The synthesis and biological characterisation of novel  -glucosidase inhibitors Amy Rawling Balliol College

Background Adam’s compound “GS1” synthesised for photolabelling experiments: This was found to be a potent inhibitor of both  -glucosidase I and II. The dinitro equivalent “SR1” was also synthesised and also found to be an inhibitor, although less potent than GS1

Objective Investigate how structural changes to the original compound affect the biological activity: by altering chain length of the linker eg n=4,5,6 by changing aromatic ring groups Measure glucosidase inhibition in a cellular assay

-N2M9G3-N2M9G3 -N 2 M 9 G 2 Fast  -glc I Fast -N2M9G1-N2M9G1  -glc II CNX/CRT GT -N2M9-N2M9 -N 2 M 8 EDEM sec61  -glc II Golgi -N 2 M 8 G 1- 3 ER lumen ER man I Slow ERAD Glycosylation maturation pathway Protein Folding Pathways in the Endoplasmic Reticulum G 1-3 M 3-5 N 2 +

Et 3 N,1,4-dioxane Dess Martin Reagent DCM NaCNBH 3 DNJ n=4, 96 % n=5, 95 % n=6, 98 % n=4, 78 % n=5, 67 % n=6, 92 % n=4, 39 % n=5, 80 % n=6, 70 % Overall yields: n=4, 29 % n=5, 51 % n=6, 63 % MeOH

n=4, (COCl) 2, DCM, then DMSO, then NEt 3, 76 % n=6, Dess-Martin Reagent, DCM. 81 % Et 3 N, 1,4-dioxane n=4, 67 % n=5, 49 % n=6, 47 % i) H 2 SO 4, NaNO 2 ii) NaN 3 85 % n=6, 58 % NaCNBH 3 DNJ MeOH Overall yield: n=6, 19 %

Et 3 N, 1,4-dioxane, ∆ n=6, (COCl) 2, DCM, then DMSO, then NEt 3, 76 % n=6, 63 % NaCNBH 3 DNJ MeOH n=6, 41 % Overall yield: n=6, 20 %

Cell homogenisation Mixed bed ion-exchange + 2-AA FOS-2-AA Labeling reductant affinity chromatography NP-HPLC fluorescence FOS-2-AA Isolation and Analysis of FOS

SR1: 6C linker As inhibitor concentration increases, triglucosylated sugars appear => SR1 is an  -glucosidase I inhibitor. Also increasing G1M5N and G2M5N levels => SR1 is an  -glucosidase II inhibitor

SR2: 4C linker No No G3M4N or G3M5N => SR2 not an  - glucosidase I inhibitor Increasing G1M5N and G1M4N levels with concentration => SR2 an  -glucosidase II inhibitor

SR1 inhibits both  -glucosidase I and II (although less potent than GS1) SR2 inhibits  -glucosidase II selectively At 100 µM:

Future work… Biological characterisation of those imino sugars already synthesised Synthesis and characterisation of full range of imino sugars where aliphatic chain n=4,5,6 and with mononitro, dinitro and azido aromatic groups

Acknowledgments Acknowledgements John Jones George Fleet Terry Butters Stuart, Anders, Shane and the rest of the Fleet group Dom, Dave, Steph and Gabi in the Butters group