Daishi Fujita

Giant hollow MnL2n spherical complexes: structure, functionalisation and applications

Drawing inspiration from the self-assembly of hollow spherical virus capsids and protein cages found in nature, a family of roughly spherical coordination polyhedra with general formula MnL2n was designed and several members of the series have been synthesised. These spherical complexes are self-assembled upon reaction of bent bis(pyridine) ligands with Pd(2+) ions. The introduction of functional side chains into the ligands is straightforward, making the synthesis of both exo- and endohedrally functionalised spherical complexes possible. Accumulation of a high density of functional groups at the periphery of the spherical framework results in an enhancement of the weak interactions used in biomolecular recognition processes and the strong and selective interaction of the complex with a variety of substrates. Discrete and well-defined environments are generated within the spherical framework by functionalisation of the interior of the complex. These environments can be used for the selective encapsulation of guest molecules, including species as diverse as simple metal ions, fluoroalkanes and fullerenes. The well-defined cavity of the spherical complexes can also be exploited for the synthesis of precisely size-controlled nanoparticles and polymers. Most recently, a protein was successfully enclosed within a hollow self-assembled spherical complex, with a long-term view towards the control of protein functions for the development of new applications.

Protein encapsulation within synthetic molecular hosts

Protein encapsulation has long attracted many chemists and biologists because of its potential to control the structure and functions of proteins, but has been a daunting challenge because of their incommensurably larger size compared with common synthetic hosts. Here we report the encapsulation of a small protein, ubiquitin, within giant coordination cages. The protein was attached to one bidentate ligand and, upon addition of Pd(II) ions (M) and additional ligands (L), M(12)L(24) coordination nanocages self-assembled around the protein. Because of the well-defined host framework, the protein-encapsulated structure could be analysed by NMR spectroscopy, ultracentrifugation and X-ray crystallography.

Self-assembly of tetravalent Goldberg polyhedra from 144 small components

Rational control of the self-assembly of large structures is one of the key challenges in chemistry, and is believed to become increasingly difficult and ultimately impossible as the number of components involved increases. So far, it has not been possible to design a self-assembled discrete molecule made up of more than 100 components. Such molecules—for example, spherical virus capsids—are prevalent in nature, which suggests that the difficulty in designing these very large self-assembled molecules is due to a lack of understanding of the underlying design principles. For example, the targeted assembly of a series of large spherical structures containing up to 30 palladium ions coordinated by up to 60 bent organic ligands was achieved by considering their topologies. Here we report the self-assembly of a spherical structure that also contains 30 palladium ions and 60 bent ligands, but belongs to a shape family that has not previously been observed experimentally. The new structure consists of a combination of 8 triangles and 24 squares, and has the symmetry of a tetravalent Goldberg polyhedron. Platonic and Archimedean solids have previously been prepared through self-assembly, as have trivalent Goldberg polyhedra, which occur naturally in the form of virus capsids and fullerenes. But tetravalent Goldberg polyhedra have not previously been reported at the molecular level, although their topologies have been predicted using graph theory. We use graph theory to predict the self-assembly of even larger tetravalent Goldberg polyhedra, which should be more stable, enabling another member of this polyhedron family to be assembled from 144 components: 48 palladium ions and 96 bent ligands.

Self-assembly of Pt(II) spherical complexes via temporary labilization of the metal-ligand association in 2,2,2-trifluoroethanol.

Thermodynamically controlled platinum(II) spherical complexes were synthesized via temporary labilization of inert Pt(II)-pyridine bonds by the addition of the strong hydrogen-bond donor 2,2,2-trifluoroethanol (TFE), which weakens the pyridine-metal interaction. The platinum complex was stably trapped after removal of TFE and showed higher acid durability than its palladium counterpart.

Geometrically Restricted Intermediates in the Self‐Assembly of an M12L24 Cuboctahedral Complex

The self-assembly of a cuboctahedral M12 L24 complex is traced by time-dependent NMR spectroscopy and mass spectrometry. The metastable intermediate structures that exist during the self-assembly process are not a chaotic mixture of numerous species, but instead are geometrically restricted. Short-lived M8 L16 (D4d ) and relatively long-lived M9 L18 (D3h ) are fully characterized as major intermediates. Employing a ligand with a smaller bend angle (112°) allows these two species to be kinetically trapped and more clearly observed by NMR spectroscopy. X-ray crystallography shows that M9 L18 has the framework topology predicted by geometric discussion.

Arrayed lipid bilayer chambers allow single-molecule analysis of membrane transporter activity

Nano- to micron-size reaction chamber arrays (femtolitre chamber arrays) have facilitated the development of sensitive and quantitative biological assays, such as single-molecule enzymatic assays, digital PCR and digital ELISA. However, the versatility of femtolitre chamber arrays is limited to reactions that occur in aqueous solutions. Here we report an arrayed lipid bilayer chamber system (ALBiC) that contains sub-million femtolitre chambers, each sealed with a stable 4-μm-diameter lipid bilayer membrane. When reconstituted with a limiting amount of the membrane transporter proteins α-hemolysin or F0F1-ATP synthase, the chambers within the ALBiC exhibit stochastic and quantized transporting activities. This demonstrates that the single-molecule analysis of passive and active membrane transport is achievable with the ALBiC system. This new platform broadens the versatility of femtolitre chamber arrays and paves the way for novel applications aimed at furthering our mechanistic understanding of membrane proteins’ function.

Permeable Self-Assembled Molecular Containers for Catalyst Isolation Enabling Two-Step Cascade Reactions

Establishment of a general one-pot cascade reaction protocol would dramatically reduce the effort of multistep organic synthesis. We demonstrate that the unique structure of M12L24 self-assembled complexes gives them the potential to serve as catalyst carriers for enabling continuous chemical transformations. A stereoselective cascade reaction (allylic oxidation followed by Diels-Alder cyclization) with two intrinsically incompatible catalysts was demonstrated. Our system is advantageous in terms of availability, scalability, and predictability.

Granulocyte macrophage colony-stimulating factor is required for aortic dissection/intramural haematoma

Aortic dissection and intramural haematoma comprise an aortopathy involving separation of the aortic wall. Underlying mechanisms of the condition remain unclear. Here we show that granulocyte macrophage colony-stimulating factor (GM-CSF) is a triggering molecule for this condition. Transcription factor Krüppel-like factor 6 (KLF6)-myeloid-specific conditional deficient mice exhibit this aortic phenotype when subjected to aortic inflammation. Mechanistically, KLF6 downregulates expression and secretion of GM-CSF. Administration of neutralizing antibody against GM-CSF prevents the condition in these mice. Conversely, administration of GM-CSF in combination with aortic inflammation to wild-type mice is sufficient to induce the phenotype, suggesting the general nature of effects. Moreover, patients with this condition show highly increased circulating levels of GM-CSF, which is also locally expressed in the dissected aorta. GM-CSF is therefore a key regulatory molecule causative of this aortopathy, and modulation of this cytokine might be an exploitable treatment strategy for the condition.

Adipose tissue-derived stem cells as a therapeutic tool for cardiovascular disease

Adipose tissue-derived stem cells (ADSCs) are adult stem cells that can be easily harvested from subcutaneous adipose tissue. Many studies have demonstrated that ADSCs differentiate into vascular endothelial cells (VECs), vascular smooth muscle cells (VSMCs), and cardiomyocytes in vitro and in vivo. However, ADSCs may fuse with tissue-resident cells and obtain the corresponding characteristics of those cells. If fusion occurs, ADSCs may express markers of VECs, VSMCs, and cardiomyocytes without direct differentiation into these cell types. ADSCs also produce a variety of paracrine factors such as vascular endothelial growth factor, hepatocyte growth factor, and insulin-like growth factor-1 that have proangiogenic and/or antiapoptotic activities. Thus, ADSCs have the potential to regenerate the cardiovascular system via direct differentiation into VECs, VSMCs, and cardiomyocytes, fusion with tissue-resident cells, and the production of paracrine factors. Numerous animal studies have demonstrated the efficacy of ADSC implantation in the treatment of acute myocardial infarction (AMI), ischemic cardiomyopathy (ICM), dilated cardiomyopathy, hindlimb ischemia, and stroke. Clinical studies regarding the use of autologous ADSCs for treating patients with AMI and ICM have recently been initiated. ADSC implantation has been reported as safe and effective so far. Therefore, ADSCs appear to be useful for the treatment of cardiovascular disease. However, the tumorigenic potential of ADSCs requires careful evaluation before their safe clinical application.

A Self‐Assembled Spherical Complex Displaying a Gangliosidic Glycan Cluster Capable of Interacting with Amyloidogenic Proteins

Physiological and pathological functions of glycans are promoted through their clustering effects as exemplified by a series of gangliosides, sialylated glycosphingolipids, which serve as acceptors for bacterial toxins and viruses. Furthermore, ganglioside GM1 clusters on neuronal cell membranes specifically interact with amyloidogenic proteins, triggering their conformational transitions and leading to neurodegeneration. Here we develop a self-assembled spherical complex that displays a cluster of the GM1 pentasaccharide, and successfully demonstrate its ability to interact with amyloid β and α-synuclein. Due to the lack of hydrophobic lipid moieties, which would stably trap these cohesive proteins or give rise to toxic aggregates, this artificial cluster enabled NMR spectroscopic characterization of the early encounter stage of protein interactions with its outer carbohydrate moieties, which were not observable with previous glycan clusters.