Multivalent lectin-glycan interactions are widespread in biology and are often exploited by pathogens to bind and infect host cells. Glycoconjugates can block such interactions and thereby prevent infection. The inhibition potency strongly depends on matching the spatial arrangement between the multivalent binding partners. However, the structural details of some key lectins remain unknown and different lectins may exhibit overlapping glycan specificity. This makes it difficult to design a glycoconjugate that can potently and specifically target a particular multimeric lectin for therapeutic interventions, especially under the challenging in vivo conditions. Conventional techniques such as surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC) can provide quantitative binding thermodynamics and kinetics. However, they cannot reveal key structural information, e.g., lectin's binding site orientation, binding mode, and interbinding site spacing, which are critical to design specific multivalent inhibitors. Herein we report that gold nanoparticles (GNPs) displaying a dense layer of simple glycans are powerful mechanistic probes for multivalent lectin-glycan interactions. They can not only quantify the GNP-glycan-lectin binding affinities via a new fluorescence quenching method, but also reveal drastically different affinity enhancing mechanisms between two closely related tetrameric lectins, DC-SIGN (simultaneous binding to one GNP) and DC-SIGNR (intercross-linking with multiple GNPs), via a combined hydrodynamic size and electron microscopy analysis. Moreover, a new term, potential of assembly formation (PAF), has been proposed to successfully predict the assembly outcomes based on the binding mode between GNP-glycans and lectins. Finally, the GNP-glycans can potently and completely inhibit DC-SIGN-mediated augmentation of Ebola virus glycoprotein-driven cell entry (with IC 50 values down to 95 pM), but only partially block DC-SIGNR-mediated virus infection. Our results suggest that the ability of a glycoconjugate to simultaneously block all binding sites of a target lectin is key to robust inhibition of viral infection.

An organocatalyst-mediated protein aldol ligation (OPAL) affords C–C linked bioconjugates at neutral pH.

A chemo-enzymatic site-specific fluorination strategy is employed to obtain glyco-nanoparticles with tuneable selectivity towards galectins.

Multivalent lectin−glycan interactions (MLGIs) are widespread and vital for biology. Their binding biophysical and structural details are thus highly valuable, not only for the understanding of binding affinity and specificity mechanisms but also for guiding the design of multivalent therapeutics against specific MLGIs. However, effective techniques that can reveal all such details remain unavailable. We have recently developed polyvalent glycan quantum dots (glycan-QDs) as a new probe for MLGIs. Using a pair of closely related tetrameric viral-binding lectins, DC-SIGN and DC-SIGNR, as model examples, we have revealed and quantified their large affinity differences in glycan-QD binding are due to distinct binding modes: with simultaneous binding for DC-SIGN and cross-linking for DC-SIGNR. Herein, we further extend the capacity of the glycan-QD probes by investigating the correlation between binding mode and binding thermodynamics and kinetics and further probing a structural basis of their binding nature. We reveal that while both lectins' binding with glycan-QDs is enthalpy driven with similar binding enthalpy changes, DC-SIGN pays a lower binding entropy penalty, resulting in a higher affinity than DC-SIGNR. We then show that DC-SIGN binding gives a single second-order k on rate, whereas DC-SIGNR gives a rapid initial binding followed by a much slower secondary interaction. We further identify a structural element in DC-SIGN, absent in DC-SIGNR, that plays an important role in maintaining DC-SIGN's MLGI character. Its removal switches the binding from being enthalpically to entropically driven and gives mixed binding modes containing both simultaneous and cross-linking binding behavior, without markedly affecting the overall binding affinity and kinetics

Pseudaminic acid (Pse5Ac7Ac) is a nonmammalian sugar present on the cell surface of a number of bacteria including Pseudomonas aeruginosa, Campylobacter jejuni and Acinetobacter baumannii. However, the role Pse5Ac7Ac plays in host-pathogen interactions remains underexplored, particularly compared to its ubiquitous sialic acid analogue Neu5Ac. This is primarily due to a lack of access to difficult to prepare Pse5Ac7Ac glycosides. Herein we decribe the in vitro biocatalytic transfer of an activated Pse5Ac7Ac donor onto glycosyl acceptors enabling the enzymatic synthesis of Pse5Ac7Ac containing glycosides. In a chemoenzymatic approach, chemical synthesis initially afforded access to a late stage Pse5Ac7Ac biosynthetic intermediate, which was subsequently converted to the desired CMP-glycosyl donor in a one-pot-two-enzyme process using biosynthetic enzymes. Finally screening a library of 13 sialyltransferases (SiaT) with the unnatural substrate enabled the identification of a promiscuous inverting SiaT capable of turnover to afford β-Pse5Ac7Ac terminated glycosides. Figure 1 Nonulosonic acid structures; the pseudaminic acid Pse5Ac7Ac 1, and sialic acid Neu5Ac 2. Scheme 2 Chemical synthesis of 6-deoxy-L-AltdiNAc 4. a) i) NaOMe, MeOH. ii) Diphenyl borinate, DIPEA, BzCl, CH 3 CN, 80% over 2steps. b) i) Tf 2 O, Pyridine, DCM, 0 °C. ii) NaN 3 , DMF, 110 °C, 58% over 2-steps. c) NaOMe, MeOH, 95%. d) i) Tf 2 O, Pyridine, DCM, 0 °C. ii) TBANO 2 , CH 3 CN, 70 °C, 46% over 2-steps. e) i) PMe 3 , NaOH, THF, 60 °C. ii) Ac 2 O, Pyridine, 69% over 2-steps. f) NaOMe, MeOH, 81%. g) CAN, CH 3 CN:H 2 O (4:1 v/v), 94%, α:β 1:3. Scheme 1 Biosynthesis of CMP-Pse5Ac7Ac 3 via the key hexose intermediate 6-deoxy-ʟ-AltdiNAc 4.