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Even after 24 hours of reaction, the yield of the product was quite low, with unoptimised yield of approximately 25%. This could be due to the use of only a slight excess of ligand that was not enough to deprotonate the NH proton of all of the imidazole ligands to allow the reaction to take place. Nevertheless, the formation of host 4.1 has been confirmed by ESI-MS, 1H and 13C NMR spectroscopy. From the ESI-
MS spectrum of host 4.1, there are peaks that correspond to the mass of the molecular ion, particularly 1019.6 for [M+Na]+, 499.3 for [(M/2)+2H+]2+ and 333.4 for
[(M/3)+3H+]3+. In the 1H NMR spectrum, the formation of host 4.1 is indicated by the
appearance of a CH2 signal that connects the tripodal core with the imidazole urea
ligands at 5.35 ppm.
Due to the low yield of host 4.1, the reaction to form host 4.2 was performed for a longer time, up to 48 hours with a higher excess of ligand 2.3 (for example 1 mole of triethylbenzenetribromide was reacted with 6 moles of ligand 2.3). After 48 hours, a yellow sticky solid was isolated from the reaction mixture. This material was evaporated to dryness using rotary evaporator giving yellow solid that upon exposure to air begin to turn sticky again. Although the reaction time was prolonged to 48 hours, the yield of host 4.2 was still low at 45% unoptimised yield. With the use of a higher excess of the ligand, the yield can be improved somewhat, but the removal of the excess ligand now becomes a challenge. Nevertheless, the formation of host 4.2 has been confirmed by ESI-MS showing molecular ion mass peak of 1185.5 for [M+Na]+,
583.6 for [(M/2)+2H+]2+ and 389.6 for [(M/3)+3H+]3+. The 1H NMR spectrum of host 4.2
also confirmed the presence of CH2 signal that connects the tripodal core with the
imidazole urea ligands at 5.19 ppm. Although the targeted product has been successfully synthesised, this method is not optimal due to the need to use excess ligand and difficulty in the separation and removal of the residual reactants since the reactants and products show similar solubility in a range of solvents such as alcohols.
Multiple recrystallisations using different types of solvents have been performed to purify compound 4.1 and 4.2 as tabulated in Table 4.1. To perform the crystallisation
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experiments, about 50 mg of host 4.1 and 4.2 were weighed into a 2 cm3 glass vials
and the vials were filled with an appropriate amount of solvent around 1-1.5 cm3
depending on the solubility. All samples were heated and sonicated until everything had dissolved. The vials were loosely sealed with lid resting on top of the vial to allow slow evaporation of the solvent. The samples were checked every couple of days until either crystal formed or non-crystalline solids formed, or a sticky oil which did not change over the course of a few weeks. Unfortunately, the crystallisation experiments only give either impure solids or impure sticky solid in which the impurities can still be seen in 1H NMR spectra of both hosts 4.1 and 4.2. Host 4.1 obtained after
recrystallisation was also washed with hot acetonitrile and the solution decanted. Washing with hot acetonitrile caused the formation of a brown viscous liquid that was then dried in the oven at 60 ⁰C for two days.
Table 4.1 Crystallisation experiments of host 4.1 and host 4.2
(Note: P = precipitate, I = Insoluble).
Hosts Solvents 4.1 4.2 Acetonitrile P P Nitromethane P P 1-propanol P P Tetrahydrofuran I I Ethyl acetate I I Acetone I I
Acetonitrile: Methanol (few drops) P P
Nitromethane: Methanol (few drops) P P
Acetonitrile: Ethanol (few drops) P P
Nitromethane: Ethanol (few drops) P P
Ethyl acetate: Ethanol (few drops) P P
Ether diffusion into methanol solution P P *P = Precipitate, I = Insoluble
On the other hand, attempts to purify host 4.2 were performed using different techniques such as membrane dialysis and column chromatography. Membrane dialysis technique was not the best solution as it decomposes the product after overnight stirring in methanol. Column chromatography was also employed to separate the residual reactants from the product using silica gel as the stationary phase and a mixture of ethyl acetate: iso-propyl alcohol: water (4:2:1) with 0.1% NH4OH as the eluent. The addition of 0.1% of NH4OH is necessary to deactivate the
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silica gel, otherwise, host 4.2 will decompose on the column. Regardless of the high polarity of the eluent system being used, the residual reactants still cannot be separated from the product as the signal of the residual reactants can still be observed in the 1H NMR spectra of the product. The use of a higher amount of excess ligand also
results in the higher percentage of impurities retained in the mixture (Figure 4.1).
Figure 4.1 1H NMR spectrum of compound 4.2.
For host 4.2, a 1H DOSY NMR (Diffusion Ordered Spectroscopy) experiment was
performed to distinguish the signals of the excess reactants and product. The 1H DOSY
NMR spectrum of 4.2 indicates the presence of two compounds proved by two distinct layers with the diffusion coefficient of 1.0 x 10-10 m2s-1 and 1.8 x 10-10 m2s-1,
corresponding to the residual reactants and product, respectively (Figure 4.1 and 4.2).
1H DOSY NMR is a very useful technique that is predominantly used for investigating
aggregation behaviour in solution. The principle of the technique is based on the diffusion coefficient parameter that is highly sensitive towards changes in the molecular or aggregate size and the number of individual molecules, which constitutes an aggregate. The molecular mass of the aggregate can then be estimated using the Stokes-Einstein equation.28 From the DOSY spectrum, the molecular mass calculated
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both in dimeric form, hence the molecular mass calculated is compared to that of theoretical molecular mass of the dimers. The estimated molecular weight of both compounds is summarised in Table 4.2.
Figure 4.2 1H DOSY NMR of compound 4.2.
Table 4.2 Estimated molecular mass (g/mol) of both mixtures found in the DMSO-d6 solution of compound 4.2.
Entry Diffusion Coefficient,
Dobs (m2s-1)
Estimated molecular mass of the dimer (g/mol)
Calculated
molecular mass of the dimer (g/mol)
Difference (%)
Host 4.2 1.0 x 10-10 2985.8 2331.6 ±28.06
Ligand 2.3 1.8 x 10-10 760.7 645 ±17.94
From the estimated molecular mass in Table 4.2, it can also be observed that the residual reactants and products are prone to form dimers in the solution as the estimated molecular mass are closer to the calculated molecular mass for both of the dimers. The compound with lower molecular weight tends to diffuse more quickly in solution, in this case, the residual reactants, in comparison with the product that is heavier, having a molecular mass of at least three times of the residual reactants. From
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this data, the peaks can be separated and assigned unambiguously, and it can be seen that the higher intensity peak of imidazole proton belongs to the residual reactants, while the lower intensity peak belongs to the product.
Due to the difficulty in the purification step, compound 4.2 was used in its impure form for qualitative anion binding studies, which is discussed in the following subsection. Due to the lower yield and the challenge in the purification of products when excess ligands were used as a base in the synthesis, a different type of inorganic base was introduced for the reaction between the triethylbenzene core with ligand 2.4. All of the synthesis attempts were monitored using LC ESI-MS in methanol for quick characterisation of the product obtained. The first attempt that was carried out in the presence of strong bases such as potassium hydroxide and sodium hydroxide, did not yield the desired product, most likely due to the insolubility of the base in organic solvent. Another alternative such as potassium carbonate also was unsuccessful in synthesising the desired compounds.
To overcome the solubility problem of the inorganic base, a phase transfer catalyst, tetrabutylammonium bromide, was also added to the reaction mixture along with potassium carbonate. However, the ESI-MS spectrum does not show any molecular ion mass that corresponds to the desired product. The use of triethylamine as a base also was not successful although triethylamine readily dissolved in the solution mixture. The reaction of 1,3,5-tri(bromomethyl)-2,4,6-triethylbenzene with ligand 2.4 also has been performed in the presence of cesium carbonate. After 48 hours, the product was isolated and dried. The ESI-MS analysis data of the product did not show the presence of a peak that corresponds to the product. The 1H NMR spectrum, however, shows
resonances that can be assigned to the desired product which are different from the chemical shifts of the resonances observed for both of the reactants suggesting that the desired product might have formed (Figure 4.3 and 4.4). The 1H NMR spectrum of
compound 4.3 shows a downfield shift compared to the free ligand of both CH protons