Ligation reactions at the anomeric center of carbohydrates have gained increasing importance in the field of glycobiology. Oxyamines are frequently used in labeling, immobilization, and bioconjugation of reducing carbohydrates. Herein, we present a systematic investigation of these ligation reactions under aqueous conditions. A series of four unprotected monosaccharides (glucose, N-acetylglucosamine, mannose, and 2-deoxyglucose) and one disaccharide (N,N'-diacetylchitobiose) was reacted with three primary and one secondary oxyamine. We monitored the concentrations of the starting materials and products by 1 H NMR spectroscopy and determined reaction times and equilibrium yields. Our experiments show that the outcome of the ligation reaction is not only dependent on the sugar and oxyamine used but also strongly on the reaction conditions. In the case of glucose, lowering the pH from 6 to 3 led to steadily increasing reaction rates, whereas the yields were decreasing at the same time. Variation of the temperature did not only influence the product ratio in equilibrium but can also have a strong impact on the equilibrium yield. In the case of reactions of a primary oxyamine, increased temperatures led to a higher proportion of acyclic products. Reaction of the secondary oxyamine with glucose unexpectedly led to lower yields at higher temperatures.

Metabolic glycoengineering (MGE) allows the introduction of unnaturally modified carbohydrates into cellular glycans and their visualization through bioorthogonal ligation. Alkenes, for example, have been used as reporters that can react through inverse‐electron‐demand Diels–Alder cycloaddition with tetrazines. Earlier, norbornenes were shown to be suitable dienophiles; however, they had not previously been applied for MGE. We synthesized two norbornene‐modified mannosamine derivatives that differ in the stereochemistry at the norbornene (exo/endo linkage). Kinetic investigations revealed that the exo derivative reacts more than twice as rapidly as the endo derivative. Through derivatization with 1,2‐diamino‐4,5‐methylenedioxybenzene (DMB) we confirmed that both derivatives are accepted by cells and incorporated after conversion to a sialic acid. In further MGE experiments the incorporated sugars were ligated to a fluorophore and visualized through confocal fluorescence microscopy and flow cytometry.

A new method for carbohydrate-oxyamine ligation starting from glycosyl amines 1 instead of the commonly used reducing sugars 2 results in tremendously increased ligation rates without the need for a catalyst, such as aniline.

Propiverine, a frequently-prescribed pharmaceutical for the treatment of symptoms associated with overactive bladder syndrome, provoked massive intranuclear and cytosolic protein inclusions in rat proximal tubule epithelium, primarily consisting of the peroxisomal targeting signal 1 (PTS1) containing protein d-amino acid oxidase (DAAO). As this type of nephropathy was also observed for other drugs, the aim was to determine whether propiverine interferes with trafficking and/or import of peroxisomal proteins. To elucidate this, DAAO- and propiverine-specific interaction partners from human HEK293 and rat WKPT cell lines and rat kidney and liver homogenate were determined using co-immunoprecipitation with subsequent nano-ESI-LC-MS/MS analyses. Corroboration of the role of DAAO- and/or propiverine-specific interaction partners in the drug-induced DAAO accumulation was sought via specific immunofluorescence staining of rat kidney sections from control and propiverine-treated rats. Above analyses demonstrated the interaction of propiverine with several protein classes, foremost peroxisomal proteins (DAAO, MFE2, HAOX2) and proteins of the protein quality control system, i.e. chaperones (HSP70 and DnaJ co-chaperones), proteases and proteasomal proteins (regulatory subunits of the 26S proteasome; Rpn1/2). The immunofluorescence analysis revealed mislocalization of many PTS1-proteins (DAAO, CAT, MFE2, ACOX1, EHHADH) in rat renal sections, strongly suggesting that propiverine primarily binds to PTS1 proteins resulting in the formation of PTS1 but not PTS2 or peroxisomal membrane protein (PMP) accumulations. Moreover, chaperones involved in peroxisomal trafficking (HSC70, DnaJB1) and peroxisomal biogenesis factor proteins (PEX3, PEX5, PEX7), also presented with distinct mislocalization patterns. Concomitantly, an increased number of peroxisomes was observed, suggestive of a compensatory mechanism for the presumably suboptimally functioning peroxisomes. Overall, the data presented suggested that propiverine interacts exclusively with DAAO or with a selected number of PTS1 proteins. The consequence of this interaction is the abrogated trafficking and peroxisomal import of PTS1 proteins concomitant with their nuclear and cytosolic accumulation due to inhibited degradation and imbalanced protein homeostasis.

The inside cover picture shows how metabolic glycoengineering can be explained in a creative way (idea: Sven Epple). Modified monosaccharides are used to engineer the cell surfaces’ glycans with functional groups. Surprisingly, bulky norbornenes can also be used as modifiers. In a second step, these modifications are “colored” through a bioorthogonal ligation reaction with a tetrazine or cyclooctyne that is linked to a label. More information can be found in the full paper by V. Wittmann et al. on page 1374 in Issue 14, 2016 (DOI: 10.1002/cbic.201600197).