Avijit Kumar Adak

We present a strategy for the irreversible and oriented immobilization of native antibodies (Abs) onto magnetic nanoparticles (MNPs) by integrating Ni²⁺-NTA chelation with diazirine (Dia)-mediated photo-crosslinking. MNPs were co-functionalized with nitrilotriacetic acid (NTA) and photoreactive Dia-2 to create a mixed monolayer NTA/Dia-2@MNPs that selectively binds the His-rich Fc domain of unmodified Abs. Short UV exposure activates Dia-2, generating reactive carbenes that covalently anchor proximal residues and permanently lock the Ab in an oriented configuration. This dual-mode immobilization preserves Fab accessibility, enhances binding performance, and prevents Ab dissociation during stringent washing. We validated the platform using two cancer therapy Abs (trastuzumab and cetuximab) and one cancer biomarker (anti-serum amyloid A, anti-SAA) in cancer cells and human serum. Anti-SAA MNPs fabricated by the NTA-Ni²⁺ method showed a 1.5-fold increase in antigen binding in the serum sample compared to the boronate affinity-based method and a significant (22-fold) improvement over random immobilization. Cetuximab-functionalized oriented MNPs by the current immobilization strategy achieved a 4.7-6-fold enhancement in EGFR pulldown efficiency from human embryonic kidney (HEK293T) and non-small cell lung cancer (NSCLC) models, compared to randomly immobilized controls. Notably, the oriented MNPs enabled co-purification of markedly high interactome coverage of >1000 proteins and differential abundance of downstream proteins. Importantly, this platform requires no prior Ab modification and is compatible with full-length native Abs and stable in complex biological samples (cell or serum). By combining chelation-guided orientation with photoinduced covalent fixation, this strategy addresses key challenges in Ab surface engineering and offers a robust, versatile solution for applications in immunoprecipitation, proteomics, and biomarker discovery.

Oriented boronate-mediated covalent immobilization of native antibodies on PDMS microchannels enhances antigen recognition and circulating tumor cell capture.

In the present work, bacterial glycosyltransferases are utilized to construct ganglioside glycans in a convergent approach via a sugar‒nucleotide regeneration system and one‐pot multienzyme reactions. Starting from β‐lactoside enables the diversification of both the glycan moieties and the linkages in the lower α‐arm and upper β‐arm. Overall, a comprehensive panel of 24 natural a‐series (GM3, GM2, GM1a, GD1a, GT1a, and fucosyl‐GM1), b‐series (GD3, GD2, GD1b, GT1b, and GQ1b), c‐series (GT3, GT2, GT1c, GQ1c, and GP1c), α‐series (GM1α, GD1aα, and GT1aα), and o‐series (GA2, GA1, GM1b, GalNAc‐GM1b, and GD1c) ganglioside glycans are prepared, which are suitable for biological studies and further applications. Moreover, a microarray is constructed with these synthesized ganglioside glycans to investigate their binding specificity with recombinant Fc‐fused Siglec‐7 and Siglec‐9, which are immune checkpoint‐like glycan recognition proteins on natural killer cells. The microarray binding results reveal that GD3 and GT1aα are specific ligands for Siglec‐7 and Siglec‐9, respectively, and this discovery can lead to the identification of appropriate ligands for investigating the roles of these Siglecs in immunomodulation.

Globo A is a neutral Globo-series glycosphingolipid (GSL) that shows natural properties of a cytotoxicity receptor NKp44 binding ligand. The highly complex heptasaccharide glycan structure of Globo A combined with its biological profile provides a unique target for the development of a synthetic method to facilitate its bioactivity studies. Here, a concise chemoenzymatic route to the synthesis of Globo A and its α1,3-galactose-linked congener Globo B is reported. The key to success was the use of a synthetic azido β-Globo H sphingosine (Globo H-βSph) as an acceptor substrate and two glycosyl transferases, an α1,3-N-acetylgalactosaminyltransferase from Helicobacter mustelae (BgtA) and a human blood group B α1,3-galactosyltransferase (h1,3GTB), for stereoselective construction of the terminal α1,3-GalNAc and α1,3-Gal linkages, respectively. The azido-Sph lipid sidechain is further elaborated by reduction and a chemoselective N-acylation to complete the total synthesis of the neutral Globo-series GSLs. In addition, the synthesis of Forssman and para-Forssman antigens were prepared. The strategy may be suitable for accessing other complex GSLs and related lipid-modified GSL derivatives. Graphical Abstract

Background: Bispecific Abs (BsAbs) are useful class of Ab‐based drugs but with difficulty to fabricate them.Aims: Cross‐linking two Abs by click chemistry.Materials & Methods: Bis‐alkynylated divinylpyrimidine (DVP) was synthesized and incorporated into an Ab, which was then cross‐linked with azido‐modified Ab to produce BsAb.Results: The heterodimeric anti‐PD‐L1‐HER2 BsAbC was prepared by CuAAC and its formation was confirmed by SDS‐PAGE. BsAbC was purified by biotinylated HER2 antigen coated magnetic bead and was used to fabricate a microarray. In the microarray binding assay, the heterodimeric anti‐PD‐L1‐HER2 BsAbC showed binding activity with PD‐L1 and HER2 antigens.Discussion: This study represents the first proof‐of‐concept that bis‐alkynylated DVP crosslinker reagent can be used for conjugating half‐antibody fragments site‐selectively by CuAAC. A preliminary characterization revealed that this reagent predominantly incorporates a bioorthogonal functional handle into the desired half‐antibody fragments with minor amounts of a re‐bridged full‐length antibody.Conclusion: This study highlights the utility of DVP crosslinker reagents and validates the use of these molecules for the rapid assembly of switchable complex antibody bioconjugates that enables the control of the intended function of fabricated BsAbC.

Structural variants of α-galactosylceramide (α-GalCer) that stimulate invariant natural killer T (iNKT) cells constitute an emerging class of immunomodulatory agents in development for numerous biological applications. Variations in lipid chain length and/or fatty acids in these glycoceramides selectively trigger specific pro-inflammatory responses. Studies that would link a specific function to a structurally distinct α-GalCer rely heavily on the availability of homogeneous and pure materials. To address this need, we report herein a general route to the diversification of the ceramide portion of α-GalCer glycolipids. Our convergent synthesis commences from common building blocks and relies on the Julia–Kocienski olefination as a key step. A cleavable fluorous tag is introduced at the non-reducing end of the sugar that facilitates quick purification of products by standard fluorous solid-phase extraction. The strategy enabled the rapid generation of a focused library of 61 α-GalCer analogs by efficiently assembling various lipids and fatty acids. Furthermore, when compared against parent α-GalCer in murine cells, many of these glycolipid variants were found to have iNKT cell stimulating activity similar to or greater than KRN7000. ELISA assaying indicated that glycolipids carrying short fatty N-acyl chains (1fc and 1ga), an unsubstituted (1fh and 1fi) or CF₃-substituted phenyl ring at the lipid tail, and a flexible, shorter fatty acyl chain with an aromatic ring (1ge, 1gf, and 1gg) strongly affected the activation of iNKT cells by the glycolipid-loaded antigen-presenting molecule, CD1d. This indicates that the method may benefit the design of structural modifications to potent iNKT cell-binding glycolipids.

LLG‐5 1 is a Neu5Gc8Me‐containing echinoderm ganglioside (EG) isolated from starfish Linckia laevigata. It showed neuritogenic activity toward the rat adrenal pheochromocytoma cell line PC‐12. The first chemoenzymatic total synthesis of the trisialoganglioside LLG‐5 1 has been accomplished by utilizing a convergent [2+3] coupling between a Neu5Gc8Meα(2→5)Neu5Gc‐linked disialoside and a C5‐amino GM3 ceramide, Neu5NH₂(2→3)Galβ(1→4)Glcβ‐Cer. A photoactivatable lactosyl phytosphingosine bearing a sulphonate moiety was developed to permit aqueous buffer solubility of the hydrophobic glycolipid acceptor for enzymatic α(2,3) sialylation. In addition, the synthetic route is flexible, allowing non‐natural LLG‐5 analogues in native lengths with or without modification at C8 of the terminal Neu5Gc and/or N‐acyl component, further expanding the diversity of LLG‐5 structures that could be achieved. The developed synthetic strategy may enable access to other EGs. magnified image

The development of a ligand‐assisted imprinting probe (i.e., LIP 1) for the site‐specific labeling of recombinant Siglec‐7 protein (rSiglec‐7) is reported. LIP 1 is a LIP bearing a ganglioside GD3 glycan, a natural ligand for Siglec‐7, that is embedded in an ortho‐hydroxyl cinnamate core, which exhibits UV‐light‐dependent conformational changes, and is appended with a clickable alkyne handle. LIP 1 enables the selective introduction of an alkyne at the threonine residue 91 (Thr91), which is proximal to the carbohydrate‐binding pocket of Siglec‐7, as identified by LC–MS/MS analysis. By using LIP 1, an azide‐containing environment‐sensitive merocyanine dye is conjugated to rSiglec‐7 by a click reaction, which is capable of directly imaging live HeLa cells as a result of highly specific interactions with cell‐surface glycoproteins containing sialic acid. Moreover, the conjugation of doxorubicin (Dox) to alkynylated rSiglec‐7 retains the original activity of protein binding to the interacting natural GD3 glycan ligand, as revealed by microarray analysis; hence, the rSiglec‐7 conjugate can serve as cargo (Dox)‐delivery vehicle to Siglec‐ligand‐expressing HeLa cells. The LIP labeling strategy described herein is expected to be a general strategy for the future development of glycoprobes for investigating lectin‐glycan interactions.

Correction for ‘Boronate affinity-based photoactivatable magnetic nanoparticles for the oriented and irreversible conjugation of Fc-fused lectins and antibodies’ by Chen-Yo Fan et al., Chem. Sci., 2019, 10, 8600–8609, DOI: 10.1039/c9sc01613a.