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Design, synthesis and studies of guanidinium-based inhibitors for isoprenoid biosynthetic pathway enzymes

University of British Columbia

In this thesis an inhibition strategy was developed to target enzymes that utilize allylic diphosphates. Positively-charged inhibitors that mimic the transition states/intermediates formed with these enzymes were synthesized. In chapter two, inhibitor 2 containing a guanidinium moiety appended to a phosphonylphosphinate was designed to mimic the transition state for the dissociation of dimethylallyl diphosphate into an allylic carbocation-pyrophosphate ion-pair. To test for the effectiveness of incorporating a guanidinium functionality into inhibitors of human farnesyl diphosphate synthase, inhibitors 3 and 4 were also prepared. Inhibitor 3 has a positive charge localized onto one atom, and inhibitor 4 is isosteric to inhibitor 2, but lacks positive charge. The inhibitors displayed IC50 values that were significantly higher than the substrate Km value, indicating that the positive charge did not result in tight binding to the enzyme. We decided to apply our inhibition strategy on other allylic diphosphate utilizing-enzymes. In chapter three, inhibitors bearing a guanidinium/amidinium moiety appended to a phosphonylphosphinate and flanked by a hydrocarbon tail (inhibitors 26 and 36) were synthesized. A neutral inhibitor 34 was also prepared as control. These inhibitors were tested against human squalene synthase (HSQS) and bacterial dehydrosqualene synthase (DSQS) from Staphylococcus aureus. It was anticipated that the lipid chain might increase the enzyme’s affinity towards the inhibitors. The positively-charged inhibitors acted as competitive inhibitors (low micromolar KI values) against DSQS. Similar trends were observed for the first half reaction of HSQS. Surprisingly, the neutral inhibitor was the most potent for both enzymes. These results indicated that the active site of both enzymes does not directly stabilize the allylic carbocation. We reasoned that these positively-charged inhibitors might be effective with enzymes that generate carbocation intermediates that are not stabilized by resonance or ion-pair interactions. Such a strategy was applied in chapter four, where inhibitors 2 – 4 were tested against isopentenyl diphosphate isomerase (IDI), and acted as competitive inhibitors of IDI. Notably, inhibitor 2 bound 400 times more tightly than its neutral isostere, inhibitor 4. We reasoned that inhibitor 2 allows for proper positioning of the positive charge in the active site, leading to favorable electrostatic interactions.

Text

2019-10-18

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/71940

Chemistry

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/