Synthesis of Multivalent Glycoconjugates Containing the Immunoactive LELTE Peptide: Effect of Glycosylation on Cellular Activation and Natural Killing by Human Peripheral Blood Mononuclear Cells

Abstract

Pentapeptide diacidic sequence LELTE, derived from the mycobacterial heat shock protein hsp65, has been recently identified as a "danger" signal of the immune system effective via specific binding to the universal leukocyte triggering receptor CD69. This sequence is not active per se, only after its presentation within the multivalent environment of its parent protein, or after artificial dimerization using a standard bifunctional reagents. Here we describe an entirely new way of presenting of this peptide based on its attachment to a cyclopeptide RAFT scaffold (K-K-K-P-G)(2) through the epsilon-amino group of lysine residues, alone or in combination with the carbohydrate epitope alphaGalNAc. The ability of such RAFT scaffolds to precipitate the target CD69 receptor or to activate CD69-positive cells is enhanced in compounds 2 and 4 possessing combined peptide/carbohydrate expression. Compounds 2 and 4 are highly efficient activators of natural killer lymphocytes, but they are completely inactive from the point of view of activation-induced apoptosis of lymphocytes by the target cells. These unique properties make the combined peptide/carbohydrate RAFTs highly suitable for future evaluation in animal tumor therapies in vivo and predict them to be readily available and efficient immunoactivators.

Dimerization of an Immunoactivating Peptide Derived from Mycobacterial hsp65 Using N-Hydroxysuccinimide Based Bifunctional Reagents Is Critical for Its Antitumor Properties

“…13 While the pentapeptide completely retained the binding activity of a larger peptide containing this sequence internally, or even the entire hsp65 subunit, it had to be presented in multimeric form to achieve optimal immunostimulating activity. Previously, we presented this sequence as a part of a complex cyclic dekapeptide raft.…”

“…13 The immunoactive pentapeptide sequence can be active on cells of the immune system per se, or as a part of a larger polypeptide or even in the environment of the entire protein.…”

“…13 These results led us to evaluate the degree of clustering or conjugation that is necessary for attainment of optimal immunological activity. Here, we describe our evaluation of the immunological activity of the active peptide and control peptide that have been conveniently dimerized using the unique amino group within their peptide sequence.…”

Synthesis of heteroglycoclusters by using orthogonal chemoselective ligations

“…Taking advantage of our experience in bioconjugation methods, we decided to explore two chemoselective strategies to achieve this purpose. We first selected the oxime ligation since we have previously used this approach successfully for the preparation of sophisticated molecular systems, such as synthetic vaccines [27–28], immunomodulators [29], lectin ligands [30] or vectors of hepatocytes [31]. As the second strategy, we have chosen the well-known copper(I)-catalyzed alkyne–azide cycloaddition (CuAAC) [32–33], which is intensively exploited for the conjugation of sugars to both molecular and biological systems [34–35].…”

Charged Hexosaminides as New Substrates for β‐N‐Acetylhexosaminidase‐Catalyzed Synthesis of Immunomodulatory Disaccharides

“…With Àlog IC 50 values approaching 10 they ranked among the best ligands ever studied, comparable only to multivalent structures. [22] Apparently, the sulfate is the most efficient moiety for increasing ligand strength (although any charged group causes an increase in binding).…”