Protein Glycosylation through Sulfur Fluoride Exchange (SuFEx) Chemistry: The Key Role of a Fluorosulfate Thiolactoside

Abstract

Protein glycosylation is the most complex post‐translational modification process. More than 50 % of human cells proteins are glycosylated, whereas bacteria such as E. coli do not have this modification machinery. Indeed, the carbohydrate residues in natural proteins affect their folding, immunogenicity, and stability toward proteases, besides controlling biological properties and activities. It is therefore important to introduce such structural modification in bioengineered proteins lacking the presence of carbohydrate residues. This is not trivial as it requires reagents and conditions compatible with the protein's stability and reactivity. This work reports on the introduction of lactose moieties in two natural proteins, namely ubiquitin (Ub) and l‐asparaginase II (ANSII). The synthetic route employed is based on the sulfur(VI) fluoride exchange (SuFEx) coupling of a lactose tethered arylfluorosulfate (Lact‐Ar‐OSO2F) with the ϵ‐NH2 group of lysine residues of the proteins. This metal‐free click SuFEx reaction relies on the properties of the fluorosulfate employed, which is easily prepared in multigram scale from available precursors and reacts chemoselectively with the ϵ‐NH2 group of lysine residues under mild conditions. Thus, iterative couplings of Lact‐Ar‐OSO2F to Ub and ANSII, afforded multiple glycosylations of these proteins so that up to three and four Lact‐Ar‐OSO2 groups were introduced in Ub and ANSII, respectively, via the formation of a sulfamoyl (OSO2‐NH) linkage.

Tyrosine Conjugation Methods for Protein Labelling

“…This sulfuryl chemistry can be sluggish with electrophiles such as arylsulfonyl fluorides (Ar‐SO 2 ‐F) and arylfluorosulfate (ArO‐SO 2 ‐F), which are kinetically stable to hydrolysis and resistant to oxidation and reduction. This specific reactivity pattern has been successfully exploited in chemical biology and medicinal chemistry for protein labelling, [46–48] after reaction with the side chain of nucleophilic AAs. Although SuFEx may be performed on a potentially wide range of AA S , chemoselectivity can only be achieved by fine‐tuning the experimental procedure or in a context‐specific environment [49] …”

Selected Research Topics of the Dondoni Group over the Last Two Decades (2000–2020)

“…Subsequent work on peptide and protein glycosylation was carried out quite recently in collaboration with Professor Nativi and Professor Fragai groups at Florence University. 33 This work was essentially based on the sulfur-fluoride exchange chemistry (SuFEx) highlighted by the Sharpless group 34 in 2014. This chemistry is based on the high reactivity of the fluorine atom of sulfonyl fluorides and fluorosulfates toward N, C, and O nucleophiles.…”

Fucosylated Ubiquitin and Orthogonally Glycosylated Mutant A28C: Conceptually New Ligands for Burkholderia Ambifaria Lectin (BambL)†

“…We therefore planned to take advantage from the seven Lys ε-NH2 residues and from the N-terminus to link up to eight residues of fucoside 7 (Scheme 2) through amide-bond formation. 25 With respect to fucosyl derivative 7 features a longer aliphatic chain, to properly exhibit the sugar moiety on protein surface, 3 ending with a NHS activated carboxylic group. Compound 7 was synthesized in four steps from carboxylic acid 5 (see Scheme 1) by treatment in DMF with HBTU and NMM, and reaction with mono-Boc protected ethylendiamine 8, to form the NHBoc derivative 9 (93%).…”

“…2 Although the function of scaffold proteins is still matter of investigation, stable and structurally determined proteins assumed a compelling interest as bio-compatible platforms to display clusters of glycosidic ligands. 3 Ubiquitin (Ub) is a fully characterized, widespread protein bearing seven lysine residues in its wild-type form (WT-Ub). Ubiquitination is a reversable modification in which the terminal glycine residue of Ub is attached to a substrate protein.…”