Bioorthogonal chemistry can be used for the selective modification of biomolecules without interfering with any other functionality that might be present. Recent developments in the field include orthogonal bioorthogonal reactions to modify multiple biomolecules simultaneously. During our research, we observed that the reaction rates for the bioorthogonal inverse-electron-demand Diels-Alder (iEDDA) reactions between nonstrained vinylboronic acids (VBAs) and dipyridyl-s-tetrazines were exceptionally higher than those between VBAs and tetrazines bearing a methyl or phenyl substituent. As VBAs are mild Lewis acids, we hypothesised that coordination of the pyridyl nitrogen atom to the boronic acid promoted tetrazine ligation. Herein, we explore the molecular basis and scope of VBA-tetrazine ligation in more detail and benefit from its unique reactivity in the simultaneous orthogonal tetrazine labelling of two proteins modified with VBA and norbornene, a widely used strained alkene. We further show that the two orthogonal iEDDA reactions can be performed in living cells by labelling the proteasome by using a nonselective probe equipped with a VBA and a subunit-selective VBA bearing a norbornene moiety.

The synthesis of N‐acetylneuraminic acid (Neu5Ac) derivatives is drawing more and more attention in glycobiology research because of the important role of sialic acids in e. g. cancer, bacterial, and healthy cells. Chemical preparation of these carbohydrates typically relies on multistep synthetic procedures leading to low overall yields. Herein we report a continuous flow process involving N‐acetylneuraminate lyase (NAL) immobilized on Immobead 150P (Immobead‐NAL) to prepare Neu5Ac derivatives. Batch experiments with Immobead‐NAL showed equal activity as the native enzyme. Moreover, by using a fivefold excess of either N‐acetyl‐D‐mannosamine (ManNAc) or pyruvate the conversion and isolated yield of Neu5Ac were significantly improved. To further increase the efficiency of the process, a flow setup was designed providing a chemoenzymatic entry into a series of N‐functionalized Neu5Ac derivatives in conversions of 48–82%, and showing excellent stability over 1 week of continuous use.

Bacterial pathogens such as Nontypeable Haemophilus influenzae (NTHi) can evade the immune system by taking up and presenting host-derived sialic acids. Herein, we report a detailed structure–activity relationship of sialic acid-based inhibitors that prevent the transfer of host sialic acids to NTHi. We report the synthesis and biological evaluation of C-5, C-8, and C-9 derivatives of the parent compound 3-fluorosialic acid (SiaNFAc). Small modifications are tolerated at the C-5 and C-9 positions, while the C-8 position does not allow for modification. These structure–activity relationships define the chemical space available to develop selective bacterial sialylation inhibitors.

Although nontypeable Haemophilus influenzae (NTHi) is a humanspecific nasopharyngeal commensal bacterium, it also causes upper respiratory tract infections in children and lower respiratory tract infections in the elderly, resulting in frequent antibiotic use. The transition from symbiotic colonizing bacterium to opportunistic pathogen is not completely understood. Incorporation of sialic acids into lipooligosaccharides is thought to play an important role in bacterial virulence. It has been known for more than 25 years that sialic acids increase resistance to complement-mediated killing; however, the mechanism of action has not been elucidated thus far. Here, we provide evidence that growth of NTHi in the presence of sialic acids Neu5Ac and Neu5Gc decreases complement-mediated killing through abrogating the classical pathway of complement activation by preventing mainly IgM antibody binding to the bacterial surface. Therefore, strategies that interfere with uptake or incorporation of sialic acids into the lipooligosaccharide, such as novel antibiotics and vaccines, might be worth exploring to prevent or treat NTHi infections.