Enzymatic Synthesis of the C-Glycosidic Moiety of Nogalamycin R

Abstract

Carbohydrate moieties are essential for the biological activity of anthracycline anticancer agents such as nogalamycin, which contains l-nogalose and l-nogalamine units. The former of these is attached through a canonical O-glycosidic linkage, but the latter is connected via an unusual dual linkage composed of C–C and O-glycosidic bonds. In this work, we have utilized enzyme immobilization techniques and synthesized l-rhodosamine-thymidine diphosphate (TDP) from α-d-glucose-1-TDP using seven enzymes. In a second step, we assembled the dual linkage system by attaching the aminosugar to an anthracycline aglycone acceptor using the glycosyl transferase SnogD and the α-ketoglutarate dependent oxygenase SnoK. Furthermore, our work indicates that the auxiliary P450-type protein SnogN facilitating glycosylation is surprisingly associated with attachment of the neutral sugar l-nogalose rather than the aminosugar l-nogalamine in nogalamycin biosynthesis.

A pharmaceutical model for the molecular evolution of microbial natural products

“…The canonical reaction of the anthracycline fourth ring polyketide cyclases such as SnoaL and AknH is to catalyze aldol condensations that determine the stereochemical configuration at C9, but interestingly SnoaL2 has also been observed to harbor minor ancestral cyclization activity . After attachment of l ‐rhodosamine by the glycosyltransferase SnogD , the subtype epitomizing C2–C5´´ bond is formed by the 2OG and nonhaem iron‐dependent oxygenase SnoK . However, a gene duplication event has led to the appearance of C4´´ epimerase SnoN from SnoK .…”

The Rieske Oxygenase SnoT Catalyzes 2′′‐Hydroxylation of l‐Rhodosamine in Nogalamycin Biosynthesis

“…The findings presented here clarify the last remaining unknown steps in the biosynthesis of 1 and elucidate how the 2''-and 4''-hydroxy groups of lnogalamine, which are important for DNA interaction, are assembled. The absence of the epimerization product 6 [16,19] confirmed the inability of SnoN to epimerize 2. I) Reaction product shown with EIC for 5, II) Reaction product shown with the EIC for consumed 2, III) authentic standard 2 lV) authentic standard 5, and V) authentic standard 6 (m/z [M + H] + 744.7).…”

“…However, because the epimerization reaction does not alter the mass of the product and the influence of the chemical modification on the retention time is unknown, the experiment did not directly reveal whether snoN had been functional. We have previously shown that 4“ epimerization does alter the retention time between 5 and 6 , which both contain the additional C2–C5” bond [16,19] . Therefore to verify if SnoN was able to epimerize 2 in vivo , the culture extract containing putatively both 2 and 3 was incubated with purified SnoK in the presence of 2‐oxoglutarate, Fe II SO 4 and l ‐ascorbate.…”

“…[12,13,14] The biosynthetic gene cluster encodes more than 30 genes [15] and many steps have been elucidated both in vivo and in vitro. [16,17,18] These studies have revealed that, surprisingly, the carbohydrate attached at C1 is initially lrhodosamine, [19] which lacks the 2''-hydroxy group and has an opposite stereochemistry at C4''. Pathway intermediates have been shown to harbor lower biological activity particularly towards topoisomerase II in comparison to 1, [15] possibly due to non-ideal interactions of the carbohydrates with DNA.…”

“…We have previously shown that 4" epimerization does alter the retention time between 5 and 6, which both contain the additional C2-C5" bond. [16,19] Therefore to verify if SnoN was able to epimerize 2 in vivo, the culture extract containing putatively both 2 and 3 was incubated with purified SnoK in the presence of 2-oxoglutarate, Fe II SO 4 and l-ascorbate. LC-MS analysis of the reaction products revealed the presence of 5 with no traces of 6 ( Figure 3C).…”