For 70 years, α-amanitin, the most cytotoxic peptide in its class, has been without a synthetic rival; through synthesis, we address the structure–activity relationships to inform the design of new amatoxins and disclose analogues that are more cytotoxic than the natural product when evaluated on CHO, HEK293, and HeLa cells, whereas on liver-derived HepG2 cells, the same toxins show diminished cytotoxicity.

Herein, we describe the efficient gram‐scale synthesis of α2,3‐ and α2,6‐sialyllactose oligosaccharides as well as mimetics from N‐acyl mannosamines and lactose in metabolically engineered bacterial cells grown at high cell density. We designed new Escherichia coli strains co‐expressing sialic acid synthase and N‐acylneuraminate cytidylyltransferase from Campylobacter jejuni together with the α2,3‐sialyltransferase from Neisseria meningitidis or the α2,6‐sialyltransferase from Photobacterium sp. JT‐ISH‐224. Using their mannose transporter, these new strains actively internalized N‐acetylmannosamine (ManNAc) and its N‐propanoyl (N‐Prop), N‐butanoyl (N‐But) and N‐phenylacetyl (N‐PhAc) analogs and converted them into the corresponding sialylated oligosaccharides, with overall yields between 10 % and 39 % (200‐700 mg.L−1 of culture). The three α2,6‐sialyllactose analogs showed similar binding affinity for Sambucus nigra SNA−I lectin as for the natural oligosaccharide. They also proved to be stable competitive inhibitors of Vibrio cholerae neuraminidase. These N‐acyl sialosides therefore hold promise for the development of anti‐adhesion therapy against influenza viral infections.