Synthesis of derivatives of potent antitumor bistramides D and A leading to the first crystal structure of natural bistramide D

We report a crystalline derivative of bistramide D synthesized from natural bistramide A, and its structure was determined by X-ray analysis.     

Spiroacetal Formation through Telescoped Cycloaddition and Carbon–Hydrogen Bond Functionalization: Total Synthesis of Bistramide A

Spiroacetals can be formed through a one‐pot sequence of a hetero‐Diels–Alder reaction, an oxidative carbon–hydrogen bond cleavage, and an acid treatment. This convergent approach expedites access to a complex molecular subunit which is present in numerous biologically active structures. The utility of the protocol is demonstrated through its application to a brief synthesis of the actin‐binding cytotoxin bistramide A.     

Synthesis of bistramide A

We have developed an efficient and highly stereocontrolled synthesis of bistramide A, a selective activator of protein kinase C isotype delta. Our synthetic strategy featured a novel bidirectional approach for spiroketal construction based on the ring-opening/cross-metathesis sequence employing a highly strained cyclopropenone acetal. The synthesis afforded the final target with the longest linear sequence of 15 steps and provided unambiguous structural determination of bistramide A, including assignment of the previously unknown C(37) stereochemistry.     

Total synthesis of bistramide A

An asymmetric synthesis of the marine metabolite bistramide A is reported. The synthesis relies on the utility of three different organosilane reagents to construct all principle fragments and 8 of the 11 stereogenic centers of the natural product. [structure: see text].     

Synthesis of a platform to access bistramides and their analogues

The platform C14-C40, which can be used to prepare bistramide C and 39-oxobistramide K, was synthesized in 19 steps with an overall yield of 6.2%. Furthermore, the chemoselective reduction of the ketone at C-39 was performed giving an easy access to bistramides A, B, D, K, and L. Finally, the versatility of the synthesis of the C14-C40 fragment can allow the preparation of a large variety of stereoisomers to produce bistramide analogues.     

Stereoselective total synthesis of bistramide A

A highly stereoselective and convergent total synthesis of bistramide A is described. The salient feature of this synthesis is the construction of the spiroketal subunit by hydrolysis of dialkylated tosylmethyl isocyanide derivative derived via alkylation of TosMIC with suitably substituted halohydrin derivatives.     

Model studies towards the bistramide D tetrahydropyran

The synthesis of a model of the bistramide D tetrahydropyran ring is achieved using a selective cross-metathesis and an intramolecular Michael addition under kinetic control.     

Total synthesis of a stereoisomer of bistramide C and assignment of configuration of the natural product

After the isolation of the bioactive polyether bistramide C from the marine ascidian Lissoclinum bistratum in 1988, NMR spectroscopic investigations over the next 12 years reduced the total number of possible stereoisomers of this compound from 1024 to 32. Based on the preparation of segments of the natural product as well as the total synthesis of a randomly selected stereoisomer of bistramide C, the stereochemical puzzle could be further simplified to eight possible stereoisomers. A convergent three-segment coupling strategy, the use of a common, D-glucose-derived intermediate for the preparation of pyran rings in two segments, a stereoselective photo-spiroketalization, and the use of azides to minimize protective group manipulations before segment couplings are highlights of the synthetic approach. The total synthesis also provided the key segments for a chiroptical analysis according to van't Hoff's principle of optical superposition, which was crucial for the assignment of a sole relative and absolute configuration of the natural product. Bistramide C represents therefore the first member of this class of structurally unusual marine polyethers whose configuration is known as a result of the combined use of synthetic and chiroptical tools.     

Nanoscale topography mediates the adhesion of f-actin

Using a controllable nanoengineered surface that alters the dynamics of filamentous actin (F-actin) adhesion, we studied the tunability of biomolecular surface attachment. By grafting aminated nanoparticles, NPs, with diameters ranging from 12 to 85 nm to a random copolymer film, precise control over surface roughness parameters is realized. The ability to selectively generate monodisperse or polydisperse features of varying size and areal density leads to immobilized, side-on wobbly, or end-on F-actin binding as characterized by total internal reflection fluorescence (TIRF) microscopy. The interaction between the surface and actin is explained by a worm-like chain model that balances the bending energy penalty required for actin to conform to topographical features with the electrostatic attraction engineered into the surface. A Myosin V motility assay demonstrates that electrostatically immobilized actin retains its ability to direct myosin motion, indicating that nanoengineered surfaces are attractive candidates for biomolecular device fabrication.     

Structural basis of swinholide A binding to actin

Marine toxins targeting the actin cytoskeleton represent a new and promising class of anti-cancer compounds. Here we present a 2.0 A resolution structure of swinholide A, a marine macrolide, bound to two actin molecules. The structure demonstrates that the actin dimer in the complex does not represent a physiologically relevant entity, for the two actin molecules do not interact with each other. The swinholide A actin binding site is the same as that targeted by toxins of the trisoxazole family and numerous actin binding proteins, highlighting the importance of this site in actin polymerization. The observed structure reveals the mechanism of action of swinholide A and provides a structural framework about which to design new agents directed at the cytoskeleton.     

Enantioselective total synthesis of bistramide A

The enantioselective synthesis of bistramide A has been achieved with a longest linear sequence of 18 steps. The synthetic strategy involves the use of a distereoselective glycolate alkylation, an aldol addition of a chlorotitanium enolate of N-acylthiazolidinthione, and a Sharpless asymmetric epoxidation to synthesize the three key fragments.     

Amphidinolide h, a potent cytotoxic macrolide, covalently binds on actin subdomain 4 and stabilizes actin filament

The actin-targeting toxins have not only proven to be invaluable tools in studies of actin cytoskeleton structure and function but they also served as a foundation for a new class of anticancer drugs. Here, we describe that amphidinolide H (AmpH) targets actin cytoskeleton. AmpH induced multinucleated cells by disrupting actin organization in the cells, and the hyperpolymerization of purified actin into filaments of apparently normal morphology in vitro. AmpH covalently binds on actin, and the AmpH binding site is determined as Tyr200 of actin subdomain 4 by mass spectrometry and halo assay using the yeast harboring site-directed mutagenized actins. Time-lapse analyses showed that AmpH stimulated the formation of small actin-patches, followed by F-actin rearrangement into aggregates via the retraction of actin fibers. These results indicate that AmpH is a novel actin inhibitor that covalently binds on actin.     

Stereoselective synthesis of the C1-C13 fragment of bistramide A

The C1-C13 fragment of bistramide A was prepared from 5-hexenoic acid in 15 linear steps and in 16% overall yield. The core 2,6-trans-tetrahydropyran ring was obtained via a kinetically controlled oxa-Michael cyclization from the corresponding chiral α,β-unsaturated hydroxyester. This precursor was prepared by using a diastereoselective alkylation reaction using Davies Superquat auxiliary and a diastereoselective Roush’s allylboration as key steps.     

Synthesis and [4 + 2]-annulation of enantioenriched (z)-crotylsilanes: preparation of the C1-C13 fragment of bistramide A

[reaction: see text]. New chiral crotylsilanes that bear a (Z)-olefin geometry were synthesized in high enantiopurity. The reagents participate in [4 + 2]-annulations with aldehydes to give stereochemically complementary pyrans (relative to (E)-crotylsilanes) bearing 2,6-cis-5,6-cis and 2,6-trans-5,6-cis relationships of peripheral functionalities. A stereoselective synthesis of the C1-C13 fragment of bistramide A is also described highlighting this annulation strategy.     

Mechanism of actin polymerization by myosin subfragment-1 probed by dynamic light scattering

Monomeric actin (G-actin) polymerizes spontaneously into helical filaments in the presence of inorganic salts. The slowest, rate-limiting step of the polymerization process is formation of actin trimers, the smallest oligomers that serve as nuclei for fast filament growth (filament elongation) by monomer addition at the filament ends. In low ionic-strength solutions, actin can be polymerized by myosin subfragment-1 (S1). In early works it has been suggested that G-actin-S1 1:1 complexes (GS) assemble into filaments according to the nucleation-filament elongation scheme. Subsequent studies indicated that one S1 molecule can bind two actin monomers, and that oligomerization of the initial complexes is a fast reaction. This has led to suggest an alternative mechanism, with a ternary G(2)S complex and its oligomers being predominant intermediates of S1-induced assembly of G-actin into filaments. We used dynamic light scattering to analyze the initial steps of S1-induced polymerization of actin. Our results suggest formation of GS complexes and their oligomers in the presence of S1 equimolar to or in excess over actin. We confirm formation of G(2)S complexes as intermediates of S1-induced polymerization in the presence of actin in excess over S1.     

A Catalyst Designed for the Enantioselective Construction of Methyl‐ and Alkyl‐Substituted Tertiary Stereocenters

Tertiary methyl‐substituted stereocenters are present in numerous biologically active natural products. Reported herein is a catalytic enantioselective method for accessing these chiral building blocks using the Mukaiyama–Michael reaction between silyl ketene thioacetals and acrolein. To enable remote enantioface control on the nucleophile, a new iminium catalyst, optimized by three‐parameter tuning and by identifying substituent effects on enantioselectivity, was designed. The catalytic process allows rapid access to chiral thioesters, amides, aldehydes, and ketones bearing an α‐methyl stereocenter with excellent enantioselectivities, and allowed rapid access to the C4–C13 segment of (?)‐bistramide A. DFT calculations rationalized the observed sense and level of enantioselectivity.     

Hierarchical self-assembly of actin bundle networks: gels with surface protein skin layers

The networklike structure of actin bundles formed with the cross-linking protein alpha-actinin has been investigated via x-ray scattering and confocal fluorescence microscopy over a wide range of alpha-actinin/F-actin ratios. We describe the hierarchical structure of bundle gels formed at high ratios. Isotropic actin bundle gels form via cluster-cluster aggregation in the diffusion-limited aggregation regime at high alpha-actinin/actin ratios. This process is clearly observed by confocal fluorescence microscopy. Polylysine is investigated as an alternative bundling agent in the high-ratio regime and the effects of F-actin length are also discussed. One particularly fascinating aspect of this system is the presence of a structured skin layer at the gel/water interface. Confocal microscopy has elucidated the full three-dimensional structure of this layer and revealed several interesting morphologies. The protein skin layer is a micron-scale structure composed of a directed network of bundles and exhibits flat, crumpled, and tubelike shapes. We show that crumpling of the skin layer results from stresses due to the underlying gel. These biologically based geometric structures may detach from the gel, demonstrating potential for the generation of biological scaffolds with defined shapes for applications in cell encapsulation and tissue engineering. We demonstrate manipulation of the skin layer, producing hemispherical structures in solution.     

Structure of small actin-containing liposomes probed by atomic force microscopy: effect of actin concentration & liposome size

Actin-containing liposomes were prepared via extrusion through 400 and 600 nm pore diameter membranes at different monomeric actin concentrations in low ionic strength buffer (G-buffer). After subjecting the liposome dispersions to high ionic strength polymerization buffer (F-buffer), topological changes in liposome structure were studied using atomic force microscopy (AFM). Paired dumbbell, horseshoelike, and disklike assemblies were observed for actin-containing liposomes extruded through 400 and 600 nm pore diameter membranes. The topology of actin-containing liposomes was found to be highly dependent on both liposome size and actin concentration. At 1 mg/mL actin, the actin-containing liposomes transformed into a disklike shape, whereas, at 5 mg/mL actin, the actin-containing liposomes retained a spherical shape. On the basis of these observations, we hypothesize that actin could either polymerize on the surface of the inner leaflet of the liposome membrane or polymerize in the aqueous core of the liposome. We explain the associated shape changes induced in actin-containing liposomes on the basis of the hypothesized mechanism of actin polymerization inside the liposomes. At higher actin concentrations (5 mg/mL), we observed membrane-induced actin self-assembly in G-buffer, which implies that G-actin is able to interact directly with lipid bilayers at sufficiently high concentrations.     

Interactions of histatin-3 and histatin-5 with actin

Background

Histatins are histidine rich polypeptides produced in the parotid and submandibular gland and secreted into the saliva. Histatin-3 and ?5 are the most important polycationic histatins. They possess antimicrobial activity against fungi such as Candida albicans. Histatin-5 has a higher antifungal activity than histatin-3 while histatin-3 is mostly involved in wound healing in the oral cavity. We found that these histatins, like other polycationic peptides and proteins, such as LL-37, lysozyme and histones, interact with extracellular actin.

Results

Histatin-3 and ?5 polymerize globular actin (G-actin) to filamentous actin (F-actin) and bundle F-actin filaments. Both actin polymerization and bundling by histatins is pH sensitive due to the high histidine content of histatins. In spite of the equal number of net positive charges and histidine residues in histatin-3 and ?5, less histatin-3 is needed than histatin-5 for polymerization and bundling of actin. The efficiency of actin polymerization and bundling by histatins greatly increases with decreasing pH. Histatin-3 and ?5 induced actin bundles are dissociated by 100 and 50 mM NaCl, respectively. The relatively low NaCl concentration required to dissociate histatin-induced bundles implies that the actin-histatin filaments bind to each other mainly by electrostatic forces. The binding of histatin-3 to F-actin is stronger than that of histatin-5 showing that hydrophobic forces have also some role in histatin-3- actin interaction. Histatins affect the fluorescence of probes attached to the D-loop of G-actin indicating histatin induced changes in actin structure. Transglutaminase cross-links histatins to actin. Competition and limited proteolysis experiments indicate that the main histatin cross-linking site on actin is glutamine-49 on the D-loop of actin.

Conclusions

Both histatin-3 and ?5 interacts with actin, however, histatin 3 binds stronger to actin and affects actin structure at lower concentration than histatin-5 due to the extra 8 amino acid sequence at the C-terminus of histatin-3. Extracellular actin might regulate histatin activity in the oral cavity, which should be the subject of further investigation.

     

The effect of pyrene labelling on the thermal stability of actin filaments

The ability of actin to form filaments is fundamental to its biological function and often characterised by various methods in vitro. One of the most frequently used methods capitalises on the observation that the fluorescence emission of a pyrene label on the Cys-374 residue of actin is enhanced by a factor of ∼20 during polymerisation. This method inherently involves the chemical modification of actin monomers with pyrene. It was reported earlier that the pyrene labelling of actin monomers has only small effect on the polymerisation and depolymerisation rates of actin, indicating that the method is suitable to characterise the effect of actin-binding proteins or peptides on the polymerisation kinetics.In our present work we tested the effect of the pyrene labelling on the thermal denaturation of actin filaments by using the method of differential scanning calorimetry (DSC). By recording the heat denaturation profiles of unlabelled and pyrene labelled actin filaments we observed that pyrene labelling shifted the melting point (T_m) of actin filaments from 66 to 68 °C. A similar effect was detected in the presence of equimolar concentration of phalloidin where the T_m shifted from 79 to 82 °C. We concluded that the observed pyrene labelling induced differences of the thermal denaturation of actin filaments were small. The DSC results, therefore, confirmed that the methods based on the measurements of pyrene intensity during actin polymerisation are suitable to characterise the polymerisation kinetics of actin under in vitro conditions.