Filipin III

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. 

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... 

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... 

In the total synthesis of filipin III (141) [95], macrocyclization under K2CO3/ 18-crown-6 conditions proceeded (Fig. 4), although the cyclization was unsuccessful under LiCl/DBU conditions, which were useful for roflamycoin (140) [94] and roxaticin [93]. The total synthesis of ulapualide A (142) was accomplished via three Wittig-type couplings [101]. 

The nature of the interaction between polyene antibiotics and membrane sterols has also been investigated using UV, fluorescence and circular dichroism spectral studies [155,159,187,188]. The rate and degree of filipin III binding to cholesterol-containing liposomes depended upon the absolute sterol concentration and the mole percent in the bilayer. Binding constants for filipin III and liposomes were also estimated. 

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. 

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. 

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... 

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