD>U/HPβCD>U/HPαCD>5FU/βCD>U/βCD>5FU/αCD>UαCD
T (°C) 299.2 262.1 260.7 258.7 254.1 253.2 252.1 239.4
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This band is attributed to the O-H stretching vibration in
symmetric and anti-symmetric modes and
is affected when the inclusion complex was formed [3]. For the
5FU/αCD, U/αCD, 5FU/HPαCD
and U/HPαCD systems there are no significant band changes at
approximately 3300 cm-1
. This band
is shifted much more for the complexes 5FU/HPβCDCD, U/HPβCD,
U/βCD and 5FU/βCD; it can
be said that ligand inclusion occurs with the involvement of a
larger number of hydrogen bonds [4].
Fig. 2.3 FT-IR spectra for: U, 5FU, cyclodextrins and inclusion
compounds formulated for 1: 1
molar ratio (ligand/CD).
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Quantum chemistry (QC) calculations were performed using the
GAMESS software package
facilities installed on an IBM cluster of 65 computing units.
The studied systems geometries were
optimized by using enhanced wave function sets: STO-3G, STO-6G,
n311-6G, and finally, zeta
triple valence (TZV) function [5, 6]. Quantum chemistry
calculations have shown that the results
obtained by applying DFT (density functional theory) confirm
that inclusion complexes have been
formed between the considered ligands (U/5FU) and the
cyclodextrins CD, CD, HPCD,
HPCD.
MPE energies (MPE = reciprocal distortion energy) are
represented for the position of the
guest molecule inside the CD cavity, for all HG structures
(CD/ligand) - Fig. 2.4. In Fig. 2.5, it was
observed for the R-configurations (the ligand molecule placed in
the center of the small opening of
the CD parallel to it), that the plane of the 5FU molecule
remained perpendicular to the Oz axis, thus
the molecule 5FU was strongly bound to the host molecule by
hydrogen bonds (Fig. 2.5).
Fig. 2.4 The variation of MPE corresponding to the position of
the guest molecule inside the CD
cavity for the HG assembly formed between: (left) - the U
molecule with CD, CD, HPCD,
HPCD and (right) - the 5FU molecule with CD, CD, HPCD, HPCD
In the case of H-5FU-R complexes, it can be seen (Figure 2.4)
that MPE (Mutual
Disturbance Energy) energies are significantly higher for HPαCD
and HPβCD. It can be argued that
5FU forms more stable complexes with modified cyclodextrins than
with native ones.
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Fig. 2.5 The final configurations of the H-5FU-R inclusion
complexes: a) CD-5FU-R, b) CD-
5FU-R, c) HPCD-5FU-R, d) HPCD-5FU-R
Based on the obtained results, it was found that:
- Both U and 5FU molecules interact weaker with αCD, and U
molecule interacts weaker than 5FU
molecule with HPβCD and βCD cyclodextrins.
- FTIR and DSC/TG data suggest inclusion complex formation of
all synthesized samples,
indicating the existence of interactions between the constituent
molecules of the inclusion complex.
- Considering calculated energies, QC results indicate higher
stability for complexes of 5FU with
modified cyclodextrins than for complexes of 5FU with native
cyclodextrins. The QC calculations
agreed with the results indicated by DSC analysis.
3. Cyclodextrin inclusion compounds with L- and D- alpha-amino
acids stereoisomers
The complexation process between the optical isomers of the
amino acids Arg, His and Trp
with αCD, βCD, HPaCD in the solid state was evaluated. It was
performed for the first time a
systematic analytical study of this type of complexes prepared
in 1:1 molar ratio by the co-
precipitation method [7]. Thermal analysis data showed that
LArg/βCD, DArg/βCD, LHis/βCD,
LTrp/βCD and DTrp/βCD are the most thermally stable. In the case
of the complexes: LArg/αCD,
LArg/HPαCD, DArg/HPαCD, LHis/αCD, LHis/HPaCD, DHis/αCD,
DHis/HPaCD, DHis/βCD,
LTrp/HPaCD have been made illustrations showing the variation of
the starting (Ton), transition (Tm)
and enthalpy (ΔH) temperatures corresponding to the melting
endotherm (Figures 3.1, 3.2 and 3.3).
Thus, comparisons could be made between these inclusion
complexes. The complex with the highest
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thermal stability could be determined, considering as criteria
the lowest value of ΔH and the highest
transition temperature.
Fig. 3.1 Variation of onset temperature, transition temperature
and enthalpy for the melting
endotherm of LArg/αCD, DArg/αCD, LArg/HPαCD
Fig. 3.2 Variation of onset temperature, transition temperature
and enthalpy for the melting
endotherm of complexes formed between His isomers and αCD, HPαCD
and for DHis/βCD
complex
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Fig. 3.3 Variation of onset temperature, transition temperature
and enthalpy for the melting
endotherm of complexes formed between Trp isomers and αCD,
HPαCD
Considering for the studied systems the correlation of the
resulted data, the following were found:
1) in the case of His, a higher affinity of cyclodextrins for
the levogir isomer is observed;
2) in the case of Arg, both isomers are preferred with higher
affinity than native cyclodextrins;
3) the most stable inclusion compounds are obtained between the
Trp and αCD isomers, βCD,
HPαCD, and the most energetically favored ones are those formed
with native cyclodextrins.
4) it can be concluded that the amino acids Arg, His and Trp
interact with αCD, βCD more strongly
than with HPαCD.
4. The study of interaction between cinchonine and
cyclodextrins
Solid form complexes formed between Cinc and cyclodextrins: βCD,
HPβCD, γCD for
molar ratios 1:1, 2:1 and 1:2 were synthesized by
co-precipitation method using a mixed water
solution of alcohol 50% Vol.
The differences regarding the thermal behavior of systems formed
between Cinc and native
βCD and γCD cyclodextrins, are shown in Fig. 4.1.
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Fig. 4.1 Variation of onset temperature, transition temperature
and enthalpy for the melting
endotherm of complexes formed between Cinc and cyclodextrins
considering the molar ratios of
1:1, 2:1 and 1:2 (guest:host)
DSC/TG analysis of the compounds formed between Cinc and HPβCD
showed the formation
of the complexes by partial inclusion of Cinc molecule into
HPβCD cavity, for the 1:1 and 1: 2
molar ratios (guest: host), (presented in Fig. 4.2 and 4.3).
220 240 260 280 300 320 340 360 380 400
HPCD
Cinc/HPCD(2:1)
Cinc/HPCD(1:2)
Cinc/HPCD(1:1)
Cinc
Flu
x c
alo
ric
(u
. a
.) E
nd
o
Temperatura (C)
Fig. 4.2 DSC curves for pure Cinc, HPβCD and
inclusion complexes formed from different molar
ratios.
Fig. 4.3 TG/DTG curves for inclusion
complexes between Cinc and HPβCD for
different molar ratios
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Based on thermal analysis data, it was found that:
• There is a possibility of favorable energy interactions
between the components of Cinc systems
and native cyclodextrins: βCD and γCD, for the 1:1 and 1:2 molar
ratios (guest:host), and for
Cinc/HPβCD(2:1), by partially inclusion of the two Cinc
molecules into the HPβCD cavity.
• The physical size of the CD cavity, the solvent and the CD rim
functionalization influence the
formation of inclusion complexes.
5. Study of interaction between doxorubicin hydrochloride (Dox)
and γ-cyclodextrin
The complexation process between Dox and γCD in the liquid and
solid state was evaluated,
performing for the first time a systematic thermodynamic study
of this interaction.
The non-covalent interaction between Dox and γCD was evaluated
in the liquid state using
pH measurements, UV-Vis spectroscopy and ITC measurements.
The pH of each Dox/γCD molar ratio was monitored when the
complex stoechiometry was
evaluated by the continuous variation method and the results are
shown in Fig. 5.1.
Fig. 5.1 Stoechiometry determination by the
continuous variation method for the Dox/γCD
system at 25 ° C (λ = 473 nm).
Fig. 5.2 Variation of temperature equilibrium
constant for Dox/γCD system.
The results showed for Dox/γCD inclusion complex the 1:1
stoichiometry. The data on the
thermodynamic processes that took place in the liquid state
confirmed that the interaction was
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21
thermodynamically favorable having a negative variation of Gibbs
free energy. It was highlighted
that the complexation process is predominantly driven by entropy
and moderately driven by
enthalpy, this was resulted from van't Hoff analysis based on
UV-Vis data and ITC measurements
(Figures 5.2 and 5.3).
Fig. 5.3 ITC data obtained for the binding interaction
between Dox (40.4 mM) and γCD (1.01 mM) in aqueous
solution at 25 ° C (p = 0.1 MPa). The upper panel shows
gross ITC data. The lower panel shows the integrated and
normalized data for the representation of heat values
according to the molar ratio, and the continuous line
represents the least squares estimated curve.
The TG/DTG analysis of pure and Dox/γCD complexes was performed
in the temperature range
from 25 °C to 450 °C, and the resulting curves are shown in Fig.
5.4 and 5.5.
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Fig. 5.4 Curbele DSC pentru substanțele pure și
pentru complexul de incluziune
Fig. 5.5 Curbele TG si DTG (inset) pentru
substanțele pure și pentru complexul Dox/γCD
The thermal behavior of the Dox/γCD complex is presented in the
DSC/TG thermograms of
Fig. 5.4 and 5.5. In the temperature range from 25 °C to 130 °C,
the dehydration process takes place
in three successive steps with a total mass loss of 2.6%. After
the dehydration phenomenon,
Dox/γCD presents a sequential decomposition process, as
evidenced by the DTG profile asymmetry
(inserted in Fig. 5.5) [8].
The morphology of pure compounds as well as the Dox/γCD complex
were evaluated using
SEM, and the obtained images are shown in Fig. 5.6.
Fig. 5.6 SEM images obtained at 25 ± 1 ° C for: a) Dox, b)
Dox/γCD inclusion complex and
c) γCD.
It can be observed that the morphology of the Dox/γCD complex
presents a new solid phase
with no presence of the morphological characteristics of Dox or
γCD.
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6. General Conclusions. Original contributions
The aim of the thesis is the thermoanalytical study of complexes
of cyclodextrins and the
correlation of the thermodynamic behavior with the structural
and morphological aspects involved
in their formation.
In the thesis, characterization of ligand-CD systems was
performed by differential dynamic
calorimetry, thermogravimetry, isothermal calorimetry titration,
and the results were supported by
spectral and morphological data.
In this work, the synthesis of inclusion complexes was
accomplished by "melting and
solution" and "coprecipitation" methods, starting from the 1: 1
molar ratio.
Ligands were chosen to belong to different structural classes to
investigate complexation
behavior under the influence of various factors such as: molar
ratio, synthesis mode, solubility.
The objectives proposed in this paper led to the following
conclusions and original contributions:
- In this thesis a systematic thermoanalytic study of the solid
powders of the complexes
formed between the ligands: uracil (U), 5-fluorouracil (5FU) and
cyclodextrins: αCD,
HPαCD, βCD și HPβCD, were done. The resulted values of the
decomposition temperatures
of the complexes were used in order to evaluate the
stability.
- Quantum chemistry calculations were done for systems formed
between U, 5FU and
cyclodextrins: αCD, βCD, HPαCD, HPβCD. The values of the
binding, deformation and
mutual perturbation energy values were established by DFT
calculations.
- The investigation of systems formed between optical isomers of
amino acids: arginine (Arg),
histidine (His), tryptophan (Trp) wih cyclodextrins: αCD, βCD,
HPαCD was accomplished.
The experimental results related to thermal, spectral and
morphological behavior solid
powders of the complexes formed between His, Arg and
cyclodextrins, were reported.
Comparative issues of the isomers of amino acids considered with
cyclodextrins have been
discussed considering the contribution of driving forces of the
complexation process.
- It was shown that native cyclodextrins αCD and βCD interact
strongly with amino acids than
HPαCD. Based on the obtained data, it was found that the size of
the CD cavity and the
existence of the functionalization of CD cavity can acting as
chiral selectors for the optical
isomers of amino acids.
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24
- The influence of structural factors on the thermodynamic
behavior was discussed for the first
time and the corresponding energetic parameters were specified
for both the aqueous
solution and solid powder complex formed between doxorubicin
(Dox) and γCD (Dox/γCD).
For a 10-5
M aqueous solution of Dox and γCD, the stoichiometry was found
to be 1:1. In
solid state, FTIR and SEM thermal data confirm that the Dox/γCD
complex was formed by
partial inclusion of the Dox molecule in the γCD cavity.
- For the first time, the complexes formed between cinconin
(Cinc) and cyclodextrins were
investigated in solid state and it was shown that the presence
of the cosolvent influences the
complexation process. Thus, the thermal analysis data and FTIR
results showed the existence
of interactions between the components without inclusion of Cinc
in the CD cavity for
systems formed between Cinc and native cyclodextrins,
considering the molar ratios of 1:1
and 1:2. An important role is played by CD functionalization,
justifying for Cinc/HPβCD the
posibility to form both the 1:1 molar ratio complex and also the
1: 2 molar ratio complex.
- In the case of ligand molecules (Cinc and Dox) made up of two
structural units, it was
observed that for the 1:1 molar ratio (in the solid state), a
partial inclusion of the ligand
molecule in the γCD cavity was happend.
- Based on the thermodynamic parameters obtained from the DSC,
comparative diagrams
were done considering the variation of onset temperature,
transition temperature and
enthalpy for the melting endotherm of the complexes formed
without inclusions into CD
cavity of the guest molecule (systems formed between Cinc or
optical isomers of the amino
acids with cyclodextrins). It was shown there is a tendency that
the lowest enthalpy values
correspond to the highest values of the start and transition
temperature of the melting
endotherm. This can be considered as a selection criterion for
the determination of stability
within the same set of complexes formed without inclusion of
guest.
- The importance of the driving forces in the complexation
process, such as: hydration,
presence of cosolvent, the possibility of self-assembly, the
size and functionalization of CD
cavity and the structural characteristics of the ligand molecule
were discussed.
- It was emphasized that the diluted aqueous solutions used in
synthesis of the complexes
determined a 1:1 stoichiometry of the complexes formed between
cyclodextrins and the
ligands: Dox, Cinc, U and 5FU.
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25
- In this thesis the SEM images corresponding to inclusion
complexes formed between the
considered ligands and cyclodextrins were analyzed for the first
time.
- In this work, the importance of characterization of pure
substances before the synthesis steps
is underlined. Considering the thermal analysis of
cyclodextrins, it is pointed out that the
presence of different thermal behavior can be induced by a
series of factors such as:
commercial variety, storage time and conditions.
- The results obtained in this thesis emphasize the importance
of methods of thermal analysis
in the study of complexes formed with cyclodextrins, together
with other methods of
investigation.
7. Perspectives of expanding and continuing research
The properties of cyclodextrins have led to the development of
many research branches, and
inclusion complexes formed with cyclodextrins constitute an
enormous research area with new
perspectives on combinatorial chemistry.
Appreciation of complexity stability and reactivity are
important in application development,
so data on energy factors are needed in areas such as molecular
engineering and supramolecular
chemistry.
The scientific data resulting from this study may be starting
points for new research such as
chiral separation and catalysis, as well as starting points for
making realistic models of more
complicated processes such as macromolecular hydration,
hydrophobic proteins, various types of
interaction etc.
The new research will allow experimental measurements to be
expanded in terms of the
influence of physicochemical factors such as: the concentration
of the constituent molecules of the
system, the concentration and type of the solvent, the pH, the
temperature, complexes stoichiometry
control etc.
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