A mechanism-based inactivator of glycoside hydrolases involving formation of a transient non-classical carbocation

Abstract

The design of mechanism-based enzyme inactivators to generate chemical probes for biological research is an important challenge in carbohydrate chemistry. Here we describe the synthesis and biological evaluation of a novel carbocyclic mechanism-based inactivator of galactosidases (glycoside hydrolase families 27 and 36). Upon catalysis of this unnatural substrate, a transient non-classical carbocation forms within the enzyme active site. We show that the inactivation event, which proceeds via a bicyclobutonium ion intermediate, leads to a single alkylation event that occurs on the enzymatic nucleophile, an aspartic acid residue in this case. We also show that the catalytic proficiencies for enzymatic hydrolysis of substrates and inactivation by our bicyclo[4.1.0]heptyl analogue of galactose differ by only a factor of 20. This inactivator has the potential for further development as a useful biological research tool for both basic research and biotechnological applications.

Structural Snapshots for Mechanism‐Based Inactivation of a Glycoside Hydrolase by Cyclopropyl Carbasugars

“…Also, hydrolysis of the Asp327‐linked carbasugar intermediate, which has only been detected previously by mass spectrometry,4 proceeds via a perturbed TS free energy pathway in the solid state. These two observations inspired us to design a second‐generation inactivator with a significantly lower reactivation rate, which would enable structural analysis of the covalently bound complex with Tm GalA in the solid state.…”

“…In both cases, pyranosylium ion like transition states (TSs), which can have half‐chair ( 4 H 3 / 3 H 4 ), boat ( 2,5 B/B 2,5 ), or envelope ( 4 E and 3 E) conformations (Figure 1 b),2, 3 are implicated. By exploiting this knowledge, we recently reported that the cyclopropyl‐containing carbasugar 1 is a mechanism‐based inactivator of an α‐ d ‐galactosidase from Thermotoga maritima ( Tm GalA; Figure 1 c) 4. Within the enzymatic active site, 1 likely forms a transient bicyclobutenium ion ( 1 + ), and enzyme inactivation occurs through alkylation of the catalytic nucleophile Asp 327.…”

“…Given the current desire for small‐molecule transition‐state analogues (TSAs) as leads for therapeutic development,5 it is important to understand how GHs stabilize cationic TSs 4. Of note, GHs are among the most catalytically proficient enzymes, since they accelerate hydrolysis of glycosidic bonds by up to 10 17 ‐fold 6.…”

“…Given the short half‐life (<1 min) for reaction of the enzyme–inhibitor complex (E:I complex) between 1 and Tm GalA in solution,4 apo Tm GalA crystals were soaked briefly (5 s to 30 min) with 1 in an effort to observe the covalent complex formed through nucleophilic trapping of the bicyclobutenium ion intermediate (Figure 1 c; 1 + ) by Asp 327. Surprisingly, these crystals failed to yield a structure with any evidence of a small molecule bound in the active site of Tm GalA.…”

“…Considering the greatly extended half‐life for reaction of the E:I Michaelis complex, it follows that the inactivator 1 undergoes a pseudo‐glycosylation reaction within the crystalline phase via a transition state that has a different activation free energy barrier to that of the equivalent reaction in aqueous solution 4. Also, hydrolysis of the Asp327‐linked carbasugar intermediate, which has only been detected previously by mass spectrometry,4 proceeds via a perturbed TS free energy pathway in the solid state.…”