Filipin

Filipin. Filipin was recently shown to be a mixture of at least four components, which were designated filipins I, II, III, and IVaccording to mass spectral studies the four compounds were present in the approximate proportion 

4 25 53 18. Rinehart and collaborators have re-examined the structure of filipin by methods similar to those employed in deducing structure (98a) for chainin. Their data are in agreement with the proposed structure (98b) for filipin III, and indicate that filipin IV is probably stereoisomeric at C-3 or C-1 with filipin III, and that filipin II has a structure similar to (98b) but lacking either a C-l or C-3 hydroxy-group. 

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Next we will describe the synthesis of filipin III (114) in greater detail, to bring to light some of the issues that arise in the total synthesis of a complex polyene macrolide [7,8]. 

The polyene macrolide filipin was isolated in 1955 from the cell culture filtrates of Sterptomyces filipinensis, and was later shown to be a mixture of four components [36]. Although too toxic for therapeutic use, the filipin complex has found widespread use as a histochemical stain for cholesterol and has even been used to quantitate cholesterol in cell membranes [37]. The flat structure of filipin III, the major component of the filipin complex, was assigned from a series of degradation studies [38]. Rychnovsky completed the structure determination by elucidating the relative and absolute stereochemistry [39]. The total synthesis plan for filipin III relied heavily on the cyanohydrin acetonide methodology discussed above. 

At this point, completion of the total synthesis required removal of the three acetonides and the two silyl protecting groups (Scheme 18). Removal of the silyl groups with TBAF and subsequent treatment to acidic deprotection conditions led to complete deprotection of 110, but failed to provide filipin III. It was sus-... 

In order to ameliorate the problem of solvolytic degradation, compound 109 was treated with TIPSOTf, to provide silyl ether 113 in 72% yield (Scheme 19). The acetonides were removed with PPTS in warm MeOH to provide a mixture of compounds in which the TBS groups were also partially removed. Exposure of this mixture to HF-pyridine successfully generated filipin III (114), in 39 % overall yield from 113. 

Norman A.W, Demel R.A., DeKmyff B., Geiuts Van Kessel W.S.M. Van Deenen L.L.M. (1972) Studies on the biological properties of polyene antibiotics comparison of the other polyenes widi filipin in their ability to interact specifically with sterol. Biochim BiophysActa, 290, 1-14. 

The inhibitory effects of in vitro sterol addition (21) showed large percentage kills (83% and 91% respectively). The in vitro addition of ergosterol (10.1 mM) to P brevis cell cultures with Filipin (1.5 mM) showed complete inhibition of the cytolytic effect. Ergosterol (10.1 mM) added to P. brevis cell cultures with cell extract showed a 10% reduction in cell mortality. The addition of ergosterol alone (control) showed no cytolytic effect at the experimental concentration (10.1 mM). 

Rochet and Lancelin50 reported revised 1H and 13C NMR assignments of the polyene antibiotic Filipin m (83). This macrolide which was isolated from Streptomyces filipinensis was reinvestigated in DMSO-dg solution using homonuclear and heteronuclear correlation spectroscopy. In addition to several corrections to previous 1H NMR... 

FIGURE 32. H NMR spectrum of filipin III, 3 mM in DMSO-dg, recorded at 400 MHz and 25 °C. The expanded region contains nine hydroxylic proton resonances that fully exchange with deuterium oxide and correspond to the nine hydroxyl groups of filipin III. No apodization functions were applied prior to the Fourier transformation. Reproduced by permission of John Wiley Sons from Reference 50... 

J. E. Schnitzer, P. Oh, E. Pinney, and J. Allard. Filipin-sensitive caveolae-mediated transport in endothelium reduced transcytosis, scavenger endocytosis, and capillary permeability of select macromolecules. J. Cell Biol. 127 1217-1232 (1994). 

P. A. Qrlandi and P. H. Fishman. Filipin-dependent inhibition of cholera toxin evidence for toxin internalization and activation through caveolae-like domains. J. Cell Biol. 141 905-915 (1998). 

The macrolide antibiotic filipin interacts with 3-p-hydroxysterols such as cholesterol in the plasma membrane to form filipin-sterol complexes (59). Subsequently, the filamentous caveolin-l-coat rapidly disassembles, which... 

Caveolae can mediate the delivery of CtxB that binds to GM1 ganglioside at the plasma membrane and is delivered to intracellular compartments. Cholera toxin, produced by Vibrio cholerae, consists of five identical subunits B and one A chain. In addition to labeled SV40 and caveolin-1-GFP, CtxB is one of the most commonly used caveolae markers. However, two groups reported that the toxin is internalized by either a clathrin-independent caveolae pathway or a clathrin-dependent uptake, bringing its selectivity/specificity into question (31,81,118). We controlled the suitability of this marker for COS-7 cells pretreated with CPZ, mpCD, and filipin and as expected, the uptake was not influenced by CPZ treatment but was strongly decreased by the latter two (data not shown). 

The following inhibiting agents are often used NEM, the PI-3 kinase inhibitors wortmannin and LY294002, filipin, nocodazole, colchicine, cyto-chalasin D, and the tyrosine kinase inhibitors herbimycin and genistein (all described above). 

Autofluorescence of cells often complicates the studies with fluorescence microscopy (especially the application of green fluorescent substances). There are different reasons for the occurrence of this phenomenon (157) (i) the fluorescent pigment lipofuscin, which settles with rising age in the cytoplasm of cells (ii) cell culture medium, which often contains phenol red that increases autofluorescence (iii) endogen substances such as flavin coenzymes [flavin-adenine dinucleotide (FDA), flavin mononucleotide (FMN) absorp-tion/emission 450/515nm], pyridine nucleotides [reduced nicotinamide adenine dinucleotide (NADH) absorption/emission 340/460nm] or porphyrine (iv) substances taken up by cells (as mentioned above filipin) and (v) preparation of the cells fixation with glutaraldehyde increases autofluorescence. 

Poiyket nolvenes amphotericins (Streptomyces spp. Actinom. Bact. from soil in Venezuela MI) filipin (Streptomyces fllipinensis from Philippine soil MI) natamycin (Streptomyces natalensis from S Africa soil candicidin from Streptomyces griseus MI). 

Filipin stock solution dissolve 12.5 mg filipin in 5 ml dimethylformamide. Store at -20 C. Filipin assay solution dissolve 20 pi filipin stock solution in 1 ml PBS. 

Let the plates come to room temperature and rinse the cover slips twice with PBS. Incubate the cover slips for 5 min with 2 ml Triton X-100 solution, remove, and rinse twice with 2 ml PBS. Remove the PBS and incubate the cover slips for 30 min with 2 ml of filipin assay solution. Remove the filipin assay solution and rinse the cover slips twice with PBS. Remove the PBS and let the cover slips dry at ambient temperature. Mount the cover slips on a microscope slide with one drop of DABCO solution... 

Lack of stimulation of cholesteryl ester formation by exogenously added LDL is a hallmark of NPC. The assay is based on the procedure described by Vanier et al. [58]. The test is only performed after a positive filipin staining test is obtained. 

Elias, P. M., Friend, D. S. and Goerke, J. 1979. Membrane sterol heterogeneity. Freeze-fracture detection with saponins and filipin. J. Histochem. Cytochem. 27, 1247-1260. 

Montesano, R., Ravazzola, M. and Orci, L. 1983. Filipin labelling of lipid droplets in lactating rat mammary gland. Cell Biol Intern Rep. 7, 194. 

Fig. 6.7 Filipin staining for free cholesterol in field CA3 of the hippocampus. A section from a normal rat, showing light staining for free cholesterol in pyramidal neurons (arrows). B section from a rat that had been injected with kainate 1 day earlier, showing intense filipin staining in affected hippocampal neurons (arrows). Scale = 250 // m. Reproduced with kind permission from Ong et al., 2003, Brain Pathology 13 250-262. Blackwell Publishing...

A combination of 2H-NMR, CD, fluorescence, and electron absorption techniques has been used to investigate the interaction of the pentene antibiotic filipin with dimyristoylphosphatidylcholine [168]. 

Rychnovsky and his group have recently developed new synthetic methods that lead to the total syntheses of the polyene macrolides roxaticin [2], roflamycoin [3], and filipin III [4]. The polyol chains of all three natural products were constructed by iterative, stereoselective alkylation of lithiated cyanohydrin acetonides and subsequent reductive decyanation, illustrated here by the synthesis of the polyol framework of filipin III (1) (Scheme I). The bifunctional cyanohydrin acetonide 2, prepared by ruthenium/BINAP catalyzed enantioselective hydrogenation of the corresponding ) -keto ester (BINAP = [ 1,1 -binaphthyl]-2,2 -diylbis(diphenylphosphane)), is deprotonated with LiNEt2 and alkylated with 2-benzyloxy-l-iodoethane. The alkylation product 3 is converted by a Finkelstein reaction into the iodide 4, which is used to alkylate a second... 

Scheme 1. Synthesis of the C(1)-C(I5) polyol fragment 8b of filipin 111 (1) according to Rychnovsky et al. Bn = henzyl, TBS = terf-hutyldimethylsilyl.

Kitajima, Y. et al. Freeze-fracture cytochemical study of membrane systems in human epidermis using filipin as a probe for cholesterol. Journal of Investigative Dermatology 84 149-153, 1985. 

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