Marc C. A. Stuart

Mechanosensitive Self-Replication Driven by Self-Organization

At Sixes and Sevens Molecular synthesis and macroscopic aggregation have often been regarded as entirely separate processes. From the researchers standpoint, once reagents have been mixed, synthesis is largely passive, whereas processes such as crystallization can be more actively manipulated. Carnall et al. (p. 1502) characterized an unusual system in which the formation of aggregated cyclic macromolecules (macrocycles) from small peptide-based building blocks was governed by intimately interdependent factors ranging from the scale of covalent bond formation all the way to micron scale fiber growth. As the macrocycles stacked against one another to form the fibers, they remained loosely bonded enough internally to incorporate or expel individual building blocks. Varying the type of mechanical force applied to the growing fibers (either through shaking or stirring the solution), alternately favored formation of either 6-membered or 7-membered covalent macrocycles. The type of mechanical agitation applied to a solution influences which of two molecular products dominate. Self-replicating molecules are likely to have played an important role in the origin of life, and a small number of fully synthetic self-replicators have already been described. Yet it remains an open question which factors most effectively bias the replication toward the far-from-equilibrium distributions characterizing even simple organisms. We report here two self-replicating peptide-derived macrocycles that emerge from a small dynamic combinatorial library and compete for a common feedstock. Replication is driven by nanostructure formation, resulting from the assembly of the peptides into fibers held together by β sheets. Which of the two replicators becomes dominant is influenced by whether the sample is shaken or stirred. These results establish that mechanical forces can act as a selection pressure in the competition between replicators and can determine the outcome of a covalent synthesis.

On the osmotic signal and osmosensing mechanism of an ABC transport system for glycine betaine

The osmosensing mechanism of the ATP‐binding cassette (ABC) transporter OpuA of Lactococcus lactis has been elucidated for the protein reconstituted in liposomes. Activation of OpuA by osmotic upshift was instantaneous and reversible and followed changes in volume and membrane structure of the proteoliposomes. Osmotic activation of OpuA was dependent on the fraction of anionic lipids present in the lipid bilayer. Also, cationic and anionic lipophilic amphiphiles shifted the activation profile in a manner indicative of an osmosensing mechanism, in which electrostatic interactions between lipid headgroups and the OpuA protein play a major role. Further support for this notion came from experiments in which ATP‐driven uptake and substrate‐dependent ATP hydrolysis were measured with varying concentrations of osmolytes at the cytoplasmic face of the protein. Under iso‐osmotic conditions, the transporter could be activated by high concentrations of ionic osmolytes, whereas neutral ones had no effect, demonstrating that intracellular ionic strength, rather than a specific signaling molecule or water activity, signals osmotic stress to the transporter. The data indicate that OpuA is under the control of a mechanism in which the membrane and ionic strength act in concert to signal osmotic changes.

Shape and release control of a peptide decorated vesicle through pH sensitive orthogonal supramolecular interactions.

A pH sensitive carrier is obtained by coating a cyclodextrin vesicle with an adamantane-terminated octapeptide through the formation of an inclusion complex. Upon lowering the pH from 7.4 to 5.0, the formation of peptide beta-sheets on the vesicle surface induces a transition of the bilayer from a sphere to a fiber. This transition is fully reversible and repeatable. The vesicles release their cargo upon fiber formation.

Sugar-Decorated Sugar Vesicles: Lectin-Carbohydrate Recognition at the Surface of Cyclodextrin Vesicles

An artificial glycocalix self-assembles when unilamellar bilayer vesicles of amphiphilic beta-cyclodextrins are decorated with maltose and lactose by host-guest interactions. To this end, maltose and lactose were conjugated with adamantane through a tetra(ethyleneglycol) spacer. Both carbohydrate-adamantane conjugates strongly bind to beta-cyclodextrin (K(a) approximately 4 x 10(4) M(-1)). The maltose-decorated vesicles readily agglutinate (aggregate) in the presence of the lectin concanavalin A, whereas the lactose-decorated vesicles agglutinate in the presence of peanut agglutinin. The orthogonal multivalent interaction in the ternary system of host vesicles, guest carbohydrates, and lectins was investigated by using isothermal titration calorimetry, dynamic light scattering, UV/Vis spectroscopy, and cryogenic transmission electron microscopy. It was shown that agglutination is reversible, and the noncovalent interaction can be suppressed and eliminated by the addition of competitive inhibitors, such as D-glucose or beta-cyclodextrin. Also, it was shown that agglutination depends on the surface coverage of carbohydrates on the vesicles.

Exciton Spectra and the Microscopic Structure of Self-Assembled Porphyrin Nanotubes

The optical properties of tubular aggregates formed by self-assembly of zwitterionic meso-tetra(4-sulfonatophenyl)porphyrin (TPPS4) molecules are studied through a combination of experimental and theoretical techniques. The interest in these systems, with diameters of 18 nm and lengths extending up to micrometers, derives from their strong interaction with light and their similarity to natural light-harvesting systems for photosynthesis. The absorption and linear dichroism spectra are obtained in the spectral region from 300 to 750 nm, which includes the exciton bands deriving from the molecular B (Soret) as well as the Q transitions. We demonstrate that a Frenkel exciton model which takes into account the four dominant molecular excited states (Bx, By, Qx, and Qy) provides a good global fit to the experimental spectra. From comparison between theory and experiment, we propose a detailed molecular structure within the nanotube.

Synthesis of Pyridinium Amphiphiles Used for Transfection and Some Characteristics of Amphiphile/DNA Complex Formation

Pyridinium amphiphiles have found practical use for the delivery of DNA into cells. Starting from 4-methylpyridine, a general synthesis has been devised for the production of pyridinium amphiphiles which allows variation in both the hydrophobic part and in the headgroup area of the compounds. By means of differential scanning microcalorimetry, zeta potential, particle size measurements and cryo electron microscopy, some characteristics of the pyridinium amphiphile/ DNA complexes have been determined.

Hydrogel Formation upon Photoinduced Covalent Capture of Macrocycle Stacks from Dynamic Combinatorial Libraries

Dynamic combinatorial chemistry was originally conceived as a method for developing synthetic receptors and ligands for biomolecules. Dynamic combinatorial libraries (DCLs) are produced by linking building blocks together using a reversible reaction, resulting in a thermodynamically controlled product distribution. Addition of a guest or a biomolecule shifts the distribution to those library members that bind best to the external target; a process referred to as templating. Recently, the first examples have appeared of DCLs in which molecular recognition does not involve an externally added template, but takes place between library members. Such self-templated DCLs constitute a promising approach for the development of new self-assembling materials. Self-assembly provides the driving force to shift the equilibrium in favor of the very molecules that self-assemble, so that these materials are in effect self-synthesizing. We now report that a selfassembled material produced by dynamic combinatorial chemistry can be further stabilized by rearranging the dynamic covalent disulfide bonds that were underlying the dynamic combinatorial process. We show how photoinitiated disulfide exchange converts fibrous stacks of macrocycles into polymeric products, enhancing the stability of the fibers and causing gelation of the aqueous solution. We recently reported how hexameric disulfide macrocycles emerge upon shaking a DCL made from dithiol 1 (Scheme 1). This building block is equipped with a short peptide sequence, predisposed to b-sheet formation by alternating hydrophobic and hydrophilic amino acid residues. While the peptide is too short to self-assemble on its own, the DCL made upon oxidizing dithiol 1 in aqueous solution contains a number of macrocycles of different ring sizes that display a different number of peptides. We reasoned that selfassembly becomes feasible for a critical size of the macrocycle that displays a sufficient number of peptides. Indeed, for a DCL made from 1 that is agitated by shaking, self-assembly of the hexameric macrocycles (16) occurs, resulting in the formation of fibers and shifting the product distribution in favor of the cyclic hexamer. The solution remains free flowing as the fibers are fragile and break when subjected to moderate shear forces. We have discovered that photoirradiation (three days, using an 8 W UV lamp, 365 nm) of a nonagitated solution containing hexamer fibers ([16] is around 0.6 mm) results in the formation of a hydrogel (Figure 1a). Oscillatory rheology experiments showed that a relatively rigid gel is formed with a ratio between storage (G’) and loss (G’’) moduli of 12 at low oscillatory frequencies (see Figure S1 in the Supporting Information). Photoirradiation of disulfides can induce their homolytic cleavage giving thiol radicals that can attack nearby disulfide bonds, resulting in disulfide exchange. This underutilized radical-mediated exchange mechanism is different from the ionic (thiolate-anion-mediated) mechanism that is typically used in dynamic covalent disulfide chemistry. We believe that this photoinduced disulfide exchange causes a Scheme 1. Shaking a dynamic combinatorial library made from dithiol building block 1 in aqueous borate buffer (50 mm, pH 8.1) gives rise to stacks of disulfide macrocycles 16, which are covalently captured upon photoirradiation (365 nm) to produce polymers/oligomers of 1.

Photoresponsive Capture and Release of Lectins in Multilamellar Complexes

The development of triggered release systems for delivery of peptides and proteins is critical to the success of biological drug therapies. In this paper we describe a dynamic supramolecular system able to capture and release proteins in response to light. The ternary system self-assembles in a dilute aqueous solution of three components: vesicles of amphiphilic cyclodextrin host, noncovalent cross-linkers with an azobenzene and a carbohydrate moiety, and lectins. The cross-linkers form inclusion complexes with the host vesicles, provided the azobenzene is in the trans state. The formation of a ternary complex with lectins requires a high density of cross-linkers on the surface of vesicles. The key innovation in this system is a photoinduced switch from a multivalent, high-affinity state that captures protein to a monovalent, low-affinity state that releases protein. By using isothermal titration calorimetry, dynamic light scattering, UV/vis spectroscopy, and cryogenic transmission electron microscopy, we demonstrate that photoinduced capture and release of lectins in dense multilamellar complexes is highly efficient, highly selective, and fully reversible.

Light-induced disassembly of self-assembled vesicle-capped nanotubes observed in real time

Molecular self-assembly is the basis for the formation of numerous artificial nanostructures. The self-organization of peptides, amphiphilic molecules composed of fused benzene rings and other functional molecules into nanotubes is of particular interest. However, the design of dynamic, complex self-organized systems that are responsive to external stimuli remains a significant challenge. Here, we report self-assembled, vesicle-capped nanotubes that can be selectively disassembled by irradiation. The walls of the nanotubes are 3-nm-thick bilayers and are made from amphiphilic molecules with two hydrophobic legs that interdigitate when the molecules self-assemble into bilayers. In the presence of phospholipids, a phase separation between the phospholipids and the amphiphilic molecules creates nanotubes, which are end-capped by vesicles that can be chemically altered or removed and reattached without affecting the nanotubes. The presence of a photoswitchable and fluorescent core in the amphiphilic molecules allows fast and highly controlled disassembly of the nanotubes on irradiation, and distinct disassembly processes can be observed in real time using fluorescence microscopy.

Molecular Recognition of Vesicles: Host–Guest Interactions Combined with Specific Dimerization of Zwitterions

The aggregation of beta-cyclodextrin vesicles can be induced by an adamantyl-substituted zwitterionic guanidiniocarbonylpyrrole carboxylate guest molecule (1). Upon addition of 1 to the cyclodextrin vesicles at neutral pH, the vesicles aggregate (but do not fuse), as shown by using UV/Vis and fluorescence spectroscopy, dynamic light scattering, zeta-potential measurements, cryogenic transmission electron microscopy, and atomic force microscopy. Aggregation of the vesicles is induced by a twofold supramolecular interaction. First, the adamantyl group of 1 forms an inclusion complex with beta-cyclodextrin. Second, at neutral pH the guanidiniocarbonylpyrrole carboxylate zwitterion dimerizes through the formation of hydrogen-bonded ion pairs. Because the dimerization of 1 depends on the zwitterionic protonation state of 1, the aggregation of the cyclodextrin vesicles is also pH dependent; the cyclodextrin vesicles do not interact at pH 5 or 9, at which 1 is either cationic or anionic and, therefore, not self-complementary. These observations are consistent with molecular recognition of the vesicles through a combination of two different supramolecular interactions, that is, host-guest inclusion and dimerization of zwitterions, at the bilayer membrane surface.