1,6-Anhydro-N-acetyl-β-D-glucosamine in the oligosaccharide syntheses: I. Synthesis of 3-acetate and 3-benzoate of 1,6-anhydro-N-acetyl-β-D-glucosamine via the 4-O-trityl derivative

3-O-Acetyl and 3-O-benzoyl derivatives of 1,6-anhydro-N-acetyl-β-D-glucosamine were synthesized via its selective tritylation followed by the 3-O-acylation and removal of the trityl protective group. Tritylium trifluoromethanesulfonate, which can easily be prepared by mixing solutions of triphenylcarbinol and trimethylsilyl trifluoromethanesulfonate in an equimolar ratio, was suggested as a reagent for the effective tritylation of a secondary hydroxyl group. This paper is dedicated to the 70th birthday of Prof. A. Ya. Khorlin.     

A new role for PGRP-S (Tag7) in immune defense: lymphocyte migration is induced by a chemoattractant complex of Tag7 with Mts1

PGRP-S (Tag7) is an innate immunity protein involved in the antimicrobial defense systems, both in insects and in mammals. We have previously shown that Tag7 specifically interacts with several proteins, including Hsp70 and the calcium binding protein S100A4 (Mts1), providing a number of novel cellular functions. Here we show that Tag7–Mts1 complex causes chemotactic migration of lymphocytes, with NK cells being a preferred target. Cells of either innate immunity (neutrophils and monocytes) or acquired immunity (CD4⁺ and CD8⁺ lymphocytes) can produce this complex, which confirms the close connection between components of the 2 branches of immune response.     

Molecular forms of alpha-1-acid glycoprotein in human blood serum. Differences in levels of di-, tri-, and tetra-antennary N-linked carbohydrate chains]

alpha 1-Acid glycoprotein isolated from healthy individuals blood was separated on Con A-Sepharose into three fractions: non-bound (AGP-1, 84%, 43.5 kDa), Con A-bound (AGP-2, 14%, 41.3 kDa), and Con A-tightly bound (AGP-3, 2%, 39.6 kDa). Amino acid compositions of these fractions were similar but carbohydrate ones differed. HPLC analysis of 7-amino-4-methylcoumarin derivatives of the oligosaccharides in combination with their sequential exoglycosidase digestion showed that AGP-1, AGP-2, and AGP-3 have the same set of oligosaccharides and differ only by their proposition. A minor quantity of agalacto-oligosaccharides (with a terminal GlcNAc residue) was identified.     

Artificial peptide and carbohydrate antigens. Immobilization of haptens and adjuvant (MDP) on polyacrylamide

To study the influence of the polyacrylamide carrier on immunogenic properties of the peptide and oligosaccharide haptens, we have prepared artificial antigens by conjugation of a synthetic hexapeptide (homologous to the fragment 95-100 of the murine H-2Db antigen heavy chain) or of an oligosaccharide (antigenic determinant of human blood groops, Lea) with polyacrylamide. In some cases the conjugates containing also a synthetic glycopeptide adjuvant, N-acetylmuramoyl-L-alanyl-D-isoglutamine (MDP), were used. Antisera against haptens were obtained by immunization of BALB/c mice with corresponding conjugates. By the enzyme-linked immunosorbent assay it was shown that these antisera had a high binding titer (up to 10 000) to corresponding hapten, and MDP immobilized on the same carrier as hapten possessed a considerable immunostimulating activity. Thus, usefulness of polyacrylamide for preparation of immunogenic artificial molecules carrying peptide and oligosaccharide haptens was demonstrated.     

Synthesis of oligosaccharides with group specificity for blood types A, B and H (Type 3)

Chemical synthesis of A, B, and H (type 3) human blood group determinant oligosaccharides (as R-glycosides, R = OCH2CH2CH2NHCOCF3) and their polymeric derivatives are reported. 4,6; 4',6'-Di-O-benzylidene derivative of Gal beta 1----3GalNAc alpha 1----R was chloroacetylated selectively at 3'-OH, the chloroacetate was alpha-fucosylated and dechloroacetylated to give protected H (type 3) trisaccharide bearing free 3'-OH. alpha-Glycosylation of the trisaccharide with 2-azido-3,4,6-tri-O-benzyl-beta-D-galactopyranosyl chloride and 2,3,4,6-tetra-O-benzyl-alpha-D-galactopyranosyl bromide gave rise to protected A and B tetrasaccharides, respectively. Deprotected R-glycosides were converted to OCH2CH2CH2NH2 derivatives. Their reaction with poly(4-nitrophenylacrylate) affords polyacrylamide-coupled conjugates with A, B, and H (type 3) specificity.     

Synthetic polymeric sialoside inhibitors of influenza virus receptor-binding activity.

Anomeric aminobenzylglycosides of Neu5Ac were coupled with the polyacrylate carrier and a number of synthetic polyvalent sialosides obtained were investigated as inhibitors of influenza virus attachment. The inhibitory activity of polymeric sialosides is highly dependent upon the Neu5Ac residue content and the nature of the carrier. The polyacrylic acid based polymer bearing 10 mol% of Neu5Ac is 3 orders of magnitude more potent inhibitor than the corresponding monovalent benzylsialoside and considerably more active than fetuin.     

The immunomodulating activity of new muramyl dipeptide derivatives in vitro.]

The action of some new MDP derivatives on functional activity of murine T-lymphocytes and macrophages was studied. The following tests have been used: proliferation of spleen cells in one-way allo-MLC; IL-1 and TNF production by peritoneal macrophages treated with the preparations. The most expressed enhancement of lymphocyte proliferative response in MLC has been exerted by beta C7H15 MDP and beta C16H33 MDP (stimulation indexes 31-69%). beta C7H15 MDP, beta C16H33 MDP and polyacrylamide-MDP (P-MDP) alone or in combination with LPS caused elevated secretion of IL-1 by macrophages. While beta C7H15 MDP was as active as MDP, beta C16H33 MDP and P-MDP manifested increased ability to stimulate IL-1 production in comparison with MDP. beta C7H15 MDP, beta C16H33 MDP, P-MDP and MDP induced similar level of TNF production by murine macrophages. However, simultaneous treatment of macrophages with beta C16H33 MDP and LPS resulted in more significant enhancement of TNF production than combination LPS + MDP.     

Glycan-binding profile of DC-like cells

Modification of vaccine carriers by decoration with glycans can enhance binding to and even targeting of dendritic cells (DCs), thus augmenting vaccine efficacy. To find a specific glycan-“vector” it is necessary to know glycan-binding profile of DCs. This task is not trivial; the small number of circulating blood DCs available for isolation hinders screening and therefore advancement of the profiling. It would be more convenient to employ long-term cell cultures or even primary DCs from murine blood. We therefore examined whether THP-1 (human monocyte cell line) and DC2.4 (immature murine DC-like cell line) could serve as a model for human DCs. These cells were probed with a set of glycans previously identified as binding to circulating human CD14low/-CD16+CD83+ DCs. In addition, we tested a subpopulation of murine CD14low/-CD80+СD11c+CD16+ cells reported as relating to the human CD14low/-CD16+CD83+ cells. Manα1–3(Manα1–6)Manβ1–4GlcNAcβ1–4GlcNAcβ bound to both the cell lines and the murine CD14low/-CD80+СD11c+CD16+ cells. Primary cells, but not the cell cultures, were capable of binding GalNAcα1–3Galβ (Adi), the most potent ligand for binding to human circulating DCs. In conclusion, not one of the studied cell lines proved an adequate model for DCs processes involving lectin binding. Although the glycan-binding profile of BYRB-Rb (8.17)1Iem mouse DCs could prove useful for assessing human DCs, important glycan interactions were missing, a situation which was aggravated when employing cells from the BALB/c strain. Accordingly, one must treat results from murine work with caution when seeking vaccine targeting of human DCs, and certainly should avoid cell lines such as THP-1 and DC2.4 cells.     

Uncharged P-selectin blockers

The blocking potency of P- and L-selectin was studied for certain small molecule mannosides and their polyacrylamide (PAA, 30 kDa) conjugates in comparison to SiaLe(x) and fucoidan. Two experimental systems were used: (1) solid phase static assay based on recombinant selectins, and (2) P-selectin dependent rat peritoneal inflammation. betaMan-SC6H4NO2- p was four times more potent P-selectin inhibitor as compared to SiaLe(x). Docking of this molecule onto the P-selectin carbohydrate-binding site demonstrated that a nitro group enabled an electrostatic interaction with residue Lys 84, while the phenyl ring and the CH2 at C-6 contacted the CH2 groups of the same Lys residue. In vivo, betaMan-SC6H4NO2- p blocked experimental inflammation better than SiaLe(x), but significantly lower than fucoidan. In vitro Man-polyacrylic acid conjugates appeared to be very potent inhibitors comparable to fucoidan, uncharged Man-PAA proved rather active, comparable to SiaLe(x)-PAA both in vitro, and in vivo, whereas mannan did not display any P-selectin blocking effect.     

Unique conformer selection of human growth-regulatory lectin galectin-1 for ganglioside GM1 versus bacterial toxins

Endogenous lectins induce effects on cell growth by binding to antennae of natural glycoconjugates. These complex carbohydrates often present more than one potential lectin-binding site in a single chain. Using the growth-regulatory interaction of the pentasaccharide of ganglioside GM(1) with homodimeric galectin-1 on neuroblastoma cell surfaces as a model, we present a suitable strategy for addressing this issue. The approach combines NMR spectroscopic and computational methods and does not require isotope-labeled glycans. It involves conformational analysis of the two building blocks of the GM(1) glycan, i.e., the disaccharide Galbeta1-3GalNAc and the trisaccharide Neu5Acalpha2-3Galbeta1-4Glc. Their bound-state conformations were determined by transferred nuclear Overhauser enhancement spectroscopy. Next, measurements on the lectin-pentasaccharide complex revealed differential conformer selection regarding the sialylgalactose linkage in the tri- versus pentasaccharide (Phi and Psi value of -70 degrees and 15 degrees vs 70 degrees and 15 degrees, respectively). To proceed in the structural analysis, the characteristic experimentally detected spatial vicinity of a galactose unit and Trp68 in the galectin's binding site offered a means, exploiting saturation transfer from protein to carbohydrate protons. Indeed, we detected two signals unambiguously assigned to the terminal Gal and the GalNAc residues. Computational docking and interaction energy analyses of the entire set of ligands supported and added to experimental results. The finding that the ganglioside's carbohydrate chain is subject to differential conformer selection at the sialylgalactose linkage by galectin-1 and GM(1)-binding cholera toxin (Phi and Psi values of -172 degrees and -26 degrees, respectively) is relevant for toxin-directed drug design. In principle, our methodology can be applied in studies aimed at blocking galectin functionality in malignancy and beyond glycosciences.