Construction of Multivalent Homo- and Heterofunctional ABO Blood Group Glycoconjugates Using a Trifunctional Linker Strategy

Abstract

The design and synthesis of multivalent ligands displaying complex oligosaccharides is necessary for the development of therapeutics, diagnostics, and research tools. Here, we report an efficient conjugation strategy to prepare complex glycoconjugates with 4 copies of 1 or 2 separate glycan epitopes, providing 4-8 carbohydrate residues on a tetravalent poly(ethylene glycol) scaffold. This strategy provides complex glycoconjugates that approach the size of glycoproteins (15-18 kDa) while remaining well-defined. The synthetic strategy makes use of three orthogonal functional groups, including a reactive N-hydroxysuccinimide (NHS)-ester moiety on the linker to install the first carbohydrate epitope via reaction with an amine. A masked amine functionality on the linker is revealed after the removal of a fluorenylmethyloxycarbonyl (Fmoc)-protecting group, allowing the attachment to the NHS-activated poly(ethylene glycol) (PEG) scaffold. An azide group in the linker was then used to incorporate the second carbohydrate epitope via catalyzed alkyne-azide cycloaddition. Using a known tetravalent PEG scaffold (PDI, 1.025), we prepared homofunctional glycoconjugates that display four copies of lactose and the A-type II or the B-type II human blood group antigens. Using our trifunctional linker, we expanded this strategy to produce heterofunctional conjugates with four copies of two separate glycan epitopes. These heterofunctional conjugates included Neu5Ac, 3'-sialyllactose, or 6'-sialyllactose as a second antigen. Using an alternative strategy, we generated heterofunctional conjugates with three copies of the glycan epitope and one fluorescent group (on average) using a sequential dual-amine coupling strategy. These conjugation strategies should be easily generalized for conjugation of other complex glycans. We demonstrate that the glycan epitopes of heterofunctional conjugates engage and cluster target B-cell receptors and CD22 receptors on B cells, supporting the application of these reagents for investigating cellular response to carbohydrate antigens of the ABO blood group system.

Heteromultivalent Glycooligomers as Mimetics of Blood Group Antigens

“…We first introduced Fuc, ac ommon monosaccharide motif found in different HBGAs, through CuAAC conjugation to the unprotected alkyne on the side chains of the DTDS. The corresponding glycooligomer was then split into four batches and further functionalized with either aG alNAc, Gal, Lac, or Sia residue after TIPS deprotection to generate glycooligomers [13][14][15][16]. In addition, ah omomultivalent all-Fuc glycooligomer 17 and an all-Gal glycooligomer 18 were synthesized forc omparison in later binding studies.…”

“…a-l-Methylfucose (MeFuc) was measured as ar eference compound. Galactosylated oligomer 18 wasu sed as an egative control, whereas homomultivalent fucosylated oligomer 17 served as ap ositive controlf or comparison of the effect of heteromultivalency in the HBGA mimetics [13][14][15][16].N egative control glycooligomer 18 showed no binding to LecB (see the Supporting Information). Ta ble 2 showst he results of the inhibition competition assayo fg lycooligomers 13-17 and a-l-methylfucose.…”

“…[11,12] However,t he questionr emains as to how the affinity and selectivity of HBGA mimetics might be altered by including not only one but several different glycanf ragments that imitate more closely the natural ligand's heterofunctional structure. [13][14][15] Indeed, it has been shown that the combination of different carbohydrate moieties in heterofunctional glycoconjugates and glycomimetic structures strongly affects their recognition process. [16] Therefore, we introduce here an ew strategyt owards obtaining glycomimetic ligandsb yu sing an oligo(amidoamine) scaffold with sequence-controlled divalent heterofunctionalg lycans ide chains that are based on different fragments of HBGA and humanm ilk oligosaccharide (HMO) ligands.…”

Anomeric Functionalization of Carbohydrates for Chemical Conjugation to Vaccine Constructs

“…Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 25 June 2018 doi:10.20944/preprints201806.0395.v1 Synthesis of multivalent ABO blood group glycoconjugates on a PEG scaffold developed by the Cairo group. A) Synthesis of amine-terminated glycans B) Synthesis of a heterotrifunctionalAlexaFluor 488 containing trivalent glycotope using iterative amine conjugation[68].…”

“…Synthesis of multivalent ABO blood group glycoconjugates using heterotrifunctional PEG linker developed by the Cairo group[68].In general, terminal alkene linkers, especially the n-4-pentenyl linker, have been popular in the carbohydrate chemistry community since the late 1980 to 2000s. While thiolinker was first introduced to study glycosylation reactions, it later proved to be a versatile functional group for total synthesis of complex glycans equipped with a functionalizable linker.…”