A Synthetic Methodology for Accessing Ceramide Based iNKT Cell Agonists
It is well established that immunological surveillance is an important method by which cancer is controlled in healthy people. Cancer immunotherapy aims to exploit the power of the host immune system for the targeted destruction of malignancies without the undesirable side effects associated with current treatment regimes. This requires training the immune system to recognise hostile cancer cells, which emit chemical signals to down-regulate the immune system. Vaccine adjuvants are thought to be critical in making such immunotherapy vaccines immunogenic enough to break through this tolerance to tumour antigens. Herein we report a robust, scalable and stereoselective synthesis of one such adjuvant β-mannosylceramide (β-ManCer, 1), with late-stage derivatisation affording a series of N-acyl analogues. Furthermore, conjugation of β-ManCer to antigenic peptide fragments is designed to create a synthetic self-adjuvanting vaccine platform for immunotherapeutic treatments. β-ManCer (1) was synthesised in 12 steps with an overall yield of 22% from D-mannose via key glycoside intermediate 85 (Figure I, A). 85 was formed by β-selective glycosylation of sulfoxide donor 73 with phytosphingosine acceptor 78 in 70% yield with an α:β ratio of 7:93. Subsequent chemoselective reduction of the azide of 85, followed by N-acylation with cerotic acid on global deprotection afforded the synthetic adjuvant 1. Alternatively, a comparable intermediate, 116, was synthesised in 62% with absolute β selectivity from mannoside donor 105 and phytosphingosine triflate 48 using a stannylene acetal mediated glycosylation approach. Global deprotection of 116 followed by N-acylation converged the synthesis, affording 1 by an expeditious route (Figure I, B). Alternatively, N-acylation with 11-(para-fluorophenyl)-undecanoic acid (143), 11- (para-(4-fluorophenoxy)phenyl)-undecanoic acid (144), and elicosadienoic acid afforded biologically relevant N-acyl analogues 140, 141, and 142 (Structures shown Figure II). An amino analogue, β-ManPhyt 50, lacking the acyl side chain was also synthesised. Investigation of a pro-drug strategy for the synthesis of self-adjuvanting vaccine constructs of the prototypical glycolipid adjuvant α-GalCer (2), afforded migrated β- ManCer (MbMC, 161) where an acid catalysed migration of the N-acyl chain to the O- 4’ hydroxyl has occurred (Figure III). The reverse O- to N- migration occurs rapidly under phosphate buffered saline (PBS) conditions to regenerate 1. Alternatively, the reverse migration can be blocked by capping the free amine of 161, in this case with a self-immolative linker scaffold terminating in a bicyclo[6.1.0]nonyne (BCN) scaffold, affording 178, with a handle for use in strain promoted azide alkyne cycloaddition (SPAAC) reactions. Conjugation of antigenic peptide fragments terminated in an alkyl azide residue afforded glycopeptide vaccine construct CI207 in 2.0% overall yield, containing an ISA peptide HPV16 E7 epitope, and CI243 in 4.1% overall yield, containing an ovalbumin (OVA) synthetic long peptide (SLP) CD4+ CD8+ epitope (Structures shown Figure IV). It is envisioned that biological evaluation of such compounds will provide critical structure activity relationship (SAR) information on this important class of invariant natural killer T (iNKT) cell agonists.
Advisor: Larsen, David; Painter, Gavin; Compton, Benjamin
Degree Name: Doctor of Philosophy
Degree Discipline: Chemistry
Publisher: University of Otago
Keywords: New Zealand; Chemistry; Glycopeptides; bMC; mannosides; beta mannosides; beta mannosylceramide
Research Type: Thesis