Abstract:
Recently, Mahmud and coworkers reported the identification of a “pseudo-glycosyltransferase” as part of the biosynthetic pathway responsible for producing Validamycin A (a fungicide that targets trehalase).[1] This pseudo-GT catalyzes transfer of a C-7 unsaturated cyclitol (valienol) moiety rather than a glycoside, but has structural and sequence similarity to known glycosyltransferase trehalose-6-phosphate synthase. From a mechanistic perspective this similarity is intriguing as it suggests this pseudo-GT may be completing cyclitol transfer by way of an allylic cation rather than the oxocarbenium ion-like transition state in traditional glycoside transfer. Since glycoside hydrolases (GHs) exploit a similar mechanism to their GT cousins (again through an oxocarbenium ion), we sought to identify GHs capable of cleaving pseudo-glycosidic linkages of valienol substrates and to ascertain whether an allylic cation was indeed implicated in their mechanism.
Valienol derivatives were prepared using known methods[2] and further derivatized to incorporate chromogenic, activated leaving groups at the C-1 position. Our efforts to establish the rates of spontaneous solvolysis for these valienol substrates revealed rates of solvolysis for valienols and their analogous glycosides were nearly identical. Inspired by the similar intrinsic reactivities, we next screened a library of glycoside hydrolases made available for the Joint Genome Institute. From this screen we identified approximately 10 enzymes with significant activity against these valienyl ethers. The mechanistic details of these reactions were further explored through enzyme kinetics and other methods. We show that valienyl substrate hydrolysis is completed by a mechanism nearly identical to that expected for the native glycosides but with evidence that supports allylic cation formation in place of the traditional oxocarbenium ion.[3] Moving forward we hope to use the enzymes identified as an evolutionary foothold to increase their activity and selectivity for these substrates.
References:
[1] S. Asamizu, J. Yang, K. H. Almabruk and T. Mahmud, J. Am. Chem. Soc. 2011, 133 (31), 12124–12135.
[2] T. K. M. Shing, Y. Chen, W. L. Ng, Synlett 2011, 9, 1318-1320.
[3] P. M. Danby and S. G. Withers, J. Am. Chem. Soc. 2017, 139 (31), 10629-10632.