UVA Chemical Filters: A Systematic Study Jacqueline F. Cawthray, B. Science (Hons) A thesis submitted for the degree of Doctor of Philosophy in The University of Adelaide Department of Chemistry February 2009
UVA Chemical Filters:
A Systematic Study
Jacqueline F. Cawthray, B. Science (Hons)
A thesis submitted for the degree of
Doctor of Philosophy
in
The University of Adelaide
Department of Chemistry
February 2009
Chapter 5
151
5 Cyclodextrin Complexation Studies
5.1 Introduction
Cyclodextrins (CDs) are cyclic oligosaccharides consisting of a number of D-
glucopyranose units that are linked by α-1,4 glycosidic bonds to form a well-defined cavity
(FIGURE 5.1). The naturally occurring CDs are α-, β- and γCD, and are composed of 6, 7
and 8 D-glucopyranose units, respectively. The CD molecule can be depicted as a
truncated cone having a relatively rigid structure. The inner cavity is lined with hydrogens
and ether-like glycosidic oxygens, making the interior of the annulus relatively
hydrophobic. The secondary hydroxyl groups are located at the wider end of the cone with
the primary hydroxyl groups at the narrow end, making the exterior hydrophilic.
O
HO
OH
O
O
OHOH
O
OOH
OH
O
OH
HO
O O
O
OHOH
O
OOH
OH
OH
OH
HO
OH
OH
OH
n
4
65
32
1
O
FIGURE 5.1: Structures of naturally occurring cyclodextrins.
Cyclodextrins are of interest due to their ability to include either all or a substantial part of
a guest molecule inside their annuli, to form inclusion complexes, otherwise known as
Primary hydroxyl rim
Secondary hydroxyl rim
n = 1 α-CD n = 2 β-CD n = 3 γ-CD
Chapter 5
152
host-guest complexes [375,376]. The inclusion complex is held together by the spatial
entrapment of a guest molecule in the CD cavity without the formation of covalent bonds.
The formation of such inclusion complexes is a reversible and dynamic process in which
free guest molecules are in thermodynamic equilibrium with included guest molecules.
There are a number of energetically favourable interactions that describe the driving force
for inclusion of a guest molecule (for reviews see Refs. [377,378]). In aqueous solution,
the hydrophobic annulus of the CD molecule is occupied by water molecules, which can be
readily replaced by an appropriate non-polar guest molecule (FIGURE 5.2). The
hydrophobic effect, therefore, is one of the main driving forces involved in complex
formation [379,380]. The presence of a guest molecule gives rise to a net gain in enthalpy
due to exclusion of the cavity-bound water and an increase in hydrophobic interactions.
Once inside the cyclodextrin cavity, the guest molecule undergoes conformational
adjustments to maximise the weak van der Waals interactions with the CD. The stability
of the inclusion complex is also influenced by steric interactions with the size of the guest
molecule relative to that of CD cavity being a determining factor [381]. Although the
inclusion complexes formed are held together by secondary bonding forces only, their
stability can be as high as 105 mol dm-3 [382].
X
Y
X
Y
+
FIGURE 5.2: Schematic representation of inclusion of an aromatic guest molecule within
the CD cavity in an aqueous environment; water is represented by the small circles.
The physicochemical properties and complexing ability of CDs can be improved by
chemical modification of naturally occurring CDs (for review see Ref. [383]). Many CD
derivatives exist in which the primary and/or secondary hydroxyl groups of the naturally
occurring CD are substituted with various functional groups to give modified CDs that
have slightly altered properties from those of the parent CD. For example, modified βCDs
such as 2-hydroxypropyl-β-cyclodextrin (HPβCD) and randomly methylated βCD in which
one or several OH groups have been replaced by the relevant alkyl substituent, have a
much higher solubility than native βCD [384]. This is due primarily to disruption of the
Chapter 5
153
intramolecular hydrogen bonding between O2-H and O3-H that, in substituted CDs, is
replaced with intermolecular hydrogen bonding with the solvent in the modified CDs.
Depending on the method of preparation, the hydroxypropyl groups are often randomly
substituted onto the hydroxyl groups of βCD shown in FIGURE 5.3. Consequently,
HPβCD and other substituted CDs are characterised by the degree of substitution, which
refers to the average number of substituents per CD molecule. The degree of substitution
and substitution pattern influences both the solubility and inclusion complex forming
ability of HPβCD in addition to the properties of the inclusion complex formed
[381,385,386].
R Solubility (mg/ml)
βCD H 18.5
HPβCD CH2CH(OH)CH3 >600
FIGURE 5.3: Structures and solubilities of βCD and HPβCD.
The formation of CD inclusion complexes can result in the advantageous modification of
certain physicochemical properties of the guest molecule including improved water
solubility and chemical stability [387]. The low toxicity of CD and its ability to act as an
excipient has meant many natural and substituted CDs have been approved by regulatory
authorities worldwide. Consequently, CDs are used in a wide range of applications in the
pharmaceutical [387-389] food, cosmetic [390,391] and chemical industries.
O
H
H
RO
H
OOR
HH
OR
7
Chapter 5
154
5.1.1 Use of Cyclodextrins with Sunscreens
It is now clear that wavelengths in the UVA region (320 – 400 nm) of the solar spectrum
can cause a wide range of detrimental biological effects [22,84,122,161]. Chemical
sunscreen filters are a popular and effective method of photoprotection against UVA [257].
An important characteristic of any effective UV chemical filter is photostability.
Photodegradation of the sunscreen filter leads to a permanent loss of protection and, in
addition, the degradation products have the potential to cause toxic or allergic reactions.
The UVA chemical filter, 4-tert-butyl-4′-methoxy dibenzoylmethane (BMDBM), is used
worldwide in sunscreen formulations [203]. However, several studies have demonstrated
that BMDBM undergoes UV-induced photodegradation leading to loss of protection
[205,225,258,392]. The degradation products of BMDBM are potentially harmful, causing
damage to relevant biological molecules [333,393,394]. As with other β-diketones,
BMDBM exists in a keto-enol tautomeric equilibrium (FIGURE 5.4) with the enol form
strongly favoured. The enol tautomer, stabilised by an intramolecular hydrogen bond,
absorbs strongly in the UVA region (λmax ~359 nm) whilst the keto absorbs in the UVB
region (λmax ~270 nm). Following UV-irradiation, ketonisation and subsequent
photodegradation of the keto tautomer leads to a permanent loss of absorbance in the UVA
region, reducing the effectiveness of the sunscreen [205,225].
FIGURE 5.4: Keto-enol equilibrium in 4-tert-butyl-4′-methoxydibenzoylmethane
(BMDBM).
Various methods have been used to prevent or minimise the photodecomposition of
BMDBM including the complexation of BMDBM with natural or modified CDs has on the
photostability of BMDBM. Of the naturally occurring CDs, βCD forms the most stable
inclusion complexes [213]. The smaller cavity of αCD can result in steric interference
O O O O
O O
Enol Keto
hv
H
Chapter 5
155
preventing stable inclusion complexes forming whilst the larger annulus of γCD generally
does not allow for optimal interactions between the guest and host. The photostability of
BMDBM is enhanced by inclusion in HPβCD [371,395] and randomly methylated βCD
[213] and forms the claim of at least one patent [396]. However, the stabilising effect of
HPβCD is reduced in lotion vehicle (oil-in-water emulsions) due to competitive
displacement of BMDBM from the CD cavity by emulsion excipients. This can by
minimised by incorporation of the inclusion complex of BMDBM and HPβCD into lipid
microparticles (lipospheres) [397]. In contrast, inclusion complexes of BMDBM and βCD
were found to have very little effect on photostability of BMDBM [289]. However it has
been demonstrated that the photodegradation products are themselves included in the CD
annulus, therefore limiting any potential toxic or allergic reactions [289,395].
The aqueous solubility of BMDBM (1.5 µg/ml [213]) is enhanced through inclusion by
natural and modified CDs. Water-soluble sunscreens are useful in cosmetic formulations
and hair care preparations [203]. Solubility studies of BMDBM and parent CDs (α-,β-,
γCD) and their derivatives (HPα-, HPβ-, HPγCD, randomly methyl βCD) show that
HPβCD and randomly methyl βCD are the most effective at increasing the aqueous
solubility of BMDBM [213,371]. The increased solubility of the inclusion complexes
formed with HPβCD over those formed with βCD can be attributed to the greater solubility
of HPβCD itself.
There are a number of published reports focused on the complexation of BMDBM with
CDs, however the focus has been on determining the influence of CD complexation with
BMDBM has on photostability, solubility and transdermal penetration
[213,289,371,395,397,398]. The methods used in previous investigations have
demonstrated the ability of CDs to form inclusion complexes with BMDBM, mostly in the
solid state, but there is little available information regarding the nature of the inclusion
complexes formed, particularly in solution. The objective of the present study is to gain
further insight into the mode of inclusion in the complexes formed in solution between
BMDBM and the cyclodextrins βCD and HPβCD.
The 1D and 2D 1H NMR techniques are widely used for providing evidence for inclusion
complexes in solution and for studying the mode of inclusion between the guest and host
CD (for review see Ref. [399]). As both techniques are particularly sensitive to changes in
the electron environment of a proton that occurs upon close contact or short-range
Chapter 5
156
association, they are particularly suitable for investigating the non-covalent interactions
present in CD inclusion complexes. When either part or all of a guest molecule is enclosed
in the CD cavity, the resonances of the CD interior protons (H3 and H5) are shifted in the
spectrum while the exterior protons (H2 and H4) remain relatively unaffected (FIGURE
5.5). In a similar manner, the resonances of the guest molecule also experiences
complexation-induced chemical shifts. The formation of inclusion complexes is a dynamic
process involving the CD moving on and off the guest molecule. Consequently, the
observed chemical shift at a given temperature in the NMR experiment is dependent on the
rate of chemical exchange. If the exchange is rapid on the NMR timescale, a single
resonance is observed whose chemical shifts is the weight average of the chemical shifts of
the individual states. Conversely, if the exchange is slow on the NMR timescale, separate
resonances for each state is observed. Intermediate rates show broad or partially averaged
resonances. Since the rate of exchange depends on the change in Gibb’s free energy (∆G),
temperature-dependent 1H NMR studies can be used to investigate the exchange process.
FIGURE 5.5: β-cyclodextrin interior and exterior protons.
The 2D NMR technique, 1H-1H ROESY (Rotating frame nuclear Overhauser Effect
SpectroscopY), is a spin-lock technique that identifies through-space interactions (NOEs)
via spin-spin relaxation. In the 1H ROESY NMR spectra, cross-peaks arise from spatially
separated protons that are ≤ 4 Å apart even in the absence of covalent bonding. Therefore, 1H ROESY NMR provides structural information regarding the functional groups of the
guest that are included within the CD annulus and how deep in the annulus the guest sits.
The intensity of the observed NOE between spatially separated protons is related to both
separation distance and concentration.
O
H
H
HO
H
OOH
HH
(H6)(H2)
(H1)
(H5)
(H3)
(H4)
OH
7
H5
H6 H6
H4
H2
H1
H3
Chapter 5
157
β-Cyclodextrin and its hydroxyalkyl derivative, HPβCD were chosen as they are
particularly suitable as the host CDs in this study as their cavity sizes and volumes are well
suited for inclusion of the hydrophobic tert-butyl and phenyl groups of BMDBM (FIGURE
5.6). The cavity volume of βCD is somewhat extended in HPβCD due to substitution by
the hydroxyalkyl group. The degree of substitution of HPβCD used in this study is 3.0-8.0
which gives an average number of 5 hydroxypropyl groups per βCD molecule. Both βCD
and HPβCD have been the subject of extensive toxicological studies and are considered
safe [400]. Both are approved for use by the Therapeutics Goods Association in Australia
making them suitable for pharmaceutical applications. They are particularly suited for
incorporation into sunscreen formulations as their large molecular size and hydrophobic
nature prevents penetration into the skin [401].
FIGURE 5.6: Dimensions of β-cyclodextrin (βCD) and hydroxypropyl-β-cyclodextrin
(HPβCD) compared with the dimensions of the phenyl group in BMDBM.
5.2 1H NMR Studies of βCD and HPβCD Complexes
The 1H and 1H ROESY NMR spectra are reported for solutions of BMDBM and either
βCD or HPβCD in which the mole ratio of BMDBM to CD is varied. A variable-
temperature 1H NMR study of the solution of BMDBM and either excess βCD or HPβCD
are also presented.
15.4 Å
6.0-6.5 Å
Cavity volume 262 Å3
7.9 Å
4.1 Å
R
HH
H H
5.0 Å
R = OCH3 or C(CH3)9
Chapter 5
158
5.2.1 βCD Complexes/NMR data of BMDBM-.
The guest molecule, BMDBM, and its βCD inclusion complexes are insufficiently water
soluble for NMR studies. Previous studies of BMDBM inclusion complexes with CDs
have either relied upon more sensitive techniques for analysis of BMDBM concentrations
such as HPLC or examined complexation in solids or suspensions [213,371,397,398]. To
achieve the concentrations required to obtain an 1H NMR spectrum, it was necessary to
prepare all solutions in 0.1 mol dm-3 NaOD/D2O such that pD ≈ 12. As the pKa of βCD is
12.20 [402], under the basic conditions employed the βCD hydroxyl groups, OH(2) and
OH(3), are partially deprotonated. This results in an increase in the solubility of βCD. The
pKa of BMDBM is 11.34 (this work; Chapter 2.5) therefore under these conditions, it will
exist predominately as the β-diketonate of BMDBM (herein called BMDBM−) (FIGURE
5.7) having only slightly improved water solubility over the parent species.
FIGURE 5.7: Generation of the β-diketonate of BMDBM (BMDBM −). Only one of the
chelated enols are shown.
Solutions were prepared by adding BMDBM to a solution of βCD in 0.1 mol dm-3
NaOD/D2O. The resulting suspension was gently heated then filtered to remove the
suspended material, which was probably BMDBM and/or its βCD complex. As a
consequence of the poor solubility of BMDBM, even under the basic condition used, the
calculated concentrations of BMDBM− were not achieved. Instead, the mole ratios of βCD
to BMDBM were determined by integration of the appropriate 1H NMR signals. In the
following text the numbering scheme shown in FIGURE 5.8, whereby the CD protons are
denoted as H1-6 and the aromatic protons of BMDBM as Ha-Hd, have been used for the
sake of clarity.
O O
O
+ OHO O
O- OH
+ H2O
BMDBM BMDBM�
H
Chapter 5
159
To enable a comparison between free and included BMDBM− the 1H NMR spectrum of
BMDBM− in 0.1 mol dm-3 NaOD/D2O was obtained (FIGURE 5.10a). To achieve the
concentration required to obtain a 1H NMR spectrum, the solution was rapidly heated to
high temperatures and the suspended material was removed by filtration. The 1H NMR
spectrum indicates that the solution consisted of a mixture of BMDBM− and degradation
products. It was possible to assign all resonances to either BMDBM− or to degradation
product by integration of the 1H NMR signals. The tert-butyl resonance appears as two
singlets, assigned as t-bu and t-bu in FIGURE 5.10a. The BMDBM− aromatic resonances
as two sets of doublets assigned as Ha-Hd and Haʹ-Hd . The methoxy resonance is
observed as two singlets appearing at δ 3.87 (assigned to degradation products) and δ 3.84.
(A comparison between the methoxy resonance of free and included BMDBM− is not
possible as it is masked by the βCD resonances and, consequently, has not been included in
the 1H NMR data presented).
FIGURE 5.8: Labelling scheme used for BMDBM − and βCD protons.
The degradation products are likely to be the result of cleavage at or near the centre of
BMDBM resulting in two fragments. There are a number of possible ways this could
occur, one such possibility is illustrated in FIGURE 5.9. Integration of the peaks assigned
to the fragments indicates that the area ratios of each fragment are significantly different.
As Fragment A, containing the hydrophobic tert-butyl group, would be somewhat less
soluble than Fragment B it is likely to have been filtered from solution. As the aim was to
obtain a 1H NMR spectrum of BMDBM− for comparison purposes, it was not deemed
necessary to investigate the degradation products further. The rapid heating to high
temperatures was not necessary when preparing solutions containing βCD and BMDBM−,
consequently there were no indications that degradation had occurred in these solutions.
O O
OHa
Hb HcHd
H5
H6 H6
H4
H2
H1
H3
Chapter 5
160
FIGURE 5.9: Two possible positions where bond cleavage of BMDBM − can occur
leading to different fragmentation patterns.
5.2.1.1 NMR data of 1 : 2 mole ratio of BMDBM− : βCD.
In the 1H NMR spectrum of a solution in which the mole ratio (determined by integration
of the 1H NMR signals) of BMDBM− and βCD was 1 : 2 (FIGURE 5.10b) the tert-butyl
resonance appears as a singlet and the BMDBM− aromatic resonances Ha-Hd as doublets
arising from AABBʹ spin-spin splitting. Unambiguous assignment of the methoxy
resonance was not possible as the βCD H3 resonance appears in the same region of the
spectrum. The tert-butyl and Ha-Hd resonances of BMDBM− appear as a sharp singlet and
well-resolved doublets respectively. The differences between the chemical shifts of free
and included BMDBM− (TABLE 5.1) are greatest for BMDBM− tert-butyl, Ha and Hb
protons. These observations are consistent with the formation of βCD·BMDBM− as either
a single includomer or two includomers in fast exchange. In processes involving fast
exchange, the observed resonances consist of the time-averaged resonances of free βCD,
BMDBM− and the inclusion complexes formed. The inclusion complexes differ in the
inclusion orientation of βCD with respect to BMDBM−, all being in thermodynamic
equilibrium. The nature of the inclusion complex formed between βCD and BMDBM− are
explored further by 1H ROESY NMR spectroscopy as detailed below.
OHa′
Hb′ Hc′Hd′
tBu′
O O
Fragment A Fragment B
OHa′
Hb′ Hc′Hd′
tBu′
O O
Fragment A′ Fragment B′
Chapter 5
161
TABLE 5.1: 1H NMR chemical shifts (ppm) corresponding to BMDBM in the absence and
presence of βCD in a 1 : 2 mole ratio in 0.1 mol dm-3 NaOD/D2O at 298 K.
δfree δcomplex (∆λa)
t-Bu 1.28 1.42 (+0.14)
Ha 7.52 7.35 (-0.17)
Hb 7.79 7.61 (-0.18)
Hc 7.82 7.72 (-0.10)
Hd 6.99 7.01 (+0.02) a Upfield displacements are negative
FIGURE 5.10: Partial 1H 600 MHz NMR spectra of BMDBM tert-butyl and aromatic Ha-
Hd resonances of (i) solution of BMDBM (Solution A) and (ii) solution of BMDBM and
βCD in a 1 : 2 mole ratio (Solution B) in 0.1 mol dm-3 NaOD/D2O at 298 K. The
degradation productions of BMDBM in (i) are denoted by Haʹ, Hb , Hc , Hd and t-Bu.
The spectra are not plotted to a constant vertical scale.
O O
OHa
Hb HcHd
6.877.27.47.67.88 1.21.5(ppm)
Hd Ha Hb Hc
Hd Ha Hb Hc
Hdʹ Hcʹ
Hbʹ Ha t-Buʹ
t-Bu
t-Bu (i)
(ii)
Chapter 5
162
The 1H ROESY NMR spectrum of a solution in which the mole ratio of BMDBM− and
βCD was 1 : 2 is shown in FIGURE 5.11.
FIGURE 5.11: 1H 600 MHz ROESY NMR spectrum of a solution BMDBM with βCD in a
1 : 2 mole ratio in 0.10 mol dm-3 NaOD at 298 K. The cross-peaks enclosed in the boxes
correspond to intermolecular interactions between the protons indicated on the F1 and F2
axes.
Strong cross-peaks are observed between BMDBM− tert-butyl resonances and those of
βCD H3, H5 and H6 protons of the annular interior. Strong cross-peaks are also observed
βC
D p
roto
ns
H4
H2
H5
H6
H3
Hc Hb Ha Hd t-Bu
βCD H1
HOD βCD H2-6
H5
H6
H6
H4
H2
H1
H3
O O
OHa
Hb HcHd
Chapter 5
163
between BMDBM− Ha and βCD H3 and H5 as well as BMDBM− Hd and βCD H3. A
somewhat weaker cross-peak can be observed between BMDBM− Hb and βCD H6. The
interactions, if any, between the methoxy protons of BMDBM− and βCD protons are not
observed as they are masked by the βCD resonances. No interactions are observed
between BMDBM− protons and the exterior βCD H1, H2 or H4 protons.
The experimental observations taken from the 1H and 1H ROESY NMR support the
formation of a 1 : 1 host-guest inclusion complex, βCD·BMDBM−, and the subsequent
formation of a 2 : 1 complex, (βCD)2·BMDBM−. The single resonances observed for each
of the tert-butyl and Ha-Hd protons supports a fast exchange between free βCD, free
BMDBM−, βCD·BMDBM− and (βCD)2·BMDBM−. As the process is rapid on the NMR
timescale, the observed resonance is a time- and weight-averaged resonance for each of the
different chemical environments.
The different possible inclusion orientations of βCD relative to BMDBM− results in a
number of includomers existing in thermodynamic equilibrium as shown in FIGURE 5.12.
A visual comparison of the cross-peak intensities in the 1H NMR ROESY, taking into
consideration the number of BMDBM− protons giving rise to the cross-peaks with βCD
protons, supports the preferential inclusion of the tert-butyl phenyl group to the methoxy
phenyl group to form βCD·BMDBM− includomer A and βCD·BMDBM− includomer Aʹ in
FIGURE 5.12. This would be facilitated by the greater hydrophobic nature of the tert-
butyl phenyl group compared with the methoxy phenyl group. Support for the proposed
mode of inclusion is provided by the chemical shift changes of BMDBM− tert-butyl group
and Ha-Hb protons. The upfield shift for Ha and Hb protons indicates a more hydrophobic
environment, consistent with the positioning of βCD over the phenyl group. The
downfield displacement of the tert-butyl protons suggests a close proximity to an
electronegative oxygen atom, placing it either near the narrow (primary) hydroxyl end
(includomer A in FIGURE 5.12) of the CD cavity or the wider (secondary) hydroxyl rim
(includomer Aʹ in FIGURE 5.12). The larger size of the βCD annulus relative to the tert-
butyl group means the tert-butyl group can insert either end without experiencing
significant steric effects. This is evident in the AM1 optimised geometry of includomer A′
shown in FIGURE 5.13 [403]. Consequently, several inclusion orientations of BMDBM−
relative to the βCD are possible and the experimental NMR spectra represents a
population-weighted average of the spectrum of βCD·BMDBM− complexes involving
different threading orientations of βCD over the tert-butyl phenyl group.
Chapter 5
164
FIGURE 5.12: Formation of βCD·BMDBM − and (βCD)2·BMDBM
− inclusion complexes
involving the possible inclusion orientations of βCD.
O O
O
O O
O
O O
O
O O
O
O O
O
O O
O
O O
O
O O
O
Includomer B
Includomer B′
Includomer D′
Includomer A′
Includomer C′Includomer C
Includomer A
Includomer D
Chapter 5
165
FIGURE 5.13: Geometry of includomer A′ optimized at the AM1 level of theory. The
carbons in βCD are coloured grey and blue in BMDBM to distinguish between the two
molecules.
Chapter 5
166
The interconversion of includomer A and Aʹ occurs through decomplexation of the
(βCD)2·BMDBM− includomer A to BMDBM− and βCD followed by formation of
βCD·BMDBM− includomer Aʹ. In the 1H NMR ROESY, the comparatively weaker cross-
peaks between BMDBM− tert-butyl protons and βCD H6 coupled with the absence of
cross-peaks between BMDBM− Ha and βCD H6 suggests the preferential formation of
βCD·BMDBM− includomer A′ over βCD·BMDBM− includomer A.
Evidence of the complexation of the methoxy phenyl group is provided by the cross-peaks
in the 1H NMR ROESY observed between BMDBM− Hd and βCD H3 protons and
between Hc and βCD H6. There are two possible inclusion orientations of βCD relative to
BMDBM−, βCD·BMDBM− includomer B and includomer Bʹ It is not possible to say with
any certainty if one includomer is preferred over the other as the methoxy interactions with
βCD protons are masked and the observed cross-peaks for Hc are either weak or noise.
This does support, however, the previously proposed notion that the tert-butyl group is
complexed in preference to the methoxy phenyl group although formation of
βCD·BMDBM− includomer A and Aʹ does not preclude formation of βCD·BMDBM−
includomer B and B.
If βCD·BMDBM− includomer Aʹ is the dominant 1 : 1 inclusion complex as previously
proposed then the variation in the intensities of the cross-peaks for Hc and Hd with βCD
protons and the absence of cross-peaks for Hd with βCD H5 can be attributed to the
presence of (βCD)2·BMDBM− includomer Cʹ in addition to includomer Dʹ where the βCD
orientation over the methoxy phenyl group is reversed. If the ROESY interactions of Hc
and βCD H6 protons were due to the presence of (βCD)2·BMDBM− includomer Dʹ only
BMDBM− Hd protons would be positioned deep in the βCD cavity and cross-peaks of
similar intensity with both βCD H3 and H5 would be observed. The difference in chemical
shift between free and included Hc and Hd of BMDBM− is much less than that observed
for Ha and Hb (TABLE 5.1), however the chemical environment of the methoxy phenyl
protons are not expected to be as sensitive to the hydrophobic environment of the βCD
cavity because of the deshielding effect of the methoxy group.
The chemical environments of the tert-butyl and Ha-Hb protons in βCD·BMDBM− are not
expected to be affected significantly by the addition of the second βCD to the methoxy
phenyl end of the molecule to form (βCD)2·BMDBM− includomers Cʹ and Dʹ.
Accordingly, only one chemical environment would be observed for the tert-butyl and Ha-
Chapter 5
167
Hb protons in βCD·BMDBM− and (βCD)2·BMDBM−. The same principle applies to the
chemical environment of the methoxy phenyl protons if the first βCD were to be positioned
over the methoxy phenyl group in βCD·BMDBM− and the subsequent complexation of a
second βCD to the tert-butyl phenyl group to form (βCD)2·BMDBM−.
5.2.1.2 NMR data of BMDBM with excess βCD.
In the 1H NMR spectrum of a solution, solution B, in which the mole ratio of BMDBM−
and βCD was 1 : 6 (as determined by integration of 1H NMR signals), the tert-butyl
resonance appears as two singlets and the BMDBM− aromatic resonances assigned to Ha-
Hd appear as three sets of doublets (FIGURE 5.14b). Assignment of the methoxy
resonance is not possible due to it having a similar chemical shift as that of the βCD H3
resonance. Integration of all BMDBM− proton signals reveals that the larger tert-butyl
resonance having an observed chemical shift of δ 1.41 is due to two tert-butyl groups
possessing coincident chemical shifts, appearing with an area ratio of 2.7 : 1. This is
possible if either they possess coincident chemical shifts or the chemical environments of
the two are similar. The 1H NMR spectrum of free BMDBM− as shown in FIGURE 5.14a
is the same as presented earlier in FIGURE 5.10a.
The 1H NMR chemical shifts of BMDBM− resonances for solutions of free BMDBM−,
BMDBM− and βCD in a 1 : 2 molar ratio and BMDBM− and βCD in a 1 : 6 mole ratio are
compared in TABLE 5.2. The distinction between the different chemical environments of
the tert-butyl phenyl and methoxy phenyl groups has been made by using different
coloured text. The chemical shifts of the dominant includomer observed for solution B
(blue text in FIGURE 5.14 and TABLE 5.2), are very similar to those of solution B. In
solution A, these resonances were proposed to be due to predominately (βCD)·BMDBM−
with a lesser amount of (βCD)2·BMDBM. In solution B, βCD is in much greater excess
and therefore it seems reasonable to assume that the major resonances (t-Bu, Ha-Hd in
FIGURE 5.14) are predominately due to (βCD)2·BMDBM−. The change in populations of
(βCD)·BMDBM− and (βCD)2·BMDBM− in solution A and B is supported by the changes
in chemical shifts.
Using 1H NMR ROESY, it was possible to assign the resonances of the minor includomers
in solution to two different tert-butyl phenyl groups and two different methoxy phenyl
groups. Using this method it was not possible to connect either of the tert-butyl phenyl
groups to a particular methoxy phenyl group and vice-versa as the distance between the
Chapter 5
168
two phenyl groups is too large to generate cross-peaks in the 1H NMR ROESY or for either
phenyl group to significantly influence the chemical environment of the other. Therefore,
the assignment of these resonances to a particular includomer cannot be made with
certainty.
FIGURE 5.14: Partial 1H 600 MHz NMR spectra of BMDBM −- tert-butyl and aromatic
Ha-Hd resonances of (i) solution of BMDBM − and (ii) solution of BMDBM − and βCD in
a 1 : 6 mole ratio (Solution B) in 0.1 mol dm-3 NaOD / D2O at 298 K. The degradation
products of BMDBM in (i) are denoted by Haʹ - Hd and t-Bu. The coloured text makes
the distinction between the resonances assigned to the different includomers. The spectra
are not plotted to a constant vertical scale.
O O
OHa
Hb HcHd
(i)
(ii)
6.877.27.47.67.88 1.21.5
(ppm)
Hd Ha Hb Hc
Hd Ha Hb Hc Hdʹ Hcʹ
Hbʹ Ha t-Buʹ
t-Bu
t-Bu t-Bu
Hd Hd Hc Hc
Hb
Hb
Ha t-Bu Ha
Chapter 5
169
TABLE 5.2: 1H chemical shifts (ppm) corresponding to BMDBM − in the absence and
presence of βCD in 0.1 mol dm-3 NaOD/D2O at 298 K .
δfree δcomplex (∆λa) δcomplex (∆λa)
1 : 2 mole ratio b 1 : 6 mole ratio t-Bu 1.28 1.42 (+0.14) 1.41 (+0.13) 1.41 (+0.13) 1.43 (+0.15)
Ha 7.52 7.35 (-0.17) 7.38 (-0.14) 7.34 (-0.18) 7.52 (+0.00)
Hb 7.79 7.61 (-0.18) 7.61 (-0.18) 7.74 (-0.05) 7.90 (+0.11)
Hc 7.82 7.72 (-0.10) 7.73 (-0.09) 7.83 (+0.01) 7.96 (+0.14)
Hd 6.99 7.01 (+0.02) 6.99 (+0.00) 6.95 (-0.04) 7.06 (+0.07) a Upfield displacements are negative b Mole ratio of BMDBM− to βCD in solution.
These observations are consistent with the formation of three distinct includomers in slow
exchange on the NMR timescale that appear in the area ratio 2.7 : 1 : 1. Evidence for the
slow exchange process is provided by the reduced resolution of the βCD resonances in
solution B as compared with those of the solution having lower βCD concentrations
(solution A) where the exchange process was rapid as shown in FIGURE 5.15. The
greatest change observed is for the resonances assigned to the interior βCD protons H3, H5
and H6. The downfield shift of the H5 resonance means it is no longer possible to assign
individual resonances to H5 and H6. There is no significant change in the resonances
assigned to the exterior βCD protons H2 and H4. Furthermore, as the concentration of
βCD in solution B is greater than for solution A, the ratios of 1 : 1 and 1 : 2 inclusion
complexes will change as will the annuli CD 1H chemical shifts.
Chapter 5
170
FIGURE 5.15: Partial 1H 600MHz NMR spectra of βCD H2-H6 resonances of (i)
solution of BMDBM − and βCD in a 1 : 2 mole ratio (solution A) and (ii) solution of
BMDBM − and βCD having a 1 : 6 mole ratio (solution B) in 0.1 mol dm-3 NaOD/D2O at
298 K. The spectra are not plotted to a constant vertical scale.
The 1H ROESY NMR spectrum of a solution of BMDBM− and an excess of βCD is shown
in FIGURE 5.16. This shows strong intermolecular interactions between the proton
resonances of BMDBM− previously assigned to the dominant includomer and those of the
βCD interior H3, H5 and H6 protons. Strong cross-peaks are observed between BMDBM−
tert-butyl protons and βCD protons H3, H5 and H6. The colour scheme used to distinguish
between includomers is the same as used previously. As the tert-butyl resonance is
coincident with the dominant tert-butyl resonance, it is not possible to ascertain if there are
interactions between the minor tert-butyl protons and βCD protons. The apparent cross-
peaks between BMDBM− tert-butyl protons and βCD exterior protons H2 and H4 are
possibly due to noise running horizontally along the spectra. Strong cross-peaks are also
observed between the BMDBM− Ha and βCD H3, H6 and H5. Cross-peaks can be seen
between BMDBM− Hb and Hc protons and βCD H5/H6 protons. Additionally, there are
interactions between BMDBM− Hd with βCD H3 protons. For the minor includomers, a
weak cross-peak is observed between BMDBM− Ha and βCD H5 and H6.
H5
H6 H6
H4
H2
H1
H3
3.43.53.63.73.83.9(ppm)
H5 H3 H6
H2 H4
H2 H4 H6 H5
H3
(i)
(ii)
Chapter 5
171
FIGURE 5.16: 1H 600 MHz ROESY NMR spectrum of a solution of BMDBM − and βCD
in a 1 : 6 mole ratio (solution B) in 0.10 mol dm-3 NaOD at 298 K. The cross-peaks
enclosed in the boxes correspond to intermolecular interactions between the protons
indicated on the F1 and F2 axes. For an expansion of BMDBM − aromatic Ha-Hd
resonances, refer to FIGURE 5.14.
The observations from the 1H NMR and the 1H ROESY NMR of solution B provides
additional evidence in support of the formation of (βCD)2·BMDBM− in which βCD is
O O
OHa
Hb HcHd
H5
H6 H6
H4
H2
H1
H3 βC
D p
roto
ns
H4
H2
H6,
H5
H3
βCD H1
HOD βCD
H2-H6
t-Bu
Chapter 5
172
positioned over the tert-butyl phenyl group whilst a second βCD envelopes the methoxy
phenyl group. In solution A, the resonances (tert-butyl, Ha-Hd) were due to a fast
exchange on the NMR timescale between (βCD)·BMDBM− and (βCD)2·BMDBM− and
having a greater population of (βCD)·BMDBM−. In solution B, the higher mole ratio of
βCD supports the observed increase in population of the 2 : 1 species, (βCD)2·BMDBM−.
The possible inclusion orientations of βCD with BMDBM− and the equilibrium between
these possible includomers is the same as discussed previously for FIGURE 5.12. In
solution A, the dominant 1 : 1 inclusion complex, (βCD)·BMDBM−, was previously
assigned to includomer A' . This was supported by chemical shift changes between free
and included BMDBM− and also by 1H ROESY NMR interactions. There is no change
observed for the chemical shifts of the resonances tert-butyl, Ha-Hd in the 1 : 1 inclusion
complex, (βCD)·BMDBM− with those in the 2 : 1 complex, (βCD)2·BMDBM−. This is
attributed to the distance between the phenyl groups, where changes in the chemical
environment of Ha and Hb are too far away to influence Hc and Hd. The relative
intensities confirm the preferential inclusion of BMDBM− tert-butyl phenyl group.
5.2.1.3 Temperature-Dependence Studies of βCD Inclusion Complexes with BMDBM
The nature of the inclusion complexes formed were investigated further by variable-
temperature 1H NMR studies (298-323 K) of a solution of BMDBM− and βCD in a 1 : 6
mole ratio. The 1H NMR spectrum obtained at 298 K and 323 K are shown in FIGURE
5.17. The entire spectrum, referenced to the HOD solvent resonance, is shifted downfield
with increasing temperature. The dielectric constant of the solvent changes as temperature
increases, influencing its shielding properties. No significant broadening of the resonances
assigned to BMDBM− tert-butyl and aromatic protons Ha-Hd can be observed with
increasing temperature. The three sets of doublets previously assigned to Ha-Hd of
BMDBM− exist in the area ratio 2.7 : 1 : 1 at 298 K indicating a major includomer and two
minor includomers where the two minor includomers exist in the same ratio. As
temperature increases to 323 K, this ratio decreases to 2 : 1 : 1 where the ratio of the two
minor includomers, relative to each other, is not influenced by temperature.
Chapter 5
173
FIGURE 5.17: Partial variable-temperature 1H 600 MHz NMR spectra of BMDBM −
tert-butyl and aromatic Ha-Hd resonances of a solution of BMDBM − and βCD in a 1 : 6
mole ratio (solution B) at (i) 298 K and (ii) 323 K in 0.1 mol dm-3 NaOD/D2O. The spectra
are not plotted to a constant vertical scale.
The observations from the variable-temperature 1H NMR studies supports the presence of a
slow exchange between the three distinct includomers of BMDBM− and βCD where the
coalescence temperature has not been reached within the temperature range studied. If
initially the exchange between the major and two minor includomers is slow on the NMR
timescale at 298 K, coalescence of the proton resonances assigned to the includomers is
expected as temperature increases. However, no coalescence is observed indicating the
rate of exchange is not increasing and the coalescence temperature, where the peaks merge,
has not been reached. The includomers are still in slow exchange even at the higher
temperatures although the relative populations of includomers are changing. The change
6.97.27.57.88.1 1.31.51.7(ppm)
Hb Hc
Hb Hb
Hc
Ha Ha
Hd Ha
Hc
Hd Hd
Hb Hc
Hc
Hb Hc Hd Ha
Ha Ha Hd Hd
t-Bu
t-Bu, t-Bu
t-Bu
t-Bu, t-Bu
(ii)
(i)
O O
OHa
Hb HcHd
Chapter 5
174
in the populations, indicated by the area ratios being 2.7 : 1 : 1 at 298 K and 2 : 1 : 1 at 323
K indicates a change in the equilibrium position. The equilibrium constant for the
exchange process, involving βCD moving on and off BMDBM−, is a function of the
temperature as indicated by:
RT
GK
∆In
−=
where ∆G is Gibbs free energy, R is the gas constant and T is temperature. That the
relative populations of the minor includomers does not change with temperature implies
these species have similar energies.
5.2.2 HPβCD Complexes
5.2.2.1 NMR data of BMDBM with HPβCD in D2O
In contrast to βCD and its inclusion complexes, the inclusion complexes of HPβCD with
BMDBM are sufficiently soluble to allow acquisition of a 1H NMR spectrum in D2O. In
the absence of HPβCD, BMDBM cannot be detected by 1H NMR spectroscopic methods
due to the limited aqueous solubility of BMDBM (1.5 µg/ml [213]). An excess of
BMDBM was added to a solution of HPβCD (0.01 mol dm-3) in D2O and stirred for 25 hrs.
The resulting suspension was filtered to remove undissolved BMDBM and, therefore the
concentration of BMDBM is not known with any accuracy. The mole ratio of BMDBM
and HPβCD, determined by integration of the 1H NMR signals, is approximately 1 : 9.
Phase solubility studies show the solubilising effect of HPβCD on BMDBM is greater than
for the parent βCD [213,371]. The increase in solubility of BMDBM with HPβCD is
attributed to the greater solubility of HPβCD itself over that of βCD.
In the 1H NMR spectrum of a solution of BMDBM and excess HPβCD in D2O (FIGURE
5.18) the tert-butyl resonance appears as two singlets. The BMDBM aromatic resonances
Ha-Hd appear as two sets of poorly resolved doublets arising from the AAʹBBʹ spin-spin
splitting pattern of the para-substituted phenyl rings. The BMDBM vinylic proton Hv of
the enol tautomer of BMDBM can be observed as a broad singlet having an area ratio less
than one due to exchange with the solvent. The enolic hydrogen involved in the
intramolecular hydrogen bond in the enol form of BMDBM is not observed for similar
Chapter 5
175
reasons. The methoxy resonance cannot be distinguished from those of the HPβCD
resonances.
FIGURE 5.18: Partial 1H 600MHz NMR spectrum of BMDBM tert-butyl and aromatic
Ha-Hd resonances and HPβCD (0.01 mol dm-3) in D2O at 298 K. The vertical scaling of
the spectra is not constant.
As with other β-diketones, BMDBM exists in a keto-enol tautomeric equilibrium (FIGURE
5.4) with the equilibrium position heavily influenced by the nature of the solvent [293].
Consequently, there are two different complexing environments of BMDBM, the keto (K)
form and the enol (E) form as shown in FIGURE 5.19. The detection of BMDBM in the 1H NMR solution may be the consequence of the formation of an inclusion compound
between the keto form and HPβCD (K·HPβCD) and/or the enol form and HPβCD
(E·HPβCD). There is also the possibility for formation of 1 : 2 (BMDBM : HPβCD)
inclusion complexes or either the keto and enol forms. It is possible to distinguish between
the two tautomers by 1H NMR based on the separate resonance signals for the vinylic and
methylene protons of the enol and keto tautomers respectively. The vinylic proton of the
1.41.56.877.27.47.67.888.28.4
(ppm)
Hd Ha Hb Hc t-Bu Hv
O O
OHa
Hb HcHd
H
Hv
Chapter 5
176
enol tautomer of BMDBM can be observed as a broad singlet whereas the methylene
resonance of the keto tautomer is not observed.
FIGURE 5.19: Keto-enol tautomerisation of BMDBM in the absence and presence of
HPβCD.
O O
O
O O
O
H
O O
O
HO O
O
E
E•HPβCD
O O
O
HO O
O
K
K•HPβCD
K•(HPβCD)2 E•(HPβCD)2
Chapter 5
177
There are two possibilities for the absence of the methylene resonance; either the keto is
not present in solution or the methylene resonance is masked by the HPβCD resonances.
In polar, protic solvents, the keto-enol equilibrium of β-diketones shifts towards the keto
tautomer as the intramolecular hydrogen bond of the enol is replaced by hydrogen bonding
with the solvent, stabilising the keto form. However, the enol form is still the dominant
tautomer. This is confirmed by investigation of the keto-enol equilibrium of BMDBM in
different solvents. The poor aqueous solubility of free BMDBM prevents acquisition of a 1H NMR spectrum of BMDBM in D2O. However, a quantitative evaluation of the
tautomeric equilibrium of BMDBM in different solvents indicates the enol form
predominates. Integration of the relative intensities of the vinylic and methylene protons
shows 100% enol in CDCl3 and 90% in d6-DMSO, which is in agreement with similar
studies of BMDBM [205]. Consequently, the absence of the keto tautomer cannot be
stated with any certainty but the enol is assumed to be the predominate form. The
geometries of both tautomers of BMDBM have been optimised at the B3LYP/6-31+G(d,p)
level [403], and while the enol is a planar molecule, the keto tautomer is not as shown in
FIGURE 5.20. Moreover, tautomerisation from the enol to the keto form requires
significant structural changes. Therefore, preferential inclusion of the enol tautomer is
expected.
FIGURE 5.20: Structures of chelated enol and keto forms of BMDBM optimised at the
B3LYP/6-31+G(d,p) level of theory [403]. (Only one of the chelated enol forms is shown).
The 1H ROESY NMR spectrum of a solution of BMDBM and an excess of HPβCD in D2O
shows intermolecular interactions between the tert-butyl resonance of BMDBM and
HPβCD H2-H6 resonances (FIGURE 5.21) indicating that the tert-butyl protons are within
~4 Å of the HPβCD interior H2-H6 protons. A weak cross-peak is observed for BMDBM
aromatic Hd resonance, however this is possibly due to interaction between BMDBM Hd
Enol Keto
Chapter 5
178
and methoxy protons rather than an interaction between BMDBM Hd protons and HPβCD
H2-6 protons. No cross-peaks are observed between BMDBM aromatic Ha-Hc and those
of HPβCD. This may be due to either no interaction or the interaction is sufficiently weak
at the present concentrations that it is not detected.
FIGURE 5.21: 1H 600 MHz ROESY NMR spectrum of a solution of BMDBM and HPβCD
in a 1 : 9 mole ratio in D2O at 298 K. The cross-peaks enclosed in the boxes correspond to
intermolecular interactions between the protons indicated on the F1 and F2 axes.
Hc Ha Hd Hb t-Bu H2-6 H1
HPβCD Methyl
H2-
6
HPβCD
HPβC
D p
roto
ns
O O
OHa
Hb HcHd
H
H5
H6 H6
H4
H2
H1
H3
Chapter 5
179
The low concentration of BMDBM in solution and corresponding absence of cross-peaks
makes it difficult to identify the mode of inclusion with any certainty. However, it seems
that there is preferential inclusion of the enol tautomer with HPβCD positioned over the
tert-butyl group. The equilibrium shown in FIGURE 5.12 for inclusion of BMDBM with
βCD is, in principle, also possible for inclusion complexes with HPβCD. From the 1H
ROESY NMR data it is likely that HPβCD·BMDBM includomer A and/or includomer Aʹ
are the dominant inclusion complexes present. It is possible that the keto tautomer can
form inclusion complexes with HPβCD. Keto-enol equilibrium studies of the β-diketone,
benzoylacetone (1-phenyl-1,3-butadione), show both the keto and enol tautomers form
inclusion complexes with βCD [404]. The planar geometry of the enol tautomer of
benzoylacetone allows deeper protrusion inside the CD cavity than the keto tautomer, it is
stabilised in the hydrophobic cavity interior of CD and the keto-enol equilibrium is shifted
to the enol tautomer.
5.2.2.2 NMR data of 1 : 1 mole ratio of BMDBM with HPβCD
For further characterisation of HPβCD inclusion complexes, it was necessary to prepare
solutions of BMDBM and HPβCD in 0.10 mol dm-3 NaOD/D2O to achieve higher
concentrations than was possible in D2O. As discussed previously for βCD solutions, the
basic conditions increase both the solubility of HPβCD and, to a lesser extent, BMDBM
with the pKa of HPβCD is expected to be comparable to the pKa of βCD (pKa 12.20 [402]).
Solutions were prepared by adding BMDBM to a solution of HPβCD in 0.1 mol dm-3
NaOD/D2O. The resulting suspension was gently heated then filtered to remove any
suspended material, which was probably BMDBM and/or its HPβCD complex. As a
consequence of the poor solubility of BMDBM, even under the basic condition used, the
calculated concentrations of BMDBM− were not achieved. Instead, the mole ratios of
HPβCD to BMDBM− were determined by integration of the appropriate 1H NMR signals.
The 1H NMR spectrum of a solution (solution A) in which the mole ratio of BMDBM− and
HPβCD was ~1 : 1 (determined by integration of NMR signals) is shown in FIGURE 5.22.
The tert-butyl resonance appears as a broad singlet and BMDBM− aromatic Ha-Hd
resonances appear as doublets with replication. There is some broadening of the aromatic
Ha-Hd resonances, which is more noticeable for Ha and Hb than for Hc and Hd. The
differences in broadening are consistent with different rates of exchange at the methoxy
and tert-butyl phenyl groups between free and included BMDBM−. The methoxy
resonances are not easily distinguished from the HPβCD H2-H6 proton resonances. Three
Chapter 5
180
separate chemical environments can be observed for BMDBM− Ha-Hd appearing in the
area ratio of ~1 : 1 : 6. Integration of the broad singlet assigned to the tert-butyl resonance
is consistent with this ratio, indicating either three chemically distinct tert-butyl groups that
possess coincident chemical shifts or the tert-butyl protons experience no significant
difference in chemical environments in the different includomers. The chemical shifts and
chemical shift differences of all BMDBM− protons in the absence and presence of HPβCD
are presented in TABLE 5.3.
FIGURE 5.22: Partial 1H 600MHz NMR spectra of BMDBM − tert-butyl and aromatic
Ha-Hd resonances of (i) solution of BMDBM − and (ii) solution of BMDBM −and HPβCD
having ~1 : 1 mole ratio (Solution A) in 0.1 mol dm-3 NaOD/D2O at 298 K. The
degradation productions of BMDBM in (i) are denoted by Haʹ - Hd and t-Bu. The
coloured text makes the distinction between the resonances assigned to the different
includomers. The spectra are not plotted to a constant vertical scale.
t-Bu′
1.21.5(ppm)
6.877.27.47.67.88
t-Bu′ t-Bu
O O
OHa
Hb HcHd
(ii)
(i)
t-Bu
Hc Hb Hc Hb
Hb Hc
Ha
Ha Ha Hd
Hd Hd
Hcʹ
Hbʹ Ha
Hc Hb Ha Hdʹ Hd
Chapter 5
181
TABLE 5.3: 1H chemical shifts (ppm) corresponding to BMDBM − in the absence and
presence of HPβCD in 0.1 mol dm-3 NaOD / D2O at 298 K.
δfree δcomplex (∆λa)
~1 : 1 mole ratiob
t-Bu 1.28 1.41(+0.13) 1.41(+0.13) 1.41(+0.13)
Ha 7.52 7.35(-0.17) ~7.34(-0.18) 7.52(+0.00)
Hb 7.79 7.63(-0.16) 7.76(-0.03) 7.91(+0.12)
Hc 7.82 7.71(-0.11) 7.81(-0.01) 7.98(+0.16)
Hd 6.99 7.01(+0.02) 6.98(-0.01) 7.06(+0.07) a Upfield displacements are negative b Mole ratio of BMDBM− : HPβCD in solution
This spectrum is similar to that of a solution of BMDBM− and βCD (1 : 6 mole ratio)
(FIGURE 5.14). As the cavity of HPβCD has a similar structure to that of the parent βCD
therefore a similar mode of inclusion is expected. A difference between the two is the
concentration of observed includomers with similar CD concentrations. At comparable
βCD concentrations (1 : 2 mole ratio BMDBM− to βCD) BMDBM− existed primarily as
βCD·BMDBM−. This can be attributed to the higher solubility of HPβCD itself relative to
βCD. The solubility of HPβCD is influenced by the nature of the substitution groups, the
degree of substitution and the pattern of substitution. The degree of substitution of HPβCD
used in this study is 3.0-8.0 which gives an average number of 5 hydroxypropyl groups per
βCD molecule.
Aided by 1H NMR ROESY, it is possible to distinguish between the three different tert-
butyl and methoxy phenyl groups, indicated by coloured text in FIGURE 5.22 and TABLE
5.3. For the minor species present in solution, it is not possible to relate the different
chemical environments of any one tert-butyl phenyl groups to a particular methoxy phenyl
group and vice-versa with any certainty. This is due to the distance between the phenyl
groups, where changes in the chemical environment of Ha and Hb are too far away to
influence Hc and Hd. Therefore, the assignment of these resonances to a particular
includomer can not be made with certainty.
The 1H ROESY NMR spectrum of a solution in which the mole ratio of BMDBM− and
HPβCD was ~1 : 1 (determined by integration of NMR signals) is shown in FIGURE 5.23.
Chapter 5
182
FIGURE 5.23: 1H 600 MHz ROESY NMR spectrum of a solution of BMDBM − and
HPβCD in a 1 : 1.3 mole ratio in 0.10 mol dm-3 NaOD at 298 K. The cross-peaks enclosed
in the boxes correspond to intermolecular interactions between the protons indicated on
the F1 and F2 axes. Not all BMDBM − resonances are labelled.
Strong intermolecular interactions are observed between the dominant BMDBM− tert-butyl
resonance and aromatic Ha resonance with HPβCD H2-H6 resonances. A weaker
interaction is observed between the dominant BMDBM− Hb and βCD H2-H6 resonances.
Cross-peaks are observed for the dominant BMDBM− aromatic Hd resonance but this may
CD
pro
tons
OC
H3
H2-
H6
Hc Hb Ha Hd
t-Bu CD H1
HOD
CD H2-6 CD
CH3 OCH3
O O
OHa
Hb HcHd
H5
H6 H6
H4
H2
H1
H3
Chapter 5
183
be due to correlation with the signal assigned to the methoxy resonance of the BMDBM−,
which overlaps with the HPβCD protons. The absence of cross-peaks between the minor
BMDBM− Ha-Hd resonances assigned to the minor includomers may be due to the low
concentration of these species. The interactions between the tert-butyl and Ha-Hb protons
with HPβCD H2-H6 protons is similar to that observed for βCD·BMDBM− (FIGURE
5.11) although no interactions are observed between BMDBM− Hc-Hd and HPβCD
protons. This supports the view that the βCD spectra represents a fast exchange process
occurring between βCD·BMDBM− and (βCD)2·BMDBM−.
These observations are consistent with BMDBM− existing primarily as HPβCD·BMDBM−
where the cavity of HPβCD is positioned over the tert-butyl and Ha protons. Again, a
similar mode of inclusion to βCD is expected for HPβCD and, therefore, the equilibrium
shown in FIGURE 5.12 and reproduced in FIGURE 5.24 is, in principle, applicable here
also. As there are two possible threading orientations of HPβCD such that either the tert-
butyl group is closest to the primary or secondary hydroxyl lined rim, two includomers
corresponding to HPβCD·BMDBM− includomer A and includomer Aʹ are possible
(FIGURE 5.24). As inclusion by HPβCD is a dynamic process, it is likely that the
observed resonances assigned to HPβCD·BMDBM− are a time- and population-average of
the individual resonances of these two includomers. This is supported by the observed
broadening of the dominant BMDBM− Ha and Hb resonances. As the HPβCD resonances
are less resolved due to substitution than those observed for the unsubstituted βCD, it is not
possible to determine if there is a preferred threading orientation of the CD. The proposed
mode of inclusion is similar to that proposed for the analogous βCD inclusion complex
(βCD·BMDBM−). This is consistent with HPβCD having a similar structure and cavity
size to that of the parent βCD.
Chapter 5
184
FIGURE 5.24: Formation of HPβCD·BMDBM − and (HPβCD)2·BMDBM
− inclusion
complexes involving the possible inclusion orientations of HPβCD.
O O
O
O O
O
O O
O
O O
O
O O
O
O O
O
O O
O
O O
O
Includomer B
Includomer B′
Includomer D′
Includomer A′
Includomer C′Includomer C
Includomer A
Includomer D
Chapter 5
185
5.2.2.3 NMR data of BMDBM with excess HPβCD
The 1H NMR spectrum of a solution of BMDBM− and an excess of HPβCD is shown in
FIGURE 5.25. The tert-butyl resonance appears as two singlets with very similar chemical
shift and the aromatics, Ha-Hd as doublets with replication. The aromatic resonances Ha-
Hd appear in the area ratio is approximately 1 : 1 : 1.5. The chemical shifts of the
replicated aromatic Ha-Hd (TABLE 5.3) are similar to those observed in the 1H NMR
spectrum of BMDBM− with equimolar HPβCD. This suggests that the βCD and HPβCD
includomers formed with BMDBM are similar and therefore, the increase in solubility of
HPβCD includomers can be attributed to the increase in solubility of HPβCD itself.
FIGURE 5.25: Partial 1H 600MHz NMR spectra of BMDBM − tert-butyl aromatic Ha-Hd
resonances of a) solution of BMDBM − and b) solution of BMDBM − with an excess of
HPβCD in 0.1 mol dm-3 NaOD/D2O at 298 K. The spectra are not plotted to a constant
vertical scale.
O O
OHa
Hb HcHd
6.877.27.47.67.88 1.31.4(ppm)
Hcʹ
Hbʹ Ha
Hc Hb Ha Hdʹ Hd
t-Bu, t-Bu, t-Bu
t-Bu
Hc
Hb
Hc Ha Ha Ha
Hd
Hb Hc Hd Hd
t-Buʹ
Hb
(ii)
(i)
Chapter 5
186
The 1H ROESY NMR spectrum of a solution of BMDBM− and an excess of HPβCD is
shown in FIGURE 5.26. Strong intermolecular interactions are observed between the tert-
butyl resonances with HPβCD H2-6 resonances. There are also strong cross-peaks
between two of the BMDBM− Ha aromatic resonances, Ha and Ha and those of HPβCD
H2-H6 protons. Additionally, a cross-peak is observed between BMDBM− Hb aromatic
resonance with the HPβCD H2-H6 resonances.
FIGURE 5.26: 1H 600 MHz ROESY NMR spectrum of BMDBM − and an excess of
HPβCD in 0.10 mol dm-3 NaOD at 298 K. The cross-peaks enclosed in the boxes
correspond to intermolecular interactions between the protons indicated on the F1
(BMDBM −) and F2 (HPβCD) axes.
H5
H6
H6
H4
H2
H1
H3
O O
OHa
Hb HcHd
CD
pro
tons
OC
H3
H2-
H6
Hc Hb Ha Hd t-Bu
CD H1
HOD
CD H2-6
CD CH3
OCH3
Chapter 5
187
5.2.2.4 Temperature-Dependence Studies of HPβCD Inclusion Complexes with
BMDBM
To study the nature of the HPβCD inclusion complexes formed further, variable-
temperature 1H NMR studies (298-323 K) were carried out on a solution of BMDBM− and
an excess of HPβCD. The 1H NMR spectrum obtained at 298 K and 323 K are shown in
FIGURE 5.27.
FIGURE 5.27: Partial variable-temperature 1H 600MHz NMR spectra of BMDBM − tert-
butyl and aromatic Ha-Hd resonances of a solution of BMDBM − with an excess of
HPβCD at (i) 298 K and (ii) 323 K in 0.1 mol dm-3 NaOD/D2O. The HPβCD methyl
resonance (δ 1.49 ppm) has been omitted for clarity. The spectra are not plotted to a
constant vertical scale.
1.31.51.7(ppm)
t-Bu, t-Bu, t-Bu
t-Bu, t-Bu, t-Bu
Ha
6.877.27.47.67.888.28.4
Hc Hb
Hc Ha Ha Hd
Hb Hc Hd Hd Hb
Ha
Hd
Hd
Hd
Ha Ha
Hc Hb
Hb Hc
Hb
Hc
(ii)
(i)
O O
OHa
Hb HcHd
Chapter 5
188
The entire spectrum, referenced to HOD solvent resonance, is shifted downfield with
increasing temperature as observed and discussed previously for the analogous studies of
BMDBM with βCD in Chapter 5.2.1.3. The area ratio of the three sets of doublets
previously assigned to BMDBM− aromatic Ha-Hd resonances changes from 1 : 1 : 1.25 at
298 K to 3 : 1 : 3 at 323 K. No significant broadening or coalescence of the resonances
assigned to BMDBM− tert-butyl or aromatic Ha-Hd protons can be observed with
increasing temperature. This indicates the coalescence temperature has not been reached
and the includomers are in slow exchange at the higher temperatures although the relative
populations of includomers are changing. The change in populations indicates a change in
the equilibrium positions where the equilibrium constants for the exchange process
involving HPβCD moving on and off BMDBM− is a function of temperature. Similar
observations were made in the analogous variable-temperature studies of BMDBM with
βCD. One difference in the two studies is that in the case of βCD the relative populations
of the minor includomers did not change with temperature whereas a change is observed
with increasing temperature for the relative populations of the HPβCD includomers.
The 1H NMR data of the inclusion complexes of βCD and HPβCD with BMDBM are in
agreement with phase solubility studies reported for these complexes [405] whereby
HPβCD forms more stable includomers. This is indicated by the association constants K of
1.44 × 103 mol dm-3 [213] and K of 2.23 × 103 mol dm-3 for formation of 1 : 1 inclusion
complexes of BMDBM with βCD and HPβCD, respectively [213,371]. Additionally, the
association constant K of 13 mol dm-3 for formation of a 1 : 2 inclusion complex of
BMDBM with HPβCD is reported whereas the analogous βCD includomer were not
detected.