Given the lack of success with aromatic diamines it was decided to continue with a similar approach to the synthesis of a potential mimic but using aliphatic amines instead of the problematic aromatic amines.
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4.2.1 Non-aromatic diamines with cross-coupling potential
With this in mind it was decided to follow a synthesis analogous to that shown in Scheme 4.4 above, but, instead of an ortho-phenylenediamine structure, to use a starting compound like that shown in Figure 4.3. Here, a CH2 spacer has
been introduced to insulate the amines from the aromatic ring. While this small molecule is not commercially available, a synthetic route, shown in Scheme 4.8, adapted from one reported for the synthesis of 4-bromophthalonitrile,219 was devised.
Figure 4.3
Scheme 4.8 Possible routes to 3,4-di(aminomethyl)bromobenzene.
The steps up to the synthesis of the diamide were completed successfully and several attempts were made to reduce this product directly to the diamine using I2/NaBH4.220 While there was evidence of the desired product shown by mass
spectrometry, the reaction did not proceed cleanly so it was decided instead to perform the conversion to the nitrile compound using phosgene. This approach proved advantageous in that the phthalonitrile compound could be obtained easily, in high yield and was relatively easy to purify by column
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chromatography. Also, given that the intent in this approach was leading towards a cross-coupling reaction with a fluorescent moiety or possible transition metal ligand, this structure provided a useful point within the overall scheme for the cross-coupling to be carried out. The nitrile groups present at this stage would be unlikely to interfere with a cross-coupling reaction whereas, if the coupling were carried out at a later stage, the functional groups present (namely amine, amide and thioester added sequentially) would possibly cause complications with the cross-coupling reaction. It was therefore decided at this stage to couple the 4-bromophthalonitrile with a bipyridine unit using a Suzuki- Miyaura coupling as shown in Scheme 4.9.
Scheme 4.9 Miyaura Borylation and Suzuki-Miyaura coupling to give a functionalised bipyridine
ligand.
At this point it would be possible to use this bipyridine unit as a ligand to form a complex with iridium before subsequent functionalisation. However, since the next step in functionalisation involves the reduction of nitriles to amines, it was decided to delay the formation of the complex until a later stage to avoid exposing it to reagents such as borane or NaBH4. However, upon the
attempted reduction of the nitriles with BH3.THF, a pure product could not be
isolated from the reaction. Similarly, attempts to perform the reduction with NaBH4 were also unsuccessful. While it seemed that some product was formed
by the reaction, the components in the mixture were highly immobile or streaky in column chromatography. It was therefore not possible to purify the product by this means, and the mixture did not seem suitable for purification by recrystallisation or distillation.
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4.2.2 Organic emitters with non-aromatic amine substituents
In another approach analogous to the synthetic route attempted with anthracene, the option of functionalising a different organic fluorophore with non-aromatic amines was investigated. The phenolic structures in fluorescein (shown in Figure 4.4) will undergo Mannich reactions to form amines and this approach had previously been used to synthesise fluorescent sensors for metal ions in biological systems.221,222 These sensors were produced using the 2,7- dichlorofluorescein derivative, to ensure that substitution occurred only at the 4 and 5 positions of the molecule. While in these reports, the Mannich reactions were performed with secondary amines, giving tertiary amine products, if this reaction would proceed with ammonia the resulting primary amines could be functionalised to give a possible mimic.
Figure 4.4 Fluorescein
Mannich reactions were therefore attempted using 2,7-dichlorofluorescein with both aqueous ammonia and ammonium chloride to give the primary amines required for functionalisation, however the products isolated from the reaction were highly insoluble and could not be positively identified as the required diamine. The only component identified was the starting material, which was detected in negative electrospray mass spectrometry. It was unclear, however, whether the material was a mixture of products including some starting material or whether there had been no reaction.
In an attempt to overcome this problem with insolubility, Mannich reactions were instead performed with benzyl amines with the intention of subsequently removing the benzyl groups by hydrogenation. These reactions were carried out using both benzylamine and dibenzylamine which would give secondary and tertiary amine products respectively. The reactions carried out with 2,7- dichlorofluorescein are summarised in Scheme 4.10.
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Scheme 4.10 Attempted Mannich reactions with 2,7-dichlorofluorescein and subsequent
deprotection of amines.
In the reaction with benzylamine the products were, again, very insoluble and could not be identified. The reaction with dibenzylamine, on the other hand, resulted in a red solid that could be purified by column chromatography, although the yield was very low. This was identified as the expected product of a Mannich reaction by mass spectrometry, however evidence that it was the correct product was less clear using NMR spectroscopy due to the large number of overlapped benzyl aromatic signals. When this product was treated with hydrogen gas in the presence of 10% palladium on activated carbon as a catalyst in methanol, a red/purple precipitate was produced. When isolated, though, this again was too insoluble to be positively identified as the desired primary diamine.
By this point there had been very little success, for various reasons, in synthesising a potential PDI mimic structure, even without the attachment of a fluorescent unit to give a finished target molecule. As discussed previously, the reactions using aromatic amines were problematic in terms of stability, while up to this point, a system with aliphatic amines suitable for functionalisation had not yet been synthesised successfully. It was therefore decided to look into alternate strategies for the synthesis of a mimic structure that would perhaps
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cause fewer complications. While it is the case that a variety of structures containing thiol groups have been shown to isomerise non-native disulfide bonds in proteins, the functional groups used by PDI itself are, of course, cysteine residues. In light of this, it seemed feasible that the amino acid cysteine could be used as the mimic part of a final target, and could be attached to a variety of structures by the formation of an amide with either an amine or a carboxylic acid.