Chemical Synthesis of All Phosphatidylinositol Mannoside (PIM) Glycans from Mycobacterium tuberculosis

Abstract

The emergence of multidrug-resistant tuberculosis (TB) and problems with the BCG tuberculosis vaccine to protect humans against TB have prompted investigations into alternative approaches to combat this disease by exploring novel bacterial drug targets and vaccines. Phosphatidylinositol mannosides (PIMs) are biologically important glycoconjugates and represent common essential precursors of more complex mycobacterial cell wall glycolipids including lipomannan (LM), lipoarabinomannan (LAM), and mannan capped lipoarabinomannan (ManLAM). Synthetic PIMs constitute important biochemical tools to elucidate the biosynthesis of this class of molecules, to reveal PIM interactions with host cells, and to investigate the function of PIMs as potential antigens and/or adjuvants for vaccine development. Here, we report the efficient synthesis of all PIMs including phosphatidylinositol (PI) and phosphatidylinositol mono- to hexa-mannoside (PIM1 to PIM6). Robust synthetic protocols were developed for utilizing bicyclic and tricyclic orthoesters as well as mannosyl phosphates as glycosylating agents. Each synthetic PIM was equipped with a thiol-linker for immobilization on surfaces and carrier proteins for biological and immunological studies. The synthetic PIMs were immobilized on microarray slides to elucidate differences in binding to the dendritic cell specific intercellular adhesion molecule-grabbing nonintegrin (DC-SIGN) receptor. Synthetic PIMs served as immune stimulators during immunization experiments in C57BL/6 mice when coupled to the model antigen keyhole-limpet hemocyanin (KLH).

Role of Phosphatidylinositol Mannosides in the Interaction between Mycobacteria and DC-SIGN

“…We have no ready explanation for the difference in outcome; however, we do note that the magnitude of the binding of PIMs to DC-SIGN reported in the study by Torrelles et al was very small for all of the species tested and thus lacked specificity. Our data are more in agreement with a recent study by Boonyarattanakalin and coworkers (12). This study on the chemical synthesis of PIMs showed a high affinity of DC-SIGN for the polar PIMs, i.e., PIM 5 and PIM 6 , and low or no affinity for the less mannosylated PIMs, i.e., PIM 2 and PIM 4 , respectively, in a fluorescence scanning assay of a PIM microarray.…”

Lipoarabinomannan and related glycoconjugates: structure, biogenesis and role inMycobacterium tuberculosisphysiology and host–pathogen interaction

“…In particular, the higher-order PIMs (PIM 5 and PIM 6 ), with terminal α(1→2)-linked Man p residues reminiscent of the mannose cap of Man-LAM, were of interest. As expected, DC-SIGN recognizes the higher-order PIMs with high affinity as compared with the lower-order PIMs (Boonyarattanakalin et al , 2008; Driessen et al , 2009;). Besides PIMs, other potential ligands in the mycobacterial cell wall for DC-SIGN have been identified: lipomannan, Man-AM and mannosylated lipoproteins 19 and 45 kDa (Pitarque et al , 2005), and more recently, capsular α-glucan (Geurtsen et al , 2009) (a list is provided in Table 3).…”

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2007–2008

“…Highest MALDI mass = 7785 (Joe, et al, 2007) (Vasan, et al, 2007) Nonasaccharide subunits of the atypical O-antigen polysaccharide from Helicobacter pylori strains TOF Synthesis by dimerization and trimerization of suitably protected trisaccharide repeating unit (Fulse, et al, 2007) Pentasaccharide repeat from Phosphatidylinositol mannosides (PIMs) from Mycobacterium tuberculosis TOF Synthesis of all PIMs including phosphatidylinositol (PI) and phosphatidylinositol mono-to hexamannosides (PIM 1 to PIM 6 ) (Boonyarattanakalin, et al, 2008) Spore-surface pentasaccharide from Bacillus anthracis Solid-phase synthesis MALDI (Werz, et al, 2007a Tetra-acylated lipid A derivatives from Porphyromonas gingivalis…”