Mycobacterium tuberculosis (Mtb) is bacterial pathogen that causes Tuberculosis (TB) and is the leading cause of death worldwide due to an infectious agent. In order to eradicate TB, new targets and faster acting treatments must be identified. One potential target is glycoside hydrolase-like phosphorylase Mtb GlgE which plays a role in α-glucan biosynthesis. The enzyme uses maltose-1-phosphate (M1P) and the growing α-glucan chain as substrates and catalyzes the glucan chain extension. Deletion of the GlgE gene in Mtb results rapid death of the organism. In order to discover improved inhibitors against this target we have utilized closely related Streptomyces coelicolor (Sco) GlgEI-V279S as a model enzyme that diffracts to high resolution during X-ray crystallography. To inhibit Sco GlgEI-V279S we designed transition state-like pyrrolidine inhibitors which incorporate a phosphonate into the pyrrolidine moiety. The targets were designed to mimic a putative late transition state for the first SN2 reaction catalyzed GlgE1 on M1P. We used a polyhydroxylated pyrrolidine we previously prepared and subjected it to a Kabachnik-Fields reaction or reductive amination to access zwitterionic pyrrolidine-phosphonates (ZPPs). One ZPP contained one carbon between the pyrrolidine nitrogen and the phosphorous (ZPP1). The other contained two carbons between the nitrogen and phosphorous (ZPP2) in order to explore optimal charge separation in the transition state. ZPP1 inhibited Sco GlgEI-V279S at Ki = 45 µM while ZPP2 showed a Ki = 95 µM. ZPP1 co-crystallized with Sco GlgEI-V279S at 3.2 Å resolution while ZPP2 co-crystallized at 2.5 Å resolution. Both transition state-like analogues were found bound in the enzyme active site. The synthesis of these targets and the structural studies will be discussed.

Zwitterionic pyrrolidine-phosphonates: Transition state mimics of the glycoside hydrolase-like phosphorylase Streptomyces coelicolor GlgEI-V279S

253rd National Meeting (2017)

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