OleD Loki as a Catalyst for Tertiary Amine and Hydroxamate Glycosylation

Abstract

We describe the ability of an engineered glycosyltransferase (OleD Loki) to catalyze the N-glycosylation of tertiary-amine-containing drugs and trichostatin hydroxamate glycosyl ester formation. As such, this study highlights the first bacterial model catalyst for tertiary-amine N-glycosylation and further expands the substrate scope and synthetic potential of engineered OleDs. In addition, this work could open the door to the discovery of similar capabilities among other permissive bacterial glycosyltransferases.

OleD Loki as a Catalyst for Hydroxamate Glycosylation

“…Using 2‐chloro‐4‐nitrophenyl β‐ d ‐glucoside (ClNP β‐ d ‐Glc) and the OleD variant Loki,– 21 representative hydroxamate‐based HDACis were evaluated as putative substrates via a simple colorimetric transglycosylation screen under the control of a catalytic amount of UDP (Figure A). Assays (1 m m hydroxamate, 2 m m ClNP β‐ d ‐Glc, 0.1 m m UDP, 25 m m Tris pH 8.0, 5 m m MgCl 2 , 0.25 μ m OleD Loki, 30 μL total volume, 30 °C, 1 h, A 410 ) were conducted in triplicate in 384 well plates.…”

“…Within this context, OleD acceptor and sugar nucleotide donor permissivity has been further enhanced via directed evolution and structure‐based approaches . An OleD‐based transglycosylation strategy has also been developed that exploits GT‐catalyzed reaction reversibility, and the ability of enhanced OleD mutants (OleD ASP, TDP16 and Loki)–– to efficiently use simple colorimetric or fluorescent substrates. This transglycosylation platform also enabled a plate‐based screen to identify new OleD substrates and improved OleD‐catalyzed syntheses via shifting the reaction equilibria toward desired glycoside product –…”

“…One such recent transglycosylation screen identified the histone deacetylase inhibitor (HDACi) trichostatin A as a new OleD Loki substrate and subsequently demonstrated the efficient OleD‐catalyzed synthesis of the corresponding glucopyranosyl hydroxamate trichostatin C (the only naturally occurring hydroxamate glycoside isolated to date, produced by Streptomyces platensis No. 145 and Streptomyces strain Y‐50) . Although HDACis have broad clinical utility, their corresponding glycosides have not been extensively studied.…”

“…Assays (1 m m hydroxamate, 2 m m ClNP β‐ d ‐Glc, 0.1 m m UDP, 25 m m Tris pH 8.0, 5 m m MgCl 2 , 0.25 μ m OleD Loki, 30 μL total volume, 30 °C, 1 h, A 410 ) were conducted in triplicate in 384 well plates. Each plate also contained a positive (4‐methylumbeliferone; 4‐MeUmb)–– and negative (DMSO) control. Seventeen preliminary primary hits were identified (Δ A 410 >3 standard deviations above the negative control) in this first‐pass screen (Figure B).…”

“…Evidence of a 1 a – 5 a β‐ O ‐glucoside derived from the key anomeric 1 H (near δ H =4.5, J =6.6–8 Hz ) and 13 C ( δ C =102–106) resonances (Tables S2–S6, Figure S4) typical of β‐ O ‐glucosides . As comparators, typical anomeric carbon 13 C resonances for C‐ and N‐glycosides range from δ C =70–85 and 85–95, respectively. It is also important to note that 1 and 3 – 5 contain only a single accessible nucleophilic OH (that of the hydroxamate).…”