UVA Chemical Filters: A Systematic Study

Transcription

1 UVA Chemical Filters: A Systematic Study Jacqueline F. Cawthray, B. Science (ons) A thesis submitted for the degree of Doctor of Philosophy in The University of Adelaide Department of Chemistry February 2009

2 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. n Primary hydroxyl rim Secondary hydroxyl rim n = 1 α-cd n = 2 β-cd n = 3 γ-cd 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 151

3 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. nce 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 10 5 mol dm -3 [382]. X + X Y 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 (PβCD) and randomly methylated βcd in which one or several 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 152

4 intramolecular hydrogen bonding between 2- and 3- 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, PβCD 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 PβCD in addition to the properties of the inclusion complex formed [381,385,386]. R R R 7 R Solubility (mg/ml) βcd 18.5 PβCD C 2 C()C 3 >600 FIGURE 5.3: Structures and solubilities of βcd and PβCD. 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 [ ] food, cosmetic [390,391] and chemical industries. 153

5 5.1.1 Use of Cyclodextrins with Sunscreens It is now clear that wavelengths in the UVA region ( 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]. owever, 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 (λ max ~359 nm) whilst the keto absorbs in the UVB region (λ max ~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]. hv Enol Keto FIGURE 5.4: (BMDBM). Keto-enol equilibrium in 4-tert-butyl-4 -methoxydibenzoylmethane 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. f the naturally occurring CDs, βcd forms the most stable inclusion complexes [213]. The smaller cavity of αcd can result in steric interference 154

6 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 PβCD [371,395] and randomly methylated βcd [213] and forms the claim of at least one patent [396]. owever, the stabilising effect of PβCD 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 PβCD 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]. owever 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 (Pα-, Pβ-, PγCD, randomly methyl βcd) show that PβCD 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 PβCD over those formed with βcd can be attributed to the greater solubility of PβCD 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 PβCD. The 1D and 2D 1 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 155

7 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 (3 and 5) are shifted in the spectrum while the exterior protons (2 and 4) 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 1 NMR studies can be used to investigate the exchange process (4) (2) (6) (5) (3) (1) 7 FIGURE 5.5: β-cyclodextrin interior and exterior protons. The 2D NMR technique, 1-1 RESY (Rotating frame nuclear verhauser Effect SpectroscopY), is a spin-lock technique that identifies through-space interactions (NEs) via spin-spin relaxation. In the 1 RESY NMR spectra, cross-peaks arise from spatially separated protons that are 4 Å apart even in the absence of covalent bonding. Therefore, 1 RESY 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 NE between spatially separated protons is related to both separation distance and concentration. 156

8 β-cyclodextrin and its hydroxyalkyl derivative, PβCD 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 PβCD due to substitution by the hydroxyalkyl group. The degree of substitution of PβCD used in this study is which gives an average number of 5 hydroxypropyl groups per βcd molecule. Both βcd and PβCD 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] Å 4.1 Å Å 7.9 Å R 5.0 Å Cavity volume 262 Å 3 R = C 3 or C(C 3 ) 9 FIGURE 5.6: Dimensions of β-cyclodextrin (βcd) and hydroxypropyl-β-cyclodextrin (PβCD) compared with the dimensions of the phenyl group in BMDBM NMR Studies of βcd and PβCD Complexes The 1 and 1 RESY NMR spectra are reported for solutions of BMDBM and either βcd or PβCD in which the mole ratio of BMDBM to CD is varied. A variabletemperature 1 NMR study of the solution of BMDBM and either excess βcd or PβCD are also presented. 157

9 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 PLC or examined complexation in solids or suspensions [213,371,397,398]. To achieve the concentrations required to obtain an 1 NMR spectrum, it was necessary to prepare all solutions in 0.1 mol dm -3 NaD/D 2 such that pd 12. As the pk a of βcd is [402], under the basic conditions employed the βcd hydroxyl groups, (2) and (3), are partially deprotonated. This results in an increase in the solubility of βcd. The pk a of BMDBM is (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 BMDBM BMDBM FIGURE 5.7: Generation of the β-diketonate of BMDBM (BMDBM ). nly one of the chelated enols are shown. Solutions were prepared by adding BMDBM to a solution of βcd in 0.1 mol dm -3 NaD/D 2. 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 1 NMR signals. In the following text the numbering scheme shown in FIGURE 5.8, whereby the CD protons are denoted as 1-6 and the aromatic protons of BMDBM as a-d, have been used for the sake of clarity. 158

10 To enable a comparison between free and included BMDBM the 1 NMR spectrum of BMDBM in 0.1 mol dm -3 NaD/D 2 was obtained (FIGURE 5.10a). To achieve the concentration required to obtain a 1 NMR spectrum, the solution was rapidly heated to high temperatures and the suspended material was removed by filtration. The 1 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 1 NMR signals. The tert-butyl resonance appears as two singlets, assigned as t-bu and t-buʹ in FIGURE 5.10a. The BMDBM aromatic resonances as two sets of doublets assigned as a-d and aʹ-dʹ. The methoxy resonance is observed as two singlets appearing at δ 3.87 (assigned to degradation products) and δ (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 1 NMR data presented). 6 6 a b c d 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 1 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. 159

11 tbu a b c d tbu a b c d Fragment A Fragment B Fragment A Fragment B FIGURE 5.9: Two possible positions where bond cleavage of BMDBM can occur leading to different fragmentation patterns NMR data of 1 : 2 mole ratio of BMDBM : βcd. In the 1 NMR spectrum of a solution in which the mole ratio (determined by integration of the 1 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 a-d as doublets arising from AAʹBBʹ spin-spin splitting. Unambiguous assignment of the methoxy resonance was not possible as the βcd 3 resonance appears in the same region of the spectrum. The tert-butyl and a-d 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, a and b 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 1 RESY NMR spectroscopy as detailed below. 160

12 TABLE 5.1: 1 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 NaD/D 2 at 298 K. δ free δ complex ( λ a ) t-bu (+0.14) a (-0.17) b (-0.18) c (-0.10) d (+0.02) a Upfield displacements are negative a b c d (i) c b a d t-bu (ii) cʹ c b a dʹ d t-bu bʹ aʹ t-buʹ (ppm) FIGURE 5.10: Partial Mz NMR spectra of BMDBM tert-butyl and aromatic a- d 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 NaD/D 2 at 298 K. The degradation productions of BMDBM in (i) are denoted by aʹ, bʹ, cʹ, dʹ and t-buʹ. The spectra are not plotted to a constant vertical scale. 161

13 The 1 RESY NMR spectrum of a solution in which the mole ratio of BMDBM and βcd was 1 : 2 is shown in FIGURE a b c d βcd 1 D βcd 2-6 c b a d t-bu 3 βcd protons FIGURE 5.11: Mz RESY NMR spectrum of a solution BMDBM with βcd in a 1 : 2 mole ratio in 0.10 mol dm -3 NaD 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 3, 5 and 6 protons of the annular interior. Strong cross-peaks are also observed 162

14 between BMDBM a and βcd 3 and 5 as well as BMDBM d and βcd 3. A somewhat weaker cross-peak can be observed between BMDBM b and βcd 6. 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 1, 2 or 4 protons. The experimental observations taken from the 1 and 1 RESY 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 a-d 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 A visual comparison of the cross-peak intensities in the 1 NMR RESY, 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 This would be facilitated by the greater hydrophobic nature of the tertbutyl 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 a-b protons. The upfield shift for a and b 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 tertbutyl 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. 163

15 Includomer B Includomer C Includomer C Includomer A Includomer A Includomer D Includomer D Includomer B FIGURE 5.12: Formation of βcd BMDBM and (βcd) 2 BMDBM inclusion complexes involving the possible inclusion orientations of βcd. 164

16 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. 165

17 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 1 NMR RESY, the comparatively weaker crosspeaks between BMDBM tert-butyl protons and βcd 6 coupled with the absence of cross-peaks between BMDBM a and βcd 6 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 1 NMR RESY observed between BMDBM d and βcd 3 protons and between c and βcd 6. 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 c 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 Bʹ. 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 c and d with βcd protons and the absence of cross-peaks for d with βcd 5 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 RESY interactions of c and βcd 6 protons were due to the presence of (βcd) 2 BMDBM includomer Dʹ only BMDBM d protons would be positioned deep in the βcd cavity and cross-peaks of similar intensity with both βcd 3 and 5 would be observed. The difference in chemical shift between free and included c and d of BMDBM is much less than that observed for a and b (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 a-b 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 a- 166

18 b 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 NMR data of BMDBM with excess βcd. In the 1 NMR spectrum of a solution, solution B, in which the mole ratio of BMDBM and βcd was 1 : 6 (as determined by integration of 1 NMR signals), the tert-butyl resonance appears as two singlets and the BMDBM aromatic resonances assigned to a- d 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 3 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 1 NMR spectrum of free BMDBM as shown in FIGURE 5.14a is the same as presented earlier in FIGURE 5.10a. The 1 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, a-d 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 1 NMR RESY, 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 167

19 two phenyl groups is too large to generate cross-peaks in the 1 NMR RESY 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. a b c d (i) b c b a d t-bu t-bu c c b a a d d t-bu (ii) cʹ c b a dʹ d t-bu bʹ aʹ t-buʹ (ppm) FIGURE 5.14: Partial Mz NMR spectra of BMDBM - tert-butyl and aromatic a-d 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 NaD / D 2 at 298 K. The degradation products of BMDBM in (i) are denoted by aʹ - dʹ and t-buʹ. The coloured text makes the distinction between the resonances assigned to the different includomers. The spectra are not plotted to a constant vertical scale. 168

20 TABLE 5.2: 1 chemical shifts (ppm) corresponding to BMDBM in the absence and presence of βcd in 0.1 mol dm -3 NaD/D 2 at 298 K. δ free δ complex ( λ a ) δ complex ( λ a ) 1 : 2 mole ratio b 1 : 6 mole ratio t-bu (+0.14) 1.41 (+0.13) 1.41 (+0.13) 1.43 (+0.15) a (-0.17) 7.38 (-0.14) 7.34 (-0.18) 7.52 (+0.00) b (-0.18) 7.61 (-0.18) 7.74 (-0.05) 7.90 (+0.11) c (-0.10) 7.73 (-0.09) 7.83 (+0.01) 7.96 (+0.14) d (+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 The greatest change observed is for the resonances assigned to the interior βcd protons 3, 5 and 6. The downfield shift of the 5 resonance means it is no longer possible to assign individual resonances to 5 and 6. There is no significant change in the resonances assigned to the exterior βcd protons 2 and 4. 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 1 chemical shifts. 169

21 (ii) (i) (ppm) FIGURE 5.15: Partial 1 600Mz NMR spectra of βcd 2-6 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 NaD/D 2 at 298 K. The spectra are not plotted to a constant vertical scale. The 1 RESY NMR spectrum of a solution of BMDBM and an excess of βcd is shown in FIGURE This shows strong intermolecular interactions between the proton resonances of BMDBM previously assigned to the dominant includomer and those of the βcd interior 3, 5 and 6 protons. Strong cross-peaks are observed between BMDBM tert-butyl protons and βcd protons 3, 5 and 6. 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 crosspeaks between BMDBM tert-butyl protons and βcd exterior protons 2 and 4 are possibly due to noise running horizontally along the spectra. Strong cross-peaks are also observed between the BMDBM a and βcd 3, 6 and 5. Cross-peaks can be seen between BMDBM b and c protons and βcd 5/6 protons. Additionally, there are interactions between BMDBM d with βcd 3 protons. For the minor includomers, a weak cross-peak is observed between BMDBM a and βcd 5 and

22 6 6 a b c d D βcd βcd t-bu 3 βcd protons 6, FIGURE 5.16: Mz RESY NMR spectrum of a solution of BMDBM and βcd in a 1 : 6 mole ratio (solution B) in 0.10 mol dm -3 NaD 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 a-d resonances, refer to FIGURE The observations from the 1 NMR and the 1 RESY NMR of solution B provides additional evidence in support of the formation of (βcd) 2 BMDBM in which βcd is 171

23 positioned over the tert-butyl phenyl group whilst a second βcd envelopes the methoxy phenyl group. In solution A, the resonances (tert-butyl, a-d) 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 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 1 RESY NMR interactions. There is no change observed for the chemical shifts of the resonances tert-butyl, a-d 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 a and b are too far away to influence c and d. The relative intensities confirm the preferential inclusion of BMDBM tert-butyl phenyl group Temperature-Dependence Studies of βcd Inclusion Complexes with BMDBM The nature of the inclusion complexes formed were investigated further by variabletemperature 1 NMR studies ( K) of a solution of BMDBM and βcd in a 1 : 6 mole ratio. The 1 NMR spectrum obtained at 298 K and 323 K are shown in FIGURE The entire spectrum, referenced to the D 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 a-d can be observed with increasing temperature. The three sets of doublets previously assigned to a-d 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. 172

24 a b c d (ii) b c b a d t-bu, t-bu c b c a a d d t-bu (i) c c b c b a d a a d d t-bu, t-bu t-bu (ppm) FIGURE 5.17: Partial variable-temperature Mz NMR spectra of BMDBM tert-butyl and aromatic a-d 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 NaD/D 2. The spectra are not plotted to a constant vertical scale. The observations from the variable-temperature 1 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. owever, 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 173

25 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: InK G = RT 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 PβCD Complexes NMR data of BMDBM with PβCD in D 2 In contrast to βcd and its inclusion complexes, the inclusion complexes of PβCD with BMDBM are sufficiently soluble to allow acquisition of a 1 NMR spectrum in D 2. In the absence of PβCD, BMDBM cannot be detected by 1 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 PβCD (0.01 mol dm -3 ) in D 2 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 PβCD, determined by integration of the 1 NMR signals, is approximately 1 : 9. Phase solubility studies show the solubilising effect of PβCD on BMDBM is greater than for the parent βcd [213,371]. The increase in solubility of BMDBM with PβCD is attributed to the greater solubility of PβCD itself over that of βcd. In the 1 NMR spectrum of a solution of BMDBM and excess PβCD in D 2 (FIGURE 5.18) the tert-butyl resonance appears as two singlets. The BMDBM aromatic resonances a-d 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 v 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 174

26 reasons. The methoxy resonance cannot be distinguished from those of the PβCD resonances. a v b c d c b a d v t-bu (ppm) FIGURE 5.18: Partial 1 600Mz NMR spectrum of BMDBM tert-butyl and aromatic a-d resonances and PβCD (0.01 mol dm -3 ) in D 2 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 The detection of BMDBM in the 1 NMR solution may be the consequence of the formation of an inclusion compound between the keto form and PβCD (K PβCD) and/or the enol form and PβCD (E PβCD). There is also the possibility for formation of 1 : 2 (BMDBM : PβCD) inclusion complexes or either the keto and enol forms. It is possible to distinguish between the two tautomers by 1 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 175

27 enol tautomer of BMDBM can be observed as a broad singlet whereas the methylene resonance of the keto tautomer is not observed. K E K PβCD E PβCD K (PβCD) 2 E (PβCD) 2 FIGURE 5.19: Keto-enol tautomerisation of BMDBM in the absence and presence of PβCD. 176

28 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 PβCD 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. owever, 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 1 NMR spectrum of BMDBM in D 2. owever, 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 CDCl 3 and 90% in d 6 -DMS, 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 Moreover, tautomerisation from the enol to the keto form requires significant structural changes. Therefore, preferential inclusion of the enol tautomer is expected. Enol Keto 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]. (nly one of the chelated enol forms is shown). The 1 RESY NMR spectrum of a solution of BMDBM and an excess of PβCD in D 2 shows intermolecular interactions between the tert-butyl resonance of BMDBM and PβCD 2-6 resonances (FIGURE 5.21) indicating that the tert-butyl protons are within ~4 Å of the PβCD interior 2-6 protons. A weak cross-peak is observed for BMDBM aromatic d resonance, however this is possibly due to interaction between BMDBM d 177

29 and methoxy protons rather than an interaction between BMDBM d protons and PβCD 2-6 protons. No cross-peaks are observed between BMDBM aromatic a-c and those of PβCD. This may be due to either no interaction or the interaction is sufficiently weak at the present concentrations that it is not detected. a b c d c b a d 1 PβCD 2-6 t-bu PβCD Methyl PβCD protons 2-6 FIGURE 5.21: Mz RESY NMR spectrum of a solution of BMDBM and PβCD in a 1 : 9 mole ratio in D 2 at 298 K. The cross-peaks enclosed in the boxes correspond to intermolecular interactions between the protons indicated on the F1 and F2 axes. 178

30 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. owever, it seems that there is preferential inclusion of the enol tautomer with PβCD 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 PβCD. From the 1 RESY NMR data it is likely that PβCD 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 PβCD. 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 NMR data of 1 : 1 mole ratio of BMDBM with PβCD For further characterisation of PβCD inclusion complexes, it was necessary to prepare solutions of BMDBM and PβCD in 0.10 mol dm -3 NaD/D 2 to achieve higher concentrations than was possible in D 2. As discussed previously for βcd solutions, the basic conditions increase both the solubility of PβCD and, to a lesser extent, BMDBM with the pk a of PβCD is expected to be comparable to the pk a of βcd (pk a [402]). Solutions were prepared by adding BMDBM to a solution of PβCD in 0.1 mol dm -3 NaD/D 2. The resulting suspension was gently heated then filtered to remove any suspended material, which was probably BMDBM and/or its PβCD 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 PβCD to BMDBM were determined by integration of the appropriate 1 NMR signals. The 1 NMR spectrum of a solution (solution A) in which the mole ratio of BMDBM and PβCD was ~1 : 1 (determined by integration of NMR signals) is shown in FIGURE The tert-butyl resonance appears as a broad singlet and BMDBM aromatic a-d resonances appear as doublets with replication. There is some broadening of the aromatic a-d resonances, which is more noticeable for a and b than for c and d. 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 PβCD 2-6 proton resonances. Three 179

31 separate chemical environments can be observed for BMDBM a-d 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 PβCD are presented in TABLE 5.3. a b c d (ii) c b a a d t-bu c b c b a d d (i) cʹ c b a dʹ d bʹ aʹ t-bu t-bu t-bu (ppm) FIGURE 5.22: Partial 1 600Mz NMR spectra of BMDBM tert-butyl and aromatic a-d resonances of (i) solution of BMDBM and (ii) solution of BMDBM and PβCD having ~1 : 1 mole ratio (Solution A) in 0.1 mol dm -3 NaD/D 2 at 298 K. The degradation productions of BMDBM in (i) are denoted by aʹ - dʹ and t-buʹ. The coloured text makes the distinction between the resonances assigned to the different includomers. The spectra are not plotted to a constant vertical scale. 180

32 TABLE 5.3: 1 chemical shifts (ppm) corresponding to BMDBM in the absence and presence of PβCD in 0.1 mol dm -3 NaD / D 2 at 298 K. δ free δ complex ( λ a ) ~1 : 1 mole ratio b t-bu (+0.13) 1.41(+0.13) 1.41(+0.13) a (-0.17) ~7.34(-0.18) 7.52(+0.00) b (-0.16) 7.76(-0.03) 7.91(+0.12) c (-0.11) 7.81(-0.01) 7.98(+0.16) d (+0.02) 6.98(-0.01) 7.06(+0.07) a Upfield displacements are negative b Mole ratio of BMDBM : PβCD 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 PβCD 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 PβCD itself relative to βcd. The solubility of PβCD is influenced by the nature of the substitution groups, the degree of substitution and the pattern of substitution. The degree of substitution of PβCD used in this study is which gives an average number of 5 hydroxypropyl groups per βcd molecule. Aided by 1 NMR RESY, it is possible to distinguish between the three different tertbutyl 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 a and b are too far away to influence c and d. Therefore, the assignment of these resonances to a particular includomer can not be made with certainty. The 1 RESY NMR spectrum of a solution in which the mole ratio of BMDBM and PβCD was ~1 : 1 (determined by integration of NMR signals) is shown in FIGURE

33 6 6 a b c d c b a d CD 1 D CD t-bu 2-6 CD C 3 C 3 C3 CD protons 2-6 FIGURE 5.23: Mz RESY NMR spectrum of a solution of BMDBM and PβCD in a 1 : 1.3 mole ratio in 0.10 mol dm -3 NaD 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 a resonance with PβCD 2-6 resonances. A weaker interaction is observed between the dominant BMDBM b and βcd 2-6 resonances. Cross-peaks are observed for the dominant BMDBM aromatic d resonance but this may 182

34 be due to correlation with the signal assigned to the methoxy resonance of the BMDBM, which overlaps with the PβCD protons. The absence of cross-peaks between the minor BMDBM a-d resonances assigned to the minor includomers may be due to the low concentration of these species. The interactions between the tert-butyl and a-b protons with PβCD 2-6 protons is similar to that observed for βcd BMDBM (FIGURE 5.11) although no interactions are observed between BMDBM c-d and PβCD 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 PβCD BMDBM where the cavity of PβCD is positioned over the tert-butyl and a protons. Again, a similar mode of inclusion to βcd is expected for PβCD 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 PβCD such that either the tertbutyl group is closest to the primary or secondary hydroxyl lined rim, two includomers corresponding to PβCD BMDBM includomer A and includomer Aʹ are possible (FIGURE 5.24). As inclusion by PβCD is a dynamic process, it is likely that the observed resonances assigned to PβCD 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 a and b resonances. As the PβCD 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 PβCD having a similar structure and cavity size to that of the parent βcd. 183

35 Includomer B Includomer C Includomer C Includomer A Includomer A Includomer D Includomer D Includomer B FIGURE 5.24: Formation of PβCD BMDBM and (PβCD) 2 BMDBM inclusion complexes involving the possible inclusion orientations of PβCD. 184