Conclusions and Future Work

This work describes the syntheses of a variety of amino acids (Section 6.1.1), cyclic dipeptides/DKPs (Section 6.1.2) and large linear CCL2 fragment peptides (Section 6.1.3). Of the selection of amino acids and dipeptides synthesised, a number have been utilised in chemical and biological applications. The linear peptides are intermediates in the total microwave assisted SPPS of CCL2, and, although this was not completed, progress was made towards developing a viable synthetic route.

6.1.1 Amino Acid Syntheses

A library of heteroaromatic amino acids has been prepared in moderate yields via an optimised Negishi cross-coupling reaction (Figure 6.1). Previously, the synthesis of these heteroaromatic amino acids was not described via this route with only one N-Boc protected derivative (of 16) reported in the literature. The developed route offers an effective alternate strategy to access heteroaromatic amino acids from a simple starting material (iodoalanine). The yields are comparable to other more established routes (e.g.

asymmetric dehydrogenation, chiral glycine equivalents and enzymatic synthesis) and in some cases may be preferable than other strategies. For example, in asymmetric hydrogenation the ee of desired product can be extremely dependant on having N- and C- protecting groups with steric bulk.

Figure 6.1: The various heteroaromatic amino acids reported in this work synthesized via Pd-catalysed Negishi cross-coupling (Chapter 2). *denotes highest isolated yield of the amino acid.

Some of the heteroaromatic amino acid building blocks were found to be tolerant to incorporation into bioactive molecules via chemical and biological syntheses. For example fluorinated 2-3-difluorotyrosine (29) was shown to be amenable to biosynthetic incorporation into the lipopeptide fengycin (Figure 6.2). Furylalanine was shown to be stable to solution and SPPS, thus, can be easily incorporated into bioactive peptides e.g. the attempted syntheses of γ-glutamyl-2-furylalanine (Figure 6.3).

Figure 6.2: A fengycin derivative lipopeptide containing a 2,3-difluorotyrosine derivative amino acid.

Figure 6.3: The chemical structure of natural product: γ-glutamyl-2-furylalanine.

A small library of thiol containing amino acids (31, 32, 33 and 34) was produced. This work included the synthesis of two different stereo-isomers of 4-substituted thio-proline via multiple synthetic routes. The thiol containing amino acids (33 and 34) were then used to provide information (in the form of pKa values and ionic species abundance (Figure 6.4)) that could indicate their reactivity towards NCL and thus, be beneficial in the development of kinetic NCL reactions.

Figure 6.4: The abundance of thiolate species (either zwitterionic or anionic) present at pH 6.8 and 7.2. The thiolate species is thought to be responsible for initial nucleophilic attack upon the thioester.*34 provided erroneous results due to oxidation.

The results show promise as a significant difference in the abundance of active deprotonated thiol species is observed over a small pH range (6.8 – 7.2). However, re-testing of all four amino acids under reducing conditions is needed so that data can be gathered on 34. This result is of interest as a major difference in reactivity in NCL has been shown between 33 and 34. To further this work, small peptides containing the terminal thiol amino acids could be synthesised and the utilised in competition experiments to correlate pKa data and NCL efficiency. Ultimately, the correlated pKa

data could lead to facile planning and execution of kinetic “one-pot” ligations with these amino acids. If successful, the work could be easily extended to a larger library of thiol-amino acids.

The aforementioned amino acids could be envisaged to be used in a variety of other peptide based molecules including the synthesis of potential CCL2 induced chemotaxis inhibiting DKPs (Section 6.1.2) and the incorporation into large peptides or proteins (Section 6.1.3).

6.1.2 DKPs and CCL2 Induced Chemotaxis Inhibition

Previous work by the Cobb group had shown potential in the use of DKPs as selective inhibitors of CCL2 induced chemotaxis. Therefore, we sought to develop an improved synthetic route and utilise this to synthesise (and analyse) a number of DKPs so that inhibitors could be intelligently designed and their mechanism of inhibition probed.

A solid-phase route to the synthesis of the target DKPs was successfully optimised to enable the production of DKPs with a significant improvement on the previous solution phase approaches. Using this solid-phase approach a single DKP could be synthesised and purified within 8 hours, compared to the solution phase synthesis that could take several days, multiple purifications and high temperatures.

In total, eleven DKPs were synthesised (from N-Boc amino acid building blocks) and underwent biological testing against inhibition of CCL2 induced chemotaxis. Of the eleven DKPs only three exhibited inhibition of <40% inhibition at a concentration of 100 μM (red, Figure 6.5) and no inhibitors were found to be significantly better than those previously described (e.g. cyclo(L-Phe-L-Pro) black, Figure 6.5). Although it was not possible to obtain crystal structures of every DKP synthesized, preliminary analysis has shown that the less active molecules (e.g. cyclo(L-Tyr-L-Pro) and cyclo(L-Pro-D -Phe)) exhibit folding of the aromatic over the core 6-membered ring (common in aromatic DKPs of this type) and the more active molecules (e.g. cyclo(L-Phe-L-Pro) and cyclo(p-fluoro-L-Phe-L-Pro)) do not. Moreover, the work provided insights into the effect on the inhibition activity of changing stereochemistry, aromatic moieties and prolyl moieties. For example, potent inhibition of CCL2 induced chemotaxis is observed from both cyclo(L-Pro-L-Trp) and cyclo(L-Phe-L-Hyp) (red, Figure 6.5). This suggests that the scaffold is tolerant to variations in heteroaromatic group and proline substitution at the 4- position. Therefore, work is currently underway to produce a 3^rd generation library of DKPs using a range of heteroaromatic and proline substituted amino acids.

Figure 6.5: Testing of 100 μM DKPs (structures shown) against CCL2 mediated chemotaxis of THP-1 cells at 10 nM CCL2 concentration.

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