Golgi-modifying properties of macfarlandin E and the synthesis and evaluation of its 2,7-dioxabicyclo[3.2.1]octan-3-one core

Abstract

Golgi-modifying properties of the spongian diterpene macfarlandin E (MacE) and a synthetic analog, t-Bu-MacE, containing its 2,7-dioxabicyclo[3.2.1]octan-3-one moiety are reported. Natural product screening efforts identified MacE as inducing a novel morphological change in Golgi structure defined by ribbon fragmentation with maintenance of the resulting Golgi fragments in the pericentriolar region. t-Bu-MacE, which possesses the substituted 2,7-dioxabicyclo[3.2.1]octan-3-one but contains a tert-butyl group in place of the hydroazulene subunit of MacE, was prepared by chemical synthesis. Examination of the Golgi-modifying properties of MacE, t-Bu-MacE, and several related structures revealed that the entire oxygen-rich bridged-bicyclic fragment is required for induction of this unique Golgi organization phenotype. Further characterization of MacE-induced Golgi modification showed that protein secretion is inhibited, with no effect on the actin or microtubule cytoskeleton being observed. The conversion of t-Bu-MacE and a structurally related des-acetoxy congener to substituted pyrroles in the presence of primary amines in protic solvent at ambient temperatures suggests that covalent modification might be involved in the Golgi-altering activity of MacE.

Fig. 1.

Known Golgi-modifying agents.

Fig. 2.

Structures of macfarlandin E, aplyviolene, and shahamin K.

Scheme 1

Analysis of 7 and 8.

Fig. 3.

MacE has a unique Golgi-modifying activity. NRK cells on coverslips were treated with DMSO, BFA (3 μg/mL), or MacE (20 μg/mL) for 60 min at 37 °C, fixed, and processed for immunofluorescence analysis with an antibody to the Golgi protein, giantin, and the DNA dye Hoechst 33342. The scale bar corresponds to 10 μM.

Scheme 2

Synthesis of 20.

Scheme 3

Synthesis of 7 and 8.

Fig. 4.

Characterization of the Golgi-modifying activity of MacE and t-Bu-MacE (A) NRK cells on coverslips were treated with DMSO, MacE (20 μg/mL), t-Bu-MacE (40 μg/mL), fixed, and analyzed by immunofluorescence with an antibody to giantin. Magnifications of the boxed areas are shown in the insets. (B) NRK cells treated for 60 min with DMSO, 20 μg/mL MacE, or 40 μg/mL t-Bu-MacE were analyzed by electron microscopy. Arrows indicate the organization of Golgi membranes under these conditions. The scale bar for DMSO and t-Bu-MacE-treated cells corresponds to 300 nm, for MacE-treated cells to 510 nm. (C) Morphometric analysis was performed to determine the average length of Golgi stacks for each experimental condition. The values for MacE or t-Bu-MacE-treated cells are statistically distinct from the DMSO control (t test: p < 0.01). (D) Cells transfected with VSV-G^tsO45-GFP were incubated for 60 min with DMSO BFA (5 μg/mL) and indicated concentrations of MacE and t-Bu-MacE prior to fixation or incubation for 60 min to 32 °C. The fraction of cells with VSV-G^tsO45-GFP at the plasma membrane was determined for > 100 cells per data point (see Fig. S4 for representative images, n = 4). (E) For BFA (5 μg/mL), MacE (20 μg/mL), t-Bu-MacE (40 μg/mL), the fraction of cells in which VSV-G^tsO45-GFP had reached the Golgi was determined in > 100 cells (n = 4).

Scheme 4

In situ observation of 26 and synthesis of 27 and 28.