Combinatorial chemistry is the study of synthetic methodologies which can be used to form large numbers of compounds in a single process. Therefore, combinatorial chemistry allows for the quick and facile building of molecular libraries, ideal for probing chemical space in arenas such as biological screening.[98]
In small-molecule synthesis terms, dynamic combinatorial chemistry uses building blocks which can react with one another in a reversible manner. Mixing these equilibrating building blocks leads to a range of products under thermodynamic control. Utilising supramolecular interactions, certain products can be favoured through strategies such as templating. For example, by the introduction of a metal templating strategy Busch et al. used nickel ions to template for macrocyclic structures 129 (Scheme 37).[99] This pioneering templating work would be built upon by Sanders and co-workers when they came to define the term “dynamic combinatorial chemistry”.[100]
Scheme 37 Metal templating in dynamic combinatorial chemistry developed by Busch et al.[99]
One of the challenges of dynamic combinatorial chemistry is that, due to all the components being in equilibrium, it is difficult to isolate products from the fluxional reaction mixture. One
87 way in which this can be overcome is to add a component to a rapidly equilibrating mixture which locks the whole system irreversibly (Scheme 38).
Scheme 38 Hypothesis behind this work
It was hypothesised that it might be possible for diol exchange to be significantly perturbed in three-component boronic acid assemblies in water as a solvent. It was postulated that a mixture of a boronic acid with a 2-formyl group along with a diol and amine would create a reversible three-component assembly. Then upon addition of a hydroxylamine, the diol would become locked and unable to exchange. Previous literature has demonstrated that hydroxylamines can use their oxygen atoms to coordinate to boron, forming a tetrahedral boronate ester, which from previous observations appears to be an irreversible process.[93f]
Scheme 39 Attempted assembly formation with 2-(2-methoxyethoxy)ethanamine
To test the theory that three-component assemblies can successfully form in aqueous environments, water-soluble components were required. To this end, FPBA 111, 3,4-dihydroxybenzoic acid 130 and 2-(2-methoxyethoxy)ethanamine 131 were targeted as the three components (Scheme 39). Upon mixing the components in a 1:1:1 ratio in D2O, it was clearly observed by 1H NMR spectroscopy that assembly formation was not occurring.
88 Overlaying the 1H HMR spectra of the individual starting components, it was clear that the mixture did not include any of the desired assembly, only a mixture of starting materials.
As 2-(2-methoxyethoxy)ethanamine 131 appeared not to be nucleophilic enough in D2O to successfully form an imine or that the equilibrium lay too far towards the starting materials, it was decided that increasing the nucleophilicity of the amine could lead to assembly formation. It is worth noting that it is hypothesised that amine and diol binding in three-component systems are cooperative, and thus without forming the imine the diol is less likely to successfully bind.[101] With this in mind the next amine trialled was methoxyamine 133 (Scheme 40). Due to the alpha effect, methoxyamine is a more potent nucleophile than a standard primary amine and thus it was postulated that even in D2O it would still be able to form an oxime.
Scheme 40 Assembly formation using methoxyamine in D2O
Upon mixing the three components 111, 130 and 133 in a 1:1:1 ratio in D2O, clear oxime resonances were observed (Figure 22). Overlaying the 1H NMR spectra of the starting materials showed that they had been fully transformed into the three-component assembly 134.
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Figure 22 1H NMR spectrum of assembly 134
Looking more closely at the 1H NMR spectrum of the assembly 134, it was noted that there were two oxime resonances: a major one at 8.17 ppm and a minor one at 8.47 ppm (Figure 22). These were attributed to the E and Z isomers of the condensed oxime. The major one was tentatively assigned as E due to sterics. This would be backed up by observations made in 11B NMR titrations detailed below.
-To probe this system (134) in more detail, several 11B- and 1H NMR titrations were carried out on the assembly. By varying the concentrations of the components, the degree of tetrahedral and trigonal boron within the assembly system could be observed.[101]
In the first titration shown below (Figure 23), the concentration of the amine 133 is increased with no diol present. In the 1H NMR titration it was observed that as the concentration of amine 133 was increased, the characteristic aldehyde resonance (9.80 ppm) from the FPBA
90 diminished whilst an imine resonance grew (8.17 ppm). As the concentration of the amine increased further, the imine resonance became broader.
Figure 23 1H and 11B NMR titration of methoxyamine 133 into 2-FPBA 111 with 3,4-dihydroxybenzoic acid 130 added at end. Spectra are numbered based upon the concentration of reagents given in Table 13.
Imine resonances
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Table 13 Titration of methoxyamine 133 into 2-FPBA 111 with 3,4-dihydroxybenzoic acid 130 added at end
Entry [FPBA] / mM [Diol] / mM [Amine] / mM
The values shown in this table refer to the spectra shown in Figure 23.
The broadening of the imine resonance in the 1H NMR titration (Figure 23) was attributed to the imine exchanging with free amine 133 in the solution. This hypothesis was supported by the observed 11B NMR titration; it was seen as the concentration of imine increased the ratio of trigonal boron (30 ppm) decreased compared to tetrahedral boron (10 ppm), thus showing that amine was being successfully transformed to imine. Once one equivalent of amine was present in solution, rapid exchange on the NMR timescale was observed within the 11B NMR spectra, with neither trigonal nor tetrahedral boron resonances present (Table 13 Entry 5, Figure 23 NMR spectrum 5). A mixture of all three components in a 1:1:1.2 (FPBA 111/diol 130/amine 133) ratio was tested for comparison (i.e. to see if the addition of diol 130 altered the appearance of NMR resonances). In the 1H NMR spectrum clear and sharp imine resonances were observed (Table 13 Entry 7, Figure 23 NMR spectrum 7), which lends evidence that the diol is successfully binding. This is due to the rate of exchange between imine and amine in solution being significantly perturbed by the presence of the diol this is due to the diol-boronic acid (3 component) assembly being more stable that the amine-boronic acid (2 component) assembly. In addition, the 11B NMR spectra showed a subtle shift
92 between spectra 1 and 7 in both resonances observed, giving further evidence of diol binding.
The subtle shift indicates that the ratio of different trigonal and tetrahedral boron-containing species is changing across the titration.
Figure 24. Possible O-B interactions in three-component boronic acid assembly 134
As can be seen in the 11B NMR titration (Figure 23), the resonance for trigonal boron dominates (30 ppm), which can be attributed to the E isomer of the imine being the major isomer in solution. The E isomer cannot position itself in a way for the oxygen to interact successfully with the boron centre (Figure 24). As this interaction cannot take place, the trigonal boron dominates (Figure 23). This also supports the hypothesis that the E imine resonance observed in the 1H NMR spectrum of the assembly dominates compared to Z.[93f]
The next titration that was carried out involved increasing the concentration of amine 133 with a constant concentration of diol 130 (Figure 25, Table 14). It was observed that the imine signal grew in as the aldehyde diminished, as was expected. Additionally, the ratio of trigonal to tetrahedral boron increased again with amine concentration. These findings support the hypothesis that successful three-component binding was occurring in D2O.
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Figure 25 1H and 11B NMR titration of methoxyamine 133 into 2-FPBA 111 and 3,4-dihydroxybenzoic acid 130.Spectra are numbered based upon the concentration of reagents given in Table 14.
7 6 5 4 3 2 1 Imine resonances
7 6 5 4 3 2 1 Trigonal boron Tetrahedral boron
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Table 14 Titration of methoxyamine 133 into 2-FPBA 111 and 3,4-dihydroxybenzoic acid 130
Entry [FPBA] [Diol] [Amine]
The values shown in this table refer to the spectra shown in Figure 25.
Following these titrations, the ability of a hydroxylamine to prevent exchange was probed (Figure 26). To do this, components 111, 130 and 133 were mixed together in D2O in a 1:1:1 ratio and an NMR spectrum was obtained (Figure 25, NMR spectrum 1) . To the assembly mixture was added 2 equivalents of hydroxylamine 135 and the assembly was heated to 40 °C for 1 hour to facilitate exchange (Figure 26, NMR spectra 2). From 1H NMR spectroscopic analysis, it was observed that full exchange to the hydroxylamine assembly 136 had occurred.
To prove that exchange to the hydroxylamine assembly was an irreversible process, a further 2 equivalents of methoxyamine 133 were added to the assembly mixture which was subsequently heated to 40 °C for another hour. Upon 1H NMR spectroscopic analysis, it was observed that the assembly remained unchanged and the hydroxylamine had not been exchanged (Figure 26, NMR spectrum 3). This proved that the assembly had been essentially locked from further exchange upon hydroxylamine addition. This experiment was also carried out at room temperature with the same outcome. However, exchange to the hydroxylamine assembly 136 took significantly longer at 2 hours before full exchange was observed.
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NMR Legend 1) Methoxyamine assembly before hydroxylamine addition. 2) Methoxyamine assembly with hydroxylamine (2 equiv.) heated. 3) Methoxyamine assembly with hydroxylamine and a further 2 equiv. of methoxyamine added heated (40
°C, 1 h). 4) Reference methoxyamine assembly. 5) Reference hydroxylamine assembly
Figure 26 Exchange experiment of three-component assembly with a hydroxylamine
The exchange experiment supported the hypothesis that a reversible three-component boronic acid assembly could be locked irreversibly with a hydroxylamine. The titrations carried out earlier in this section give evidence towards boronic acid-diol binding successfully occurring in the presence of methoxyamine in D2O. This shows that the assembly is amenable to water/D2O, a solvent which can compete for diol binding.
5
4
3
2
1
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3.3.1 Summary
This project demonstrated that the Bull-James three-component boronic acid assembly concept can be carried out in D2O and thus aqueous environments when the correct components are selected.[102] The coordination environment of the boron centre was probed via 11B NMR titrations and it was found that trigonal boron dominates in D2O. Evidence of amine-imine exchange and diol binding was seen in 1H NMR titrations. Subsequent experiments utilising hydroxylamines showed that exchange within the system could be stopped, thus potentially irreversibly locking a diol and boronic acid together from a previously fluxional mixture. Further work on these systems is being carried out within the Anslyn group and it is hoped that these studies will prove applicable to dynamic combinatorial chemistry. Experimental work in this section was carried out by the author of this thesis and the primary investigator was Professor Eric Anslyn (UT Austin).