SYNTHESIS AND EVALUATION OF LABELED PHOSPHATIDYLGLYCEROL PROBES TO ELUCIDATE MECHANISMS BEHIND CHOLESTEROL TRAFFICKING IN NIEMANN-PICK TYPE C DISEASE
Niemann-Pick Type C (NPC) disease is a rare lysosomal storage disorder that occurs in about 1/89,000 to 1/120,000 live births and is characterized by an aberrant accumulation of cholesterol within the late endosome/lysosome of cells. Symptoms of this disease include splenomegaly, neurological deterioration, and often death before adulthood. Mutations in the membrane bound NPC1 or luminal NPC2 proteins lead to a decrease in cholesterol efflux within the lysosomes by which excess cholesterol crystallizes within membranes resulting in cell death. It has been demonstrated that increasing the amount of the lysosomal specific phospholipid Bis(monoacylglycerol)phosphate (BMP), also known as Lysobisphosphatidic acid (LBPA), in cells increases the rate of cholesterol transport in npc1-/- cells, but not in npc2-/- cells, indicating a strong synergistic relationship between the NPC2 protein and the lysosomal membranes. Increasing the amount of phosphatidyl glycerol (PG), a hypothesized precursor to BMP, has also shown an increase in cholesterol egress. While it is hypothesized that the increase in cholesterol clearance in the latter is due to the biosynthesis of LBPA from PG, there is no study to directly confirm this phenomenon. Therefore, we set out to synthesize diastereochemically pure PG containing isotopically labeled oleyl acyl chains to examine LBPA levels using lipidomic analysis of npc1-/- cells post treatment with PG.
Initially, efforts centered around the use of phosphoramidite methodology commonly encountered in DNA oligonucleotide synthesis. While this route proved to be successful in making PG in modest yield (52%), reproducibility of this route with consistent yields was hindered due to the use of tetrabutylammonium fluoride (TBAF) in the final global deprotection step. Thus, we set out to discover a phosphorylated intermediate that did not require TBAF in the final step or contain easily hydrolysable protecting groups. It was discovered that H-phosphonate methodology using diphenyl phosphite for phosphorylation of the glycerol headgroup and backbone proved to be robust enough for PG synthesis. In this strategy, PG can be isolated in two steps from the final protected intermediate by first oxidizing the H-phosphonate from PIII to PV followed by deprotection of the glycerol head group under acidic conditions. Additionally, the H-phosphonate strategy also allowed us to omit headgroup modification prior to phosphorylation which reduced the number of synthetic steps from 11 steps to 7 steps. As a result, we were able to synthesize diastereochemically pure PG more consistently than the previous route in 75% yield. The route was further modified further to incorporate asymmetric acyl chains allowing the selective installation of a labeled acyl chain on the sn-1 or sn-2 positions of the phosphoglycerol backbone. The results from the lipidomic experiments indicate that increased LBPA concentrations in cells rise upon incubation with labeled PG. Additionally, increases in lyso-PG and acyl-PG are also observed leading to several hypotheses on how LBPA might be synthesized from PG. Future directions on this effort include identification of phospholipid species made from PG containing asymmetrically labeled acyl chains. Synthesis of photoaffinity labeled PG is also underway to determine the protein partners involved in PG metabolism.