Exploiting aldolase variants in the synthesis of fluorinated analogues of N-acetyl neuraminic acid

This thesis reports the use of NAL variants to catalyse the reaction between a range of aldehyde analogues and fluoropyruvate, generating two new stereogenic centres. These reactions were catalysed by variants of N-acetyl neuraminic acid lyase (NAL). Wild-type NAL catalyses the aldol reaction between pyruvate and N-acetyl mannosamine (ManNAc) to give N-acetylneuraminic acid (Neu5Ac) whereas the NAL variants were evolved to catalyse the aldol reaction between pyruvate and the aldehyde analogue DHOB ([2R,3S]‐2,3‐dihydroxy‐4‐oxo‐N,N‐dipropylbutanamide) to
give DPAH ([5R,6R]‐7‐(dipropylamino)‐4,5,6‐trihydroxy‐2,7‐dioxoheptanoic acid). This work has sought to explore the effect on the activity and stereoselectivity of the NAL
variants with a broader range of substrates. A major challenge was the identification of the products of the NAL variant catalysed reactions and to determine the selectivity of each NAL variant. 19F NMR spectroscopy was invaluable in aiding characterisation of the enzyme reaction products.
Synthesis of the enzyme substrate where X=NHAc has been of particular focus within this work and has provided further insight into the potential importance of the Nacetyl
group in directing the binding of the aldehyde, therefore directing the diastereoselectivity. This work has also discovered that the introduction of fluorine may have an effect on the energy barrier to the transition state leading to the (4R) and (4S)-epimers of the products. However, in the case of more sterically demanding
aldehydes, the diastereoselectivity is predominantly driven by the initial binding mode.
This work has provided a more in-depth understanding of the effect of changing both the donor and acceptor substrates of the NAL-catalysed reaction. It has also evaluated
the viability of using NAL variants in the synthesis of fluorinated analogues of N-acetyl neuraminic acid.