B. cell epitopes, inducing strong and practical anti-Tn IgG Lazabemide antibodies in mice actually in the absence of external adjuvants.16C18 More recently, Tn and other carbohydrate antigens have been chemically linked to MPLA19C21 and -GalCer22,23 glycolipids, yielding novel types of di-component glycoconjugates that induced Tn-specific IgG antibodies when administered in liposomes. Besides the related mucin-like PS A-1).30 However, despite its promise,31 saponin-derived adjuvants have not been exploited so far for chemical conjugation to Lazabemide synthetic antigens, making the development of such saponin-based molecular vaccines unprecedented. Therefore, we wanted to leverage the encouraging properties of our minimal synthetic saponin analogues to apply them, for the first time, in the building of novel, minimalistic adjuvantCantigen glycoconjugates as potential self-adjuvanting vaccine candidates. Adjuvant-active saponin variants have been previously demonstrated to be internalized by dendritic cells,32 which play a role in adjuvant activity and are involved in the uptake, processing and demonstration of antigens to T cells, leading to activation of adaptive immunity. Consequently, we hypothesized that, by colocalizing both parts in the same molecule through covalent linkage of the Tn antigen to the saponin scaffold, this adjuvant platform could assist in delivering the conjugate create (and consequently the attached antigen) to the same antigen showing cell, therefore enhancing its uptake and demonstration to the adaptive immune system and potentially increasing the immune response. In developing this fully synthetic approach, streamlined Lazabemide synthesis, structural simplicity and facile conjugation chemistry were our key design criteria to facilitate convenience, full chemical characterization and easy manipulation of the producing glycoconjugate construct. Herein, we statement the 1st chemical synthesis and initial immunological evaluation of a novel carbohydrate-based vaccine design consisting of two minimal saponinCTn conjugates (2 and 3) HAX1 (Fig. 1b), which leverage ideal triterpene cores and a well-established site for changes (acyl chain terminus) with retention of saponin adjuvant activity. Initial immunological studies suggest that this 1st dicomponent design induces a moderate antibody response against the weakly immunogenic carbohydrate antigen Tn. Notably, the versatility of the chemical strategy as well as the presence of important sites for chemical changes in the saponin scaffold provides additional opportunities for further structureCactivity optimization currently underway in our laboratories. In terms of chemical design Lazabemide of the candidates, our previous development of unimolecular adjuvant conjugates by covalently linking the immunopotentiator tucaresol to saponin adjuvants at the side chain terminal amine6 indicated that these saponins are amenable to significant structural changes in the acyl chain terminus without impairing adjuvant activity. Therefore, we decided to exploit this privileged position in our minimal saponin scaffolds for covalent attachment of well-defined carbohydrate antigens to generate novel glycoconjugate vaccine constructs. To test our hypothesis and probe the self-adjuvanting potential of our saponin platform, we selected the Tn antigen as the prototypical TACA for the synthetic building of minimalistic saponinCTn glycoconjugates 2 and 3. As saponin scaffolds, we selected 6-aminohexanoic amide variants 9 and 1010 (Plan 1), which are derived from our sugar-truncated lead compounds comprising the quillaic acid (QA) and echinocystic acid (EA) triterpenes, respectively.11 In place of the longer carboxylic acid terminating side chain present in the lead saponins, analogues 9 and 10 incorporate a six-carbon acyl chain with a main amine at its end that enables chemoselective functionalization of the fully deprotected molecule with conveniently activated counterparts. Therefore, we envisioned the Tn antigen could be attached to the terminal amino group of the saponins by amidation of a properly derivatized Tn(Thr) glycoamino acid building block (8) (Plan 1). Open in a separate window Plan 1 Synthesis of (a) spacer-containing Tn antigen 8 and (b) saponinCTn conjugates 2 (QACTn) and 3 (EACTn). This compound was equipped with a triethyleneglycol (TEG) spacer to improve water solubility of the producing conjugate and was readily synthesized in three methods starting from a previously reported precursor (4)17 (Plan 1a). Briefly, Fmoc-protected Ac3-GalNAc–O-Thr acid 4 was first elongated with amino-PEG3-amidation under cautiously controlled conditions to avoid racemization (HATU, collidine, CH2Cl2, 0 C, 1 h), providing spacer-containing Tn intermediate 6 in 82% yield. Fmoc removal (piperidine, DMF) and subsequent acetylation (pyridine, Ac2O) of the threonine amino group afforded compound 7 in 93% yield over two methods. Finally, deprotection of the sugars hydroxyl organizations (hydrazine, MeOH) followed by removal of the linker activator in the presence of DIEA like a foundation, providing the desired QA and EA-derived saponinCTn conjugates (2 QACTn and 3 EACTn) in 85% and 84% yield,.