This work was financially supported by Grant-in-Aid for Scienti&#64257;c Research B (24350072, 25288036) and Grant-in-Aid for Scienti&#64257;c Research on Innovative Areas (24108723) from MEXT and JSPS, Japan. T.S. acknowledges Grant-in-Aid for JSPS Fellows (25763), the GCOE Program of Osaka University and Grants for Excellent Graduate Schools, MEXT, Japan.

1. Preparation of Single Crystals Composed of Primary Ammonium Triphenylacetates
<ol>
	<li>Prepare organic salts, primary ammonium triphenylacetates (Figure 1a).
	<ol>
		<li>Dissolve triphenylacetic acid (TPAA, 0.10 g, 0.35 mmol) and primary amine: n-butylamine (nBu, 2.5 x 10-2 g, 0.35 mmol), isobutylamine (isoBu, 2.5 x 10-2 g, 0.35 mmol), t-butylamine (tBu, 2.5 x 10-2 g, 0.35 mmol), or t-amylamine (tAm, 3.0 x 10-2 g, 0.35 mmol), together in methanol (20 ml) in TPAA : amine = 1:1 molar ratio for preparation of binary organic salts.</li>
		<li>In the case of ternary organic salts, dissolve TPAA (0.10 g, 0.35 mmol) and two kinds of primary amines: nBu (1.3 x 10-2 g, 0.17 mmol)-tBu (1.3 x 10-2 g, 0.17 mmol), nBu (1.3 x 10-2 g, 0.17 mmol)-tAm (1.5 x 10-2 g, 0.17 mmol), isoBu (1.3 x 10-2 g, 0.17 mmol)-tBu (1.3 x 10-2 g, 0.17 mmol), or isoBu (1.3 x 10-2 g, 0.17 mmol)-tAm (1.5 x 10-2 g, 0.17 mmol), together in methanol (20 ml) in TPAA : amine-1 : amine-2 = 2:1:1.</li>
		<li>Evaporate all of the solutions by rotary evaporators (40 &#176;C, 200 Torr), affording organic salts: TPAA-nBu, TPAA-isoBu, TPAA-tBu, TPAA-tAm, TPAA-nBu-tBu, TPAA-nBu-tAm, TPAA-isoBu-tBu, and TPAA-isoBu-tAm.</li>
	</ol>
	</li>
	<li>Prepare single crystals composed of supramolecular clusters.
	<ol>
		<li>Dissolve each of the organic salts (5.0 mg) in a glass vial in toluene (0.30 ml) as a non-polar good solvent, which was selected because the supramolecular clusters are preferably constructed in non-polar environment. For the organic salts TPAA-tBu, TPAA-tAm, TPAA-isoBu-tBu, and TPAA-isoBu-tAm, heat toluene up to 40 &#176;C to dissolve them.</li>
		<li>Add hexane: 0.5 ml, 0.5 ml, 0.5 ml, 2 ml, 2 ml, 1 ml, and 0.5 ml, to the solution of the organic salts of TPAA-nBu, TPAA-isoBu, TPAA-tAm, TPAA-nBu-tBu, TPAA-nBu-tAm, TPAA-isoBu-tBu, and TPAA-isoBu-tAm, respectively, as a poor solvent to decrease solubility of the organic salt, except for the solution of the organic salt TPAA-tBu.</li>
		<li>Keep the solution stable at room temperature in the glass vial, affording single crystals within a day.</li>
	</ol>
	</li>
	<li>Confirm the organic salt formation by Fourier transform infrared (FT-IR) spectra35,36.
	<ol>
		<li>Mix the single crystals of the organic salt with potassium bromide (KBr) in 1:100 weight ratio.</li>
		<li>Grind the mixture by an agate mortar until it becomes homogeneous powdered mixture.</li>
		<li>Fill up a round die (diameter: 5 mm) with the powdered mixture and make a pellet by pressing it with a pellet press.</li>
		<li>Put the pellet into a FT-IR spectrometer, and perform measurements (cumulative number: 16, resolution: 1 cm-1).</li>
	</ol>
	</li>
</ol>
2. Crystallographic Studies
<ol>
	<li>Pick up a high quality single crystal of the organic salt TPAA-tAm from the glass vial into paraffin on a glass plate. The crystal looks uniform under a stereomicroscope, meaning the crystal is not assemblies of multiple crystals but a single crystal,&#160; and has a crystal size around 0.3 to 1 mm without cracks.</li>
	<li>Put the single crystal on a loop.</li>
	<li>Set the loop with the single crystal in single crystal X-ray diffraction equipment.</li>
	<li>Select a collimator: 0.3, 0.5, 0.8, or 1 mm, depending on the maximum size of the single crystal.</li>
	<li>Start a preparatory measurement of single crystal X-ray diffraction to collect X-ray diffraction patterns37,38 from the single crystal using the single crystal X-ray diffraction equipment (radiation source: graphite monochromated CuK&#945; (&#955; = 1.54187 &#197;), exposure time: 30 sec (determine based on the crystal size), detector: e.g. imaging plate, crystal-to-detector distance: 127.40 mm, temperature: 213.1 K, number of frames: 3).</li>
	<li>Determine possible crystal parameters and set conditions: exposure time, angles of X-ray exposure: &#969;, &#967;, and &#966;, and number of frames, for following measurement based on the result of the above preparatory measurement.</li>
	<li>Start a regular measurement of single crystal X-ray diffraction to collect X-ray diffraction patterns37,38 from the single crystals using the single crystal X-ray diffraction equipment under the conditions (radiation source: graphite monochromated CuK&#945; (&#955; = 1.54187 &#197;), detector: e.g. imaging plate, crystal-to-detector distance: 127.40 mm, temperature: 213.1 K).</li>
	<li>Solve a crystal structure from the diffraction patterns by direct methods, SIR200439 or SHELXS9740, and refine by a full-matrix least-squares procedure using all the observed reflections based on F2. Refine all the non-hydrogen atoms with anisotropic displacement parameters, and place the hydrogen atoms in idealized positions with isotropic displacement parameters relative to the connected non-hydrogen atoms and not refined. Perform these calculations using a software such as the CrystalStructure41.</li>
	<li>Repeat the procedures from steps 2.1 to 2.8 with some modifications of the conditions: collimator size, exposure time of X-ray for preparatory measurements and regular measurements, and angles of X-ray exposure: &#969;, &#967;, and &#966;, and number of frames, for the single crystals of organic salts: TPAA-nBu-tBu, TPAA-nBu-tAm, TPAA-isoBu-tBu, and TPAA-isoBu-tAm to reveal their crystal structures.</li>
	<li>Retrieve crystal structures of the organic salts: TPAA-nBu (refcode: MIBTOH)22, TPAA-isoBu (refcode: GIVFEX)24, and TPAA-tBu (refcode: GIVFIB)23, from Cambridge Structural Database42 using a software, Conquest43, or a request form44.</li>
	<li>Investigate the supramolecular clusters in the crystal structures by computer graphics using software such as Mercury45-48&#160;and Pymol49; Determine point group symmetries of hydrogen-bonding patterns in the supramolecular clusters by comparing the obtained patterns with the previously classified ones (Figure 1b) and chiral conformations of the trityl groups as &#923; or &#916; (Figure 1a).</li>
	<li>Characterize polyhedral features of the supramolecular clusters in each of the crystal structures of the organic salts.
	<ol>
		<li>Delete all of the atoms in the supramolecular clusters except for carbon and nitrogen atoms of the component carboxylate anions and ammonium cations.</li>
		<li>Make bonds between the carbon and nitrogen atoms of which original carboxylate and ammonium cations are connected by hydrogen bonds.</li>
		<li>Measure distances between carbon-carbon and nitrogen-nitrogen atoms, and set boundaries for making further bonds (5.3 and 4.1 &#197; for carbon-carbon and nitrogen-nitrogen distances, respectively, in this study).</li>
		<li>Make further bonds between carbon-carbon and nitrogen-nitrogen atoms of which distances are less than 5.4 and 4.2 &#197;, respectively.</li>
		<li>Determine the resulting polyhedrons of the organic salts: TPAA-isoBu, TPAA-tBu, TPAA-tAm, TPAA-isoBu-tBu, and TPAA-isoBu-tAm, as trans-bicapped octahedron (tbo), triangular dodecahedron (td), td, td, and td, respectively, by considering rotational axis (C3 or C2) as well as numbers of sides of the polyhedrons.</li>
		<li>Make additional bonds to the resulting polyhedrons of the organic salts: TPAA-nBu, TPAA-nBu-tBu, and TPAA-nBu-tAm, by considering number of sides, symmetry elements, and intermolecular interactions in the supramolecular clusters because they have less sides than ideal ones: td, tbo, and square antiprism (sa).</li>
		<li>Determine the polyhedron of the TPAA-nBu salt as sa by making two addition bonds due to its C2 symmetry and 14 original bonds. Determine the other polyhedrons of the organic salts TPAA-nBu-tBu and TPAA-nBu-tAm as td based on their C2 symmetry and &#34;bands&#34; of the trityl groups around the supramolecular cluster. Namely, the trityl groups in the supramolecular clusters of the ternary organic salts form &#34;bands&#34; by intermeshing their three phenyl rings and the polyhedron td has sides connecting four acids (Figure 1c(ii)), meaning they have similar structural features.</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

A series of supramolecular clusters with closed hydrogen-bonding networks was successfully constructed and characterized from viewpoints of chirality and polyhedral features using organic salts of TPAA, which has a trityl group, and various kinds and combinations of primary amines. In this method, the critical steps are introduction of a molecule with a bulky trityl group and recrystallization of organic salts composed of the molecule and counter ions from non-polar solvents. This is because the supramolecular cluster has an inverted-micellar structure, that is, ionic hydrogen bonds and hydrophobic hydrocarbons are inside and outside, respectively. Therefore, the supramolecular cluster is stable and is selectively constructed in non-polar environments.
Although the supramolecular cluster always has four ammoniums and four triphenylacetates, it is possible to introduce two kinds of ammoniums as well as a kind of ammonium, giving the supramolecular clusters variety by forming ternary supramolecular clusters in addition to binary ones. This flexibility in forming the supramolecular clusters leads to effective systematical symmetric studies for clarifying dependence of not only kinds but also numbers of components and suggests a potential to achieve multi-functionalization of the supramolecular clusters. It is also noteworthy that high quality single crystals of the organic salts are easily obtained, and thus their crystal structures were revealed by single crystal X-ray analysis. On the contrary, only a few structures of octameric water clusters, of which hydrogen-bonding networks are similar to those of the supramolecular clusters and their topology was classified theoretically13, have been confirmed experimentally50,51. This advantage is attributable to facts that the supramolecular clusters are stable due to robust charge-assisted hydrogen bonds and are packed effectively in crystalline states due to their cube-like shapes.
A method using organic salts is one of the most useful approaches to achieve prediction and/or control of supramolecular and crystal structures based on systematical studies. In this study, it is demonstrated that introduction of trityl groups results in selective construction of supramolecular clusters. The supramolecular clusters with specific symmetric properties can be constructed after clarification of their counter ion-dependent symmetric features. For example, it is reported that supramolecular clusters composed of triphenylmethylammonium sulfonates construct diamondoid porous organic salts52. The results obtained in this study show a possibility to control construction of the diamondoid and another unknown types of porous structures by changing components. In addition, introduction of another specific moieties (e.g. linear long alkyl chains) can lead to construction of another unique supramolecules (e.g. supramolecular layers)29-31. Systematical symmetric studies on these supramolecules can contribute to achieve component-based symmetry control of the supramolecules and crystals. Furthermore, although the supramolecular clusters composed of carboxylates and ammoniums are limited to clusters with eight components: four ammoniums and four carboxylates, it is possible to tune the component numbers by using phosphonic acids instead of carboxylic acids. This is because phosphonic acids have two acidic protons, and thus they can form more diverse hydrogen-bonding networks than carboxylic acids, which have only one acidic proton. Discoveries and systematical symmetric studies of further new and functional supramolecular clusters will bring an exciting future in material sciences as well as crystal engineering.

Supramolecules are fascinating and important research targets because of their unique functions, such as construction of supramolecular architectures, sensing of ions and/or molecules, and chiral separations, originated from their molecular recognition abilities using flexible non-covalent bonds1-11. In molecular recognitions, symmetry of supramolecular assemblies is one of the most important factors. Despite the importance, it is still difficult to design supramolecules with desired symmetries due to flexibility in numbers and kinds of the components as well as angles and distances of non-covalent bonds.
Clarification of correlations between symmetries of supramolecules and their components based on systematical studies is useful strategy to achieve construction of desired supramolecules. For this purpose, supramolecular clusters were selected as research targets because they are composed of limited number of components and are evaluable theoretically12-14. However, contrary to metal complexes, there are a limited number of reports constructing supramolecular clusters due to low stability of non-covalent bonds for sustaining the supramolecular structures15,16. This low stability also becomes a problem in obtaining a series of supramolecular assemblies which have the same kinds of structures. In this study, charge-assisted hydrogen bonds of organic salts, which are one of the most robust non-covalent bonds17-20, are mainly employed to construct specific supramolecular assemblies preferentially21-32. It is also noteworthy that organic salts are composed of acids and bases, and thus numerous kinds of organic salts are easily obtained just by mixing different combinations of acids and bases. Especially, organic salts are useful for systematic studies because combinations of a specific component with various kinds of counter ions result in the same types of supramolecular assemblies. Therefore, it is possible to compare structural differences of supramolecular assemblies based on kinds of counter ions.
In previous works, supramolecules with 0-dimensional (0-D), 1-dimensional (1-D), and 2-dimensional (2-D) hydrogen-bonding networks by primary ammonium carboxylates were confirmed and characterized from a viewpoint of chirality32. These multi-dimensional supramolecules are important research targets in hierarchical crystal design27 as well as applications exploiting their dimensionality. In addition, characterization of the hydrogen-bonding networks would give important knowledge about roles of biological molecules because all of amino acids have ammonium and carboxylic groups. Providing guidelines to obtain these supramolecules separately gives them further opportunities in applications. In these supramolecules, construction of supramolecular clusters with 0-D hydrogen-bonding networks is relatively difficult as demonstrated in statistical study28. However, after clarification of factors for constructing the supramolecular clusters, they were selectively constructed, and a series of the supramolecular clusters was obtained21-25,32. These works make it possible to conduct systematical symmetric study on the supramolecular clusters to clarify component-dependent symmetric characteristics of the supramolecular clusters. For this purpose, the supramolecular clusters of primary ammonium triphenylacetates have interesting features, that is, their topological variety in hydrogen-bonding networks24,32, which would reflect their symmetric features as well as chiral conformations of the component trityl groups (Figure 1a and 1b). Here methodologies for constructing a series of supramolecular clusters using primary ammonium triphenylacetates and for characterizing symmetric features of the supramolecular clusters are demonstrated. Keys for the construction of the supramolecular clusters are introduction of bulky trityl groups and recrystallization of the organic salts from non-polar solvents. Binary and ternary primary ammonium triphenylacetates were prepared for the construction of the supramolecular clusters. Crystallographic studies from viewpoints of topologies of the hydrogen-bonding networks24,32, topographies (conformations) of trityl groups33,34, and molecular arrangements as analogues of octacoordinated polyhedrons12 (Figure 1c) revealed component-dependent symmetric characteristics of the supramolecular clusters25.

<table><tbody><tr><td>Triphenylacetic acid</td><td>Aldrich</td><td>T81205-10G</td><td></td></tr><tr><td>&lt;em&gt;n&lt;/em&gt;-Butylamine</td><td>TCI</td><td>B0707</td><td></td></tr><tr><td>Isobutylamine</td><td>TCI</td><td>I0095</td><td></td></tr><tr><td>&lt;em&gt;tert&lt;/em&gt;-Butylamine</td><td>TCI</td><td>B0709</td><td></td></tr><tr><td>&lt;em&gt;tert&lt;/em&gt;-Amylamine</td><td>TCI</td><td>A1002</td><td></td></tr><tr><td>Methanol</td><td>Wako</td><td>131-01826</td><td>hazardous substance</td></tr><tr><td>Toluene</td><td>Wako</td><td>204-01866</td><td>hazardous substance</td></tr><tr><td>Hexane</td><td>Wako</td><td>085-00416</td><td></td></tr><tr><td>KBr</td><td>Wako</td><td>165-17111</td><td></td></tr></tbody></table>

construction and systematical symmetric studies of a series of supramolecular clusters with binary or ternary ammonium triphenylacetates

Functions of clusters in nano or sub-nano scale significantly depend on not only kinds of their components but also arrangements, or symmetry, of their components. Therefore, the arrangements in the clusters have been precisely characterized, especially for metal complexes. Contrary to this, characterizations of molecular arrangements in supramolecular clusters composed of organic molecules are limited to a few cases. This is because construction of the supramolecular clusters, especially obtaining a series of the supramolecular clusters, is difficult due to low stability of non-covalent bonds compare to covalent bonds. From this viewpoint, utilization of organic salts is one of the most useful strategies. A series of the supramolecules could be constructed by combinations of a specific organic molecule with various counter ions. Especially, primary ammonium carboxylates are suitable as typical examples of supramolecules because various kinds of carboxylic acids and primary amines are commercially available, and it is easy to change their combinations. Previously, it was demonstrated that primary ammonium triphenylacetates using various kinds of primary amines specifically construct supramolecular clusters, which are composed of four ammoniums and four triphenylacetates assembled by charge-assisted hydrogen bonds, in crystals obtained from non-polar solvents. This study demonstrates an application of the specific construction of the supramolecular clusters as a strategy to conduct systematical symmetric study for clarification of correlations between molecular arrangements in supramolecules and kinds and numbers of their components. In the same way with binary salts composed of triphenylacetates and one kind of primary ammoniums, ternary organic salts composed of triphenylacetates and two kinds of ammoniums construct the supramolecular clusters, affording a series of the supramolecular clusters with various kinds and numbers of the components.

Organic salt formation of TPAA and primary amines were confirmed by FT-IR measurements. Crystal structures of the organic salts were analyzed by single crystal X-ray diffraction measurements. As a result, the same kinds of the supramolecular clusters, which are composed of four ammoniums and four triphenylacetates by charge-assisted hydrogen bonds (Figure 1a), were confirmed in all of the single crystals of the organic salts regardless of kinds and numbers of the component ammoniums (Table 1, Figure 2). This result is applicable to systematical symmetric study to clarify component-dependent symmetric varieties of the supramolecular clusters.
According to systematical symmetric studies on the supramolecular clusters, it was found that chirality and polyhedral features of the supramolecular clusters (Figure 1b and 1c) depend on kinds and numbers of the component primary ammoniums. The binary supramolecular cluster of TPAA-nBu has an achiral D2d hydrogen-bonding network from a topological viewpoint. The D2d is a symbol of the Sch&#246;nflies notation and denotes that there are three two-fold axes which are perpendicular to each other as well as two vertical mirror planes which pass between two of the two-fold axes. In the supramolecular cluster, its components are arranged in a square antiprism (sa) manner with two-fold rotational axes or C2 symmetry (Figure 3a). This polyhedral feature is clarified by replacing the component molecules with representative atoms: carbon atoms of the carboxylate anions and nitrogen atoms of the ammonium cations, and making new connections between carbon-carbon, carbon-nitrogen, and nitrogen-nitrogen atoms depending on distances of the atoms. The supramolecular cluster has racemic, &#923; : &#916; = 2 : 2, trityl groups. The binary supramolecular clusters of TPAA-tBu and TPAA-tAm, which have achiral S4 hydrogen-bonding networks, also have racemic, &#923; : &#916; = 2 : 2, trityl groups (Figure 3c and 3d). In these cases, their components are arranged in triangular dodecahedron (td) manners which have C2 symmetry according to the same analysis with the case of the TPAA-nBu supramolecular cluster. Contrary to these cases, the binary supramolecular cluster of TPAA-isoBu has an achiral CS hydrogen-bonding network, which has not a two-fold rotational axis but a pseudo-three-fold rotational axis (Figure 3b). This symmetry results in chiral trityl groups as &#923; : &#916; = 1:3 (and vice versa). Namely, when the trityl group with &#923; conformation is on the pseudo-three-fold rotational axis, the other trityl groups with &#916; conformation are around the axis (and vice versa). In addition, the molecular arrangement in the supramolecular cluster belongs to a trans-bicapped octahedron (tbo) manner with a three-fold rotational axis, or C3 symmetry. From these investigations, it can be said that if the hydrogen-bonding network (or the polyhedron) has C2 or (pseudo-)C3 symmetry, the trityl groups prefer to form racemic &#923; : &#916; = 2:2, or chiral &#923; : &#916; = 1:3 (and vice versa) conformations, respectively, in the binary supramolecular cluster.
Contrary to the case of the binary supramolecular clusters, the ternary supramolecular clusters of TPAA-nBu-tBu, TPAA-nBu-tAm, TPAA-isoBu-tBu, and TPAA-isoBu-tAm, which have C2/C2&#39;, D2d, D2d, and D2d hydrogen-bonding networks, respectively, have different characteristics (Figure 4). All of them have two-fold rotational axes, or C2 symmetries, in hydrogen-bonding network, but the trityl groups have chirality as &#923; : &#916; = 0:4 (and vice versa). This result indicates that introduction of the third component leads to symmetry reduction in the supramolecular clusters, demonstrating usefulness of organic salts for inducing chirality. It should be noted that chirality of the trityl groups are used in chiral columns33,34. Further investigations on the ternary supramolecular clusters revealed that the trityl groups were intermeshed each other in the supramolecular clusters (Figure 5). This intermesh could be one of the main factor to form chiral &#923; : &#916; = 0:4 (and vice versa) conformations by delivering chirality of one of the trityl group to the others. This connectivity among four TPAA anions are similar with a structural feature of the td (Figure 5b), and thus the polyhedrons of the ternary supramolecular clusters are easily determined as the td by combination of the same analysis with the case of the binary supramolecular cluster.
<img alt="Figure 1" src="/files/ftp_upload/53418/53418fig1.jpg" /> 
Figure 1: Characteristics of hydrogen-bonding supramolecular clusters. (a) Formation of the hydrogen-bonding supramolecular clusters. Chemical structures of (i) triphenylacetates with chiral trityl conformations: left- (&#923;) and right-handed (&#916;), and (ii) primary ammoniums employed in this study. (b) Topological classification of the hydrogen-bonding networks of supramolecular clusters. Point group symmetries are described by Sch&#246;nflies notation. (c) Polyhedral arrangements: (i) regular cube (rc) (ii) triangular dodecahedron (td) (iii) trans-bicapped octahedron (tbo), and (iv) square antiprism (sa). This figure has been modified and adapted with permission from Cryst. Growth Des. 15 (2), 658 - 665, doi: 10.1021/cg5013445 (2015). Copyright (2015) American Chemical Society.
<img alt="Figure 2" src="/files/ftp_upload/53418/53418fig2.jpg" /> 
Figure 2: Structures of supramolecular clusters. Structures of supramolecular clusters of (a) binary organic salts: (i) TPAA-nBu (ii) TPAA-isoBu (iii) TPAA-tBu, and (iv) TPAA-tAm, and (b) ternary organic salts: (i) TPAA-nBu-tBu (ii) TPAA-nBu-tAm (iii) TPAA-isoBu-tBu, and (iv) TPAA-isoBu-tAm. All of the supramolecular clusters are constructed by ion pairs of the TPAA anions and the primary ammonium cations. Hydrogen bonds are represented in light blue by dotted lines. This figure has been modified and adapted with permission from Cryst. Growth Des. 15 (2), 658 - 665, doi: 10.1021/cg5013445 (2015). Copyright (2015) American Chemical Society.
<img alt="Figure 3" src="/files/ftp_upload/53418/53418fig3.jpg" /> 
Figure 3: Polyhedral supramolecular clusters of binary organic salts. Structures of polyhedral supramolecular clusters of binary organic salts (a) TPAA-nBu (sa) (b) TPAA-isoBu (tbo) (c) TPAA-tBu (td), and (d) TPAA-tAm (td). (i) The full structure of the supramolecular cluster and (ii) polyhedral arrangement of the components, which is represented by carbon atoms of the carboxylate groups and nitrogen atoms of the ammonium groups, in the supramolecular cluster. Trityl groups with &#923; or &#916; conformations are colored in light pink or light blue, respectively. A symbol &#39;C2*&#39; means that the two-fold rotational axis is pseudo symmetry. Ammoniums are colored in green. Hydrogen atoms are omitted for clarity. This figure has been modified and adapted with permission from Cryst. Growth Des. 15 (2), 658 - 665, doi: 10.1021/cg5013445 (2015). Copyright (2015) American Chemical Society.
<img alt="Figure 4" src="/files/ftp_upload/53418/53418fig4.jpg" /> 
Figure 4: Polyhedral supramolecular clusters of ternary organic salts. Structures of polyhedral supramolecular clusters of ternary organic salts (a) TPAA-nBu-tBu (td) (b) TPAA-nBu-tAm (td) (c) TPAA-isoBu-tBu (td), and (d) TPAA-isoBu-tAm (td). (i) The full structure of the supramolecular cluster and (ii) polyhedral arrangement of the components, which is represented by carbon atoms of the carboxylate anions and nitrogen atoms of the ammonium cations, in the supramolecular cluster. Trityl groups with &#923; or &#916; conformations are colored in light pink or light blue, respectively. Ammoniums are colored in green for nBu and isoBu and orange for tBu and tAm. Hydrogen atoms are omitted for clarity. This figure has been modified and adapted with permission from Cryst. Growth Des. 15 (2), 658 - 665, doi: 10.1021/cg5013445 (2015). Copyright (2015) American Chemical Society.
<img alt="Figure 5" src="/files/ftp_upload/53418/53418fig5.jpg" /> 
Figure 5: Arrangements of trityl groups in the ternary supramolecular clusters (TPAA-isoBu-tAm). Manners of intermesh between two out of four trityl groups (numbered from 1 to 4): (i) 1 and 2, (ii) 2 and 3, (iii) 3 and 4, and (iv) 4 and 1. (b) Schematic representation of a &#39;band&#39; structure composed of the intermeshing trityl groups and that in a triangular dodecahedron. The trityl groups with &#916; conformation are colored in light blue. The ammoniums: isoBu and tAm, are colored in green and orange, respectively. This figure has been modified and adapted with permission from Cryst. Growth Des. 15 (2), 658 - 665, doi: 10.1021/cg5013445 (2015). Copyright (2015) American Chemical Society.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Entries</td>
			<td>Topology</td>
			<td>Trityl chirality</td>
			<td>Polyhedron</td>
			<td>Ref. code or CCDC no.</td>
		</tr>
		<tr>
			<td>TPAA-nBu</td>
			<td>D2d</td>
			<td>&#923; : &#916; = 2 : 2</td>
			<td>sa</td>
			<td>MIBTOH[a]</td>
		</tr>
		<tr>
			<td>TPAA-isoBu</td>
			<td>CS</td>
			<td>&#923; : &#916; = 1 : 3 
			&#923; : &#916; = 3 : 1</td>
			<td>tbo</td>
			<td>GIVFEX[b]</td>
		</tr>
		<tr>
			<td>TPAA-tBu</td>
			<td>S4</td>
			<td>&#923; : &#916; = 2 : 2</td>
			<td>td</td>
			<td>GIVFIB[c]</td>
		</tr>
		<tr>
			<td>TPAA-tAm</td>
			<td>S4</td>
			<td>&#923; : &#916; = 2 : 2</td>
			<td>td</td>
			<td>973395[d]</td>
		</tr>
		<tr>
			<td>TPAA-nBu-tBu</td>
			<td>C2/C2&#39;</td>
			<td>&#923; : &#916; = 4 : 0 
			&#923; : &#916; = 0 : 4</td>
			<td>td</td>
			<td>973399[d]</td>
		</tr>
		<tr>
			<td>TPAA-nBu-tAm</td>
			<td>D2d</td>
			<td>&#923; : &#916; = 4 : 0 
			&#923; : &#916; = 0 : 4</td>
			<td>td</td>
			<td>973398[d]</td>
		</tr>
		<tr>
			<td>TPAA-isoBu-tBu</td>
			<td>D2d</td>
			<td>&#923; : &#916; = 4 : 0 
			&#923; : &#916; = 0 : 4</td>
			<td>td</td>
			<td>973397[d]</td>
		</tr>
		<tr>
			<td>TPAA-isoBu-tAm</td>
			<td>D2d</td>
			<td>&#923; : &#916; = 4 : 0 
			&#923; : &#916; = 0 : 4</td>
			<td>td</td>
			<td>973396[d]</td>
		</tr>
	</tbody>
</table>
Table 1: Characteristics of the supramolecular clusters.&#160;[a]-[d]: See refs. 22, 24, 23 and 25, respectively. This table has been modified and adapted with permission from Cryst. Growth Des. 15 (2), 658 - 665, doi: 10.1021/cg5013445 (2015). Copyright (2015) American Chemical Society.

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Construction and Systematical Symmetric Studies of a Series of Supramolecular Clusters with Binary or Ternary Ammonium Triphenylacetates

This article describes construction of a series of hydrogen-bonding supramolecular clusters in crystals using primary ammonium triphenylacetates, which are recrystallized from non-polar solvents. This selective construction of the supramolecular clusters leads to effective systematical symmetric studies about a correlation between the supramolecular clusters and their components.

Functions of clusters in nano or sub-nano scale significantly depend on not only kinds of their components but also arrangements, or symmetry, of their ...

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Nanyang Technological University

Research

JoVE Journal

Chemistry

8.1K Views.  Osaka University. This article describes construction of a series of hydrogen-bonding supramolecular clusters in crystals using primary ammonium triphenylacetates, which are recrystallized from non-polar solvents. This selective construction of the supramolecular clusters leads to effective systematical symmetric studies about a correlation between the supramolecular clusters and their components.

Video: Construction and Systematical Symmetric Studies of a Series of Supramolecular Clusters with Binary or Ternary Ammonium Triphenylacetates

Controlling the Size, Shape and Stability of Supramolecular Polymers in Water

For aqueous based supramolecular polymers, the simultaneous control over shape, size and stability is very difficult1. At the same time, the ability to do so is highly important in view of a number of applications in functional soft matter including electronics, biomedical engineering, and sensors. In the past, successful strategies to control the size and shape of supramolecular polymers typically focused on the use of templates2,3, end cappers4 or selective solvent techniques5. 
Here we disclose a strategy based on self-assembling discotic amphiphiles that leads to the control over stack length and shape of ordered, chiral columnar aggregates. By balancing electrostatic repulsive interactions on the hydrophilic rim and attractive non-covalent forces within the hydrophobic core of the polymerizing building block, we manage to create small and discrete spherical objects6,7. Increasing the salt concentration to screen the charges induces a sphere-to-rod transition. Intriguingly, this transition is expressed in an increase of cooperativity in the temperature-dependent self-assembly mechanism, and more stable aggregates are obtained. 
For our study we select a benzene-1,3,5-tricarboxamide (BTA) core connected to a hydrophilic metal chelate via a hydrophobic, fluorinated L-phenylalanine based spacer (Scheme 1). The metal chelate selected is a Gd(III)-DTPA complex that contains two overall remaining charges per complex and necessarily two counter ions. The one-dimensional growth of the aggregate is directed by &pi;-&pi; stacking and intermolecular hydrogen bonding. However, the electrostatic, repulsive forces that arise from the charges on the Gd(III)-DTPA complex start limiting the one-dimensional growth of the BTA-based discotic once a certain size is reached. At millimolar concentrations the formed aggregate has a spherical shape and a diameter of around 5 nm as inferred from 1H-NMR spectroscopy, small angle X-ray scattering, and cryogenic transmission electron microscopy (cryo-TEM). The strength of the electrostatic repulsive interactions between molecules can be reduced by increasing the salt concentration of the buffered solutions. This screening of the charges induces a transition from spherical aggregates into elongated rods with a length &gt; 25 nm. Cryo-TEM allows to visualise the changes in shape and size. In addition, CD spectroscopy permits to derive the mechanistic details of the self-assembly processes before and after the addition of salt. Importantly, the cooperativity -a key feature that dictates the physical properties of the produced supramolecular polymers- increases dramatically upon screening the electrostatic interactions. This increase in cooperativity results in a significant increase in the molecular weight of the formed supramolecular polymers in water.

The goal of this experiment is to determine and control the size, shape and stability of self-assembled discotic amphiphiles in water. For aqueous based supramolecular polymers such level of control is very difficult. We apply a strategy using both repulsive and attractive interactions. The experimental techniques applied to characterize this system are broadly applicable.

For aqueous based supramolecular polymers, the simultaneous control over shape, size and stability is very difficult1. At the same time, the ability to do ...

Engineering

Formation of Ordered Biomolecular Structures by the Self-assembly of Short Peptides

In nature, complex functional structures are formed by the self-assembly of biomolecules under mild conditions. Understanding the forces that control self-assembly and mimicking this process in vitro will bring about major advances in the areas of materials science and nanotechnology. Among the available biological building blocks, peptides have several advantages as they present substantial diversity, their synthesis in large scale is straightforward, and they can easily be modified with biological and chemical entities1,2. Several classes of designed peptides such as cyclic peptides, amphiphile peptides and peptide-conjugates self-assemble into ordered structures in solution. Homoaromatic dipeptides, are a class of short self-assembled peptides that contain all the molecular information needed to form ordered structures such as nanotubes, spheres and fibrils3-8. A large variety of these peptides is commercially available.
This paper presents a procedure that leads to the formation of ordered structures by the self-assembly of homoaromatic peptides. The protocol requires only commercial reagents and basic laboratory equipment. In addition, the paper describes some of the methods available for the characterization of peptide-based assemblies. These methods include electron and atomic force microscopy and Fourier-Transform Infrared Spectroscopy (FT-IR). Moreover, the manuscript demonstrates the blending of peptides (coassembly) and the formation of a &#34;beads on a string&#34;-like structure by this process.9 The protocols presented here can be adapted to other classes of peptides or biological building blocks and can potentially lead to the discovery of new peptide-based structures and to better control of their assembly.

This paper describes the formation of highly ordered peptide-based structures by the spontaneous process of self-assembly. The method utilizes commercially available peptides and common lab equipment. This technique can be applied to a large variety of peptides and may lead to the discovery of new peptide-based assemblies.

In nature, complex functional structures are formed by the self-assembly of biomolecules under mild conditions. Understanding the forces that control ...

Atomic Scale Structural Studies of Macromolecular Assemblies by Solid-state Nuclear Magnetic Resonance Spectroscopy

Supramolecular protein assemblies play fundamental roles in biological processes ranging from host-pathogen interaction, viral infection to the propagation of neurodegenerative disorders. Such assemblies consist in multiple protein subunits organized in a non-covalent way to form large macromolecular objects that can execute a variety of cellular functions or cause detrimental consequences. Atomic insights into the assembly mechanisms and the functioning of those macromolecular assemblies remain often scarce since their inherent insolubility and non-crystallinity often drastically reduces the quality of the data obtained from most techniques used in structural biology, such as X-ray crystallography and solution Nuclear Magnetic Resonance (NMR). We here present magic-angle spinning solid-state NMR spectroscopy (SSNMR) as a powerful method to investigate structures of macromolecular assemblies at atomic resolution. SSNMR can reveal atomic details on the assembled complex without size and solubility limitations. The protocol presented here describes the essential steps from the production of 13C/15N isotope-labeled macromolecular protein assemblies to the acquisition of standard SSNMR spectra and their analysis and interpretation. As an example, we show the pipeline of a SSNMR structural analysis of a filamentous protein assembly.

Structures of supramolecular protein assemblies at atomic resolution are of high relevance because of their crucial roles in a variety of biological phenomena. Herein, we present a protocol to perform high-resolution structural studies on insoluble and non-crystalline macromolecular protein assemblies by magic-angle spinning solid-state nuclear magnetic resonance spectroscopy (MAS SSNMR).

Supramolecular protein assemblies play fundamental roles in biological processes ranging from host-pathogen interaction, viral infection to the ...

Biochemistry

Synthesis and Characterization of Supramolecular Colloids

Control over colloidal assembly is of utmost importance for the development of functional colloidal materials with tailored structural and mechanical properties for applications in photonics, drug delivery and coating technology. Here we present a new family of colloidal building blocks, coined supramolecular colloids, whose self-assembly is controlled through surface-functionalization with a benzene-1,3,5-tricarboxamide (BTA) derived supramolecular moiety. Such BTAs interact via directional, strong, yet reversible hydrogen-bonds with other identical BTAs. Herein, a protocol is presented that describes how to couple these BTAs to colloids and how to quantify the number of coupling sites, which determines the multivalency of the supramolecular colloids. Light scattering measurements show that the refractive index of the colloids is almost matched with that of the solvent, which strongly reduces the van der Waals forces between the colloids. Before photo-activation, the colloids remain well dispersed, as the BTAs are equipped with a photo-labile group that blocks the formation of hydrogen-bonds. Controlled deprotection with UV-light activates the short-range hydrogen-bonds between the BTAs, which triggers the colloidal self-assembly. The evolution from the dispersed state to the clustered state is monitored by confocal microscopy. These results are further quantified by image analysis with simple routines using ImageJ and Matlab. This merger of supramolecular chemistry and colloidal science offers a direct route towards light- and thermo-responsive colloidal assembly encoded in the surface-grafted monolayer.

A protocol for the synthesis and characterization of colloids coated with supramolecular moieties is described. These supramolecular colloids undergo self-assembly upon the activation of the hydrogen-bonds between the surface-anchored molecules by UV-light.

Control over colloidal assembly is of utmost importance for the development of functional colloidal materials with tailored structural and mechanical ...

Structural Studies of Macromolecules in Solution using Small Angle X-Ray Scattering

Protein-protein interactions involving proteins with multiple globular domains present technical challenges for determining how such complexes form and how the domains are oriented/positioned. Here, a protocol with the potential for elucidating which specific domains mediate interactions in multicomponent system through ab initio modeling is described. A method for calculating solution structures of macromolecules and their assemblies is provided that involves integrating data from small angle X-ray scattering (SAXS), chromatography, and atomic resolution structures together in a hybrid approach. A specific example is that of the complex of full-length nidogen-1, which assembles extracellular matrix proteins and forms an extended, curved nanostructure. One of its globular domains attaches to laminin &#947;-1, which structures the basement membrane. This provides a basis for determining accurate structures of flexible multidomain protein complexes and is enabled by synchrotron sources coupled with automation robotics and size exclusion chromatography systems. This combination allows rapid analysis in which multiple oligomeric states are separated just prior to SAXS data collection. The analysis yields information on the radius of gyration, particle dimension, molecular shape and interdomain pairing. The protocol for generating 3D models of complexes by fitting high-resolution structures of the component proteins is also given.

Here, we present how Small Angle X-Ray Scattering (SAXS) can be utilized to obtain information on low-resolution envelopes representing the macromolecular structures. When used in conjunction with high-resolution structural techniques such as X-Ray Crystallography and Nuclear Magnetic Resonance, SAXS can provide detailed insights into multidomain proteins and macromolecular complexes in-solution.

Protein-protein interactions involving proteins with multiple globular domains present technical challenges for determining how such complexes form and ...

Synthesis and Characterization of 1,2-Dithiolane Modified Self-Assembling Peptides

This report focuses on the synthesis of an N-terminus 1,2-dithiolane modified self-assembling peptide and the characterization of the resulting self-assembled supramolecular structures. The synthetic route takes advantage of solid-phase peptide synthesis with the on-resin coupling of the dithiolane precursor molecule, 3-(acetylthio)-2-(acetylthiomethyl)propanoic acid, and the microwave-assisted thioacetate deprotection of the peptide N-terminus before final cleavage from the resin to yield the 1,2-dithiolane modified peptide. After the high-performance liquid chromatography (HPLC) purification of the 1,2-dithiolane peptide, derived from the nucleating core of the A&#946; peptide associated with Alzheimer&#39;s disease, the peptide is shown to self-assemble into cross-&#946; amyloid fibers. Protocols to characterize the amyloid fibers by Fourier-transform infrared spectroscopy (FT-IR), circular dichroism spectroscopy (CD) and transmission electron microscopy (TEM) are presented. The methods of N-terminal modification with a 1,2-dithiolane moiety to well-characterized self-assembling peptides can now be explored as model systems to develop post-assembly modification strategies and explore dynamic covalent chemistry on supramolecular peptide nanofiber surfaces.

A protocol for the synthesis of a 1,2-dithiolane modified peptide and the characterization of the supramolecular structures resulting from the peptide self-assembly.

This report focuses on the synthesis of an N-terminus 1,2-dithiolane modified self-assembling peptide and the characterization of the resulting ...

Synthesis of Information-bearing Peptoids and their Sequence-directed Dynamic Covalent Self-assembly

This protocol presents the use of Lewis acidic multi-role reagents to circumvent kinetic trapping observed during the self-assembly of information-encoded oligomeric strands mediated by paired dynamic covalent interactions in a manner mimicking the thermal cycling commonly employed for the self-assembly of complementary nucleic acid sequences. Primary amine monomers bearing aldehyde and amine pendant moieties are functionalized with orthogonal protecting groups for use as dynamic covalent reactant pairs. Using a modified automated peptide synthesizer, the primary amine monomers are encoded into oligo(peptoid) strands through solid-phase submonomer synthesis. Upon purification by high-performance liquid chromatography (HPLC) and characterization by electrospray ionization mass spectrometry (ESI-MS), sequence-specific oligomers are subjected to high-loading of a Lewis acidic rare-earth metal triflate which both deprotects the aldehyde moieties and affects the reactant pair equilibrium such that strands completely dissociate. Subsequently, a fraction of the Lewis acid is extracted, enabling annealing of complementary sequence-specific strands to form information-encoded molecular ladders characterized by matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS). The simple procedure outlined in this report circumvents kinetic traps commonly experienced in the field of dynamic covalent assembly and serves as a platform for the future design of robust, complex architectures.

A protocol is presented for the synthesis of information-encoded peptoid oligomers and for the sequence-directed self-assembly of these peptoids into molecular ladders using amines and aldehydes as dynamic covalent reactant pairs and Lewis acidic rare-earth metal triflates as multi-role reagents.

This protocol presents the use of Lewis acidic multi-role reagents to circumvent kinetic trapping observed during the self-assembly of information-encoded ...

Computation of Atmospheric Concentrations of Molecular Clusters from ab initio Thermochemistry

The computational study of the formation and growth of atmospheric aerosols requires an accurate Gibbs free energy surface, which can be obtained from gas phase electronic structure and vibrational frequency calculations. These quantities are valid for those atmospheric clusters whose geometries correspond to a minimum on their potential energy surfaces. The Gibbs free energy of the minimum energy structure can be used to predict atmospheric concentrations of the cluster under a variety of conditions such as temperature and pressure. We present a computationally inexpensive procedure built on a genetic algorithm-based configurational sampling followed by a series of increasingly accurate screening calculations. The procedure starts by generating and evolving the geometries of a large set of configurations using semi-empirical models then refines the resulting unique structures at a series of high-level ab initio levels of theory. Finally, thermodynamic corrections are computed for the resulting set of minimum-energy structures and used to compute the Gibbs free energies of formation, equilibrium constants, and atmospheric concentrations. We present the application of this procedure to the study of hydrated glycine clusters under ambient conditions.

Computation of Atmospheric Concentrations of Molecular Clusters from ab initio Thermochemistry

The atmospheric concentrations of weakly bound molecular clusters can be computed from the thermochemical properties of low energy structures found through a multi-step configurational sampling methodology utilizing a genetic algorithm and semi-empirical and ab initio quantum chemistry.

The computational study of the formation and growth of atmospheric aerosols requires an accurate Gibbs free energy surface, which can be obtained from gas ...

Thermochemical Studies of Ni(II) and Zn(II) Ternary Complexes Using Ion Mobility-Mass Spectrometry

This article describes an experimental protocol using electrospray-ion mobility-mass spectrometry (ES-IM-MS) and energy-resolved threshold collision-induced dissociation (TCID) to measure the thermochemistry of the dissociation of negatively-charged [amb+M(II)+NTA]- ternary complexes into two product channels: [amb+M(II)] + NTA or [NTA+M(II)]-&#160;+ amb, where M = Zn or Ni and NTA is nitrilotriacetic acid. The complexes contain one of the alternative metal binding (amb) heptapeptides with the primary structures acetyl-His1-Cys2-Gly3-Pro4-Tyr5-His6-Cys7 or acetyl-Asp1-Cys2-Gly3-Pro4-Tyr5-His6-Cys7, where the amino acids&#39; Aa1,2,6,7 positions are the potential metal-binding sites. Geometry-optimized stationary states of the ternary complexes and their products were selected from quantum chemistry calculations (presently the PM6 semi-empirical Hamiltonian) by comparing their electronic energies and their collision cross-sections (CCS) to those measured by ES-IM-MS. From the PM6 frequency calculations, the molecular parameters of the ternary complex and its products model the energy-dependent intensities of the two product channels using a competitive TCID method to determine the threshold energies of the reactions that relate to the 0 K enthalpies of dissociation (&#916;H0). Statistical mechanics thermal and entropy corrections using the PM6 rotational and vibrational frequencies provide the 298 K enthalpies of dissociation (&#916;H298). These methods describe an EI-IM-MS routine that can determine thermochemistry and equilibrium constants for a range of ternary metal ion complexes.

Thermochemical Studies of NiII and ZnII Ternary Complexes Using Ion Mobility-Mass Spectrometry

This article describes an experimental protocol using electrospray-ion mobility-mass spectrometry, semi-empirical quantum calculations, and energy-resolved threshold collision-induced dissociation to measure the relative thermochemistry of the dissociation of related ternary metal complexes.

This article describes an experimental protocol using electrospray-ion mobility-mass spectrometry (ES-IM-MS) and energy-resolved threshold ...

Constructing Cyclic Peptides Using an On-Tether Sulfonium Center

In recent years, cyclic peptides have attracted increasing attention in the field of drug discovery due to their excellent biological activities, and, as a consequence, they are now used clinically. It is, therefore, critical to seek effective strategies for synthesizing cyclic peptides to promote their application in the field of drug discovery. This paper reports a detailed protocol for the efficient synthesis of cyclic peptides using on-resin or intramolecular (intermolecular) bisalkylation. Using this protocol, linear peptides were synthesized by taking advantage of solid-phase peptide synthesis with cysteine (Cys) and methionine (Met) coupled simultaneously on the resin. Further, cyclic peptides were synthesized via bisalkylation between Met and Cys using a tunable tether and an on-tether sulfonium center. The whole synthetic route can be divided into three major processes: the deprotection of Cys on the resin, the coupling of the linker, and the cyclization between Cys and Met in a trifluoroacetic acid (TFA) cleavage solution. Furthermore, inspired by the reactivity of the sulfonium center, a propargyl group was attached to the Met to trigger thiol-yne addition and form a cyclic peptide. After that, the crude peptides were dried and dissolved in acetonitrile, separated, and then purified by high-performance liquid chromatography (HPLC). The molecular weight of the cyclic peptide was confirmed by liquid chromatography-mass spectrometry (LC-MS), and the stability of the cyclic peptide combination with the reductant was further confirmed using HPLC. In addition, the chemical shift in the cyclic peptide was analyzed by 1H nuclear magnetic resonance (1H NMR) spectra. Overall, this protocol aimed to establish an effective strategy for synthesizing cyclic peptides.

This protocol presents the synthesis of cyclic peptides via bisalkylation between cysteine and methionine and the facile thiol-yne reaction triggered by the propargyl sulfonium center.

In recent years, cyclic peptides have attracted increasing attention in the field of drug discovery due to their excellent biological activities, and, as ...

Construction and Systematical Symmetric Studies of a Series of Supramolecular Clusters with Binary or Ternary Ammonium Triphenylacetates

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