Chemoenzymatic synthesis and lectin recognition of a selectively fluorinated glycoprotein

Abstract

A chemoenzymatic glycosylation remodeling method for the synthesis of selectively fluorinated glycoproteins is described. The method consists of chemical synthesis of a fluoroglycan oxazoline and its use as donor substrate for endoglycosidase (ENGase)-catalyzed transglycosylation to a GlcNAc-protein to form a homogeneous fluoroglycoprotein. The approach was exemplified by the synthesis of fluorinated glycoforms of ribonuclease B (RNase B). an interesting finding was that fluorination at the C-6 of the 6-branched mannose moiety in the Man3GlcNAc core resulted in significantly enhanced reactivity of the substrate in enzymatic transglycosylation. A structural analysis suggests that the enhancement in reactivity may come from favorable hydrophobic interactions between the fluorine and a tyrosine residue in the catalytic site of the enzyme (Endo-A). SPR analysis of the binding of the fluorinated glycoproteins with lectin concanavalin A (con A) revealed the importance of the 6-hydroxyl group on the α-1,6-branched mannose moiety in con A recognition. The present study establishes a facile method for preparation of selectively fluorinated glycoproteins that can serve as valuable probes for elucidating specific carbohydrate-protein interactions.

Specificity of Donor Structures for endo‐β‐N‐Acetylglucosaminidase‐Catalyzed Transglycosylation Reactions

“…These enzymes are well known for their use in the structural elucidation of glycoproteins through hydrolysis activity and for the preparation of glycoproteins with homogeneous glycan structures owing to their transglycosylation activity . Recently, the introduction of chemically modified glycans into proteins by using ENGase has been actively investigated . Depending on the modified position of the substrate, the enzyme may or may not recognize it.…”

“…[19,20] Recently,t he introduction of chemically modified glycansi nto proteinsb yu sing ENGase has been actively investigated. [21][22][23][24][25] Depending on the modified position of the substrate, the enzyme may or may not recognize it. For that reason, ad etailed analysis of ENGase-substrate specificity related to donor structure is necessary.Kadowaki et al isolated endo-b-N-acetylglucosaminidase from Mucor hiemalis (endo-M).…”

Development of a Broadly Applicable Assay for Measurement of Glycan-Directed Enzymatic Activity

“…From these data, the bio-RNAseB K d for ConA was found to be 1.13 ± 0.11 µM, well in line with literature data. 17 In addition, the kinetic parameters k on and k off were calculated to be 7.86 ± 92.9 × 10 3 M -1 s -1 and 0.00927 ± 6.37 × 10 -5 s -1 , respectively. To determine the rate of product formation at different bio-RNAseB concentrations, 0, 150, 450, and 1300 nM bio-RNAseB were incubated with 3 U/µL of PNGaseF, or buffer as a control, at room temperature for 30 min.…”

Towards the next generation of biomedicines by site-selective conjugation

“…A chemoenzymatic glycosylation remodeling method was also applied for the synthesis of selectively fluorinated glycoproteins. 242 The chemically assembled fluoroglycan oxazoline was used as a donor substrate for endoglycosidase (ENGase)-catalyzed transglycosylation to a GlcNAc-protein. Interestingly, it was observed that at the C-6 of the 6-branched mannose moiety in the Man3GlcNAc core resulted in significantly enhanced reactivity of the substrate in enzymatic transglycosylation.…”