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660a22a5e9ebbb4db917b00e | 20 | Investment data from Pitchbook 216 is indicative of growing commercial interest in CNTs over the last decade (Figure ). In terms of geographical distribution, almost half of the capital invested (45%) between 2013-2023 appears to be in 82 companies located in the USA (Figure ), followed by China. Luxembourg (LUX) accounts for 12% of capital invested, which can be attributed to OCSiAl, largest known manufacturer of commercially available CNTs (Tuball). Other countries/regions showing commercial interest in CNTs include South Korea, Finland (FIN), the United Kingdom, Australia (AUS), Israel (ISR), Canada and Japan. |
660a22a5e9ebbb4db917b00e | 21 | Breakdown across primary industry groups indicates interest in CNTs across a wide range of industry types ranging from semiconductors, energy and biomedical (Figure ) with the latter accounting for a smaller fraction. Amongst the top 50 deals (in terms of deal size) in the area of CNTs over the last decade, majority appear to be in the material resources, information technology and business to business segments (Figure ). Energy and healthcare sectors account for very few deals of large magnitudes. In the first quarter of 2022, OCSiAl received the largest capital investment of nearly 300 million USD from a group of venture capitalists. Similarly, significant capital investments but of a smaller magnitude were also received by JEIO Co and Nano-C in 2022. JEIO Co, a South Korean company manufactures CNTs specializing in their applications in batteries (Li-ion batteries) while Nano-C appears to specialize in SWCNT manufacturing with possible use/applications in sensors and electronics. In addition, Nano-C also appears to manufacture SWCNT ink that can be deposited on the surface using a variety of printing methods. 217 |
660a22a5e9ebbb4db917b00e | 22 | We have used natural language processing to identify the most prominent and emerging applications, associated materials, and properties of carbon nanotubes in journal and patent publications from 2020-2022. We have combined this with our in-depth landscape analysis to show that polymer-matrix composites, sensors, biomedical applications, and batteries have been the most prominent applications over last 20 years. Among these, battery applications have a significantly higher patent-to-journal publication ratio, indicating relatively higher commercial R&D activity in this area. |
660a22a5e9ebbb4db917b00e | 23 | Tracking the frequency of publications related to CNT synthesis, CVD is by far the most common method used to make CNTs, outnumbering other methods by a factor of 4-5. In general, publications related to the synthesis of CNTs have decreased since 2006, however, there is still significant research interest in achieving greater control of CNT structure through synthesis parameters. |
660a22a5e9ebbb4db917b00e | 24 | While there appeared to have been a plateau in the number of yearly publications on CNTs around 2014-2017, this number has generally increased since that time. To determine the trends that are driving this increase, we calculated the rate of change of specific applications, materials, and properties in CNT publications identified using NLP analysis. The results of these calculations were used to make a conceptual map of areas of high research areas involving CNTs. The main applications that appear to be driving growth include energy storage and conversion, sensors (particularly human motion and wearable sensors), environmental remediation and CO2 reduction, and applications where CNTs are combined with other nanoscale materials such as MXenes. |
660a22a5e9ebbb4db917b00e | 25 | Through these applications, the unique properties of CNTs, including strength, electrical and thermal conductivity, and ability for functionalization, are being leveraged to address the most critical problems and promising research areas currently known. Understanding the landscape of key emerging trends in this area, specifically the fast-growing applications, properties, and materials used in combination with CNTs, will be important for researchers who are driving further growth in CNTs and nanoscale materials in general. |
65f285c39138d231616bbedc | 0 | As the ubiquitous driving force in nature, starting from the molecular level building blocks like L-amino acids, D-sugars to nanoscale helical structures found in DNA, RNA and macroscale in conch and snails, chirality is indispensable in our daily life. It is no wonder that the concept of molecular chirality in terms of asymmetric synthesis and catalysis is at the forefront in the development of pharmaceuticals and functional molecules. Moreover, molecular chirality is important because it is closely linked to biomimetics at the cellular level to produce functional chiral materials. Originally described by Lehn as "chemistry beyond molecules," supramolecular chemistry makes use of noncovalent interactions to facilitate the spontaneous organizing of constituents into structures or patterns without the need for human involvement. In the case of non-symmetric extended spatial packing, which can be caused by the aggregation of molecular building blocks (with or without chiral centers), the resulting architecture would be chiral at the supramolecular level, also known as supramolecular chirality. Notably, chirality transfer, chiral amplification, and asymmetry breaking of either chiral or achiral components upon selfassembly can all cause supramolecular chirality to develop. In addition to improving our comprehension of the inherent homochirality observed in complex biological systems, rationalizing supramolecular chirality in multicomponent systems has significant implications in the pursuit of developing functional chiral materials. In this review article, we attempt to present a comprehensive account of the recent development of the supramolecular chirality within the self-assembled systems over the past few years, with a particular emphasis on the aspects of (i) chiroptical properties and (ii) enantio-selective separation in multicomponent systems. Notably, multicomponent systems are more complicated than single-component systems due to the possibility of integrated co-assembly, narcissistic self-sorting, heterojunctions and their mixtures, especially when assembled from achiral molecules, making chirality determination difficult. As it is a rapidly growing field with numerous examples reported regularly, we intend to enclose our discussion to the field of co-ordination self-assembled systems namely porous metal-organic cages (MOCs) only. First and foremost, our primary objective revolves around the characterization of supramolecular chirality. Subsequently, we delve into the intricate processes of chirality transfer and amplification, specifically examining the induction and transmission mechanisms from the constituent organic ligands to the self-assembled metallacages. The establishment of subtle chiral microenvironments enables chiral hosts to selectively interact with one enantiomer in comparison to the other, thereby bestowing exceptional capabilities in chiral separation and recognition. Later, the potentialities, prospects, and critical concerns pertaining to this domain are examined. directionality of such non-covalent interactions determines the ultimate geometry of the assembly, but to design a selfassembled structure only based on the non-covalent interactions is a tedious job given the dynamic and reversible nature. Therefore, stronger interactions like dynamic covalent bonding or metal-ligand coordination are required to minimize the dynamic nature of the final assembly to a greater extent and stabilize the final geometry. The discrete coordination assemblies are generated from metal ions and organic ligands, mainly focusing on the generation of convex polyhedra, that are surrounded by organic ligands clipped together via the metal-ligand coordination bonds. Featuring enclosed 3D space, such polyhedral structures can be mainly divided into two main classes, (i) Platonic solid, comprised of only one type of identical polyhedral edges or faces, and (ii) Archimedean solids, which are formed by at least two kinds of different polygonal planes (Figure ). |
65f285c39138d231616bbedc | 1 | By the means of point-group symmetry classification, the presence of mirror planes, and/or inversion centres make the five Platonic solids achiral, whereas the Archimedean solids can be classified into thirteen types, mostly with Td, Oh, or Ih symmetry groups, except the snub cube (O) and snub dodecahedron (I), where the absence of inversion or mirror symmetries and the presence of only axial symmetries afford the chiral attributes. Generally, the MOCs expand around the design and application of the Platonic geometries (Figure ), with single ligands defining the edges and the faces of the polyhedron (homoleptic cages). However, biomolecules and high-value synthetic compounds are rarely highly symmetrical species, which urges the construction of MOCs with interior cavities of reduced symmetry, incorporating multiple ligands into the structurally intriguing Archimedean solids. However, a particular challenge in this approach is to ensure that different building blocks integrate into a single product (integrative self-sorting) rather than segregating into simpler structures (narcissistic sorting) or a statistical distribution of products (social sorting ). However, for this review, we mainly focus on the homoleptic tetrahedral, cubic, and octahedral assemblies. |
65f285c39138d231616bbedc | 2 | Originally described by Stang and co-workers, this approach deals with two basic structural requirements; (i) the complementary precursor units must be structurally rigid with predefined bite angles, and (ii) the appropriate stoichiometric ratio of the precursors must be used. The donor building blocks are usually organic/metalloorganic ligands with two or more binding sites possessing angular orientations ranging from 0° to 180°, and the acceptors are metal-containing subunits, usually structurally rigid with fixed pre-defined coordination sites. For example, a tetrahedron can be broken down into four 60° tritopic (blue) and six ditopic (red) 180° subunits, where the tritopic units represent the ligand on the edges, resulting a M4L6 tetrahedron. Conversely, a M6L4 tetrahedron can be generated from linear metal units (ditopic) on the edges and tritopic ligand on the vertices (Figure ). |
65f285c39138d231616bbedc | 3 | In this approach, the symmetry of the cage is defined by the geometric relationship between the metal and ligand, as presented by the coordinate vector, from the strong binding affinity and coordination mode of chelating ligands, along with the inherent symmetry of the coordination sites available on the naked metal centre acting as driving force for the assembly process. For example, in a M4L6 tetrahedron, the three coordinate vectors of each metal complex form a chelate plane containing a C3 axis and an angle of 70.6° is required between the coordinate vectors within each ligand to bridge the metal centres (Figure ). |
65f285c39138d231616bbedc | 4 | Pioneered by Fujita and coworkers, this approach involves the assimilation of square planar complexes with a 90° angle achieved by blocking two of the coordination sites with another cis-coordinating ligand (e.g. ethylenediamine) and 2D panels (e.g. triangles, squares, and pentagons) representing the organic ligands (Figure ).for example, a tetrahedron can be designed by stitching together four triangular panel, while an octahedron can be generated by bringing together eight such triangular panels (Figure ). |
65f285c39138d231616bbedc | 5 | The arrangement of individual components greatly influences the optical output in the resultant assembly, regardless of whether the supramolecular chirality results from the combination of chiral/achiral subcomponents. While achiral molecules in a mixture of chiral and achiral components can be forced into chiral assemblies under the influence of strong interaction between both, chiral molecules generally prefer to form distinct chiral structures with specified supramolecular chirality. On the other hand, assemblies with exclusively achiral molecules, can generate supramolecular chirality by their stereospecific arrangements in 3D space, but generally are racemic. Thus, proper characterization of the resulting assemblies to gain insight into their absolute configuration and enantiomeric excess (ee) is of utter importance. The direct observation of chiral morphology in supramolecular assemblies has become feasible due to the rapid development in sophisticated microscopic techniques, including scanning tunnelling microscopy (STM), atomic force microscopy (AFM), scanning electron microscopy (SEM) and transmission electron microscopy (TEM). In contrast, spectroscopic techniques such as Raman optical activity (ROA), circular dichroism (CD), and vibrational circular dichroism (VCD) offer insights into the direct stereochemical imprint. Although single-crystal X-ray structural analysis is one of the most viable methods of determining the absolute configuration and understanding the self-assembly process, obtaining appropriate single crystals of these assemblies is a potential problem. Herein, we emphasize on the CD spectroscopic analyses, a frequently employed technique for the assessment of chirality. |
65f285c39138d231616bbedc | 6 | Circular Dichroism (CD) refers to the differential absorption of left-vs. right-circularly polarized light. Since CD spectrum is recorded as a function of wavelength, if a molecule lacks a chromophore or if the absorption occurs outside the relevant wavelength range, it is not possible to detect any CD signal. Thus, CD spectroscopy is usually considered together with UV-vis absorption spectroscopy. CD spectroscopy is particularly valuable in the analysis of self-assembled systems due to its ability to provide insights into the formation dynamics, absolute configurations, molecular conformations, and even molecular interactions. Typically, a CD spectral measurement yields two distinct spectra, as depicted in Figure . In the CD spectrum, the occurrence of a peak or valley is referred to as the Cotton effect. This effect is characterized by a distinct alteration in the circular dichroism around an absorption band (l0) in the UV-vis spectrum, while the chirality of the supramolecular assemblies determines the sign of the CD. Mirror images should be observed if the enantiomers are measured, thus successful determination of the absolute configuration of the system and enantiomeric excess (ee) value, if both the enantiomers are present. From the coordination-driven self-assembly aspect, usually, the ligands consist of aromatic sidewalls, enabling the stereo-specific information of the system to be monitored through CD spectroscopy. For a quantitative understanding, herein we describe two cases, i) chiral induction or resolution by stereospecific interaction between a racemic host and chiral guest, and ii) stereochemical communication. |
65f285c39138d231616bbedc | 7 | 3.1.1. Chiral induction/resolution: if two chromophores, each from the host and the guest successively brought into proximity and the chiral group in the guest forces the chromophores to oriented in a chiral fashion, induced CD signals with the split-like cotton effects are generated, which might be used to assign the absolute configuration of the system based on the signs of the split Cotton effects (mechanism A, Figure ). On the other hand, when the host is racemic, and the chiral guest has different binding affinities to both the enantiomers, two enantiomers are induced to exist in different concentrations, accessed by a conformational selection mechanism by the guest (mechanism B, Figure ). However, for the relatively rigid hosts, upon binding to a chiral guest, a chiral conformation of the host is induced, which is otherwise no detectable in the free state (induced fit mechanism, mechanism C, figure ). 41 |
65f285c39138d231616bbedc | 8 | Originally developed by Aida and Yashima for the assembly of porphyrin aggregates and helical polymers, "chiral memory" employs the interesting aspect of generating a preferred configuration by using a chiral component in the system, which after complete removal and replacement by an achiral component "memorize" the earlier configuration, preserving the stereochemical information possessed by the system. This interesting aspect endows an interesting approach to generating amplified supramolecular chirality in a selfassembled structure, especially in the enantioselective preparation of MOC systems, as a combined effect of (i) cooperative communication between the metal centres, and (ii) relative arrangement of the ligands around the metal centres dictating its absolute configuration. |
65f285c39138d231616bbedc | 9 | In self-assembled systems, supramolecular chirality is produced by a plethora of non-covalent interactions. However, it is noteworthy that metal-coordination has emerged as a highly efficacious approach in this regard. This can be attributed to the inherent capability of coordination assemblies to manifest symmetrical organization, while simultaneously offering meticulous control over the attributes of shape (morphology), size (dimensions), and functionality. By capitalizing on the remarkable stability, diversity, and directionality of metalligand interactions, a wide array of coordination assemblies have been investigated to exploit both metal-and ligandcentred chiralities. For the purpose of designing stable enantiopure ensembles with potential applications in stereoselective molecular recognition and sensing, separation, stabilization of reactive species and as asymmetric reaction vessels, a quantitative understanding of the mechanisms underlying the processes that induce, maintain, switch, and relay chirality within assembled architectures is necessary. In general, chirality within the coordination architectures can be attained via two principal pathways, (i) by using the enantiopure building blocks; and (ii) employing achiral building blocks where the relative orientation of the ligands and coordination environment around the metal centres determine the overall stereochemical outcome. In addition, the introduction of an appropriate guest molecule within the coordination assemblies or the environment, i.e., presence of a chiral counterion or solvent sometimes offers a crucial approach to disrupt the symmetry of metallo-supramolecular architectures, resulting in their chirality. The domain of chiral metal-supramolecular assemblies has experienced substantial expansion in recent decades, giving rise to an abundance of noteworthy discoveries and phenomena. To provide a general insight into the recent progress of discrete MOCs, this paper presents a summary of notable and recent development in this area. The subsequent discussion is organized based on the aforementioned design strategies. |
65f285c39138d231616bbedc | 10 | As the enantiopure precursors predetermine the chirality of the coordination species, from the perspective of molecular symmetry, employing this approach leads to the MOCs, which are unlikely to racemise. Generally, in such an approach chirality is either induced by the spatial arrangement of the tetrahedrally or octahedrally coordinated metal centres (vertices-directed assemblies), or by optically pure organic ligands that are sustained by strong covalent bonds (edge-or face-directed assemblies). |
65f285c39138d231616bbedc | 11 | Using self-assembly from triangular pyridine ligand 1,3,5tris-[(4-pyridyl)ethynyl]benzene with [(R)-(+)-BINAP]Pd II / Pt II bis (triflates), Stang and colleagues first demonstrated the discrete chiral three-dimensional M6L4-type MOCs with Tsymmetry (MOC-1) in the year 1997. Notably, the absence of four faces removes the usual C4 and inversion center (i) needed for a regular octahedron with Oh space group; additionally, the chiral Pd II or Pt II metal corners also preclude the presence of mirror planes in order for Td space group. Consequently, T space group with well-defined chirality features is afforded to MOC-1 (Figure ) granted by the reserved 4C3 + 3I4 symmetry, and other chiral polyhedra with D3, I or O symmetry can also be realized from the same starting materials. Using a similar approach, the Fujita group has achieved the incorporation of chirality into the periphery of their well-known ''panelling" M6L4-type MOCs by substituting the ethylenediamine capping ligands with chiral diamine auxiliaries on the Pd(II) corners. 66 Wang and colleagues pioneered the diastereoselective synthesis of heterometallic enantiopure chiral cubic cages in 2015. They accomplished this by combining heterotopic ligands with stereomeric or racemic amines used in situ with Zn II and Pd II ions concurrently to produce heteronuclear cubic cages (MOC-2-3, Figure ). The chirality of the C* centers in the chiral amines can be transferred to all the Zn(II) vertices, resulting in the formation of O-symmetric homochiral cubic cages (MOC-2). On the other hand, the achiral amines lead to the formation of racemic assemblies of MOC-3. |
65f285c39138d231616bbedc | 12 | Through the assembly of [cis-(tmeda)Pd(NO3)2] acceptors with 1,4-di(1H-tetrazol-5-yl)benzene and [1,3,5-tri(1Htetrazol-5-yl)benzene], respectively, an interesting case of two aqua-soluble edge-and face-directed tetrahedral cages were generated, followed by a post-metalation process utilizing cisblocked Pd II . Follow-up work in this direction employed changing the central ligand core from benzene to naphthalene and biphenyl, to obtain water-soluble enantiopure assemblies (MOC-4-6) with larger cavities (Figure ). 69 MOC-6 with largest hydrophobic cavity showed a certain selectivity toward 2,2'-diethoxy-1,1'-binapthalene (EtB), where the L4 - MOC-6 showed a preference for R-EtB over S-EtB (75:25), attributed to the superior fit inside the chiral cavity (L/R pair), whereas D4 -MOC-6 preferred S-EtB over R-EtB with similar selectivity (D/S ratio = 73:27). For all these cases, the chiralities were derived from the spatial arrangement of the triazole linkers, which caused the final assemblies to adopt a specific geometry characterized by a loss of symmetry, and to be oriented in the same handedness (DDDD or LLLL) of the vertices, which ultimately conferred chirality. By employing this methodology, a pair of enantiopure Tsymmetric tetrahedral Fe4L6 cages (MOC-7) were synthesized through the utilization of a subcomponent selfassembly technique, as demonstrated by Nitschke et al. The chiral glyceryl group modification defines helical twists of the organic ligands on six edges of the tetrahedron, giving rise to three C2-symmetry axes (Figure ). Additionally, it dictates the stereochemistry of the handedness of the apical Fe II centers, resulting in same handedness for all four vertices. Gu and colleagues extended and modified the chiral auxiliary strategy by using chiral amine-modified organic ligands that were formed in-situ through a diastereoselective subcomponent self-assembly process. This led to the creation of chiral tetrahedral Fe4L6 cages, which demonstrated successful spin-crossover features and exhibited structural interconversion. |
65f285c39138d231616bbedc | 13 | A 3D polycage network, denoted as [Zn8L4I8], was generated by employing enantiopure pyridyl-functionalized metallosalalen units with Zn II iodide. This network was interconnected by pentahedral cages, which were created by neighboring pentamers. The amphiphilic helical cavity, adorned with chiral NH functionalities, can selectively interact with enantiomeric saccharides in solution, exhibiting enantioselectivity factors ranging from 2.480-4.943. In the solid state, it also demonstrates enantioselective fluorescence enhancement ratios of 1.30 to 3.60 when interacting with five enantiomeric amines. |
65f285c39138d231616bbedc | 14 | Lützen and co-workers exploited flexible BINOL-based bis(pyridine) ligands to assemble enantiopure endo-or exofunctionalized M 6 L 12 -type or M 12 L 24 -type cages (MOC-8-9), respectively, based on the position of the substitution on the BINOL core (with the exception of the edge-directed construction of chiral M 4 L 6 tetrahedral cages). 72 A subsequent investigation was undertaken to examine the analogous flexible BINOL-based ligand assembly with Pd II , with the anticipation of obtaining a homochiral Pd2L4 complex, However, instead, chiral Pd4L8 (MOC-10) assembly was obtained. Single-crystal X-ray diffraction analysis revealed a unique stretched tetrahedron structure, where four Pd(II) ions occupy the vertices. Among the eight ligands, four exhibit a twisted C-shaped configuration, while the other four adopt a W-shaped configuration. The unique arrangement of the assembly led to the formation of three cavities: two peripheral cavities accommodated two BF4 anions, while a central cavity with a hydrophilic nature was occupied by acetonitrile molecules (Figure ). Furthermore, a series of chiral metallacycles and cages were successfully synthesized by Stang and co-workers employing a chiral BINOL-based Recently, Nitschke group reported an enantiopure Fe4L6 cage (MOC-11), where the stereochemistry is defined by the incorporation of a chiral formylpyridine subcomponent (Figure ). Subsequent fullerene (C60) encapsulation and chemo-, regio-and enantio-selective Diels-Alder cycloaddition with the cage's anthracene panels resulted in the inherently chiral functionalization patterns of homo-and hetero-substituted C60 analogs. |
65f285c39138d231616bbedc | 15 | The first examples of chiral tetrahedral lanthanide MOCs with fascinating self-sorting behavior and characteristic Eu III luminescence features were described by Sun et al. in 2015. Two sets of enantiopure R-and S-ligands, i.e., bis(tridentate) L1 and tris(tridentate) L2 containing pyridine-2,6dicarboxamide units are devised, which after coordination with Eu III leads to Eu4(L1)6 and Eu4(L2)4, which are topologically chiral tetrahedral cages. During the process of self-assembly, the ligand's chirality is transmitted to the apical Eu III centers, resulting in either DDDDor LLLL-stereomeric tetrahedral configurations. Incorporation of an electron-deficient enantiopure ligand with Fe II afforded a Fe II 4L4 tetrahedron (MOC-12). This tetrahedron exists as a pair of distinct diastereomers adopting either an all Δ or Λ configuration of metal centers. However, encapsulation of an electron-rich chiral cryptophane-111 (CRY-A) dictated the diastereocontrol of the self-assembly process (Figure ). Whereas the Δ4 configuration was favoured by the MM-CRY, PP-CRY preferred the Λ4 configuration. |
65f285c39138d231616bbedc | 16 | The intriguing interplay of chirality, degree of chelation (denticity), and hydrogen bonding enables the diastereoselective formation of diverse asymmetrical and lowsymmetric architectures (MOC-13-16) through subcomponent self-assembly (Figure ). Enantiopure components bearing H-bond donors and acceptors (S-A) form M4L4 assemblies (MOC-13), whereas racemic mixtures give rise to M3L3 stacks (MOC-14). Chiral amplification was observed within MOC-14 enantiomers upon utilization of 2:1 ratio of R-and S-subcomponent enantiomers. By altering the solvent (from acetonitrile to methanol) or ligating unit (from bidentate 2-formylpyridine (P 1 ) to tridentate 2-formylphenanthroline (P 2 ), the range of structures that can be derived from these building blocks was expanded. This resulted in the selective formation of novel M3L2 (MOC-15) and M2L2 (MOC-16) assemblies. |
65f285c39138d231616bbedc | 17 | In a coordination-driven self-assembly process, diastereomeric coordination cages can arise from the stereochemical inversion of specific octahedral metal vertices, or less frequently, entire triangular faces (which may assume an anticlockwise or clockwise orientation). Diastereomers may also result from the handedness of ligand faces relative to that of octahedrally coordinated metal centers in face-capped tetrahedral cages. However, inevitable racemization in the self-assembly process while using achiral precursors, without any chiral source remains a formidable challenge. Self-assembly of 72 subcomponents produced a series of chiral 20-nucleii Co-imidazolate cages with a racemic tetartoidal (tetragonal pentagonal dodecahedron) structure. Chiral induction of the D-and L-enantiomers of menthol can yield both homochiral D and L cages chiral cages, respectively. The presence of the 2-methyl substituent on imidazolyl is essential for the development of a tetaroidal cage; in the absence of this steric effect, a cubic cage will result. |
65f285c39138d231616bbedc | 18 | The homochirality of the tetrahedral cages of general formula M4L6 can be attributed to the stereochemical coupling occurring between the metal centers, which is facilitated by the arylene rings comprising the cages' edges. In a study incorporating a series of C2-symmetric methylated pdiaminoterphenyls Nitschke group inferred that the diastereoselectivity of such self-assembly process is highly dependent on the substituents of the bridging edges, and the type of conformations they impose upon metal-coordination. Utilizing diamine ligands which preferentially adopt antiarrangement results in the preferential generation of homochiral T-symmetric cages. Conversely, ligands that adopt a syn arrangement tend to favour the formation of S4symmetric cages. |
65f285c39138d231616bbedc | 19 | In 2019, Nitschke group reported the exclusive formation of a pair of T-symmetric truxene-derived Zn4L4 tetrahedral enantiomers. These enantiomers have faces and vertices which possess a single stereochemical orientation. The ethyl chains that surround the inner cavity allow the smallest hydrocarbon guests (CH4 and C2H6) in solution to bind and may also deform easily enabling gaseous guests to diffuse into the cavity. |
65f285c39138d231616bbedc | 20 | Depending on the arrangement of the panels around the cage; the diastereomers obtained thereafter can result in contrasting guest-binding affinities to the hosts. While the cage constructed using a 1,6-pyrene scaffold offers an adequately isolated microenvironment for effective binding for large aromatic (coronene, perylene, pyrene triphenylene) and hydrophobic guests (C60 and C70), the 2,7-pyrene-edged cage fails to encapsulate these neutral guests due to its insufficient enclosure, which limits its ability to confine potential guests within its cavity. |
65f285c39138d231616bbedc | 21 | Recently, the spontaneous resolution of a racemic M6L8 octahedral cage (MOC-17) with three types of chirality has been reported in the absence of any external resolving agents (Figure ). As a prochiral moiety, once the free flip of the truxene core is locked after metal coordination, planar chirality of the consequent cage is determined based on the different orientation of the truxene core, i.e., anticlockwise (A) or clockwise (C). This is in addition to the axial chirality resulting from the R-or S-configuration achieved by twisting pyridyl groups and propeller chirality derived from the L and D arrangement of Py around the Cu II ions. For the purpose of investigating the intricate guest binding behavior, rational design of low-symmetry host systems that possess inherent conformational adaptability is utterly important. Subcomponent self-assembly of tritopic aniline subcomponent (A) with 3-nitropiconaldehyde (B) and Fe(NTf2)2 generated a Fe II 4L4 cage of S4 symmetry (MOC-18), in which all the Fe centers exhibit a mer stereochemical configuration, with equal distribution of D and L handedness (Figure ). In contrast, using 6-methyl-2formypiconaldehyde (C), a high-spin Fe II 3L2 sandwich species (MOC-22) has been generated, with the HS character resulting from the steric conflict between methyl groups and the neighbouring pyridyl rings around the Fe centers. Interestingly, treatment of the S4-symmetric MOC-18 with MM-CRY resulted in the formation of a T-symmetric cage (MOC-20), with all the metal centers adopting fac-D, whereas PP-CRY resulted in the isomers with fac-L metal vertices. On the other hand, optically active D-TRISPHAT housed inside the cavity resulted in the cage (MOC-21) with two C3symmetric diastereomers, with the major having a 3:1 fac:mer metal centre configuration. This unique example elucidates a case where the enantiopure guest molecule can lead to different stereoisomers of the host. |
65f285c39138d231616bbedc | 22 | For the cases involving enantiopure ligands, metal-organic assembly usually yields a single diastereomer, with diastereoselectivity of the self-assembly process minimally influenced by external variables. Different guestbinding preferences are shown by diastereomeric cages due to variations in the size and enclosure of the cavities or the arrangement of hydrogen bond donors. Nitschke group reported that, by simply changing the solvent, a single enantiopure porphyrin-based ligand was able to self-assemble with octahedral metal ions to form two different diastereomers of a M II 8L6 (MOC 22, M = Zn II or Co II ) cage, with the metal centers taking on a preferred Δ or Λ configuration (Figure ). In acetonitrile, the major diastereomer was assigned as (S)24-L8, with a d.r. of 3.8:1, whereas the diastereomer with opposite metal handedness, (S)24-D8, developed preferentially in nitromethane, with a d.r. of 1:6, which was further confirmed by CD spectroscopic analysis. In the presence of a chiral template guest D-TRISPHAT (G), which possesses a configuration that complements the propeller-shaped pentapyrrole backbone, a truncated rhombohedral structure Zn II 15L6 (MOC-23) was formed exclusively (Figure ), inferred to have a structure similar to its Co II congener. Comparing the average dihedral angle of ~82° (between the pentagonal faces along a common edge), with the dihedral angle of ~88° (between two tetrachlorocatecholate groups of G), stereoselectivity of the cage formation process was confirmed through CD spectroscopic analysis. Although the handedness of the Zn vertices could not be confirmed due to the absence of MLCT (metal-to-ligand charge transfer) transitions, a similar CD spectrum for its Co congener inferred that the Zn vertices possess D handedness. Thus, G not only served as a template to stabilize the otherwise disfavoured Zn II 15L6 but also controlled the enantioselectivity in the self-assembly process. Interestingly, in absence of such chiral modifier, the dodecahedron Zn20L12 (MOC-24) transforms to give a larger Zn30L12 icosidodecahedron (MOC-25) through subcomponent exchange, or an unprecedented pseudotruncated octahedral structure Zn20L8 (MOC-26). Apart from the nature of the subcomponents present in the reaction medium, their concentration can also be used to govern the self-assembly outcome of the system. The selective formation of a Fe12L12 icosahedron, a Fe2L3 helicate and Fe4L4 tetrahedron occurred from the same triazatriangulenium component in CH3CN with the reaction concentrations of 4.4 mM and 2.2 mM, respectively for the icosahedron and helicate. Interestingly, when larger anions like CB11H12 -or B12F12 2-were added to the helicate solution, complete conversion into tetrahedron Fe4L4 was witnessed, where the larger anions act as the templates to bring four cationic ligands together, overcoming inter-ligand Columbic repulsions. |
65f285c39138d231616bbedc | 23 | Exploiting the inherent molecular cavities of the metallosupramolecular cages, the breadth of applications in this field of research is too broad to cover comprehensively. However, chiral molecular separations (enantio-separation) employing the non-covalent interactions and functionalities of the host molecules present a formidable yet intriguing challenge. As Enantioselective separation of atropisomeric molecules, such as R/S-Binol and R/S-spirodiol, was accomplished by the Su group utilizing enantiopure Pd6(RuL3)8 (MOC-27) octahedra D-3 and L-3 (Figure ). These enantiomeric cages were produced via self-assembly of the pre-resolved chiral metalloligands (based on Ru II ) and Pd(BF4)2 and encapsulated >10 guests per cage. Extraction of these atropisomeric guests suggested a stereoselectivity of D-3 for R-isomers and L-3 for S-isomers, which ultimately led to D-3 resolving R-Binol with an ee of 34%. However, changing the inert Ru II to more labile Fe II prevented the ligand resolution before the cage preparation (Figure ). The structural analysis discloses that each cage in the absence of any chiral factor led to equal amounts of Dand L-FeL3 2+ to form FeL3 2+ racemate, which after stereoselective secondary assembly with Pd II resulted in the equivalent homochiral D8 and L8 cage (MOC-28) as racemic mixture. However, when R-Binol was present during the process of self-assembly, rapid precipitation of the reaction mixture and slow crystallization resulted in metal cages with opposite absolute stereochemistry. precipitation of the reaction mixture and slow crystallization resulted in metal cages with opposite absolute stereochemistry. As molecular recognition and separation entirely depend on the specific non-covalent interactions between the host and guest, it is rather necessary to emphasize on the specific functionalities of the ligand backbone. In 2020, the Mukherjee group reported that the presence of a chiral modifier like R-or S-Binol induced a dynamic mixture of different stereoisomers (MOC-30, Figure ) into different enantiopure octahedral cages, in a process called "reverse chiral recognition", where the handedness of the host is determined by the guest molecule. Likewise, Nitschke group emphasized on the hydrogenbonding capability of the dialdehyde component as a determining factor in the stereochemical outcome of a selfassembly into one homochiral (LL) cage, compared to the heterochiral meso component (LD) in the case of its methylated congener. Similarly, the template effect of the hydrogen bonding donor R/S-Binol or R/S-Spol induced the stereoselective self-assembly of D4or 4-type Eu4L4 cages, whereas the non-covalent confinement effect of pyrene led to a racemic mixture of enantiopure cages. Structural adaptability due to the reversibility of the coordinative bonds, 107-109 cage-in-cage complexes, can be used to confine small molecules like methane or xenon, in addition to cations and anions. In 2019, Nitschke group reported that the handedness of the tetrahedron host framework of a Fe4L4 8+ capsule (MOC-31), obtained from a triazatruxene-based subcomponent, is fixed by the enantiopure cryptophane-111 (CRY) stereochemistry, i.e., racemic mixture of PP-CRYÌD4-MOC-31 and MM-CRYÌL4-MOC-31 was formed. 111 Interestingly, prolonged heating of a mixture of MM-CRYÌL4-MOC-31 and MM-CRYÌD4-MOC-31 converted the mixture to MM-CRYÌD4-MOC-31 (Figure ), which dictates that the diastereomer interconversion involves a high energy barrier and a notable extent of cage disassembly and reassembly. Furthermore, upon the displacement of the chiral CRY by the achiral fullerene C60 within MOC-31, the pronounced cotton effect observed in the MLCT region indicates that the retention of stereochemical memory arises from the structural integrity of the complete tetrahedral framework. The interconversion between the diastereomeric forms is further pronounced for the Zn congeners of MOC-31. Based on either anticlock-or clockwise orientations of the triazatruxene moieties within the faces of MOC-31, each trischelated octahedral vertex of the tetrahedron might assume either L or D handedness. At 25°C, the distinct diastereomeric pairs (C4D4/A4L4, T1) and (A4D4/C4L4, T2) were obtained in the ratio of 66:34, which changed to 17:83 at 80°C and reversed back by cooling for 12 hours. Interestingly, the optimized structures revealed that T2 has larger pores than T1, and can house larger guests like calix [4]arene compared to the smaller guest like adamantane. Altogether, these examples show a stimulus-directed breathable system, which can be in the context of switchable catalytic systems. Notably, employing the spherical cavity of the parent cage MOC-31, and the terminal triethylene glycol functionalization enabled a reversible phase transfer strategy in D2O/CD3NO2 biphasic medium via anion metathesis, thereby enabling the purification of a broader spectrum of polyaromatic hydrocarbons. Employing the chiral auxiliary synthesis approach, by using enantiopure R/S-1-phenylethylamine or R/S-1cyclohexylethylamine afforded the enantiopure MOC-32. The arrangement of the triazatruxene moiety in this compound is dictated by the capacity of the amines to induce single handedness at the metal vertices through steric influences. Interestingly, these enantiopure hosts were found to exhibit excellent enantio-and diastereoselectivity between spironolactone (SP) and its epimer (ESP). Although they only differ in one stereogenic center, the remarkable diastereoselectivity of D4-MOC-32 and L4-MOC-32 evokes the ability to distinguish two epimers, displaying relative affinity ratios of 4.2:1 and 1:100, respectively (Figure ). Due to their highly twisted appearance, helicene-based subunits can be utilized to construct distinct coordination cages that demonstrate intriguing effects driven by chirality on their assembly. In 2018, Clever and co-workers reported the first example of a series of homochiral Pd2L4 cages (MOC-33-34), in addition to the interpenetrated Pd4L8 dimer (MOC-35), made up of eight interlocked helicenes. While rac-L 1 exclusively generates MOC-33 meso incorporating both L 1 P and L 1 M in a 1:1 ratio, rac-L 2 leads to a statistical mixture of all possible stereoisomers (PPPM/MMMP/PPMM/MMPP/PMPM/PPPP/MMMM) of MOC-34, shown in Figure . However, using enantiopure ligands, the stereochemical information of the self-assembled cages was determined by the ligand's original conformation. While the limited size of MOC-34 restricted the uptake of any 1-R-/S-camphorsulfonate anions (G1), MOC-35 showed chiral discrimination between G1 R and G1 S , along with a higher binding affinity for its diastereomeric combinations. Combination of a fluorene-backbone with emissive properties with helicene-based homochiral ligand resulted in a series of multifunctional Pd2L2L ' 2 heteroleptic cages (MOC-36) (Figure ). 118 These cages exhibit chiroptical properties that arise from the cooperative interactions between all the components. The homochiral helicene-based ligands introduce a twist to the overall structure, transmitting chiral information and leading to circularly polarized luminescence (CPL) emission from the achiral fluorene-based ligand, which is further pronounced by binding an aliphatic bis-sulfonate ligand. |
65f285c39138d231616bbedc | 24 | The class of structurally and functionally diverse MOCs that are easily assembled through coordination-driven self-assembly is particularly intriguing due to their chiroptical characteristics, distinct chiral phenomena, and chemical tunability. The examples presented in this review showcase the potential of rational chemical manipulations in exploring large and complex metal-organic cages (MOCs) as mimics of biological systems. These MOCs can be utilized in various applications, ranging from sensing to catalysis, thanks to the generation of subtle chiral microenvironment created via coordination. However, there remains a substantial gap in our understanding of the mechanisms underlying the effects of chirality and the most effective approaches for exploring resulting biochemical and biomedical applications. |
65f285c39138d231616bbedc | 25 | Despite notable advancements in the rational design of well-defined chiral metallosupramolecular cages, there are still many unknowns related to the development of lowersymmteric Archimedian solids, as well as the achievement of globally chiral supramlecules. Additionally, the development of heteroleptic architectures remain a formidable challenge, as narcissistic self-sorting stands as a potential problem while ensembling multiple different molecules in a cooperative manner into a single integrated system. |
65f285c39138d231616bbedc | 26 | It is evident that the study of chiral MOCs will remain a dynamic field of research and a crucial facet of supramolecular chemistry and materials science in the future. At the fundamental level, rational design of the chiral building blocks with precise positioning of functional groups is necessary to achieve the preparation of functional MOFs. Additionally, the topic of inducing chirality in MOCs by the assimilation of achiral building blocks is emerging, and while its promise is just beginning to be shown, a major barrier still stands in the way due to the lack of appropriate generic chemical manipulations. |
65f285c39138d231616bbedc | 27 | On the application level, the ease of excellent solution processability confers to the possibilities to be explored in biological applications, including trans-membrane transport, biological imaging and drug delivery. Overcoming biocompatibility, water solubility, and retention of structural integrity in a complex biological environment are the major obstacles, though. On the other hand, manipulations at the molecular level to tune the emissive properties and their effect on the enantiopure environment is a compelling way to address the challenges related to the development of chiroptical materials in the field of circularly polarised luminescence. In a more ambitious manner, it would be utterly captivating to explore the molecularly dispersed materials in order to fabricate chiral MOC-embedded membranes or thin films, with the ultimate objective of tackling the formidable task of enantio-separation through membrane-based techniques. It is foreseen that endeavors shall persist in the intensification of exploration into the vast expanse and aesthetic allure of metallo-supramolecular chemistry, thereby enabling synthetic chemists to fabricate materials of heightened utility for practical applications. |
65a6f6f3e9ebbb4db9510a74 | 0 | Differences in pH within the cellular environment are associated with several cellular processes. Examples include the intracellular acidification associated with apoptosis; 2-3 reduced protein glycosylation that results from impaired acidification of the Golgi compartment; 4 the pHdependent regulation of synaptic activity in neurons. Intracellular pH varies by from 0.3 to 0.5 units during the process of mitosis. Intracellular pH has a large role in the activity of many proteins, including enzymes that exhibit pH dependencies on catalysis, providing clear connections with the associations between pH and cellular processes. Mutations can affect an enzyme's pH dependency in several ways, and the potential effects of disease-associated mutations could arise from the resulting dysregulation of key enzymes in the cell. |
65a6f6f3e9ebbb4db9510a74 | 1 | The straightforward view is that the pH dependency of enzymatic catalysis arises from the pKa values of catalytic residues taking part in the chemical steps. However, the pH dependency can vary between enzymes in the same family even when they share identical catalytic residues due to several factors that cause the kinetic pKa values displayed in a pH-rate profile to be perturbed from their intrinsic, thermodynamic values. For example, differences in the local environment can alter the pKa of catalytic residues. Additionally, the kinetic pKa values reflected in a pH-rate profile are often distorted from the intrinsic, or thermodynamic, pKa values of catalytic residues. For example, we recently showed that a point mutation to a noncatalytic residue in the protein tyrosine phosphatase (PTP) YopH does not significantly affect its maximal turnover number, but broadens its pH-rate dependency, making the variant a much faster enzyme at low pH than the native enzyme. A computational analysis showed this catalytic change arose from mutation-induced changes in conformational equilibria. Point mutations can also alter the pKa of neighboring residues by altering the electrostatic environment, or, by inducing changes in hydrogen bonding and solvation networks. Because of such possibilities the effect on enzymatic catalysis of any mutation is better assessed by assaying rate across a pH range rather than at a single pH. Here, we report findings from a kinetic and computational investigation of point variants of another PTP, SHP-1, that were reported to exhibit faster catalysis than the WT at pH 5. The catalytic activities of PTPs have bell-shaped pH-dependencies and show optimal activity within a narrow range of pH. The bell-shaped pH-rate profiles result from a mechanism that requires specific protonation states of two conserved catalytic residues, an aspartic acid and a cysteine (Figure ). The first step of the PTP mechanism requires the protonated form of the aspartic acid and the deprotonated cysteine nucleophile. Conformational changes of the PTP active site also contribute to activity and the formation of the catalytically competent enzyme-substrate complex (Scheme 1). Classical PTPs exhibit two major conformers that differ in the major orientation of a solvent accessible loop motif, the WPD-loop. In substrate-bound classical PTPs a WPD-loop closed, catalytically active conformation predominates, while in the free enzymes the WPD-loop is primarily found in an open, catalytically nonproductive conformation. Catalysis in PTPs has been correlated with protein motions. In particular, the dynamics of the WPD-loop involving the chemical step, and the variability of these dynamics with pH, can affect the populations of catalytically active enzyme-substrate complexes. Mutations that affect loop equilibrium can alter the pH-rate profile of catalysis in PTPs. 9 The WPD-loop is shown in orange, and the P-loop is shown in green. Reproduced from Ref. with permission from the Royal Society of Chemistry. Originally published under a CC-BY-NC-CD license. |
65a6f6f3e9ebbb4db9510a74 | 2 | SHP-1 plays a critical role in human immune systems, especially in regulating B-cell and Tcell signal transduction pathways. Mutations and impaired SHP-1 activities have been linked to the murine motheaten disease and Familial hemophagocytic lymphohistiocytosis, featuring SHP-1 as an intriguing research target for clinical studies. The phosphatase activity of the full- length SHP-1 is autoinhibited at rest by the insertion of an SH2 domain to its active site, while the enzyme resumes the catalytically active conformation upon cellular stimulation. Similar to two of the most characterized PTPs, YopH and PTP1B, SHP-1 contains a mobile WPD-loop, and a static P-loop. The overall secondary structure of SHP-1 is highly superimposable to that of PTP1B according to X-ray studies, especially the signature active site motifs, the WPD-loop and the Ploop. |
65a6f6f3e9ebbb4db9510a74 | 3 | X-ray data indicate that SHP-1 shares a mobile WPD-loop with other classical PTPs. It has been inferred that SHP-1 and other PTPs with mobile WPD-loops exhibit the same correlation between loop dynamics and catalysis as seen in PTP1B and YopH. This study investigated the pH-dependent enzymatic activities of several point variants of the catalytic subunit of SHP-1 that were reported to exhibit elevated turnover rates and efficiencies at pH 5 in solutions containing 40% glycerol. Glycerol raises both kcat and KM of SHP-1 catalysis and increases susceptibility to proteolysis, effects tentatively ascribed to relaxation of structural components. We were interested in whether these variant SHP-1 proteins had higher activities in general and how their pH-dependencies of catalysis compared to the native enzyme. Our kinetic studies were carried out in aqueous solution, more reflective of biological conditions, and to avoid complicating effects of glycerol. Glycerol is a potent viscosigen, and the co-solvent will also affect pH by altering the pKa of buffering species, and affect the pKa and nucleophilicity of catalytic residues. These kinetic studies are supported by molecular dynamics simulations of wild-type and variant SHP-1 in the unliganded and phosphoenzyme intermediate states, to explore the impact of the substitutions on the dynamics of the WPD-loop. Curiously, our computational results show minimal impact of the mutations on loop dynamics, suggesting that the observed shifts in pH-dependency are due to chemical effects (primarily through modulating the pKa of the catalytic aspartic acid), in contrast to YopH where altered pH-dependencies were shown to be due to altered dynamics of the WPDloop. 9 |
65a6f6f3e9ebbb4db9510a74 | 4 | Dithiothreitol (DTT), kanamycin monosulfate, lysozyme, and ampicillin (AMP) were purchased from GoldBio. Protease-inhibitors aprotinin, pepstatin A, and leupeptin were purchased from Sigma-Aldrich. HisPur™ Ni-NTA Resin was purchased from Thermo Fisher. All other buffers and reagents were purchased from Sigma-Aldrich or Thermo Fisher. The substrate pnitrophenyl phosphate (pNPP) was synthesized using a published method. |
65a6f6f3e9ebbb4db9510a74 | 5 | The DNA was transformed into BL21-DE3 cells and grown overnight at 37 °C on an LB culture plate containing 100 ng/µL kanamycin monosulfate for WT SHP-1, or 100 ng/µL ampicillin for the SHP-1 variants. One colony was selected and placed into 10 mL of SOC media containing 100 ng/µL kanamycin monosulfate or ampicillin and grown overnight. The following morning, 1 L of LB media containing 100 ng/µL kanamycin monosulfate or ampicillin was inoculated with the 10 mL of overnight growth and shaken at 170 rpm at 37 °C until the OD600nm reached 0.6-0.8. After the optimal OD was reached, the 1 L growth was induced by 0.1 mM isopropyl β-d-thiogalactoside (IPTG) and shaken at 170 rpm at room temperature overnight. The cells were harvested by centrifugation at 12,000g for 30 min at 4 °C and stored at -80 °C. |
65a6f6f3e9ebbb4db9510a74 | 6 | WT SHP-1 cells were thawed on ice and resuspended in 10 mL equilibration buffer, consisting of 25 mM Tris pH 8.0, 1 mM DTT, and 200 mM NaCl with 2 mg lysozyme, 0.5 mg/mL aprotinin, 0.7 mg/mL pepstatin A, and 0.5 mg/mL leupeptin. SHP-1 variant cells were thawed and resuspended in 50 mM BisTris pH 6.5, 1 mM EDTA, 3 mM DTT, and 10% glycerol with the same protease inhibitors. The cells were lysed by sonication at 70% amplitude for 30 seconds and then mixed on ice for 1 min and repeated 5-6 times until completely lysed. The cell lysate was centrifuged at 4 °C at 30,000g for 30 min. |
65a6f6f3e9ebbb4db9510a74 | 7 | The WT SHP-1 lysate was purified via a 1 ml HisPur™ Ni-NTA column. Cleared lysate was applied to the column resin, followed by a wash step with 10 column volumes of equilibration buffer and 10 column volumes of wash buffer, consisting of 25 mM Tris pH 8.0, 1 mM DTT, 200 mM NaCl, and 20 mM imidazole. Elution for WT SHP-1 was processed using 15 mL elution buffer containing 25 mM Tris pH 8.0, 1 mM DTT, 200 mM NaCl, and 500 mM imidazole. Eluted fractions were tested with pNPP for phosphatase activity. Fractions that showed activity were pooled and dialyzed in 2L equilibration buffer with 0.5 mg TEV protease overnight at 4 °C with gentle stirring. Dialyzed and untagged SHP-1 was applied to a second Ni-NTA column, followed by 10 column volumes of equilibration buffer and 10 column volumes of wash buffer. The flowthrough containing untagged SHP-1 was pooled and concentrated to <12 mL, loaded onto a preequilibrated HiLoad 26/60 Superdex 200 prepgrade column (GE), and purified with 25 mM Tris pH 8.0, 150 mM NaCl, 0.5 mM TCEP. Fractions were assayed with pNPP for activity and purity on a 15% SDS-PAGE gel. Pure protein was concentrated to 10-20 mg/mL, diluted with 10% glycerol, frozen with liquid nitrogen, and stored at -80 °C in aliquots. |
65a6f6f3e9ebbb4db9510a74 | 8 | All SHP-1 variants, SHP-1 H422Q, SHP-1 E427A, and SHP-1 S418A were purified via a 5 mL HiTrap Q HP column attached above a 5 mL HiTrap SP HP column using an FPLC filtration system. Both columns were equilibrated with lysis buffer. The cell lysate was loaded onto the columns, the HiTrap Q HP column was removed after loading, and the HiTrap SP HP column was washed with lysis buffer until the absorbance at 280 nm was baselined. Elution for SHP-1 variants was processed using a 100% gradient with elution buffer containing 500 mM NaCl, 50 mM BisTris pH 6.5, 1 mM EDTA, 3 mM DTT, and 10% glycerol. Eluted fractions exhibiting absorbance at 280 nm were collected and tested with pNPP for phosphatase activity. Fractions that showed activity were assayed for purity on a 15% SDS-PAGE gel. |
65a6f6f3e9ebbb4db9510a74 | 9 | The active fractions were pooled and concentrated to <12 mL, loaded onto a pre-equilibrated HiLoad 26/60 Superdex 200 prepgrade column (GE), and purified using 10 mM BisTris buffer pH 6.5, with 25 mM NaCl, 0.2 mM EDTA, and 3 mM DTT. Fractions were assayed with pNPP for activity and purity on a 15% SDS-PAGE gel. Pure protein was concentrated to 10-20 mg/mL and either kept on ice to immediately set up crystal trays or diluted with 10% glycerol and frozen with liquid nitrogen and stored at -80 °C in aliquots. |
65a6f6f3e9ebbb4db9510a74 | 10 | Steady-state kinetic parameters were measured at 25 °C. Concentrated protein aliquots were thawed on ice and diluted with a buffer base mix (BBM) containing 50 mM sodium acetate, 100 mM Tris, and 100 mM bis-Tris from pH 4.0 to pH 7.5. This buffer system maintains constant ionic strength throughout the pH range examined. A 50 mM solution of pNPP was prepared in the buffer base mix. The reactions were run on a 96-well plate using substrate concentrations from 0.76 to 10.61 mM. Reactions were allowed to proceed for 2 -8 min for WT SHP-1 and the SHP-1 variants. |
65a6f6f3e9ebbb4db9510a74 | 11 | The reactions were quenched using 50 μL of 2.5 M NaOH, and the amount of the product pnitrophenol was assayed from the absorption at 400 nm using the molar extinction coefficient of 18,300 M -1 cm -1 . Reaction blanks were made using identical conditions replacing the enzyme with buffer to correct for non-enzymatic hydrolysis of the substrate. The amount of product released and elapsed time were used to calculate initial rates. These data were fitted to the Michaelis-Menten equation to obtain steady-state kinetic parameters. Kinetic data were obtained on both variants as a function of pH to obtain pH-rate profiles which were fitted to Eqs. 1 and 2. In Eq. 2 KS2 was set to the second ionization constant of the substrate pNPP (pKa = 4.96). These equations relate the dependence of the observed values of kcat, or kcat/KM, to their maximal, or limiting, values as a function of pH, where catalysis is dependent on two ionizable enzymatic residues, one protonated and the other deprotonated. |
65a6f6f3e9ebbb4db9510a74 | 12 | Molecular Dynamics (MD) simulations were carried out using the GROMACS simulation package v. 2022.05, together with the ff14SB 29 force field and TIP3P water model for the parameters of protein atoms and water molecules, respectively. We used two initial crystal structures (PDB ID: 4HJP Parameters for the phosphoenzyme intermediate were obtained from prior work, see the Supporting Information of ref. . Point mutations were introduced into the wild-type structures using PyMol. Residues 315 to 317 in PDB ID: 4HJP were missing from the crystal structure, and were manually introduced by overlay with PDB ID: 4GRZ. Missing residues at the N-and C-termini were omitted from our simulations (Gly241, Ser242, Val525 and Gln526 in both structures, and Gln527 and Ser528 in PDB ID: 4GRZ 31 ). PROPKA 3.0 was used to check for anomalous pKa values for ionizable residues, and, based on this evaluation all systems were kept in their standard protonation states at physiological pH. The protein was then placed inside a truncated octahedral water box of TIP3P water molecules, extending 10Å from the solute in all directions, and the system was neutralized with Na + counterions. |
65a6f6f3e9ebbb4db9510a74 | 13 | Energies were minimized using the steepest descent algorithm. The system was heated to 300 K over 100 ps using velocity rescaling. This was followed by 100 ps NPT production at 300 K and 1 atm, using a Parinello-Rahman barostat. For each system, 8 replicas of 1.5 µs long MD simulations were carried out, for a total of 12 µs for each of the 16 systems, and 192 µs total simulation time over all systems. The convergence of the simulations can be seen in Figures S1. |
65a6f6f3e9ebbb4db9510a74 | 14 | MD simulations were run with a time step of 2 fs (by making use of the SHAKE algorithm for constraining hydrogen bonds ) and a cut-off 10 Å for non-bonded interactions, while long-range electrostatics were calculated with the particle-mesh Ewald method. Temperature and pressure were controlled by means of the modified Berendsen thermostat (with velocity rescaling 36 of 0.1 ps) and a Parinello-Rahman barostat (with pressure fluctuations at equilibrium every 2 ps). All simulation analyses were carried out using CPPTRAJ and MDAnalysis based on snapshots saved every 50 ps of MD simulation time. |
65a6f6f3e9ebbb4db9510a74 | 15 | Empirical valence bond (EVB) simulations of wild-type SHP1 and variants were performed at the phosphoenzyme intermediate preceding the rate-limiting hydrolysis step, using the same protocol as in prior work, using the OPLS-AA force field, and the TIP3P water model. PDB ID 4GRZ was used to describe the closed catalytic state, while the different variants were generated in silico by truncation of the corresponding side chains or substitution for the highest probability rotamer using the Dunbrack 2010 Rotamer Library. A list of ionized residues in our simulations (necessary for describing the system using the Surface Constrained All Atom Solvent Model, SCAAS, (see prior work ) is found in Table . All simulations were performed in 30 individual replicates per system, with each trajectory involving an initial 30 ns of equilibration followed by 10.2 ns of EVB simulation (51 EVB mapping frames x 200 ps simulation time per frame), as in prior work, to a total simulation time of 1.206 μs per system and 4.824 μs cumulatively across all systems. Convergence of the EVB equilibration runs is shown in Figure . The EVB mapping parameters shown to obtain calculated activation free energies are identical to those used in prior work. |
65a6f6f3e9ebbb4db9510a74 | 16 | A previous kinetic study of a set of SHP-1 alanine variants reported higher phosphatase activities than the WT enzyme, speculated to arise from increased WPD-loop flexibility and stability. One of these mutations was to the residue immediately following the general acid D421, and it was speculated that the side chain length of the D+1 residue affected the catalytic rate by altering the dynamic rate of the WPD-loop. There is precedent for a mutation in the D+1 position affecting turnover by altering WPD-loop dynamics. In the D+1 position in PTP1B, the mutation F182Q results in a decrease of about an order of magnitude in kcat with no shift in the pH optimum but a broader maximum than the native enzyme, and retention of the basic limb. Because of the effect on the pH dependency of catalysis in that case, we sought to investigate some of the previously reported SHP-1 variants across a pH range to more broadly examine the effect of the mutations. We collected and compared kinetic data for the catalytic activities of three SHP-1 variants, S418A, H422Q, and E427A, with those of WT-SHP-1. Residues S418 and E427 reside on the N-terminal and the C-terminal hinge of the WPD-loop, respectively, and H422 is the D+1 residue. |
65a6f6f3e9ebbb4db9510a74 | 17 | Figure shows the pH-dependent catalytic activity with the substrate pNPP for WT SHP-1 and the variants H422Q, E427A, and S418A. The parameter kcat/KM reflects the part of the overall mechanism up to and including the first irreversible step, formation of the phosphoenzyme intermediate, the first step in Figure . The overall rate-determining step in the SHP-1 reaction, reflected in kcat, is the hydrolysis of this intermediate, in which D421 acts as a base to deprotonate a nucleophilic water molecule. The pH dependency of kcat/KM is broader in the variants, reminiscent of the variability of pH dependencies within the PTP family. For example, YopH catalysis shows a narrower pH profile for compared to PTP1B. These PTPs share catalytic residues, but differ in residues neighboring the catalytic ones and within their WPD-loops. In the present case, the broader pH profile reflects the fact that the E427A variant has higher catalytic efficiency at low and high pH. The acidic limbs of the kcat pH-rate profiles and the maximal activities showed modest changes between the variants and the native enzyme, but the basic limbs show more significant effects. Each of the variants has higher turnover numbers above pH 6 than the native enzyme. At pH 7, H422Q and E427A have turnover numbers approximately 5-fold higher than WT SHP-1. |
65a6f6f3e9ebbb4db9510a74 | 18 | Focusing on kcat, which reflects the rate-determining step in turnover, the variants show alterations in the pH optimum, in their pH-independent limiting kcat values, and the kinetic pKa values obtained from fits to Eq. 1 (Table ). The kinetic pKa values for the aspartic acid for the variants are each ~1 unit higher than the WT, reflected in their broader pH-rate profiles. The native SHP-1 has a kinetic pKa of the aspartic acid of 5.6, while in the variants, it is increased to 6.9, 6.7, and 6.5 for H422Q, E427A, and S418A, respectively. Though the variants and WT have similar activities at their optimal pH, all of the variants have faster turnover at pH > 6. In contrast, the pH-independent limiting kcat values show minimal difference between the different enzyme variants, an observation also supported by empirical valence bond simulations (Table ). For comparison, Table lists the previously reported kcat values obtained in acetate buffer at pH 5.0 with added 40% glycerol. The pKa of carboxylic acid moves higher in nonaqueous solvents and in cosolvent mixtures. The pKa of acetic acid in glycerol/water mixtures has not been reported to our knowledge, but is increased by a full unit in 50% ethanol. It is possible that the aqueous glycerol conditions were at an effective pH of 6 or above, in the range where our aqueous solution kcat values for the variants are higher than WT (Figure a The pKa1 value arises from the cysteine nucleophile and pKa2 from the aspartic acid. Shown here for comparison are reported kcat values (s -1 ), in 40% glycerol solution, pH 5. 10, 25 b Data from ref. . c Data from ref. . d Calculated activation free energies, in kcal mol -1 , presented as average values and standard error of the mean across 30 independent EVB trajectories propagated for each system, performed as described in the Materials and Methods. |
65a6f6f3e9ebbb4db9510a74 | 19 | Point mutations on noncatalytic WPD-loop residues in PTPs rarely affect the secondary structures of the active sites, however, differences in the WPD-loop dynamics are likely to be observed. 9, 14 A previous study of point variants of corresponding residues on the WPD-loops of PTP1B and YopH demonstrated that a single mutation changes the pH-dependency of catalysis, loop dynamics, and loop equilibrium. Residues S418, H422, and E427 are located on the Nterminal, central portion, and C-terminal of the WPD-loop in SHP-1. Although SHP-1 has a modest difference in the backbone positioning of its WPD-loop compared to YopH and PTP1B (Figure ), residues S418, H422, and E427 are likely to experience, as well as affect, significant conformational dynamics between loop closed and open forms. In PTP1B, the central portion of the WPD-loop and residues E186 and S187 exhibits relatively high mobility compared to the rest of the loop. Due to the high sequence identity and structural similarity of the WPD-loop of SHP-1 and PTP1B, it would not be unreasonable to assume that SHP-1 exhibits similar dynamics and flexibility of the loop to PTP1B, and that these SHP-1 mutations might alter pH dependency by changing loop conformational equilibria. 9 (SHP-1 PDB ID: 4GRY, 31 PTP1B PDB ID: 3I80, YopH PDB ID: 2I42 57 ). |
65a6f6f3e9ebbb4db9510a74 | 20 | variants in this study result from alterations in WPD-loop conformational equilibria, or other causes, simulations of the wild-type enzyme as well as the H422Q, E427A, and S418A variants were performed, in both unliganded and phosphoenzyme intermediate forms of each variant, and with simulations initiated from both the loop open and loop closed conformations of these variants. |
65a6f6f3e9ebbb4db9510a74 | 21 | For each system, we evluated the root mean square displacement (RMSD) of both the Cα-atoms of just the WPD loop of the different SHP-1 variants (Figure ) as well as the root mean square fluctuations of these atoms across the full enzyme scaffold (Figure ). Based on this data, there are no statistically significant differences between wild-type SHP-1 and the variants as determined by pairwise t-tests conducted at each residue index followed by a Benjamin-Hochberg correction (apart from small local differences in flexibility in residue A513 E427 and P428 between the wildtype and E427A variant). This is in contrast to our prior studies of PTP1B and YopH, where a single point swap between T177G (PTP1B) and G352T (YopH) on the WPD-loop both significantly impacted loop dynamics and the pH-dependency of catalysis. For comparison, the PTP1B F182Q point mutation similarly has no significant impact on loop dynamics, but in this case the pH-dependency of catalysis is unaffected. We have successfully used the empirical valence bond (EVB) approach to model chemistry in both wild-type PTP1B and YopH and variants. In the present case, while the H422Q, E427A and S418A mutations all impact the rates of catalysis by the SHP-1 catalytic domain, from a thermodynamic perspective, the difference in activation free energy between the variants is very small (within 0.5 kcal mol -1 change compared to wild-type), and thus EVB (or other) simulations of the chemical step of catalysis are unlikely to capture the origins of these small energy differences. Nevertheless, our EVB simulations ( resolution, the differences in calculated activation free energies between the variants is minimal, in agreement with the pH-independent limiting kcat values (Table ). Interestingly, examination of the number of water molecules within 3.5Å of the side chain of D421 (measured as the distance between the Cγ-atom of the D421 side-chain and the oxygen atoms of the surrounding water molecules) during our simulations of the phosphoenzyme intermediate with a closed WPD-loop (Figure and Table ) suggest subtle differences in solvation of the aspartic acid that could rationalize the differences in pKa of this residue. Further, the mean P-Onuc distance during our simulations of wild-type SHP1 and variants in the phosphoenzyme intermediate state is very subtly shifted to a tighter P-O distance, which would be expected to be favorable for catalysis, although this difference is within the standard deviation of the calculations (Figure and Table ). . |
65a6f6f3e9ebbb4db9510a74 | 22 | A prior investigation of these SHP-1 variants at pH 5.0 in a medium containing 40% glycerol observed faster rates than the WT enzyme, which were hypothesized to arise from enhanced WPD-loop dynamics. This study shows the variants do not have broadly faster rates in aqueous solution, but their pH dependencies have been altered and, as a result, some of the variants exhibit faster turnover than WT at pH >6, reflected in a higher kinetic pKa of the catalytic residue D421. Further, our molecular dynamics simulations indicate that there are no statistically significant differences in loop dynamics, and any differences in pKa of the aspartic acid likely arise from differences in solvation of this side chain in the SHP-1 variants. The modeling results and structural analysis thus far suggest that the origins of shifted pH dependencies among these SHP-1 variants results primarily from changes in solvation and hydrogen-bonding networks that affect the pKa of the D421 residue. This explains why the changes in pH-rate profiles for kcat in these variants are observed on the basic limb. These findings contrast with the observations from mutations in YopH and PTP1B, whose pH dependencies for kcat were more broadly affected on both acidic and basic limbs. In summary, the results here combined with our previous work show multiple avenues exist by which mutations to noncatalytic residues affect the pH dependency of catalysis in PTPs. This phenomenon provides a means for nature to tune the activity of enzymes in particular environments, and for point mutations to alter an enzyme's activity in pH-dependent cellular processes, potentially with pathogenic consequences. The findings also emphasize the need to characterize the catalytic effect of enzyme mutations beyond comparative kinetic measurements at a single pH. |
62721ddad048ed83a45b797c | 0 | Figure : Origin of the magne6c force between chiral molecules. Chirality: Two polarizable chiral molecules, depicted as springs, undergo correlated fluctua6ons in their electron clouds (blue arrows). Charge: At the peak of one of these fluctua6ons, both molecules behave as charged dipoles aligned head-to-tail (6lted line). The correlated intramolecular electron currents that give rise to the transient dipole are subject to spin selec6vity, and cause a par6al excess of one spin orienta6on at one end of the molecule and a par6al deple6on at the other end. Spin: When the molecules are homochiral (enan6omer, leb), the unpaired spins at the point of intermolecular contact are of opposite polarity (singlet), causing a small a]rac6ve magne6c force. With molecules of opposite chirality (racemic, right), spins of the same polarity face each other (triplet), and the force is repulsive. Reversing the direc6on of the intramolecular current inverts spin polari6es but does not change the sign of the forces. Viscometry Typical viscometry experiments using a bob-and-cup arrangement require sample volumes of 20 ml or more. For enan6omerically pure chemicals, this quan6ty is prohibi6vely expensive for at least one enan6omer. Unusually among commonly available chemicals, lac6c acid (2-hydroxypropanoic acid) is available in quan6ty as either enan6omer with good chemical and enan6omeric purity. Pure lac6c acid is solid, but 90% solu6ons in water are liquid. Viscosity falls steeply with dilu6on, and variability in water content causes small differences in the viscosity of commercial lac6c acid solu6ons at the same nominal concentra6on. The lac6c acid enan6omers used in these experiments had slightly different viscosi6es. We did not adjust them by dilu6on insofar as the effect of mixing was unambiguous. |
62721ddad048ed83a45b797c | 1 | For simplicity and low cost, we used a bob and cup rotary viscometer and controlled temperature by immersing the cup in a 5-liter water bath kept at room temperature (19-21C). Temperature varia6ons were less than 0.2 C during the course of an experiment. Bob rota6on speed was 6 rpm, and viscosity data was updated every 10 seconds during experimental runs las6ng 1.5 hours. The results are shown in figure . Remarkably, in every run the viscosity of the 50-50 racemic mixture was lower either of the two enan6omers. were used without viscosity adjustments from the stock solu6ons. S is slightly more viscous than R. The raw data from the viscometer recordings is shown. Each panel shows one series of measurements in which the viscosi6es of R, S, and the freshly mixed racemic mixture of lac6c acid were measured. Measurements are made every ten seconds. Abscissa is in minutes. Aber an ini6al transient due in part to the viscometer performing a running average between successive measurements, the traces se]le to values varying by ≈2% during the period of measurement. Variability between and within experiments is likely due to imperfect temperature control in the simple setup used. In all experiments, the viscosity of the racemic mixture lies below the viscosity of either enan6omer. Assuming that racemic viscosity, absent any other effects, should be midway between R and S viscosi6es, the viscosity deficit is 3-5 mPa.s Molecular Dynamics. In order to rule out purely geometric factors poten6ally affec6ng viscosity, we performed molecular dynamics simula6ng a rotary viscometer. The bob is an alpha helix of 20 serines terminated with NH3+ and COO-groups. Serine was chosen to maximize hydrogen-bonded interac6ons between its side-chain hydroxyls and the solvent. The cup is a cubical box 7nm on the side, and the serine bob was placed in its center with its axis parallel to one face. To compare viscosi6es, we rotate the helix on its axis and monitor the resul6ng torque for both enan6omers of lac6c acid and for the racemic mixture. Viscous flow dissipates mechanical energy to heat, and in the linear regime, total energy dissipated is the integral of torque x angle. The results are shown in figure . An alpha helix of 20 serines is posi6oned at the center of a cube 7 nm on the side, its axis parallel to the bo]om face. The cube is filled with the appropriate enan6omer of lac6c acid or the racemic mix. For clarity, only half the lac6c acid molecules are shown. Right: The graph shows torque fluctua6ons in six separate MD runs of the serine helix in R-lactate solvent. Two regimes are visible. For the first 30 or so degrees (blue region), torque increases linearly with displacement, indica6ng elas6c behavior. Thereaber, a viscous regime is established and rota6on causes random changes in torque around a mean. integra6on of the torque curves yields work for each run. |
62721ddad048ed83a45b797c | 2 | . LeP: averaged torques for six "R" runs (red), 4 "S" runs (blue) and 5 racemic runs (purple). No systema6c effect of the enan6omeric composi6on is visible. Right: integrated averages. The racemic curve lies midway between R and S. The inset shows the curves within the do]ed box at higher magnifica6on with the racemic line midway between the enan6omers. |
62721ddad048ed83a45b797c | 3 | Discussion: A literature search revealed that the anomalously low viscosity of racemic solu6ons of hydrogen-bonded liquids had in fact been known for well over a century, though apparently unmen6oned in recent decades . At the 6me the effect was a]ributed to hypothe6cal racemate complexes forming in the solu6on, but these were never found. Our simple experimental setup confirms the viscosity anomaly, while the MD simula6ons are further evidence against the no6on that some form of associa6on between enan6omers could explain it. |
62721ddad048ed83a45b797c | 4 | What else could account for our results? 1-The solu6ons of lac6c acid we used were not pure, since some water must be present for lac6c acid to be a liquid. The amounts of water in the two samples were slightly different, or their viscosi6es would have been iden6cal. The water concentra6on in the racemic must therefore be midway between those of the two enan6omers, and there could conceivably exist a local viscosity minimum. There is, however, no evidence of any anomaly in the monotonic viscosity vs dilu6on curves of lac6c acid . 2-The forma6on of a small amount of lac6c acid polymers affec6ng the viscosity cannot be ruled out, but it is hard to see how mixing them could yield the observed effect. 3-Differen6al effects of chirality on enan6omer drib in viscous flow are known , but none would lead to a low racemic viscosity even if some enan6omer separa6on occurred at the low shear values used in these experiments. In conclusion, the most parsimonious explana6on of the effect we observe is a decrease in intermolecular force similar to the one predicted by SDCR and seen in atomic force measurements . It may be possible to detect a reduc6on in hydrogen bond strength in racemates directly by vibra6onal spectroscopy . If its origin in SDCR is established, the anomalous viscosity of racemates would be a classroom demonstra6on of the first fluid-mechanical effect since superfluidity that requires a quantum mechanical explana6on. |
62721ddad048ed83a45b797c | 5 | Methods: 90% R and S lac6c acid solu6ons were obtained from Musashino Chemical Laboratory Ltd, Tokyo. Viscosity measurements are made with a Baoshishan NDJ-5s Viscometer (Vevor UK) with a 25 ml bob-and-cup #0 rotor accessory. Data acquisi6on was via an RS-232 interface and wifi-enabled AirDrive Serial Logger Pro accessed via an iPhone. The cup was immersed in a 5 l water bath at room temperature for thermal iner6a. MD simula6ons were done with Gromacs 22 , using the CHARMM27 force field for the helix. Compa6ble parameters for the lac6c acid were obtained using SwissParam . Lennard-Jones and electrosta6c interac6ons were truncated at 1 nm. The 6mestep was 2 fs. Each system was equilibrated during 30 ps of MD in the NVT ensemble at room temperature , followed by 30 ps in the NPT ensemble at atmospheric pressure and room temperature . During equilibra6on, the heavy atoms of the helix were harmonically restrained. Each produc6on run consisted of 4 ns in the NPT ensemble. Helix rota6on was achieved using the pivot-free isotropic poten6al method with a spring constant of 50 kJ/(mol nm 2 ). The helix was rotated along its axis at a rate of 0.36 degree/ps and the torque required to do so was recorded every 0.2 ps. |
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