Antibody−drug conjugates (ADCs) are an important class of therapeutic agents that harness the highly specific antigen targeting property of antibodies to deliver toxic drugs for targeted cell killing. Site-specific conjugation methods are highly desirable for constructing homogeneous ADCs that possess a welldefined antibody-to-drug ratio, stability, ideal pharmacological profile, and optimal therapeutic index. We report here a facile synthesis of functionalized glycan oxazolines from free sialoglycans that are key donor substrates for enzymatic Fc glycan remodeling and the application of an efficient endoglycosidase mutant (Endo-S2 D184M) for site-specific glycan transfer to construct homogeneous ADCs. We found that by a sequential use of two coupling reagents under optimized conditions, free sialoglycans could be efficiently converted to selectively functionalized glycan oxazolines carrying azide-, cyclopropene-, and norbornene-tags, respectively, in excellent yield and in a simple one-pot manner. We further demonstrated that the recently reported Endo-S2 D184 M mutant was highly efficient for Fc glycan remodeling with the selectively modified glycan oxazolines to introduce tags into an antibody, which required a significantly smaller amount of glycan oxazolines and a much shorter reaction time than that of the Endo-S D233Q-catalyzed reaction, thus minimizing the side reactions. Finally homogeneous ADCs were constructed with three different click reactions. The resulting ADCs showed excellent serum stability, and in vitro cytotoxicity assays indicated that all the three ADCs generated from the distinct click reactions possessed potent and comparable cytotoxicity for targeted cancer cell killing.

Drug repurposing is considered a promising strategy to fight antimicrobial resistance (AMR). Methotrexate (Mtx), a classical anticancer drug, could strongly inhibit bacterial dihydrofolate reductase (DHFR). However, its poor permeability into bacteria and potent human cytotoxicity make it unsuitable as an antibacterial. Herein, we reported the conjugation of Mtx with a siderophore to construct "Trojan horse" antibacterials. The most potent conjugate 8 with nanomolar minimum inhibitory concentration (MIC) values exhibited over 1.00 × 10 3 -fold improved activity against Gram-positive Streptococcus pneumoniae (S. pneumoniae) and Gram-negative Yersinia enterocolitica (Y. enterocolitica) compared with Mtx, while possessing 2.31 × 10 3 -fold reduced human cytotoxicity, resulting in 2.08 × 10 6 -fold improvements in the therapeutic index. This proof-of-principle study verifies that siderophore conjugation is an effective strategy for developing new antibacterials from anticancer drugs.

A robust glycosylation protocol was fixed to construct the GlcN–(1α→4)-GlcA/IdoA linkagesen routeto heparin oligosaccharides.