Insight into trifluoromethylation – experimental electron density for Togni reagent I

3,3-Dimethyl-1-(trifluoromethyl)-1,3-dihydro-1-λ3,2-benziodoxole represents a popular reagent for trifluoromethylation. The σ hole on the hypervalent iodine atom in this “Togni reagent” is crucial for adduct formation between the reagent and a nucleophilic substrate. The electronic situation may be probed by high resolution X-ray diffraction: the experimental charge density thus derived shows that the short intermolecular contact of 3.0 Å between the iodine and a neighbouring oxygen atom is associated with a local charge depletion on the heavy halogen in the direction of the nucleophile and visible polarization of the O valence shell towards the iodine atom. In agreement with the expectation for λ3-iodanes, the intermolecular O⋯I–C_aryl halogen bond deviates significantly from linearity.

The experimentally observed electron density for the “Togni reagent” explains the interaction of the hypervalent iodine atom with a nucleophile.

A “halogen bond” denotes a short contact between a nucleophile acting as electron density donor and a (mostly heavy) halogen atom as electrophile;^1,2 halogen bonds are a special of σ hole interactions.^3–5 Such interactions do not only play an important role in crystal engineering;^6–11 rather, the concept of a nucleophile approaching the σ hole of a neighbouring atom may also prove helpful for understanding chemical reactivity.The title compound provides an example for such a σ hole based reactivity: 3,3-dimethyl-1-(trifluoromethyl)-1,3-dihydro-1-λ3,2-benziodoxole, 1, (Scheme 1) commonly known as “Togni reagent I”, is used for the electrophilic transfer of a trifluoromethyl group by reductive elimination. The original articles in which the application of 1¹² and other closely related “Togni reagents”¹³ were communicated have been and still are highly cited. Trifluoromethylation is not the only application for hypervalent iodine compounds; they have also been used as alkynylating¹⁴ or azide transfer reagents.^15,16 The syntheses of hypervalent iodanes and their application in organofluorine chemistry have been reviewed,¹⁷ and a special issues of the Journal of Organic Chemistry has been dedicated to Hypervalent Iodine Catalysis and Reagents.¹⁸ Recently, Pietrasiak and Togni have expanded the concept of hypervalent reagents to tellurium.¹⁹Open in a separate windowScheme 1Lewis structure of the Togni reagent, 1.Results from theory link σ hole interactions and chemical properties and indicate that the electron density distribution associated with the hypervalent iodine atom in 1 is essential for the reactivity of the molecule in trifluoromethylation.²⁰ Lüthi and coworkers have studied solvent effects and shown that activation entropy and volume play relevant roles for assigning the correct reaction mechanism to trifluoromethylation via1. These authors have confirmed the dominant role of reductive elimination and hence the relevance of the σ hole interaction for the reactivity of 1 in solution by ab initio molecular dynamics (AIMD) simulations.^21,22 An experimental approach to the electron density may complement theoretical calculations: low temperature X-ray diffraction data of sufficient resolution allow to obtain the experimental charge density and associate it with intra- and inter-molecular interactions.^23–25 Such advanced structure models based on aspherical scattering factors have also been applied in the study of halogen bonds.^26–30 In this contribution, we provide direct experimental information for the electronic situation in Togni reagent I, 1; in particular, we analyze the charge distribution around the hypervalent iodine atom.Excellent single crystals of the title compound were grown by sublimation.^§ The so-called independent atom model (IAM), i.e. the structure model based on conventional spherical scattering factors for neutral atoms, confirms the solid state structure reported by the original authors,¹² albeit with increased accuracy. As depicted in Fig. 1, two molecules of 1 interact via a crystallographic center of inversion. The pair of short intermolecular O⋯I contacts thus generated can be perceived as red areas on the interaction-sensitive Hirshfeld surface.³¹Open in a separate windowFig. 1Two neighbouring molecules of 1, related by a crystallographic center of inversion. The short intermolecular I⋯O contacts show up in red on the Hirshfeld surface³² enclosing the left molecule. (90% probability ellipsoids, H atoms omitted, symmetry operator 1 − x, 1 − y, 1 − z).The high resolution of our diffraction data ^¶ for 1 allowed an atom-centered multipole refinement^33,34 and thus an improved model for the experimental electron density which takes features of chemical bonding and lone pairs into account. Fig. 2 shows the deformation density, i.e. the difference electron density between this advanced multipole model and the IAM in the same orientation as Fig. 1.^|| The orientation of an oxygen lone pair (blue arrow) pointing towards the σ hole of the heavy halogen in the inversion-related molecule and the region of positive charge at this iodine atom (red arrow) are clearly visible. Single-bonded terminal halides are associated with one σ hole opposite to the only σ bond, thus resulting in a linear arrangement about the halogen atom. Different geometries and potentially more than a single σ hole are to be expected for λ3-iodanes such as our target molecule, and as a tendency, the resulting halogen bonds are expected to be weaker than those subtended by single-bonded iodine atoms.³⁵ In agreement with these theoretical considerations, the closest I⋯O contacts in 1 amount to 2.9822(9) Å. This distance is significantly shorter than the sum of the van-der-Waals radii (I, 1.98 Å; O, 1.52 Å (ref. 36)) but cannot compete with the shortest halogen bonds between iodine and oxygen^37,38 or iodine and nitrogen.^39–41Fig. 1 and ​and22 show that the C_aryl–I⋯O contacts are not linear; they subtend an angle C10–I1⋯O1′ of 141.23(3)° at the iodine atom. On the basis of theoretical calculations, Kirshenboim and Kozuch³⁵ have suggested that the split σ holes should be situated in the plane of the three substituents of the hypervalent atom and that halogen and covalent bonds should be coplanar. Fig. 3 shows that the C_aryl–I⋯O interaction in 1 closely matches this expectation, with the next oxygen neighbour O1′ only 0.47 Å out of the least-squares plane through the heavy halogen I1 and its three covalently bonded partners C1, O1 and C10.Open in a separate windowFig. 2Deformation density for the pair of neighbouring molecules in 1; the dashed blue and red arrows indicate regions of opposite charge. (Contour interval 0.10e Å⁻³; blue lines positive, red lines negative, green lines zero contours, symmetry operator 1 − x, 1 − y, 1 − z).Open in a separate windowFig. 3A molecule of 1, shown [platon] along O1⋯C1, and its halogen-bonded neighbour O1ⁱ. Symmetry operator 1 − x, 1 − y, 1 − z.The Laplacian, the scalar derivative of the gradient vector field of the electron density, emphasizes local charge accumulations and depletions and it allows to assess the character of intra- and inter-molecular interactions. A detailed analysis of all bonds in 1 according to Bader''s Atoms In Molecules theory⁴² is provided in the ESI.^‡ We here only mention that the electron density in the bond critical point (bcp) of the short intermolecular I⋯O contact amounts to 0.102(5)e Å⁻³; we are not aware of charge density studies on λ3-iodanes, but both the electron density and its small positive Laplacian match values experimentally observed for halogen bonds involving O and terminal I in the same distance range.⁴³The crystal structure of 1 necessarily implies additional contacts beyond the short halogen bond shown in Fig. 1 and ​and2.2. The shortest among these secondary interactions is depicted in Fig. 4: it involved a non-classical C–H⋯F contact with a H⋯F distance of 2.55 Å.Open in a separate windowFig. 4C–H⋯F contact in 1; additional information has been compiled in the ESI.^‡ Symmetry operators i = 1 − x, 1 − y, 1 − z; ii = 1 − x, 1 − y, −z.The topological analysis of the experimental charge density reveals that this non-classical C–H⋯F hydrogen bond and all other secondary contacts are only associated with very small electron densities in the bcps. Table S8 in the ESI^‡ provides a summary of this analysis and confirms that the I⋯O halogen bond discussed in Fig. 1 and ​and22 represents by far the most relevant intermolecular interaction.The relevance of this halogen bond extends beyond the crystal structure of 1: Insight into the spatial disposition of electrophilic and nucleophilic regions and hence into the expected reactivity of a molecule may be gained from another electron-density derived property, the electrostatic potential (ESP). The ESP for the pair of interacting molecules in 1 is depicted in Fig. 5.Open in a separate windowFig. 5Electrostatic potential for a pair of molecules in 1 mapped on an electron density isosurface (ρ = 0.5e Å⁻³; program MoleCoolQt^44,45). Fig. 5 underlines the complementary electrostatic interactions between the positively charged iodine and the negatively charged oxygen atoms. One can easily imagine to “replace” the inversion-related partner molecule in crystalline 1 by an incoming nucleophile.The ESP tentatively obtained for a single molecule in the structure of 1 did not differ significantly from that derived for the inversion-related pair (Fig. 5), and even the results from theoretical calculations in the gas phase for an isolated molecule²⁰ are in good qualitative agreement with our ESP derived from the crystal structure. In the absence of very short contacts, polarization by neighbouring molecules only has a minor influence on the ESP. The experimentally observed electron density matches the proven reactivity for the title compound, and we consider it rewarding to extend our charge density studies on related hypervalent reagents.     

Oxa-Michael-based divergent synthesis of artificial glutamate analogs

Herein we report stereoselective generation of two skeletons, 1,3-dioxane and tetrahydropyranol, by oxa-Michael reaction as the key reaction from δ-hydroxyenone. The construction of the 1,3-dioxane skeleton, achieved through hemiacetal formation followed by oxa-Michael reaction from δ-hydroxyenone, was exploited to access structurally diverse heterotricyclic artificial glutamate analogs. On the other hand, formation of a novel tetrahydro-2H-pyranol skeleton was accomplished by the inverse reaction order: oxa-Michael reaction followed by hemiacetal formation. Thus, this study succeeded in showing that structural diversity in a compound collection can be acquired by interchanging the order of just two reactions. Among the skeletally diverse, heterotricyclic artificial glutamate analogs synthesized in this study, a neuronally active compound named TKM-50 was discovered in the mice in vivo assay.

By interchanging the order of reactions, two types of skeletons were created and a neuroactive artificial glutamate analog was developed.

Ionotropic glutamate receptors (iGluRs) mediate the majority of the excitatory neurotransmissions such as learning, memory, and nociception in the mammalian central nervous system (CNS).¹ To study and control the function of iGluRs, specific glutamate analogs have been developed in natural product chemistry² and in medicinal chemistry.^3,4 IKM-159 (Fig. 1A) is an artificial glutamate analog designed and developed based on dysiherbaine^5,6 and kainic acid⁷ in our laboratories as an antagonist selective to (S)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA)-type iGluR.^8–12 The AMPA receptor consists of four subunits: GluA1, GluA2, GluA3, and GluA4.³In vitro, IKM-159 selectively inhibits the GluA1/GluA2 heterodimer and GluA4 homodimer.¹⁰In vivo, IKM-159 inhibits voluntary action of mice for 50 min to several hours upon intracerebroventricular injection. The potency and selectivity of IKM-159 are, however, not very satisfactory to selectively modulate the function of AMPA-type iGluR. As an attempt to improve the biological profiles of IKM-159, we have been studying its structural modification.Open in a separate windowFig. 1Background (A) and summary (B) of this work. (A) AMPA-type iGluR antagonist IKM-159. (B) Artificial glutamate analogs 1 and 2, generated by oxa-Michael-based transformations (this work). The gray circle in 1 denotes the position for the structural diversity.From the first-generation studies on structure–activity relationships (SARs) of IKM-159, it had been shown that the ring size and the heteroatom of the C-ring were important for neuroactivity of IKM-159.^10,13 We then studied the second-generation SAR on the oxa analogs generated by a Prins-Ritter three-component coupling strategy, although all analogs were found to lose the original neuronal activity of IKM-159.¹⁴ Herein, we report our continuous effort along this line employing the homoallylic alcohol such as 5 and 7 (see Scheme 2) as the common intermediates.¹⁵Open in a separate windowScheme 2Preparation of the common intermediate 7 (racemate).One of the strategies in this work is the thermodynamically controlled, stereoselective formation of 1,3-dioxane (1 in Fig. 1B) by hemiacetal formation followed by oxa-Michael reaction from δ-hydroxyenone derivative that we recently developed (Scheme 1).¹⁶ The other strategy is the novel stereoselective formation of tetrahydropyranol (2 in Fig. 1B) by the inverse reaction order; oxa-Michael reaction followed by hemiacetal formation (see Scheme 5). Thus, this study succeeded in showing that structural diversity in a compound collection can be acquired by interchanging the order of just two reactions; hemiacetal formation and oxa-Michael reaction. Among the skeletally diverse, heterotricyclic artificial glutamate analogs thus synthesized, a compound named TKM-50 (1ar) was discovered to be neuronally active in the mice in vivo assay.Open in a separate windowScheme 1Our recent work regarding stereoselective 1,3-dioxane formation.¹⁶ For clarity and comparison, enantiomers of the reported compounds are shown in this scheme.Open in a separate windowScheme 5The heterotricyclic artificial glutamate analog 2, constructed by intermolecular oxa-Michael reaction of MeOH followed by acetalization.The substrate used for the 1,3-dioxane formation was prepared from the known dimethyl ester 5 (Scheme 2).¹⁷ Exposure of dimethyl ester 5 to hydrochloric acid (6 M) at 65 °C provided dicarboxylic acid 6.¹⁷ Without purification, dicarboxylic acid 6 was treated with BnBr and Cs₂CO₃ to furnish the common intermediate 7 in 72% yield (2 steps).¹⁸The alkene 7 was subjected to cross metathesis with methyl vinyl ketone (8) mediated by Hoveyda-Grubbs second generation catalyst (9)¹⁹ to provide enone 10 in 82% yield (Scheme 3). Upon exposure to paraformaldehyde as an equivalent of formaldehyde and 1,3,5-trioxane¹⁶ in the presence of MsOH, 1,3-dioxane ring formed smoothly by oxa-Michael reaction to give rise to desired (7R*)-heterotricycle 11r and the (7S*) epimer 11s (structure not shown) in the ratio of >9 : 1, as well as the N-hydroxymethylated product 12r (see Scheme 3) and the (7S*) epimer 12s (structure not shown). Since we had found that alkaline hydrolysis is of use to remove the N-hydroxymethyl group, the mixture of hemiaminals (12r/12s) and free amides (11r/11s) was treated with ammonium hydroxide²⁰ to obtain free amide 11r in 73% isolated yield (2 steps), and free amide 11s in 10% yield (estimated by NMR, 2 steps). The formation of 1,3-dioxane ring of 11r was determined by the HMBC correlations (Fig. 2A), and the stereochemical configuration was established by a ³J_H,H value and NOESY correlations denoted in Fig. 2B. Both configuration and conformation of 11r are identical to those we observed recently in the simple case (3 → 4, see Scheme 1),¹⁶ showing that the 1,3-dioxane formation in this study is also thermodynamically controlled (see below for the mechanism).Open in a separate windowScheme 3Stereoselective 1,3-dioxane formation leading to heterotricyclic artificial glutamate analog 1ar.^{a a}dr denotes the diastereoselectivity in the 1,3-dioxane formation.Open in a separate windowFig. 2Structure analysis of 1,3-dioxane 11r. (A) HMBC correlations in 11r indicates formation of the 1,3-dioxane ring. (B) Small ³J_H,H value and NOESY correlations show the configuration and the conformation of 11r.The proposed mechanism for the 1,3-dioxane formation is shown in Scheme 4A. Reaction of alcohol 10 and paraformaldehyde would form hemiacetal intermediate A under acidic conditions, which, then undergoes intramolecular oxa-Michael reaction to give 11r and 11s. Since the second conjugate addition is generally a thermodynamically controlled, reversible process, production of more stable (7R*) isomer 11r predominated over the (7S*) epimer 11s, as discussed also in our preliminary study.¹⁶ It should be also noted here that, in that preliminary study employing a simple substrate, the (7S) epimer had not been obtained.¹⁶ Generation of the less stable (7S*) epimer 11s in this study would be due to unfavorable steric interactions between the acetyl group and the benzyl ester on the near side in 11r (Scheme 4B), that make the energy difference between the two diastereomers (11r and 11s) smaller.Open in a separate windowScheme 4The plausible mechanism of 1,3-dioxane ring formation. (A) Stepwise mechanism that consists of hemiacetal formation followed by intramolecular oxa-Michael reaction. (B) The steric repulsion included in the stable isomer 11r.Then two benzyl groups of 11r were removed by hydrogenolysis²¹ to cleanly provide glutamate analog 1ar ((2R*,7R*)-TKM-50) in 86% yield (Scheme 3).With the same reaction sequences for 1ar (Scheme 3), two more analogs 1br and 1cr were furthermore synthesized (Fig. 3). The marked decrease in diastereoselectivity in these oxa-Michael reactions (see Fig. 3) suggests that the steric repulsion between the pentyl/methoxyphenyl group and the benzyl ester on the near side is extremely large. The minor (7S*) diastereomers obtained in these oxa-Michael reactions were also isolated and deprotected to give 1bs and 1cs (see the ESI^†), which were subjected to in vivo assay (see below).Open in a separate windowFig. 3Other 1,3-dioxane analogs synthesized by the intramolecular oxa-Michael reaction.^{a a}dr denotes the diastereoselectivity in the 1,3-dioxane formation.We also found that another skeleton can be constructed from δ-hydroxyenone being used for 1,3-dioxane formation, under alkaline hydrolytic conditions. Thus, as shown in Scheme 5, the δ-hydroxyenone 13 derived from homoallylic alcohol 5 by cross metathesis was selectively transformed into cyclic hemiacetal 2 in 53% yield (1 M LiOH in water, MeOH, rt). In this transformation, dimethyl ester and δ-hydroxyenone moiety independently suffer hydrolysis and cyclization, respectively, to generate glutamate analog 2 efficiently. The configuration of 2 was determined by combined analysis of NMR and DFT calculation (see the ESI^†).²²The plausible mechanism for the hemiacetal formation is shown in Scheme 6. In view of the fact that the hydroxy and carbonyl groups are located apart in 13, the six-membered-ring formation should take place after saturation of the trans-alkene. It is, therefore, supposed that oxa-Michael reaction of MeOH to enone 13 first generates saturated ketone Cvia enolate B.²³ Under alkaline conditions, the alkoxide C intramolecularly attacks carbonyl group to give rise to hemiacetal 2. Considering the fact that oxa-Michael reaction and the acetalization are thermodynamically controlled, reversible processes, energetically favorable diastereomer 2 would have been obtained predominantly (see the ESI^† for discussions on thermodynamic stability of 2). A related example had been reported in 1992 by Shing et al.²⁴Open in a separate windowScheme 6The plausible mechanism for hemiacetal formation under alkaline conditions.Behavioral activities of all six compounds upon intracerebroventricular (i.c.v.) injection were evaluated in mice (Fig. 4).²⁵ Injection of 1ar (TKM-50, 50 μg per mouse) resulted in loss of voluntary motor activity for 10 min after injection and then ataxia-like motions were recorded, thus annotated as hypoactive. The hypoactivity observed for 1ar (TKM-50) is thus somewhat weaker than IKM-159 which causes loss of mice spontaneous activity for up to 4 h.¹² Other congeners, however, did not cause any noticeable behavioral changes at the same dose tested.Open in a separate windowFig. 4The in vivo activities on mice.     

Microwave-assisted synthesis of mutually embedded Rh concave nanocubes with enhanced electrocatalytic activity

Novel mutually embedded Rh concave nanocubes were synthesized by reducing Rh(acac)₃ in tetraethylene glycol in the presence of benzyldimethylhexadecylammonium, KI and polyvinylpyrrolidone under microwave irradiation for 120 s. KI and HDBAC were crucial to the formation of mutually embedded nanostructures. The as-prepared Rh nanocrystals exhibited higher electrocatalytic activity and stability.

Mutually embedded Rh concave nanocubes were synthesized by reducing Rh(acac)₃ with tetraethylene glycol (TEG) as both a solvent and a reducing agent under microwave irradiation for 120 s.

As important catalytic materials, the controlled syntheses of platinum group metal nanocatalysts have attracted wide attention for many years. The catalytic performances of platinum group metal nanomaterials are highly dependent upon their morphologies, compositions and surface structures. Their nanocrystals with controlled shapes have been extensively explored in order to promote their catalytic performance and reduce their cost because of their scarcity and high prices. As an important platinum group metal, rhodium (Rh) is often used as a typical catalyst with high activity and selectivity in hydroreduction,¹ hydroformylation,^1f,2 NO_x reduction,³ CO oxidation,⁴ cross coupling,⁵ and fuel cell^1g,4d,6 and other chemical reactions.⁷ In addition, Rh has a strong resistance to acids and bases as well as a high melting point. However, nanoscaled Rh exhibits high thermodynamic instability owing to its high surface free energy, although it is more stable than many other catalytically active metals. So, the shape-controlled synthesis of Rh nanocrystals is still one of the challenges in this field, though Pt and Pd nanocrystals with many different morphologies have been obtained. In the past decade, great efforts have been devoted to tailoring the sizes, morphologies and surface structures of Rh nanoparticles to improve their catalytic efficiency because of the scarcity and preciousness. Up to now, many Rh nanomaterials with various morphologies such as nanosheets,^1f,4a,8 nanotetropods,⁹ hierarchical dendrites,⁶ hyperbranched nanoplates,^1g,10 ultrathin nanosheet assemblies,^1e,7a and cubic,¹¹ tetrahedral,^1d icosahedral,¹² tetrahexahedral,^4d concave cubic,¹³ concave tetrahedral nanocrystals,¹⁴ as well as cubic nanoframes,^5b,5c truncated octahedral nanoframes,¹⁵ multipods¹⁶ and mesoporous³ Rh nanoparticles have been successfully synthesized. Moreover, all the obtained Rh nanoparticles were monodispersed and displayed enhanced catalytic activities. Although various nanoparticles with concave, frame, branched, or hierarchical structures as well as normal flat or convex surfaces have been created, it is still worthy to develop further novel Rh nanostructures.Herein, we report a facile microwave-assisted strategy for a one-pot synthesis of mutually embedded Rh concave nanocubes, a unique hierarchical nanostructure with several identical concave nanocubes embedded in each other. The co-adsorption of I⁻ ions and benzyldimethylhexadecylammonium (HDBAC) was dominantly responsible for the generation of the hierarchical concave cubic Rh nanocrystals. The as-prepared Rh nanocrystals displayed an enhanced electrochemical activity for formic acid electro-oxidation. Fig. 1a and b as well as Fig. S1 (ESI^†) display TEM images of the typical Rh nanocrystals synthesized under microwave irradiation for 120 s in the presence of PVP and an appropriate amount of KI and HDBAC. Interestingly, as can be seen, the resulting Rh nanocrystals were present in mutually embedded concave nanocubic morphologies under TEM, showing a unique hierarchical nanostructure feature. This hierarchical structure was consisted of at least two concave cubes (Fig. 1b) which were embedded in each other. If considering an individual concave nanocube, the average diameter was about 85 nm. The high-resolution TEM image, as shown in Fig. 1c and d, showed the lattice fringes with an interplanar spacing of 0.192 and 0.135 nm, which can be indexed to the (200) and (220) planes of face-centered cubic (fcc) Rh. The corresponding fast Fourier transform (FFT) pattern for the selected box area in Fig. 1c is shown in the inset in Fig. 1d, indicating a single crystal structure and good crystallinity. In fact, the as-prepared concave cube demonstrated octapod characteristics because of its showing both concave faces and concave edges. The SEM image further confirmed hierarchical structure feature. As shown in Fig. 1e and the inset for partially enlarged picture as well as Fig. S2 (ESI^†), the concave nanocubic structures and their mutually embedded feature are present clearly.Open in a separate windowFig. 1TEM (a and b), HRTEM (c and d) and SEM (e) images of the as-prepared mutually embedded Rh concave nanocubes. (d) shows the selected box area in (c). The insets in (d and e) show the FFT pattern and a partially enlarged SEM picture, respectively.The typical XRD pattern of the as-synthesized mutually embedded Rh concave nanocubes is shown in Fig. 2. The characteristic peaks at 41.28, 48.05, 70.18 and 84.45° are corresponding well with the (111), (200), (220) and (311) lattice planes according to the standard diffraction file (JCPDS 05-0685), respectively. The sharp and strong (111) diffraction peak, which indicated the preferential orientation of (111) planes and the consistency with the HRTEM observation, suggested its high purity and crystallinity of the obtained Rh nanocrystals. In addition, XPS measurement demonstrated the binding energy of Rh 3d_5/2 and Rh 3d_3/2 at 307.16 and 311.91 eV (Fig. 3), respectively, with an interval of 4.75 eV, which was coincident with the reference values (307.0 and 311.75 eV),¹⁷ indicating Rh⁰ with zero oxidation for the as-prepared mutually embedded concave nanocubes.Open in a separate windowFig. 2XRD pattern of the typical mutually embedded Rh concave nanocubes.Open in a separate windowFig. 3XPS spectrogram of the typical mutually embedded Rh concave nanocubes.It was worth noting that the use of KI was much essential for creating the mutually embedded Rh concave nanocubes. As shown in Fig. 4a, except flower ball structures connected with each other, neither concave cube nor hierarchical structure was observed in the absence of KI. When 0.4 mmol of KI was added, the embedded Rh concave nanocubes accompanying with some irregular nanostructures were generated (Fig. 4b). With further increasing the amount of KI from 0.8 to 1.2 mmol, complex inter-embedded nanostructures with obscure polyhedral outlines were formed (Fig. 4c and d). Accordingly, an excessive amount of KI was unfavorable for the generation of the mutually embedded Rh concave nanocubes. According to the previous report,¹⁸ the addition of KI would manipulate the reducing kinetics to generate Rh concave nanostructures under microwave irradiation. In the presence of KI, the precursor was transformed to a more stably coordinated anion [RhI₆]³⁻. As a result, the reducing rate of Rh(iii) to Rh(0) as well as both the nucleation and growth rate of Rh nanoparticles decreased, which would be favorable for the oriented growth of Rh concave cubes. The role of I⁻ ions was elucidated by using an equivalent amount of KBr or KCl in stead of KI, respectively, under the same other conditions. As can be seen (Fig. S3, ESI^†), no single or embedded concave nanocubes but amorphous Rh nanoparticles with agglomeration were observed under these two alternative experiments. These results suggested that different halides would result in different Rh nanostructures and the existence of an appropriate amount of I⁻ ions was beneficial for the formation of the mutually embedded Rh concave nanocubes. According to the literature,¹⁹ halides tend to selectively adsorb to {100} planes. Generally, the six surfaces of a Rh cube are 〈100〉 oriented. So, we suggested that the selective adsorption of I⁻ ions on Rh {100} planes confined a growth along 〈100〉 direction and facilitated the formation of Rh concave structures with growth along {111} facets.Open in a separate windowFig. 4TEM images of the products prepared with different amount of KI while keeping the same other conditions. (a) Without KI; (b) 0.4 mmol KI; (c) 1.2 mmol KI; (d) 1.6 mmol KI.Moreover, the effect of HDBAC on the creation of the embedded Rh concave nanocubes was also investigated. As shown in Fig. 5a, no shaped Rh nanocrystal was produced except agglomerated nanoparticles without using HDBAC. While 0.1 mmol of HDBAC was used relatively to the parameters in the typical experimental procedure, Rh nanostructures with an nonuniform cross-sectional dimension and overly embedded each other were generated (Fig. 5b). With further increasing the amount of HDBAC from 0.4 to 0.6 mmol, the cross-section dimension and the concavity of the concave Rh nanocubes decreased gradually though embedded Rh nanostructures were still generated (Fig. 5c and d). Whereas no concave nanostructure was observed with using an equivalent amount of CTAB or CTAC in stead of HDBAC, respectively (Fig. S4, ESI^†). These results implied that the formation as well as the size and surface structure of the mutually embedded Rh concave nanocubes were dependent upon the confinement effect of HDBAC. On the one hand, the existence of HDBAC would contribute to creation of the concave cubes and their mutually embedded structures, on the other hand, the growth of shaped Rh nanoparticles was confined and the adsorption of I⁻ ions on Rh {100} planes was disturbed, resulting in less concavity and smaller size, due to an excessive amount of HDBAC.Open in a separate windowFig. 5TEM images of the products prepared with different amount of HDBAC while keeping the same other conditions. (a) Without HDBAC; (b) 0.1 mmol HDBAC; (c) 0.4 mmol HDBAC; (d) 0.6 mmol HDBAC.In order to investigate whether its formation was related to oxidation etching of Rh surface by I⁻ ion/O₂,^19,20 nitrogen was filled into the reaction bottle to remove oxygen before reaction and the same results were obtained. So, the oxidative etching can be negligible, which may be ascribed to the extremely short time under microwave irradiation.According to the previous report,²¹ based on the results of the experiments with dependent I⁻ ions and HDBAC, the formation of the mutually embedded Rh concave nanocubes may be ascribed to symmetry breaking due to asymmetric passivation and attachment of Rh nuclei. I⁻ ions were responsible for retarding the growth of {100} and {110} facets of cubic nuclei and promoting the preferential overgrowth on {111} planes, resulting in the formation of the concave structure with concave faces and edges. However, the existence of an appropriate amount of HDBAC may retard the deposition of Rh atoms on one or two corners with (111) facets due to the confinement, resulting in symmetry breaking. Meanwhile, with the confinement of HDBAC, the inevitable collision of nuclei leads to attachment of nuclei one another along the confined corners due to surface defects or dislocations. As a result, the mutually embedded Rh concave nanocubes would be generated with the growth of nuclei.The electrochemical performances of the as-synthesized Rh nanocrystals were examined by electrocatalytic oxidation of formic acid. The specific current density was normalized to the electrochemical surface area (ECSA). According to the cyclic voltammetry (CV) curves (Fig. S5^†), the ECSAs were calculated as 66.5 and 52.3 m² g⁻¹ for the mutually embedded Rh concave nanocubes and the commercial Rh black, respectively. Fig. 6a shows the CV curves for the electro-oxidation of formic acid in HClO₄ by the as-prepared mutually embedded Rh concave nanocubes and the commercial Rh black. The peak current density for the mutually embedded Rh concave nanocubes was measured to be 2.988 mA cm⁻² at 0.667 V, while it was 1.379 mA cm⁻² at 0.674 V for Rh black. The electrocatalytic activity of the hierarchical Rh nanostructures, though with a larger size, was about 2.2 times that of Rh black. Obviously, the mutually embedded Rh concave nanocubes exhibited an enhanced electrocatalytic activity for formic acid comparing with the commercial Rh black, which should be ascribed to their special surface structure with more edges, corners and terraces. Fig. 6b shows the CA curves of the electrocatalytic oxidation of formic acid for these catalysts. Compared with Rh black, a slower current attenuation as well as a higher retention of current after 800 s was observed for the as-prepared embedded Rh concave nanocubes, revealing a good electrochemical stability.Open in a separate windowFig. 6The CV (a) and CA (b) curves of the mutually embedded Rh concave nanocubes and Rh black in 0.1 M HClO₄ + 0.5 M HCOOH solutions with the cyclic potential between −0.2 and 1.0 V at a sweep rate of 50 mV s⁻¹.In summary, a novel hierarchical Rh nanostructure with several concave nanocubes embedded mutually could be rapidly prepared by reducing Rh(acac)₃ in TEG under microwave irradiation for 120 s in the presence of PVP and an appropriate amount of KI and HDBAC. In the preparing process, TEG was used as both a solvent and a reducing agent. The existence of KI and HDBAC was critical to the formation of the mutually embedded Rh concave nanocubes. The as-prepared mutually embedded Rh concave nanocubes demonstrated higher electrocatalytic activity and stability than commercial Rh black in the electro-oxidation of formic acid.     

Three-component assembly of stabilized fluorescent isoindoles

The tandem addition of an amine and a thiol to an aromatic dialdehyde engages a selective three-component assembly of a fluorescent isoindole. While an attractive approach for diversity-based fluorophore discovery, isoindoles are typically unstable and present considerable challenges for their practical utility. We found that introduction of electron-withdrawing substituents into the dialdehyde component affords stable isoindole products in one step with acceptable yields and high purity.

The tandem addition of an amine and a thiol to an aromatic dialdehyde engages a selective three-component assembly of a stabilized fluorescent isoindole.

Since the preparation of the first isoindole (1, Fig. 1) in 1951 (ref. 1) and the isolation of the parent unsubstituted isoindole (2) in 1972,² the relative instability of this heterocyclic ring system has been an important impediment to the discovery of new chemical transformations and biological applications. The position of the equilibrium between the two tautomeric forms 2 and 3, respectively 2H- and 1H-isoindoles, could be invoked to assess the stability of the 2H-form. It has been found that substituents on the isoindole ring system play a key role. For example, it appears that electron-donating groups, such as methyl groups in 4, destabilize the 2H-isoindole,³ whereas electron acceptors in 5⁴ and 6⁵ improve stability.Open in a separate windowFig. 1 N-methylisoindole (1) and a selection of the first isoindoles prepared 4–6 along with a depiction of the substituent-based tautomeric preferences.Multicomponent reactions^6,7 that enable three or more discrete molecules to combine into one product not only curtail synthetic operations but also advance the complexity viable within a single operation. To date, many of our fluorescent probes are prepared by two-component processes wherein moiety A is attached to moiety B to generate a fluorescent probe. Conventionally, this is achieved by adding dye A to a biological molecule B, however methods have been established that generate the probe motif as part of the coupling process.^8,9 The latter, referred to as turn-on labeling, advantageously removes potential non-fluorescent impurities as only the desired product displays the proper fluorescence. While rare, advance of multicomponent turn-on labeling strategies offers a robust ability to improve the selectivity of labeling as well as to further expand the diversity possible within a labeling reaction. Here, we turn our attention to explore a three-component strategy to prepare isoindoles with improved stability.An early report of the formation of fluorescent species when o-diacetylbenzene was exposed to proteins¹⁰ led to the discovery of a multicomponent reaction between o-phthalaldehyde (7), amines 8 and thiols 9, which yields highly fluorescent isoindole 10 (Fig. 2).^11–16 This reaction now forms the basis for the quantitative determination of amino acids and is used in commercial amino acid analyzers.¹⁷ The method is characterized by high sensitivity, although the lack of mechanistic understanding has led researchers to optimize the conditions primarily empirically.Open in a separate windowFig. 2The three-component isoindole reaction. (a) Reaction of phthalaldehyde (7) or 2,3-naphthalenedicarboxaldehyde (11) with amines (8) and thiols (9) to afford fluorescent isoindoles 10 and 12, respectively. (b) Proposed mechanism of the reaction with 7 where the initial formation of imine 7a is followed by an attack by a thiol 9 to form acetal-like species 7b. This undergoes cyclization to give hemiaminal 7c with a subsequent elimination of water to result in isoindole 10.An important drawback of the method is the lack of stability of the product isoindoles 10 (Fig. 2), which, once form, undergo further conversions introducing inaccuracies due to unstable fluorescence. In general, the highest fluorescence must be achieved within 5–25 min and remain time-independent for another 20–30 min, the conditions which are hard to fulfill as the rate of isoindole formation and its stability depend on an individual amino acid.^18,19 Naphthalene-1,2-dicarboxaldehyde (11) was recommended as an alternative reagent with claims that the product isoindoles 12 would be more stable, but the proposal seemingly has not received acceptance from the scientific community as the increases in stability are probably not significant to justify the cost of this reagent.¹⁷Our initial attempt to characterize the product of the three-component reaction between phthalaldehyde 7, amines and thiols (Fig. 3) met with significant synthetic challenges, as evident by the low stability of 10a. This compound is unstable at room temperature and rapidly decomposed when column chromatography purification was attempted. In order to obtain an analytical sample, 10a was repeatedly recrystallized from cold CH₃CN, dried in vacuum at 0 °C and immediately analyzed by NMR. Isoindole 12a, derived from 2,3-naphthalenedicarboxaldehyde (11), turned out to be only slightly more stable in comparison to product 10a and also rapidly decomposed when column chromatography purification was attempted.Open in a separate windowFig. 3One-pot synthesis of fluorescent 1-thio-2H-isoindoles and related structures from aromatic dialdehydes, butylamine and N-(tert-butoxycarbonyl)-l-cysteine methyl ester (Boc-Cys-OMe).Electron-deficient dialdehydes 13, 15, 17 and 19 (Fig. 3) on the other hand, all gave stable isoindoles 14a, 16a (structure assigned by NOESY analyses, see ESI^†), 18a and 20a respectively (Fig. 3), when reacted with butyl amine and protected cysteine. These isoindoles could be purified by column chromatography and were considerably easier to handle.Next, we turned our attention to evaluate if n-butyl- and n-octylphthalimidic phthalaldehydes 17 and 19 produced readily isolated products from a variety of aliphatic or aromatic amines and thiols. As shown in Fig. 4, isoindoles 18b–d and 20b–f can be obtained by adding a thiol and amine to the solution of 17 or 19 in CH₃CN at 0 °C. Subsequent simple removal of the volatiles on the rotary evaporator and purification of the product, facilitated by its fluorescence on TLC, gave the desired 18b–d and 20b–f in a straightforward manner. These reactions could be conducted with a 1 : 1 : 1 ratio of dialdehyde, amine, and thiol in acceptable isolated yields (48–66%, Fig. 4). The yields did not seem to be affected by the electronic properties of the reacting amines or thiols and were similar for reactions involving electron-rich (18b, c, 20c–f) vs. electron-deficient (18d, 20b) amines or electron-rich (18c, 20d) vs. electron-deficient (18b, d, 20b, c, e, f) thiols.Open in a separate windowFig. 4One-pot synthesis of fluorescent 1-thio-2H-isoindoles from n-butyl- and n-octylphthalimidic phthalaldehydes 17 and 19, aliphatic and aromatic amines, and aliphatic and aromatic thiols.While an attractive analytical tool and fluorophore, practical applications of isoindoles suffer due to their rapid degradation. Proposed early on by Simons and Johnson,²⁰ the problem arises from nucleophilic attack by ROH (water or alcohols) at C1 (Fig. 5) resulting in the loss of the thiol and formation of the corresponding γ-lactam 21b.²¹ This is further complicated by the potential for self-dimerization by a Diels–Alder reaction as well as cycloaddition with oxygen, the latter of which results in the incipient formation of a rapidly degraded endoperoxide. In 1981, a team led by Simmons and Ammon reported the first stable isoindole by the reaction with dimethylene acetylenedicarboxylate.²² Here, the product of the Diels–Alder cycloaddition opened to deliver a stabilized isoindole by indirect functionalization at C3. While a practical discovery, the overall analytical utility of isoindoles would benefit from discovery of a material with bench stability.Open in a separate windowFig. 5Stability analyses on isoindole 18b were evaluated using time course NMR studies. Selected spectra are shown with expansion of the aromatic region from 6.50–8.50 ppm. The predominant product of this degradation step found to be 21b. Expansions and spectra from treating 18a, 18c and 18d under the same conditions are provided in the ESI.^†Overall, we were able to prepare and collect spectral data on 13 new isoindoles (Fig. 3 and ​and4).4). These materials were sufficiently stable for purification and spectral analyses. While we were able to prepare and isolate 10a and 12a, spectra on these materials needed to be collected immediately after preparation. NMR analyses were most challenging due to the fact that trace acidic or basic materials in the solvents led to rapid decomposition ultimately leading to the use of acetone-d₆ for NMR data collection.To further evaluate their utility, samples of 18a–d were monitored for their stability neat and in solution. Subjecting these samples to the same protocol (see ESI^†), isoindoles 18c and 18d were not sufficiently stable in neat form to endure >30 days at −20 °C followed by 48 h at 23 °C (conditions that modelled compound storage). Under the same conditions, 18a and 18b (Fig. 5a) were stable when stored (>30 days at −20 °C followed by 48 h at 23 °C) and when dissolved acetone-d₆ (Fig. 5a) and kept in the dark (Fig. 5b). Exposure to light, however, led to decomposition of both 18a and 18b (Fig. 5c). These observations indicated that steric bulk within the amine and thiol components contributed to the products stability. Additionally, the presence of alkene functionality within the amine was not tolerated, as given by the comparison of unstable 18d to stable 18a.Synthesis of isoindoles²³ through a three-component coupling provides a robust tool to rapidly access diverse fluorescent materials as recently demonstrated by adaptation for a Click-like processes²⁴ or crosslinking,²⁵ called Flick. Here, we describe how the addition of electron withdrawing groups effectively stabilizes the materials as demonstrated by 18a and 18b. While this data suggests that stability can be achieved, the light sensitivity of these agents suggests a future potential as photochemical sensors or modifiers. Efforts are now underway to explore this application.     

Asymmetric total synthesis of four bioactive lignans using donor–acceptor cyclopropanes and bioassay of (−)- and (+)-niranthin against hepatitis B and influenza viruses

The asymmetric total synthesis of four lignans, dimethylmatairesinol, matairesinol, (−)-niranthin, and (+)-niranthin has been achieved using reductive ring-opening of cyclopropanes. Moreover, we performed bioassays of the synthesized (+)- and (−)-niranthins using hepatitis B and influenza viruses, which revealed the relationship between the enantiomeric structure and the anti-viral activity of niranthin.

The total synthesis of four lignans including (−)- and (+)-niranthin has been achieved utilizing cyclopropanes. Based on bioassays of the (+)- and (−)-niranthins using HBV and IFV, we speculated the bioactive site of niranthin against HBV and IFV.

Lignans are attracting considerable attention due to their widespread distribution in plants and their varied bioactivity.^1–5 For example, matairesinol,² dimethylmatairesinol,³ yatein,⁴ and niranthin⁵ are found in nature and exhibit e.g., cytotoxicity,^2b,d,3b,4b anti-bacterial,^2c anti-allergic,^3c anti-viral,^4d,5b,e anti-leishmanial,^5d and strong insect-feeding-deterrent activity.^4c Among these compounds, anti-viral compounds have received significant attention owing to the worldwide pandemic of coronavirus disease 2019 (COVID-19). Although niranthin exhibits anti-viral activity toward the hepatitis B virus (HBV),^5b,e the enantiomeric SAR (structure–activity relationship) for the anti-viral activity of niranthin has not been revealed so far. To examine the SAR for a pair of enantiomers, an independent asymmetric synthesis of both enantiomers is necessary. However, the alternative synthesis of (−)- or (+)-niranthin has not been reported.⁶ During our recent studies on the transformation of cyclopropanes,⁷ we have reported a reductive ring-opening of enantioenriched donor–acceptor (D–A) cyclopropanes and its application to an asymmetric total synthesis of yatein.⁷ⁱ As a further extension of this synthetic method, we disclose here the asymmetric total synthesis of (−)-dimethylmatairesinol, (−)-matairesinol, (+)-niranthin, and (−)-niranthin. Moreover, the results of bioassays using (+)-niranthin and (−)-niranthin against HVB and influenza virus (IFV) are described (Scheme 1).Open in a separate windowScheme 1Some examples of bioactive dibenzyl lignans. Scheme 2 outlines the enantioselective synthesis of optically active lactones 5a and 5b. Following our previous report,⁷ⁱ we attempted to synthesize the enantio-enriched bicyclic lactones 4a and 4b.Open in a separate windowScheme 2Enantioselective synthesis of key intermediates 5a and 5b.Initially, the cyclopropanation of enal 1 with dimethyl α-bromomalonate 2 using the Hayashi–Jørgensen catalyst afforded the desired optically active cyclopropylaldehydes 3a and 3b in good to high yield with high ee.^{7c,e,h–j,8,9} The reduction of the aldehydes to alcohols and subsequent lactonization with p-TsOH afforded lactones 4a and 4b in high yield with high ee. The optical purity of lactones 4a and 4b were determined using HPLC analyses on a chiral column, and the ee values of the enantioselective cyclopropanations were estimated based on these HPLC analyses. Next, treatment of bicyclic lactones 4a and 4b with hydrogen in the presence of a catalytic amount of Pd–C in AcOEt at 0 °C resulted in a regioselective reductive ring-opening to furnish benzyloxylactones 5a and 5b in good to high yield with high dr and high ee. In the hydrolysis step, debenzylation of the benzyloxyaryl group did not occur under these mild conditions, i.e., in AcOEt at 0 °C.⁷ⁱFor the α-benzylation of 5a and 5b to afford 6a–c, the corresponding substituted benzylhalides were necessary. 3-Methoxy-4-benzyloxybenzylbromide and 3,4-dimethoxybenzylbromide were easily prepared by known methods (for details, see the ESI^†); however, the preparation of 3,4-methylenedioxy-5-methoxybenzylbromide (16) required a modified procedure that involves the regioselective protection of the hydroxy group at the 3-position of 3,4,5-trihydroxybenzene (Scheme 3). The methylenedioxylation of gallic acid (10) during the first step resulted in a low yield of 11.¹⁰ Consequently, we successfully synthesized 12 using a cyclic boron-ester system.¹¹ Arylmethylbromide 16 was derived from ester 12 in two steps in good to high yield.Open in a separate windowScheme 3Preparation of substituted benzylbromide 16.Next, enolates were generated from lactones 5a and 5b using K₂CO₃ in DMF, and successfully attacked the benzylhalides on the less-hindered side to afford α-benzyl lactones 6a–c with excellent dr values (Scheme 4).^7i,12 The trans-α,β-disubstituted lactones 7a–c were obtained via the hydrolysis of the α-methoxycarbonyllactones 6a–c followed by decarboxylation. The transformation from the enol form to the keto form gave the thermodynamically favored trans products (7a–c) with excellent dr values.^7i,12 Finally, the debenzylation of 7a using a catalytic amount of Pd–C in methanol under a hydrogen atmosphere afforded matairesinol (7d) in 96% yield. Thus, the total syntheses of dimethylmatairesinol (7b) and matairesinol (7d) were achieved, and spectral data of these natural products were consistent with reported data.^2e,3d The absolute configuration of these compounds were determined using the known data of optical rotation values. The reduction of lactone 7c using LAH afforded diol 8 in 91% yield (Scheme 4). Subsequent dimethylation of the resulting diol 8 using NaH and MeI furnished (−)-niranthin in 89% yield with 95% ee.¹³ Spectral data of (−)-niranthin was also consistent with reported data.^5b,e,6 Following the total synthesis of (−)-niranthin, we also achieved the total synthesis of (+)-niranthin via an alternative enantioselective cyclopropanation using a different enantiomeric Hayashi–Jørgensen catalyst derived from d-proline instead of l-proline (Scheme 5).¹⁴Open in a separate windowScheme 4Alternative asymmetric total synthesis of dimethylmatairesinol, matairesinol, and (−)-niranthin.Open in a separate windowScheme 5Alternative asymmetric total synthesis of (+)-niranthin.(−)-Niranthin has been reported to exhibit anti-HBV activity.^5b,e Aiming to shed light on the relationship between its enantiomeric structure and activity, we performed a bioassay on the synthesized (−)- and (+)-niranthin against not only HBV, but also the influenza virus (IFV). The anti-HBV activity results are summarized in Fig. 1 and ​and2,2, while the anti-IFV activity is summarized in Fig. 3 (for details, see the ESI^†).Open in a separate windowFig. 1HBV-infection assay using (−)- and (+)-niranthin.Open in a separate windowFig. 2HBV-replication assay using (−)- and (+)-niranthin.Open in a separate windowFig. 3Growth-inhibition assay of IFV using (−)- and (+)-niranthin.Based on the assays using HBV-infected HepG2-hNTCP-C4 cells and HBV-replicating Hep38.7-tet cells, the amount of HBs antigen decreased in a concentration-dependent manner without apparent cytotoxicity. The 50% inhibition concentration (IC₅₀) in the HBV-infected cells was calculated to be 14.3 ± 0.994 μM for (−)-niranthin and 9.11 ± 0.998 μM for (+)-niranthin (Fig. 1), while the IC₅₀ in the HBV-replicating cells was calculated to be 16.2 ± 0.992 μM for (−)-niranthin and 24.2 ± 0.993 μM for (+)-niranthin (Fig. 2). These results show that (−)-niranthin and (+)-niranthin exhibit anti-HBV activity, and that there is no remarkable difference between the anti-HBV activity of both enantiomers. In contrast, based on the bioassay of (−)- and (+)-niranthins against IFV using MDCK cells, cytotoxicity of (−)-niranthin appears at >400 μM judging that cell viability without IFV is less than 80%, and (−)-niranthin inhibited IFV-infection to cells in a concentration-dependent manner on the concentration range of non-cytotoxicity, and exhibits anti-IFV activity at 200–400 μM judging that cell viability with IFV is over 50% (Fig. 3). However, (+)-niranthin does not exhibit anti-IFV activity, and similarly to (−)-niranthin, cytotoxicity appears at >400 μM. Thus, the anti-IFV activity between (−)- and (+)-niranthins is clearly different. Our findings suggest that the enantiomeric site in niranthin endows (−)-niranthin with more potent anti-IFV activity than (+)-niranthin. We speculated that the anti-HBV active site of niranthin might be a part of the molecular structure such as aromatic groups which are far from chiral centers. In contrast, anti-IFV active site of niranthin might be closer to the chiral centers (Scheme 6).Open in a separate windowScheme 6A speculation for the bioactive site of niranthin against HBV and IFV.     

Guided cellular orientation concurrently with cell density gradient on butterfly wings

A simple method to create guided cellular orientation is illustrated by assembling fibroblasts on the dorsal side of M. menelaus wings. Moreover, by inserting the wing into tendon fibroblasts suspension at a tilt angle, guided cellular orientation concurrently with the cell density gradient is formed on the butterfly wings.

A simple method to create guided cellular orientation concurrently with cell density gradient on tilt butterfly wings was illustrated.

In normal in vivo microenvironments, cellular organization, including cellular orientation and cell density, can induce different extracellular matrix components, which in turn lead to different cell growth, proliferation and apoptosis.¹ By introducing different cell culture materials, such as substrates patterned with grooves or ridges, cell alignment could be induced effectively.² Moreover, by employing mechanical loading,³ inkjet printing,⁴ chemical modification⁵ and other means,⁶ spatial variations in the cell density gradient can also be generated in many ways.⁷ However, some technologies tend to suffer certain drawbacks, such as low time efficiency and even decrease in cell viability during manipulation.⁸ Moreover, it is still difficult to obtain guided cellular orientation concurrently with a cell density gradient, which is a major concern in the recreation of many types of tissue junctions.It has been suggested that many photonic architectures occur naturally in certain insects, such as butterfly wings.⁹Morpho butterflies have impressed many researchers due to the amazing natural colours on their wings.¹⁰ It was found that the brilliant coloration originates from complex nanostructures located on the surface of a Morpho butterfly wings.¹¹ Those periodic nano-ridge structures can control the propagation of photons, thus presenting butterflies with a brilliant structural color.¹² Benefitted from the development of nanotechnology and materials science, many researches have fabricated different artificial wing-like photonic structures to duplicate the complex shape of the nanostructure of butterflies.¹³ However, they are fairly expensive, time consuming and could not meet the requirements of mass production.¹⁴ Considering this, the natural structures from Morpho butterflies has become an optimal choice for many researches. For example, Morpho butterfly wings have been used directly as vapour sensor and also in surface-enhanced Raman spectroscopy for many years.¹⁵Herein, we propose a simple, inexpensive and reproducible method to create guided cellular orientation concurrently with a cell density gradient by assembling fibroblasts on butterfly wings. We found that the parallel periodic nanoridges on the butterfly wings could induce oriented alignment of cells. This is demonstrated by both NIH-3T3 fibroblasts and tendon fibroblasts. Moreover, by tilting the butterfly wings into a homogeneous tendon fibroblast suspension, the number of sedimentary cells above the wings alters accordingly, leading to naturally increasing cell density from one side of the butterfly wing to the other. Thus, the cell density gradient can be easily produced without any chemical modification or mechanical process. It is noteworthy to mention that the tendon fibroblasts with a gradient density still exhibited guided orientation after sedimentation. Therefore, both guided cellular orientation and gradient cell density assembled simultaneously in a convenient way. This simple and robust method will have potential applications in tissue engineering.The wings of Morpho menelaus (M. menelaus) were selected as substrates for cell culture (Fig. 1a). First, scanning electron microscopy (SEM) was performed to investigate the micro/nanostructure of the wing. Fig. 1b illustrates the view of the ground scale of the composition of periodic parallel ridges. This anisotropic structure is the foundation of the structural colour of the M. menelaus wings. Atomic force microscopy (AFM) observations also show the same result for the micro/nanostructure on the wing (Fig. 1c). Normally, the original M. menelaus wings are strongly hydrophobic to protect the butterfly from rain and other natural disasters (Fig. 1d). However, this character might not be appropriate for direct applications in cell culture. Therefore, to improve the capacity for cell culture, the surface of M. menelaus wings should be treated prior to the cell experiment.Open in a separate windowFig. 1(a) Optical image of a M. menelaus wing; (b) SEM image of the nanostructure of the M. menelaus wing; (c) AFM image of the nanostructure of the M. menelaus wing; (d) the water contact angle of the M. menelaus wing before plasma treatment.For the case study, the M. menelaus wing was pre-treated to become more biocompatible for use in cell culture (Fig. 2a). First, the wing was treated by oxygen plasma to generate hydroxyl, achieving a more hydrophilic surface (Fig. 2b). Inorganic salts were then removed from the wing using hydrochloric acid solution, followed by the removal of pigments and other proteins using sodium hydroxide solution. The wing turned transparent after the chemical treatments, indicating that the pigments and other proteins were removed (Fig. 2c). Here, the treated wing retained the slight blue structural colour, suggesting that main structure may not be destroyed after the treatment. SEM as well as AFM results confirmed that the parallel nanostructure is retained in the wing despite a little deformation of the ridges (Fig. 2d and e).Open in a separate windowFig. 2(a) Scheme of the construction of the M. menelaus wing pre- and post-treatment; (b) the water contact angle of the M. menelaus wing after the plasma treatment; (c) optical image of the M. menelaus wing after chemical treatments; (d) SEM image of the nanostructure of the M. menelaus wing after treatment; (e) AFM image of the nanostructure of the wing after treatment.To investigate the biocompatibility, the treated M. menelaus wing was then prepared for cell culture. NIH-3T3 fibroblasts were chosen as their anisotropic behaviour would make results easy to observe. After a long culturing period, we found that the 3T3 fibroblasts assembled on the dorsal side of wing exhibited same proliferation tendency as those on the traditional culture dish (Fig. 3a). The number of these fibroblasts reached the peak amount within one week interval and gradually decreased in the following days, matching with the standard cell survival curve. Cell activity assay was also applied after 48 h culturing to evaluate the cell activities between the wing and cell culture plate. The results indicate that the 3T3 fibroblasts assembled on the treated wing demonstrate a slightly higher viability compared with the cells cultured on culture plate (Fig. 3b). Therefore, the treated M. menelaus wings possess excellent biocompatibility and are thus appropriate for cell adhesion and survival.Open in a separate windowFig. 3(a) A long culturing period of NIH-3T3 fibroblast cells on the M. menelaus wing and cell culture plate as control; (b) cell activities of NIH-3T3 fibroblast cells cultured on the M. menelaus wing and culture plate as control.To check if the parallel periodic nanoridges on the dorsal side of the butterfly wings affect cell orientation, NIH-3T3 fibroblasts were cultured on the treated wing for a certain period. Since the M. menelaus wings are primarily made of chitin in most cases, a cell culturing system on a chitinous hybrid inverse opal substrate, on which the parallel strips were absent, was investigated for comparison. Meanwhile, cells cultured on the cell culture plate were also analyzed as a control. By taking the advantage of Calcein AM staining, it was found that the orientation of the cells on the parallel stripes of the butterfly wing became uniformly aligned (Fig. 4a), while cells on the culture plate and chitinous-modified substrate without parallel strips had disordered microfilaments (Fig. 4b and c). This was further confirmed by the corresponding SEM results. The fibroblasts exhibited induced orientation with distinct cell bipolarizations along the parallel stripes of the wing. In contrast, the fibroblasts on the culture plate as well as chitinous hybrid substrate patterned without parallel strips exhibited random orientation. The main possibility is that the stripes from the M. menelaus wing could induce cell arrangement by contact guidance. In addition, it was found that the guided cell orientation could also be achieved by using the inverse opal substrate, on which the surface was structured with stripe-like pattern (Fig. S1^†). However, the parallel stripe structures located on the inverse opal substrate may suffer from batch-to-batch variations, thus affecting the cell performance.Open in a separate windowFig. 4(a) Fluorescence microscopy image and SEM image of NIH-3T3 fibroblasts cultured on the treated M. menelaus wing after 48 h; (b) fluorescence microscopy image and SEM image of NIH-3T3 fibroblasts cultured on the hybrid substrate as a control, inset is the detail of the surface pattern of the hybrid substrate; (c) fluorescence microscopy image and SEM image of NIH-3T3 fibroblasts cultured on a cell culture plate.To further confirm the trend of cell orientation, the angles between the growth direction of fibroblasts and the parallel stripes were measured and analyzed (Fig. 5). The results demonstrate that about 80% of NIH-3T3 fibroblasts exhibited an orientation within 30° of the stripes'' direction. In contrast, no particular cell orientation was observed on the cell culture plate and chitinous hybrid substrate. Cell viabilities on those substrates were also investigated and no significant difference in cell viabilities were detected. All these results when considered together indicate that the M. menelaus wings could provide an appropriate environment for induced cell culture.Open in a separate windowFig. 5Orientation angle frequency distribution of NIH-3T3 fibroblasts on the butterfly wing, culture plate and hybrid substrate.Traditionally, cell density gradients are typically regulated by performing hemocytometer, which is time consuming and labour intensive.¹⁶ Alternatively, chemical and electrical means could be employed to establish cell density gradients.¹⁷ However, these methods are still plagued by poor reproducibility or low controllability. Recently, a simple method involving a glass substrate tilted in cell suspension for rapidly establishing cell density gradients was conducted.¹⁸ In this method, various cell gradient patterns could be assembled in a convenient way. Herein, we intend to gather cells with gradient density by utilizing the M. menelaus wings. To prevent floating above the cell suspension, the dorsal side of the clipped wing was attached to a polystyrene (PS) substrate. Having been used previously as the raw material for standard Petri dish and multi-well plates in cell culturing, PS was chosen as the supporting substrate since it has an adequate mechanical strength.¹⁹ Further, the wing-PS composite was then inserted into the cell suspension to gather the sediment cells.In this case study, tendon fibroblasts were chosen since they originated from connecting tissue, in which the gradient in cell types, organizations as well as extra cellular matrix represent a common feature. The wing-PS composite was then tilted at an angle of 45 degree and inserted into the tendon fibroblast suspension in such way that one end of the wing stripes were against the bottom of the cell culture container. The tilting angle of 45 degree was chosen since a clear observation of cell density gradient could be achieved at this angle as compared with the others (Fig. 6a). Cell sedimentation experiments with different tilting angles were also performed and the results are shown in Fig. S2.^† The homogeneous suspension of tendon fibroblasts was set at a concentration of 3 × 10⁵ mL⁻¹ in order to obtain a clear observation of cell density distribution after fluorescence staining. After 2 hours of setting, cell suspension became clear, indicating that most cells adhered on either the wing or at the bottom of the culture container. Following 24 h culturing time, the wing-PS composite was then extracted and rinsed briefly with a culture medium to wash away the unattached cells.Open in a separate windowFig. 6(a) Fluorescence images of gradient in cell density generated on the M. menelaus wing; (b) illustration of the wing-PS composite inserted in the cell suspension; (c) cell density calculation along the wing.Normally, the clipped wings of M. menelaus are around 4 cm in length. The highest point on the wing is denoted as the starting point and the lowest point is denoted as the position of 4 cm. The total volume of the fibroblast suspension was set to barely immerse the starting point of the wing (Fig. 6b). Therefore, gradient patterns started from a density of zero at the starting point (that is there were no cells at this position available for sedimentation). Fluorescence staining was performed subsequently to help visualize the cells and determine whether the sedimentation process has influenced the cell viability. The cells at the position of 0.5 cm, 1.5 cm, 2.5 cm and 3.5 cm were examined (Fig. 6a). As expected, a clear gradient of cell density can be observed on the wing. Upon calculating, the number of fibroblasts adhered at the position of 0.5 cm was 6 × 10³, followed by 9 × 10³ at 1.5 cm, 19 × 10³ at 2.5 cm and 34 × 10³ at 3.5 cm. The highest number of cells was around 40 × 10³ at the position of 4 cm. Thus, a clear gradient pattern of cell density is formed (Fig. 6c). Moreover, cells retained good viabilities after sedimentation, showing the great advantage of this method over other gradient generation approaches. It is worth mentioning that owing to the parallel stripes on the M. menelaus wing, those fibroblasts maintained guided orientation along the parallel stripes of the wing. Thus, guided cellular orientation concurrently with cell density gradient was formed easily on the butterfly wings without any complex procedures.     

High-contrast mechanochromic benzothiadiazole derivatives based on a triphenylamine or a carbazole unit

Four triphenylamine or carbazole-based benzothiadiazole fluorescent molecules have been successfully synthesized and characterized. Interestingly, the donor–acceptor (D–A) type luminogens 1, 2, 3 and 4 showed different solid-state fluorescence. Furthermore, the four compounds exhibited reversible high-contrast mechanochromism characteristics.

Four triphenylamine or carbazole-based benzothiadiazole dyes were synthesized. Interestingly, the four dyes exhibited high-contrast mechanochromism characteristics.

Stimuli-responsive materials receive much attention currently due to their academic importance and potential applications in optoelectronic devices and fluorescent sensors,^1–7 especially organic smart materials whose solid-state luminescence can be tuned by external stimuli.^8–13 Mechanochromic fluorescence materials, as a class of smart materials, are also receiving increasing attention.^14–20 To date, a number of mechanofluorochromic organic molecules have been reported.^21–24 In contrast, examples of high-contrast mechanochromic luminescence materials are still inadequate. Indeed, many traditional organic materials are aggregation caused quenching (ACQ)-active, and these materials are weakly emissive or nonluminescent in the solid state due to the presence of strong intermolecular electronic interactions in their aggregated state, which promotes the formation of exciplexes and excimers.^25–27 Obviously, the ACQ effect is unbeneficial to gain high-contrast mechanofluorochromic materials.^28–30 It is no doubt that mechanochromic molecules with a bright solid-state fluorescence emission are easier to achieve high-contrast mechanofluorochromic phenomenon. Therefore, the corresponding highly emissive smart luminophors have attracted considerable attention.^31,32In general, the emission characteristics of mechanochromic luminescence materials depend strongly on their molecular structures and intermolecular interactions.^33–35 Therefore, it is an effective method for the realization of mechanofluorochromic materials to change the morphological structures by means of external mechanical stimulus.³⁶Benzothiadiazole-based derivatives are regarded as attractive candidates for the organic π-conjugated fluorescent dyes owing to their strongly electron-withdrawing feature.^37–41 Meanwhile, the benzothiadiazole unit is also advantageous to the construction of donor–acceptor (D–A) type molecules, which have emerged as a significative class of optical materials finding potential value in some areas such as in fluorescent sensors and displays.^42,43 Motivated by the fact that triphenylamine or carbazole fluorogen has been broadly applied in the field of emissive materials,^44,45 we attempted to link one triphenylamine or carbazole group to one benzothiadiazole moiety. As a result, we have obtained four D–A type fluorescent molecules on the basis of a combination of the electron-donating triphenylamine or carbazole unit and the electron-accepting benzothiadiazole unit (Fig. 1). Compound 1, 2, 3 or 4 contains rotatable aromatic rings, and thus their molecular structures are nonplanar, which is advantageous to the radiative decay in the aggregated state. Indeed, compounds 1, 2, 3 and 4 showed bright solid-state fluorescence with different emission colors. In addition, we found that the D–A type luminogens 1, 2, 3 and 4 applying the triphenylamine or carbazole moiety as an electron donor and the benzothiadiazole moiety as an electron acceptor exhibited various mechanochromic fluorescence characteristics with good reversibility. Furthermore, luminogen 1 showed mechanofluorochromic behavior involving color change from orange to rare red.Open in a separate windowFig. 1Molecular structures of the compounds 1, 2, 3 and 4.To investigate the solid-state fluorescence behaviors of compounds 1, 2, 3 and 4 in detail, the corresponding solid-state emission spectra were studied initially. As shown in Fig. 2, the fluorescence spectrum of triphenylamine-containing benzothiadiazole derivative 1 exhibited one emission band with the λ_max at 575 nm, and the fluorescent molecule exhibited strong orange luminescence with the fluorescence quantum yield (Φ) of 7.13%, and triphenylamine-containing compound 2 exhibited strong yellow luminescence (Φ = 7.43%) with the λ_max at 567 nm. In contrast, the emission spectrum of carbazole-based benzothiadiazole derivative 3 exhibited one emission band with the λ_max at 504 nm, and the luminogen exhibited bright green fluorescence with the quantum yield of 16.10%, and carbazole-based compound 4 also exhibited bright green fluorescence (Φ = 16.53%) with the λ_max at 498 nm. Therefore, the photoluminescence (PL) behaviors of compounds 1, 2, 3 and 4 could be adjusted via introducing various fluorogens containing triphenylamine and carbazole. In addition, the fluorescence lifetimes of 1, 2, 3 and 4 were also measured. As shown in Fig. 3, the average lifetime of fluorescent molecule 1 was 0.82 ns, the average lifetime of 2 was 1.56 ns, the average lifetime of 3 was 3.00 ns, and the average lifetime of 4 was 1.41 ns.Open in a separate windowFig. 2Solid-state emissive spectra of the compounds 1, 2, 3 and 4, and the related fluorescence images under 365 nm UV light.Open in a separate windowFig. 3(a) Time-resolved luminescence (575 nm) of solid sample 1. Excitation wavelength: 365 nm. (b) Time-resolved luminescence (567 nm) of solid sample 2. Excitation wavelength: 365 nm. (c) Time-resolved luminescence (504 nm) of solid sample 3. Excitation wavelength: 365 nm. (d) Time-resolved luminescence (498 nm) of solid sample 4. Excitation wavelength: 365 nm.Subsequently, the mechanochromic fluorescence characteristics of compounds 1, 2, 3 and 4 were investigated. As shown in Fig. 4, the solid sample of luminogen 1 showed a bright orange fluorescence. Interestingly, the orange luminescence was changed to the red luminescence with the λ_max at 593 nm upon treating with mechanical force stimulus. Furthermore, the initial orange emission could be restored after treatment of the ground compound 1 with fuming dichloromethane for 1 min. Therefore, 1 showed reversible high-contrast mechanofluorochromic behavior with color change from orange to red, which is a relatively rare color conversion among all mechanochromic fluorescence phenomena.Open in a separate windowFig. 4(a) PL spectra of solid sample 1 at different conditions. Excitation wavelength: 365 nm. (b) Fluorescence image of the unground sample 1 under 365 nm UV light. (c) Fluorescence image of the ground sample 1 under 365 nm UV light. (d) Fluorescence image of the ground sample 1 after treatment with dichloromethane under 365 nm UV light.Similarly, as shown in Fig. 5, compound 2 also showed reversible high-contrast mechanochromic fluorescence behavior. Moreover, the reversible mechanochromic fluorescence of 1 or 2 could be repeated four times between the orange or yellow and red or orange emissions without obvious changes by alternating grinding and dichloromethane treatments. To date, this mechanochromic luminescence conversion of some reported mechanochromism compounds with superior performance is also repeated three or four times,^46–48 and thus the reversibility of the mechanochromic fluorescence effect of 1 or 2 is good (Fig. 6). On the other hand, as shown in Fig. 7, when sample 3 were ground in an agate mortar with a pestle, the green emission was changed to the yellow-green fluorescence with the λ_max at 533 nm. Moreover, the yellow-green emission could also revert to the original green emission after a 1 min treatment of the ground powder with fuming dichloromethane vapor. Furthermore, as shown in Fig. 8, compound 4 also showed similar mechanochromic fluorescence behavior.Open in a separate windowFig. 5(a) PL spectra of solid sample 2 at different conditions. Excitation wavelength: 365 nm. (b) Fluorescence image of the unground sample 2 under 365 nm UV light. (c) Fluorescence image of the ground sample 2 under 365 nm UV light. (d) Fluorescence image of the ground sample 2 after treatment with dichloromethane under 365 nm UV light.Open in a separate windowFig. 6(a) Repetitive experiment of mechanofluorochromic effect for compound 1. (b) Repetitive experiment of mechanofluorochromic effect for compound 2.Open in a separate windowFig. 7(a) PL spectra of solid sample 3 at different conditions. Excitation wavelength: 365 nm. (b) Fluorescence image of the unground sample 3 under 365 nm UV light. (c) Fluorescence image of the ground sample 3 under 365 nm UV light. (d) Fluorescence image of the ground sample 3 after treatment with dichloromethane under 365 nm UV light.Open in a separate windowFig. 8(a) PL spectra of solid sample 4 at different conditions. Excitation wavelength: 365 nm. (b) Fluorescence image of the unground sample 4 under 365 nm UV light. (c) Fluorescence image of the ground sample 4 under 365 nm UV light. (d) Fluorescence image of the ground sample 4 after treatment with dichloromethane under 365 nm UV light.As can be seen in Fig. 9, the reversibility of the mechanofluorochromic behavior of compound 3 or 4 is also excellent. Next, the powder X-ray diffraction (XRD) patterns were studied in order to ensure the morphological characteristics. As can be seen in Fig. 10, the XRD patterns of compound 1 or 2 exhibited a number of sharp reflection peaks, suggesting that the unground compound 1 or 2 was crystalline in nature. However, the ground powder sample became amorphous, with a lack of sharp diffraction peaks. Therefore, the change in fluorescence of compound 1 or 2 could be attributed to the conversion from a crystalline state to an amorphous state. On the other hand, when the ground sample was exposed to dichloromethane vapor for 1 min, the sharp and intense peaks reappeared, indicative of the recovery of the crystalline nature. As presented in Fig. 11, the structural transition of the powder sample of compound 3 or 4 was similar to that of 1 or 2. Based on the above mentioned analysis, the powder XRD results demonstrated that the interesting mechanochromic fluorescence characteristics of compounds 1, 2, 3 and 4 were ascribed to the switchable morphology transition between the crystalline state and the amorphous state.Open in a separate windowFig. 9(a) Repetitive experiment of mechanofluorochromic effect for compound 3. (b) Repetitive experiment of mechanofluorochromic effect for compound 4.Open in a separate windowFig. 10(a) Powder XRD patterns of compound 1 in different solid states. (b) Powder XRD patterns of compound 2 in different solid states.Open in a separate windowFig. 11(a) Powder XRD patterns of compound 3 in different solid states. (b) Powder XRD patterns of compound 4 in different solid states.In conclusion, in this work, four triphenylamine or carbazole-based benzothiadiazole fluorescent molecules were successfully synthesized. The compounds 1, 2, 3 and 4 belonged to the highly solid-state emissive donor–acceptor (D–A) type luminescent molecules. It is noteworthy that the four D–A type luminogens exhibited high-contrast mechanofluorochromic characteristics. Furthermore, the reversibility of their mechanochromic phenomena is good. The results of powder XRD experiments confirmed that this switchable morphology transformation is responsible for the reversible mechanochromic fluorescence characteristics of 1, 2, 3 and 4. This work is valuable for designing high-contrast mechanochromic materials involving red light-emitting feature.     

Antibacterial nanoparticles: enhanced antibacterial efficiency of coral-like crystalline rhodium nanoplates

This paper deals with the newly found antibacterial efficiency of coral-like crystalline Rh nanoplates. Rh nanoplates with rough surface morphology synthesized by inverse-directional galvanic replacement exhibited highly enhanced antibacterial efficiency compared to Rh³⁺ ion and Rh nanospheres. The observed antibacterial efficiency was comparable to Ag nanoplates, a well-known anticancer nano-agent. Results clearly demonstrate that the composition and morphology of a nanostructure play significant roles in antibacterial effects.

This paper deals with the newly found antibacterial efficiency of coral-like crystalline Rh nanoplates.

Bacteria live everywhere, covering most of where we live. Although some bacteria such as lactobacilli are beneficial to humans, the risks of chronic infection and sepsis caused by a bacterial infection also exist.¹ Although the use of antibiotics is widely accepted as a general countermeasure against infections, drug overuse has brought about the emergence of super bacteria, multidrug-resistant bacterial strains that can be considered as a major crisis.² As a new direction in developing antibiotics, approaches using antibacterial nanoparticles have attracted much attention.³ Typical examples include Gram-positive antibacterial metal oxides such as zinc oxide (ZnO) and titanium dioxide (TiO₂),^4,5 and Gram-negative antibacterial metal nanoparticles such as silver (Ag) nanoparticles.⁶ Antibacterial effects of nanoparticles are known to rely on bacterial cell membrane disruption,⁷ reactive oxygen species generation,⁸ and intracellular antibacterial effects.⁹ For nanoparticles have such abilities, small dimensions and positive surface charge tend to be more favorable, but the exact features of inorganic nanostructures responsible for antibacterial effect remain to be elucidated. Therefore, the discovery of antibacterial nanoparticles based on new elements, morphology, composition, and physicochemical properties still merits efforts.Synthesis via surface control or post-synthetic treatments are generally utilized to manufacture fascinating nanostructures with various shapes and elemental compositions.¹⁰ Among them, galvanic replacement based on spontaneous redox reactions between oxidation-prone nanotemplates and reduction-prone replacing metal cations is generally used, since it is environment-friendly and convenient to perform.¹¹ In typically such galvanic replacement setups, Ag or copper (Cu) for nanotemplates, and gold (Au), platinum (Pt), and palladium (Pd) for replacing cations, many of which are closely related to antibacterial properties.¹² Therefore, we expected galvanic replacement can be a promising way to develop new antibacterial nanoparticles.In this study, we identified the antibacterial property of coral-like crystalline rhodium nanoplates (RhNPs), which recently emerged for its new possibilities in biomedical applications.¹³ To verify the enhanced antibacterial activity of RhNPs based on the morphology and composition, a comparative experiment was performed using Ag nanoplates (AgNPs), conventionally small Rh nanospheres (RhNSs; 5 nm diameter), and Rh³⁺ ions (Fig. 1).¹⁴ Quantitative analysis showed that RhNPs exhibited a slightly improved effect when compared to AgNPs (typically used as an antibacterial nanomaterial),¹⁵ and free Rh ions and Rh nanospheres were not effective. The results not only demonstrate a new possibility of using metal elements beyond Ag for antibacterial nanostructures, but also show that structural properties play an important role.Open in a separate windowFig. 1Schematic illustration of the antibacterial effect of Rh nanoparticles in spherical and coral-like plate morphologies.RhNP preparation was accomplished using a two-step procedure: seed-mediated synthesis of sacrificial Ag nanoplate templates, followed by inverse-directional galvanic replacement with Rh³⁺. The high-temperature (190 °C) reaction along with the reducing agent and ethylene glycol (EG) co-solvent condition, which caused galvanic replacement depending on the shape of the AgNPs. A subsequent outward surface growth resulted in the formation of coral-like RhNPs. Similarly, control RhNS synthesis was accomplished via reductant-based synthesis under the same conditions without Ag nanotemplates. According to the transmission electron microscopy (TEM) and scanning electron microscopy (SEM) images, approximately 120 nm long (transverse length) RhNPs with rough surface morphologies were produced successfully (Fig. 2a and S1^†). The formation of RhNPs was accomplished by rapid galvanic replacement of AgNPs template with Rh³⁺ ions by spontaneous redox reaction at high temperature. The manufactured nanostructures showed a nanoplatelet structure caused by rapid galvanic replacement of template and outward-grown coral-like morphology by subsequent secondary growth. Coral-like nanoplates with high surface-to-volume ratio and many surface irregularities have a high potential for intimate interactions with bacterial or mammalian cell membranes.Open in a separate windowFig. 2Characterization of prepared RhNPs. (a) TEM, (b) UV-Vis spectrometer, (c) DLS and zeta-potential, and (d) XRD pattern exhibited the successful synthesis of crystalline monodisperse RhNPs. The scale bar is 200 nm.The extinction spectrum of RhNP identified through UV-visible-near infrared spectroscopy showed a weak UV-localized plasmon resonance peak at 273 nm and uniformly high optical density over the whole wavelength region with weak and broad bands at around 320–1100 nm, which probably originated from the coral-like two-dimensional interconnected complex structure (Fig. 2b). The hydrodynamic diameter of RhNP was observed to be 68.1 nm for maximum number distribution, which is smaller than that measured using the TEM images, because calculations carried out using the Stocks–Einstein equation assume a spherical shape. Although the polydispersity index (PDI) was excellent (0.146 ± 0.016), it did not satisfy the recommended parameters for antibacterial nanoparticle efficacy due to the highly negative surface charge (ζ-potential: −27.1 ± 0.3) and relatively large sizes (120 nm; Fig. 2c).High-resolution (HR)-TEM observations and fast Fourier transforms of the images provided highly crystalline face-centered cubic (fcc) structures (Fig. S2^†). Moreover, a slight shift from the X-ray diffraction pattern was observed possibly due to the partially-presenting Rh–Ag alloy composition (Fig. 2d).To investigate the antibacterial effect of RhNP further, three additional control materials were prepared and their antibacterial properties against Escherichia coli were tested. AgNPs, a well-known antibacterial material and a template for galvanic replacement, were prepared using the same particle count as RhNPs to identify constituent element-based differences. In addition, Rh³⁺ ions and RhNS were prepared with the same Rh content via inductively-coupled plasma mass spectroscopy. All nanoparticles were prepared at a similar degree of surface negative charge, which excluded the antibacterial effect from the charge property (Fig. S3 and S4^†).Compared to untreated control (negative), RhNPs showed remarkable inhibition of E. coli growth; the inhibition was even slightly better than AgNPs (positive) when treated at the same concentration. Contrastingly, free Rh³⁺ ions and RhNSs did not show noticeable effects even though they had the same Rh contents. From these results, both the constituent element and morphological characteristics were confirmed to be crucial factors (Fig. 3a). Similar results were also obtained for the dose-dependent antibacterial effects of RhNPs (Fig. 3b).Open in a separate windowFig. 3Growth curve of E. coli as an optical density (OD₆₀₀) plot. Comparison against (a) nanomaterials and (b) RhNP concentrations exhibited significant antibacterial efficiency.SEM images of RhNP-treated E. coli provided more detailed information regarding the observed antibacterial effects. Surface adsorption of a large number of nanoplates was observed in E. coli treated with RhNPs when compared to control (Fig. 4a and b). Magnified SEM images (Fig. 4c and d) show that RhNPs were adsorbed in a form of clusters on the surfaces of E. coli. The results also indicated that RhNPs bigger than a certain size were required for interaction with E. coli surfaces, and that relatively small RhNPs were ineffective due to difficulties in accessing and interacting with the E. coli surface. Negatively-charged particles may seem hard to adhere to cells due to the negatively-charged cell membrane, so the adhesion presumably occurred between RhNPs and protein corona clusters. Such adsorption of RhNPs resulted in cell membrane weakening (Fig. 4c and d; red arrows) or disruption (Fig. 4d; yellow arrow), which account for the observed antibacterial property.Open in a separate windowFig. 4SEM images of (a) control- and (b) RhNP-treated E. coli clearly exhibiting surface adsorption of the nanoplates. The red (c) and blue (d) boxes are enlarged images of the red- and blue-boxed regions in (b). Red arrows indicate the surface interaction between E. coli and RhNPs, and the yellow arrow indicates a ruptured E. coli cell. The scale bar is 500 nm.     

Characterization of biosurfactant lipopeptide and its performance evaluation for oil-spill remediation

Biosurfactant lipopeptide is a promising dispersant over varieties of chemical ones in oil-spill remediation. The toxicity, biodegradability and performance of the biosurfactant lipopeptide are studied in this paper.

Biosurfactant lipopeptide is a promising dispersant over varieties of chemical ones in oil-spill remediation.

Dispersants were globally applied to physico-chemically enhance the dispersion of oil in water and were assumed to stimulate oil biodegradation by indigenous microorganisms and to reduce the environmental impact of oil spills.^1,2 Since the 1960s,^3,4 chemical dispersants have been applied as an emergency response to oil spills in marine ecosystems,⁵ and have showed effectiveness at removing oil slicks from the coast.^3,6,7 However, most of the chemically synthesized dispersants are inherently toxic to various aquatic species and hardly biodegradable in the natural environment.^2,8 The application of chemical dispersants in the 2010 Gulf of Mexico oil spill also raised concerns regarding the toxicity and the potential environmental impact,^9,10 and caused a debate about the effectiveness of chemical dispersants on the rates of oil biodegradation.¹¹ Biosurfactants are promising dispersants in oil-spill remediation, owning to their environmentally friendly and biodegradable properties.¹² Chemical surfactants could be replaced with biosurfactants and this change would diminish the environmental impact of traditional dispersants.^8,13,14 Lipopeptide produced by microorganisms is one of the representative biosurfactants and has showed great potential applications in food,¹⁵ medicine,¹⁶ microbial enhanced oil recovery,¹⁷ and other fields.¹⁸ Nevertheless, the knowledge about the application of biosurfactant lipopeptide in marine oil-spill remediation is still limited.In the present work, the dispersion effectiveness, aquatic toxicity, biodegradability and environmental compatibility of the biosurfactant lipopeptide were determined using recognized standardized methods,^19–23 and the biosurfactant lipopeptide used as a bio-dispersant for marine oil-spill remediation were studied, which is, to the best of our knowledge, the first report about biosurfactant lipopeptide used in oil-spill remediation.The lipopeptide samples were isolated from cell-free broth of B. subtilis HSO121 at our laboratory.²⁴ The typical chemical structure of the lipopeptide used in the study was shown in Fig. 1 and its critical micelle concentration (CMC) was 8.69 × 10⁻⁵ mol L⁻¹.Open in a separate windowFig. 1Typical chemical structure of lipopeptides (a) and the surface tensions of lipopeptides respect to concentrations (b).Dispersion effectiveness (DE) of lipopeptides was examined at different surfactant-to-oil ratios (SORs), temperatures, pH values, and salinities. It indicated in Fig. 2 that DE of lipopeptides reached 70.23% at SORs of 1 : 10 (w/w) at 25 °C, pH 7 and the present of 3% NaCl (w/v). It should be noticed that the DE of lipopeptides was almost kept when SORs dropped to 1 : 250 w/w. The increase in DE with increasing SORs can be attributed to the generation of emulsions with smaller droplets and lower rising velocity.² Sharp drop off in DE was observed for lipopeptides when SORs below 1 : 500 (w/w), and DE value was 36.45% at an extreme SORs of 1 : 1250 (w/w). It had been reported that the abrupt decline for 80 : 20 lecithin : Tween 80 (w/w) surfactant happened when SORs below 1 : 100 v/v, from 77% (SORs 1 : 100 v/v) to 15% (SORs 1 : 200 v/v),²⁵ indicating a lower SOR in lipopeptides usage could reach its maximum effectiveness. Lipopeptides exhibited >70% DE values with temperatures ranged from 15 °C to 25 °C, and an increasing DE values when pH values raised, the largest DE was 77.45% at pH 11. DE of lipopeptides increased from 56.12% to 71.14% with increase in salinity. Higher DE at higher salinity was observed for anionic biosurfactants, which can be attributed to the electrostatic repulsion between polar head groups reduced by ions, and a close-packed arrangement of surfactant molecules at the oil–water interface were formed.²Open in a separate windowFig. 2The dispersion effectiveness (DE) of lipopeptides under different SORs (), temperatures (), pH values () and salinities ().Mortalities of zebrafish under different concentrations of different surfactants were shown in Fig. 3. It was evident that the toxicity of lipopeptides was far less than those of sodium dodecyl sulfate (SDS) and 3-(N,N-dimethyl palmityl ammonio) propane sulfonate (Betaine). The 24 h median lethal concentration (LC₅₀) values were calculated and showed in 26 in which an 96 h LC₅₀ of 1.9 mg L⁻¹ for Cyprinodon variegatus was reported. Low toxicities of lipopeptides on whiteleg shrimp and copepods were also evaluated that the 96 h LC₅₀ of lipopeptides from Bacillus sp. GY19 were 1050 mg L⁻¹ and 1174 mg L⁻¹, respectively.²⁷Open in a separate windowFig. 3Mortality of zebrafish (M_z) after a 24 h exposure to Betaine, SDS or lipopeptides.Ecotoxicity of tested surfactants to the zebrafish

Large-scale synthesis of a monophosphonated tetrathiatriarylmethyl spin probe for concurrent in vivo measurement of pO2, pH and inorganic phosphate by EPR

Low-field electron paramagnetic resonance spectroscopy paired with pTAM, a mono-phosphonated triarylmethyl radical, is an unmatched technique for concurrent and non-invasive measurement of oxygen concentration, pH, and inorganic phosphate concentration for in vivo investigations. However, the prior reported synthesis is limited by its low yield and poor scalability, making wide-spread application of pTAM unfeasible. Here, we report a new strategy for the synthesis of pTAM with significantly greater yields demonstrated on a large scale. We also present a standalone application with user-friendly interface for automatic spectrum fitting and extraction of pO₂, pH, and [P_i] values. Finally, we confirm that pTAM remains in the extracellular space and has low cytotoxicity appropriate for local injection.

We report a new strategy for the synthesis of a mono-phosphonated triarylmethyl radical spin probe and a standalone application with a user-friendly interface for automatic spectrum fitting and extraction of pO₂, pH, and [P_i] values.

Low-field Electron Paramagnetic Resonance (EPR) with the use of a molecular spin probe is a powerful technique to non-invasively measure important physiological parameters in a living animal.^1–4 EPR combines high sensitivity and good penetration depth. Stable tetrathiatriarylmethyl radicals (TAMs or trityls) are ideal spin probes for in vivo EPR applications. They exhibit unprecedented stability in vivo and ultra-narrow linewidths, which result in a high signal-to-noise ratio.⁵ TAM structures with spectral sensitivity to oxygen,⁶ pH,^7,8 thiol concentration,^9,10 microviscosity,¹¹ ROS,^12–14 or redox^15,16 have been developed. We recently reported on a mono-phosphonated tetrathiatriarylmethyl radical pTAM (Fig. 1) whose EPR spectrum is sensitive to multiple parameters, namely oxygen concentration, pH, and inorganic phosphate concentration, [P_i].^17–20 This multifunctional probe was utilized to profile the tumor microenvironment (TME) in various mouse models of cancer.¹⁹ The unmatched capability to measure [P_i] has resulted in the identification of this biomarker as a new TME marker for tumor progression.¹⁹ Moreover, the ability to measure pO₂, pH, and [P_i] concurrently using the same probe allows for the direct correlation of these important parameters independent of the probe distribution, providing insight into the biological processes occurring in the TME.Open in a separate windowFig. 1(A) Structure of pTAM spin probe and ionic forms at physiological pH. (B) L-band full spectrum (top) of pTAM at pH = 7.13 showing both ionic forms present in the spectrum and zoom on the high field component (bottom). The molar fraction of the acidic form P_aversus basic form P_b is a function of the pH of the solution while the linewidths are functions of the oxygen concentration. Inorganic phosphate modulates the exchange rate between the two ionic forms and the A/B distance. Spectral simulation allows the three parameters to be extracted from the spectrum.While this spin probe has proven to be of great importance for the study of tissue microenvironment in vivo, its current synthesis suffers from a very low yield. Indeed, the published synthesis¹⁷ (Scheme 1) uses a lithiation of tetrathiatriarylmethanol 1 and subsequent reaction with a (2 : 1) mixture of diethyl carbonate and diethyl chlorophosphate. This reaction leads to a statistical mixture of mono-, di- and tri-phosphonated tetrathiatriarylmethanol 2n that requires tedious purification and drastically decreases the yield of the desired 2b. After hydrolysis of the ethyl esters using sodium hydroxide and deprotection of the phosphonic acid by TMSBr, the final pTAM probe was isolated with a yield of less than 5% from 1. The published procedure allowed for isolating milligram quantity of the probe for limited in vivo studies.¹⁹ However, more extensive utilization of this probe would require a synthetic method that enables gram-scale synthesis of pTAM.Open in a separate windowScheme 1The first reported synthesis of pTAM from 1.¹⁷Hereby we report an efficient protocol for the large-scale production of the pTAM probe as well as a MATLAB application for the automatic fitting of the EPR spectra and determination of the physiological parameters, namely pH, pO_2, and [P_i]. Our new strategy takes advantage of a reaction of ipso nucleophilic substitution of an aromatic hydrogen or a carboxyl group on tetrathiatriarylmethyl derivatives reported previously.^21,22 Our synthesis starts with the deuterated Finland trityl (dFT) which can be synthesized at large scale without chromatography (Fig. 2A).^23–25 The one-electron oxidation of dFT with one equivalent of potassium hexachloroiridate(iv), K₂IrCl₆, in water leads to the trityl carbocation dFT⁺, which is immediately treated with ten equivalents of trimethyl phosphite. The nucleophilic addition of the phosphite in the para-position of the aryl ring triggers an oxidative decarboxylation, leading to the mono-phosphonic ester pTAM-(OMe)₂ in 35% conversion, as determined by HPLC/MS (Fig. 2B and S6^†). Importantly, the HPLC/MS chromatogram shows that dFT radical was also generated back from the trityl carbocation dFT⁺ in 65% yield, consistent with preferential oxidation of intermediate 3 by dFT⁺ in line with previous reports.^21,22dFT can therefore be recycled for future reactions. In addition, <5% of quinone methide (QM) was also generated from the nucleophilic addition of water on the trityl cation (see ESI^† for mechanism). The use of additional equivalents of K₂IrCl₆ did not increase the yield of pTAM-(OMe)₂ but did lead to higher conversion to the QM, TAMs with multiple phosphonates, and unidentified products. The preferential oxidation of 3 by dFT⁺ explains 50% of the back conversion of the trityl radical from the cation. The slightly higher formation of dFT observed (65%) could be the result of the direct reduction of the trityl cation by the trimethyl phosphite.Open in a separate windowFig. 2(A) Synthesis of pTAM-(OMe)₂ from dFT and (B) HPLC/MS chromatogram and m/z ratio of the products after addition of P(OMe)₃.The mono-phosphonated derivatives pTAM-(OMe)₂, and dFT can be separated using a C18 column in 30% and 60% yield, respectively. Finally, the phosphonic acid was deprotected by treatment of pTAM-(OMe)₂ with TMSBr in 95% yield (Scheme 2).Open in a separate windowScheme 2Deprotection of the phosphonic acid leading to pTAM.However, for a multigram scale, we found the separation of dFT and pTAM-(OMe)₂ to be more challenging. The use of other phosphites with longer alkyl chains (triethyl-, triallyl- or tributyl phosphite), allowed for easier purification but led to smaller conversion (15–25%). On a large scale (tens of grams), the quantitative esterification of the carboxylic acids using methyl iodide and sodium carbonate in DMF directly on the dFT/pTAM-(OMe)₂ mixture (Scheme 3) allowed for easy purification by flash chromatography on silica gel. The esterified pTAM-(OMe)₄ was isolated in 35% yield from dFT starting material alongside with dFT-(OMe)₃ (63%). Then, the phosphonic acid was deprotected by TMSBr in DCM, and the methyl esters hydrolyzed using lithium hydroxide in 1,4-dioxane/water leading to pTAM in 95% yield after purification on a C18 column. dFT-(OMe)₃ was also hydrolyzed, leading to dFT in 99% yield with no purification needed. The relatively low conversion of dFT to the monophosponated ester is compensated by the recovery of the starting material. The calculated yield based on the recovery of the starting material reaches 92%. Our large scale synthesis allowed for the selective mono-phosphorylation of dFT in 4 steps and two purifications. The key step is the nucleophilic quenching of the trityl cation by trimethyl phosphite leading to the mono-phosphonated derivative.Open in a separate windowScheme 3Esterification of the carboxyl groups to allow for large-scale separation of pTAM-(OMe)₄ and dFT-(OMe)₃. Then the carboxyl and phosphonic acids are deprotected, leading to pTAM and dFT.The extraction of pO₂, pH, and [P_i] from the spectrum can be achieved using spectral fitting of the whole spectrum (see Fig. 1B, top) or only the high or low field EPR lines (Fig. 1B, bottom) using a homemade MATLAB algorithm as reported previously.^18,19 However, to provide a user-friendly interface for those unfamiliar with MATLAB, we developed a graphical user interface for fitting the spectra and deriving the values for pO₂, pH, and [P_i]. Fig. 3 demonstrates the use of the standalone application to fit a spectrum of pTAM administered into the mammary gland of a MMTV-PyMT mouse (see ESI^† for calibration and use of the app).Open in a separate windowFig. 3Screenshot of the pTAM spectrum fitting app developed in-house with a spectrum measured of pTAM injected directly in the mammary gland of a MMTV-PyMT mouse. Values of pO₂ = 84.21 mmHg, pH = 7.07 and [P_i] = 1.91 mM are automatically calculated from the experimental spectrum.When applied in vivo, the charged nature of the probe and its large size (MW = 1073 g mol⁻¹) is expected to prevent its diffusion through the cell membrane. In order to verify that pTAM cannot enter the cytosol, pTAM (200 μM) was incubated with 8.5 × 10⁶ MDA-MB-231 cells (human triple negative breast cancer cells) with and without 10 mM Gd-DTPA, a paramagnetic extracellular broadening agent.⁷Fig. 4 shows a large broadening of the EPR lines of pTAM upon addition of Gd-DTPA and no residual narrow component confirming the absence of pTAM spin probe in the intracellular compartment. In vivo, the physiological parameters reported by pTAM are therefore the extracellular ones.Open in a separate windowFig. 4X-band EPR spectra of pTAM (200 μM, 100 μL) incubated with 8.5 × 106 MDA-MB-231 cells without (black) and with 10 mM of Gd-DTPA (red) as extracellular broadening agent.Next we assessed pTAM cell toxicity using the MTT assay for cell viability and proliferation. MDA-MB-231 cells at 60–70% confluency were incubated with various concentration of pTAM for 24 h. The result (Fig. 5) shows that up to 1 mM, the probe is well tolerated with ∼80% cell viability after 24 h. It is worth noting that only a few hundred micromolar range is required for in vivo L-band spectroscopy and the MTT results show no significant difference between 100 μM of probe and the control. Moreover, pTAM was incubated 24 h with cells while in vivo the probe is cleared from the tissue in less than 1 h.²⁶ Therefore, the probe can be considered as non-toxic upon local injection, which is the mode of delivery for pTAM.¹⁹Open in a separate windowFig. 5MTT assays for pTAM at various concentration incubated with MDA-MB-231 cells for 24 h. (n = 3, *p < 0.05, **p < 0.01).     

Electronic energy levels of porphyrins are influenced by the local chemical environment

Self-assembled islands of 5,10,15,20-tetrakis(pentafluoro-phenyl)porphyrin (2HTFPP) on Au(111) contain two bistable molecular species that differ by shifted electronic energy levels. Interactions with the underlying gold herringbone reconstruction and neighboring 2HTFPP molecules cause approximately 60% of molecules to have shifted electronic energy levels. We observed the packing density decrease from 0.64 ± 0.04 molecules per nm² to 0.38 ± 0.03 molecules per nm² after annealing to 200 °C. The molecules with shifted electronic energy levels show longer-range hexagonal packing or are adjacent to molecular vacancies, indicating that molecule–molecule and molecule–substrate interactions contribute to the shifted energies. Multilayers of porphyrins do not exhibit the same shifting of electronic energy levels which strongly suggests that molecule–substrate interactions play a critical role in stabilization of two electronic species of 2HTFPP on Au(111).

Self-assembled islands of 5,10,15,20-tetrakis(pentafluoro-phenyl)porphyrin (2HTFPP) on Au(111) contain two bistable molecular species that differ by shifted electronic energy levels.

The packing of two-dimensional self-assembled monolayers is facilitated by molecule–surface and molecule–molecule interactions. At low coverage, molecules with repulsive intermolecular interactions can adsorb at preferred surface-adsorption sites,^1–4 while at higher concentrations the preferred surface site may be sacrificed to increase density and maximize intermolecular interactions.^3–6 Surface adsorption can change the conformational structure of a molecule to maximize these interactions resulting in a conformational shape not found in bulk crystals or solvated molecules.^4,7–10 Rational manipulation of shape and local environment can fine tune the electronic properties of the adsorbate.Porphyrins consist of four pyrrole-like moieties connected with methine bridges. The high level of conjugation, with 18 π electrons in the shortest cyclic path, allow porphyrins to strongly absorb light in the visible region, 400–700 nm.^11–13 Porphyrins are known as the color of life molecule.^14,15 Heme, an iron–porphyrin complex, is responsible for transporting oxygen through the bloodstream and gives red blood cells their bright color.¹⁶ Chlorophyll, a partially hydrogenated porphyrin, is responsible for the green color in plants and plays a critical role in photosynthesis.¹⁶ The strong interaction with light, overall chemical stability, and diversity through functionalization makes pyrrolic macrocycles ideal candidates for use in photovoltaics,^17–21 chemical sensors,^22–25 catalysis,^26–28 and molecularly based devices.^29–32The existence of bistable switchable states is the first step towards use of porphyrins in applications of molecular memory storage or binary devices.³³ Pyrrolic macrocycles have been shown to exhibit bistable switching behavior on surfaces including induced conformational switching,^34–36 orientational flip-flopping,^37–41 and tautomerization.^42–46 Examples include subphthalocyanine arrays that adsorb with a shuttlecock shape and exhibit scanning tunneling microscopy (STM) induced reversible orientational switching on Cu(100),³⁷ and current from the STM tip that has been used to induce hydrogen tautomerization in naphthalocyanine on an NaCl bilayer on Cu(111).⁴²Confinement to two dimensions through adsorption of 2HTFPP onto a surface creates a complex heterogeneous environment with influences from the substrate and neighboring molecules. In this study, we characterize the local density of states of 2HTFPP adsorbed on Au(111), and found that the local chemical environment can cause the energies of molecular orbitals to shift. This creates an environment where chemically identical molecules exist as two species on the surface.The sample was prepared using an Au(111) substrate on mica (Phasis) that was cleaned through several rounds of annealing and argon sputtering. Images were collected at ultra-high vacuum and 77 K with a low-temperature scanning tunnelling microscope (LT-STM, Scienta Omicron) under constant current mode. The tips were mechanically etched 0.25 mm Pt80/Ir20 wire (NanoScience Instruments). The sample was prepared at room temperature by depositing 1 mM solution of 2HTFPP dissolved in dichloromethane via pulsed-solenoid valve in a vacuum chamber onto the clean gold or 2HTFPP dissolved in dimethylformamide via drop-casting and annealing to 200 °C.⁴⁷The electronic structure of 2HTFPP, neutral TFPP, and dianionic TFPP were calculated using density functional theory (DFT). The structures were optimised using the B3LYP^48,49 density functional approximation and the 6-311+G(d) basis set. A harmonic vibrational frequency analysis confirmed the structure was in a local minimum on the ground state potential energy surface without imaginary frequencies. All calculations were performed on Gaussian 16 Rev. A.03.⁵⁰ Simulated STM images were computed using the Tersoff and Hamann approximation^51,52 as done by Kandel and co-workers.^53,54 The tunneling current was modeled as a function of tip position, and the surface was modeled as a featureless and constant density of states to reproduce a constant current experiment. Through the Tersoff and Hamann approximation the tunneling current leads to information on the local density of states at a specific energy and a discrete location.Pulse-depositing 2HTFPP in vacuum via a pulsed-solenoid valve resulted in ordered islands of the adsorbate, Fig. 1. The formation of close-packed islands, despite submonolayer coverage, indicates attractive intermolecular interactions between adsorbates. 2HTFPP has a packing density of 0.64 ± 0.04 molecules per nm² with a 1-molecule unit cell and rectangular packing.Open in a separate windowFig. 1(a–c) Unoccupied and (d–f) occupied electron states of 2HTFPP. All scale bars are 5 nm. (e) Two bistable species, diffuse square outlined in green and a double-dot feature outlined in white. (f) 2HTFPP appears as double-dot features. The white box indicates the 1-molecule, rectangular unit cell. Images taken with a tunneling current of 5 pA.Changing the polarity of the bias voltage of the STM sample allows unoccupied (positive voltage) or occupied (negative voltage) molecular orbitals to be imaged along with the topography of the surface. The unoccupied electron states, Fig. 1a–c, have an even distribution of electron density across the molecule and appear as diffuse squares. As the magnitude of the bias voltage is increased, a larger number of electronic energy levels are included in the measurement. Increasing the magnitude of the negative bias voltage from −0.25 V to −1.5 V, Fig. 1d and f, results in a change in contrast from a diffuse square to a bright double-dot feature. Imaging at −0.75 V, an intermediate voltage between these two energy levels, we observe two species—a mixture of diffuse squares and double-dot features, Fig. 1e. These species are differentiated from one another by having the same ground state molecular orbitals at shifted energies. If all the molecules on the surface were equivalent, we would expect to image the same molecular orbitals of each molecule at each bias voltage. At −0.75 V we do not observe the same orbital shape for each molecule, but we observe influence of the HOMO−2 and HOMO−3 energy levels in approximately 60% of that adsorbed 2HTFPP. Interaction with the local chemical environment causes the energy levels to be shifted enough to be included at a bias voltage with a smaller magnitude.We investigated the possibility that the two species were chemically different. If a gold atom formed a complex with the porphyrin, we would expect the displacement of the inner pyrrolic hydrogens. It was also possible that the presence of the STM tip was altering the adsorbates. Smykalla et al. reported that the STM tip can reversibly remove and replace the pyrrolic hydrogens which changes the shape and contrast of the porphyrin in the STM images.⁵⁵ Deng and Hipps reported a small shift in orbital energies of 0.04 eV for vapor-deposited metallo-porphyrins on Au(111) due to the height of the tip.⁵⁶ We observed a march larger shift of orbital energies and used DFT to calculate shapes of the occupied and unoccupied molecular orbitals of gas phase 2HTFPP, TFPP with the inner pyrrolic hydrogens removed, dianionic TFPP with the inner pyrrolic protons removed, and Au-TFPP with a gold atom coordinated to the porphyrin, Fig. 1 in ESI.^† We used the electronic structure calculations to make theoretical STM images. We found that the double-dot feature was only present in the occupied orbitals of 2HTFPP, Fig. 2. Theoretical STM images of Au-TFPP, Fig. 2 in ESI,^† does not show the double-dot feature. This suggests that intact 2HTFPP molecules make up the close-packed islands, and both detected species are chemically equivalent. The shift in electronic energy levels is due to the heterogeneous local chemical environment.Open in a separate windowFig. 2DFT, theoretical STM images, and comparison with experimental STM images of 2HTFPP. HOMO and HOMO−1 have a diffuse square shape, while HOMO thru HOMO−3 exhibit the double dot feature. Both of these shapes were experimentally observed at −0.75 V.The packing of adsorbates is a balance between molecule–molecule and molecule–substrate interactions. We observed various packing densities and unit cells that were dependent on sample preparation. Preparation with the pulsed-solenoid valve at room temperature created a 1-molecule unit cell with a packing density of 0.64 ± 0.04 molecules per nm², Fig. 1. Annealing the sample to 200 °C resulted in a 1-molecule unit cell with a packing density of 0.38 ± 0.03 molecules per nm², Fig. 4. Annealing altered both the adsorption sites and intermolecular ordering of 2HTFPP, and we no longer observed two electronic species on the surface. Annealing to 200 °C may remove excess solvent and provide the energy needed for the adsorbed species to leave the preferred adsorption site and maximize intermolecular interactions. This suggests that the local chemical environment plays a critical role in the creation of two bistable species. The surface of Au(111) undergoes herringbone reconstruction with packing of the surface gold atoms.^57,58 The surface consists of both face-centered cubic packing (fcc) and hexagonally-close packed (hcp) gold atoms with strained transition areas. The three different domains create a heterogeneous surface electronic structure.⁵⁹ To investigate the relationship of species 1 and species 2 with the underlying substrate we characterized the images using Fast Fourier Transform (FFT). FFT converts an image from real space to frequency space and can reveal longer-range ordering of molecular assemblies.Open in a separate windowFig. 42HTFPP after annealing to 200 °C. The unit cell has dimensions of 1.6 nm × 1.6 nm and the monolayer has a packing density of 0.38 molecules per nm². The bright double-dot features are doubly stacked porphyrins, the start of a second layer. The image was taken with a bias voltage of −10.0 mV and a tunneling current of 10 pA.In order to focus the FFT on one species at a time, white circles were placed over each double dot feature, Fig. 3a and b. FFT were taken of the images with the circles to determine the packing of the adsorbates, Fig. 3c and d. Hexagonal ordering as well as longer-range rectangular packing can be seen in the FFT, Fig. 3c, of the locations of the double-dot features in the image showing two species of 2HTFPP, Fig. 3a. We observed rectangular packing of 2HTFPP when the magnitude of the bias voltage was large enough for all of the adsorbed porphyrins to exhibit bright double-dot features, Fig. 3b and d. The presences of hexagonal packing for one species, but not the overall self-assembled island suggests that the adsorption site on the underlying gold herringbone reconstruction is playing a role in the shift of electronic energy levels. We did not observe the two species after annealing to 200 °C, suggesting that solvent and intermolecular interactions may influence the location of the adsorption site on Au(111). We also observed that the double-dot features in Fig. 3a were more likely to exist around molecular defects and edges of the close-packed island suggesting that intermolecular interactions also play a role in the shift of the electronic energies of the molecular orbitals.Open in a separate windowFig. 3(a) Circles placed over each 2HTFPP molecule with bright double dot features at −0.75 V and (b) −1.5 V (c) is FFT of (a) showing hexagonal order (d) is FFT of (b) showing rectangular order.Pulse deposition with a solenoid valve into vacuum has been shown to create meta-stable molecular motifs due to the rapid evaporation of solvent.⁵³ We planned to directly compare room-temperature pulse deposition with drop-casting but were unable to resolve drop-cast porphyrins prior to annealing. Drop-casting followed by annealing resulted in an alternate packing of 0.21 molecules per nm², Fig. 3 in ESI.^† Despite the fluorinated phenyl group inverting the polarity of the ring, we observe π stacking of pentafluorophenyl rings after pulse-depositing and annealing to 200 °C, Fig. 4. Although this is unexpected, Blanchard et al. have previously observed face-to-face π stacking in the crystal structure of pentafluorophenyl substituted ferrocenes.⁶⁰Several molecules on top of the self-assembled island, the start of a second layer, appear with bright double-dot features, Fig. 4. We did not observe the coexistence of two electronic species in the second layer of 2HTFPP.In conclusion, we observed two bistable species of 2HTFPP on Au(111) with non-degenerate electronic energy levels. DFT and theoretical STM images suggest that the two species are both 2HTFPP and not chemically altered through adsorption or interaction with the STM tip. The species with the affected electronic energy levels appear near molecular vacancies and exhibit longer-range hexagonal packing indicating that the two species are created through molecule–substrate and molecule–molecule interactions. Annealing to 200 °C decreases the packing density and alters the molecule–molecule and molecule–surface interactions. Adding a second layer of adsorbate only exhibits one electronic species which strongly suggests that the local chemical environment plays a critical role in the shift of the electronic energies of the molecular orbitals of 2HTFPP.     

A julolidine-fused anthracene derivative: synthesis,photophysical properties,and oxidative dimerization

We describe the synthesis and characterization of a julolidine-fused anthracene derivative J-A, which exhibits a maximum absorption of 450 nm and a maximum emission of 518 nm. The fluorescent quantum yield was determined to be 0.55 in toluene. J-A dimerizes in solution via oxidative coupling. Structure of the dimer was characterized using single crystal X-ray diffraction.

A julolidine fused anthracene derivative with unique photophysical and redox properties was presented.

Julolidine¹ is a popular structural subunit in various fluorescent dyes (Chart 1).² The restricted motion and strong electron-donating capability of the fused julolidine moiety are quite effective for improving the photophysical properties. For instance, julolidine-fused fluorophores normally display desirable photophysical characteristics, such as high quantum yield, red-shifted absorption and emission, and good photostability. Recently, julolidine derivatives have been widely exploited in various applications such as sensing,³ imaging,⁴ and nonlinear optical materials.⁵ Several julolidine dyes have been used in dye-sensitized solar cells due to their large π-conjugated system and the promising electron donating property.⁶Open in a separate windowChart 1The structure of julolidine, J-A and DAA.In this paper, we report a julolidine-fused anthracene derivative J-A, which exhibits attractive photophysical properties not observed in DAA, a dimethyl-amino substituted analogue. Both the absorption and emission of J-A show a dramatic red-shift (ca. 74 and 131 nm, respectively), compared with the unmodified anthracene (Fig. 2). The fluorescence quantum yield of J-A was determined to be 0.55 in toluene, while the emission of DAA was completely quenched. The observed spectral properties were rationalized by DFT calculations. In addition, we found that J-A was stable in the solid state, but reactive in solution. J-A dimer was formed through oxidative coupling at the para-position of the N-atom in a dichloromethane solution under air atmosphere. The structure of the dimerized product was characterized using single crystal X-ray diffraction, which unambiguously reveals the structural feature of the julolidine-fused anthracene compound. Preparation of J-A is shown in Scheme 1. Detailed synthesis and characterizations are provided in the ESI.^†Open in a separate windowScheme 1Synthetic route of J-A.Open in a separate windowFig. 2(A) Absorption spectra of J-A, AN, and DAA (1 × 10⁻⁴ mol L⁻¹ in dichloromethane); (B) emission spectra of J-A, AN, and DAA (1 × 10⁻⁵ mol L⁻¹ in dichloromethane). Excitation wavelength: 350 nm. 1 cm cuvette was used in both of the experiments. Inset: visualized fluorescence in solution was shown. ¹H-NMR signals of J-A shift to the high-field significantly, compared with DAA (Fig. 1), which indicates that the fused structure of J-A facilitates electron delocalization from the nitrogen atom to the anthracene moiety, and thus resulting in a stronger shielding effect. In the case of DAA, however, electron delocalization from the dimethyl amino group to the anthracene core is essentially inhibited due to steric hindrance, which will explain the fact that it displays a spectral feature similar to that of the unmodified anthracene.Open in a separate windowFig. 1Comparison of the ¹H-NMR spectrum between J-A and DAA in CDCl₃. Partial resonance signals in aromatic region are shown.Fusing with julolidine will exert significant effects on the photophysical properties of anthracene. The absorption and fluorescence spectra of J-A, DAA, and anthracene are shown in Fig. 2. The maximum absorption of J-A is 450 nm, which displays a red-shift of about 70 nm compared with the unmodified anthracene. J-A emits green light (^maxλ_em = 518 nm, Φ = 0.55), while anthracene emits blue light (^maxλ_em = 380 nm, Φ = 0.22). In contrast, the absorption of DAA essentially overlaps with that of anthracene, with only a minor red shift of ca. 10 nm, but its fluorescence is quenched significantly (Fig. 2). This spectral feature indicates that the dimethyl amino group is electronically separated from the anthracene moiety in the ground state, a result in good accordance with the ¹H-NMR data shown above. The quenched fluorescence of DAA may result from the photo-induced electron transfer⁷ from the lone pair of the nitrogen atom to the anthracene moiety in the excited state.The observed photophysical properties of J-A were reproduced by DFT calculations. The HOMO and LUMO orbitals are evenly distributed over the anthracene moiety and the N-atom in the julolidine, indicating the existence of a conjugated structure. The HOMO–LUMO transition (f = 0.10) corresponds to the absorption band at 450 nm. The sharper absorption at 390 nm can be assigned to the HOMO to LUMO + 1 transition. In contrast, the HOMO and LUMO orbitals of DAA resemble those of anthracene, because the dimethyl-amino group is orthogonal to the conjugated π-system (Fig. 3).Open in a separate windowFig. 3Molecular orbitals of J-A and DAA calculated at the B3LYP/6-31G(d) level of theory (iso value = 0.02). Orbital energies were given in parentheses. Excitation energies were computed by TD-DFT at the same level. Values in parentheses represent the oscillator strengths (f).J-A is stable in the solid state, but reactive in solution. The cyclic voltammetry (CV) diagram of J-A shows an irreversible oxidation potential at 0.007 V (vs. Fc/Fc⁺), indicating that J-A is easy to be oxidized (Fig. S3^†). Single crystals suitable for X-ray diffraction study were obtained by slow evaporation of a dichloromethane solution of J-A under air atmosphere. To our surprise, instead of J-A, X-ray data discloses the formation of a dimeric product (Scheme 2) of J-A. We hypothesized that the dimeric compound 5 formed via oxidative coupling reaction, a mechanism well-documented for the dimerization of the dimethylaniline compounds.⁸ The ¹H-NMR spectrum of 5 is distinct from that of J-A. All protons of the anthracene part (b′–d′) appear as a group of multiplet resonance signals (6.93–7.04 ppm) (Scheme 2). In addition, mass spectrometric analysis indicates that two hydrogen atoms were removed after the dimerization of J-A. Compound 5 exhibits a maximum absorption at 460 nm and a very weak emission (^maxλ_em = 530 nm, Φ = 0.03, Fig. S1^†). Two quasi-reversible oxidation waves were identified in the CV diagram of 5 at −0.010 V and 0.135 V (vs. Fc/Fc⁺), respectively (Fig. S5^†).Open in a separate windowScheme 2Oxidative dimerization of J-A. Inset: partial ¹H-NMR of 5 is shown.X-ray structure of 5 is shown in Fig. 4. The two connected anthracene planes are found to be orthogonal to each other with a dihedral angle of 90.17°, as a result of steric repulsion. Specifically, the bond length of N–C3 is 1.389 Å, which is similar to those of the other reported julolidine compounds (1.359–1.393 Å), while significantly shorter than that of the dimethyl-amino anthracene (1.433 Å).⁹ This result testifies the presence of electron delocalization between the fused julolidine nitrogen and the anthracene π-plane, which is in good agreement with the DFT calculations (Fig. 3). However, the two anthracene π-planes connected by the single bond (C4–C5, 1.489 Å) might not exhibit electron delocalization because of the orthogonal conformation. The fused julolidine ring-i and -ii are symmetric to each other, and both of them adopt an “envelope” conformation. The fused julolidine is nonplanar (bond angle, C3–N–C2, 115.73°, C3–N–C1, 115.92°, C1–N–C2, 113.81°). 5 is closely packed in the crystal (Fig. 4B), and no intercalated solvent molecules were observed. The closest distance between two adjacent anthracene planes is 3.922 Å, indicating a weak π–π stacking. Detailed crystal data are summarized in Table S5.^†Open in a separate windowFig. 4(A) Single crystal X-ray structure of 5. (B) View along a-axis.In summary, we report the synthesis and characterizations of a julolidine-fused anthracene derivative J-A, which demonstrates significantly red-shifted absorption (^maxλ_ab = 450 nm) and emission (^maxλ_em = 518 nm, Φ = 0.55), compared with the unmodified anthracene. The photophysical properties of J-A also contrast dramatically with a dimethyl-amino analogue DAA, which were rationalized by DFT calculations. In addition, J-A could be transformed into 5, a dimeric product, whose single crystal X-ray structure unambiguously confirmed the structural feature of the julolidine-fused anthracene.     

Correction: High iodine adsorption performances under off-gas conditions by bismuth-modified ZnAl-LDH layered double hydroxide

Correction for ‘High iodine adsorption performances under off-gas conditions by bismuth-modified ZnAl-LDH layered double hydroxide’ by Trinh Dinh Dinh et al., RSC Adv., 2020, 10, 14360–14367, DOI: 10.1039/D0RA00501K.

The authors regret that the reference for Fig. 1 was omitted. The reference has been added below.Open in a separate windowFig. 1Schematic of the device for the iodine adsorption experiments in static air.¹The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers.     

Ultra-high adsorption of cationic methylene blue on two dimensional titanate nanosheets

In this work, we examined the performance of 2D titanate nanosheets for dye adsorption. Their adsorption capacity for methylene blue (MB) is up to 3937 mg g⁻¹, which is more than 10 times higher than active carbon and occupies the highest place among all the reports.

2D titanate nanosheets show ultra-high adsorption for methylene blue.

Dyes are important materials in many industries such as the textile, paper, leather, printing, and plastic industries. However, the dye effluents released by these industries give rise to major environmental issues because the dyes are generally toxic and/or carcinogenic to human beings.¹ Dye removal from the wastewater is essential before release in these industries. There are several methods to remove dye contaminants from wastewaters, such as adsorption, coagulation, chemical oxidation, membrane separation processes, etc.¹ Among the technologies, the adsorption process has been generally considered to be the most efficient method of quickly lowering the concentration of dissolved dyes in an effluent.² Various adsorbents have been studied for dye removal purposes, such as activated carbons (ACs),^3–6 clay,^7,8 sawdust,⁷ beer brewery waste,² chitosan,⁹ metal oxides,^10–12 carbon nanotubes and graphene oxide.^13,14 A general strategy to enhance the dye adsorption is to increase the surface area of the adsorbents, so the nanosized materials have been attracting much attention for the dye removal application because they exhibit much large surface area.^14–20Two dimensional (2D) materials are gaining great attention since discovery of graphene.^21–23 The 2D materials consist of large number of groups, such as graphene, transition metal dichalcogenides (TMDs), transition metal oxides (TMOs), layered double hydroxides (LDHs), MXene et al.^21,24–26 One may divide the 2D materials into three groups based on the charge of the host layer: neutral, negatively charged, and positively charged. The charged 2D materials must couple with counterions to compensate the charges. As a sample, 2D titanium oxide, also called titanate nanosheet (TONS) is negatively charged. The TONS is generally synthesized by the chemical exfoliation.^27,28 These chemical exfoliated TONS is monolayer nanocrystals with crystallographic thickness of 0.75 nm.²⁹ The nanosheets in aqueous solution behave as individual molecular entities, and they are surrounded by positively charged ions (TBA⁺ and H⁺).^28,30,31 The colloidal system is governed by electrostatic interaction.³¹As an emerging class of materials, the charged 2D materials are potentially useful for treatment of the dyes with opposite charges. This has motivated us to study the performance of the titanate nanosheets, a charged 2D material, for dye treatment.We synthesized the titanate nanosheets via top down approach report by Sasaki et al.^32,33Fig. 1a and b show the XRD pattern and SEM image of the protonated titanate. The XRD pattern is consistent with the previous report by Sasaki et al.^32,33 The SEM image shows that the crystal size is up to 20 μm. Fig. 1c shows the UV-Vis spectrum of the exfoliated titanate nanosheets. The peak is located at 265 nm, which is consistent with the value in the previous reports.^28,29,34Fig. 1d shows the STEM image of the nanosheets obtained by chemical exfoliation, and provides an evidence for the successful exfoliation. The AFM image of the nanosheets transferred to Si substrate by simple adsorption shown in Fig. S1^† shows the lateral size of the adsorbed nanosheets is generally less than 1 μm.Open in a separate windowFig. 1The XRD (a), SEM image (b) of protonated titanate; UV-Vis spectrum (c) and STEM image (d) of 2D titanate nanosheets. The dash line in (c) indicates the position of 265 nm.We first evaluated the adsorption capacity of the exfoliated nanosheets comparing with its bulk counterparts K_0.8[Ti_1.73Li_0.27O₄] (KLTO) and its protonated form (HTO). Fig. 2a shows that the adsorption capacity and adsorption percentage of the TONS in the function of the initial concentration of MB. The observed adsorption capacity increased up to 2236 mg g⁻¹ as the initial MB concentration was 4000 mg l⁻¹. It is noted that this value is underestimated because we didn''t consider the MB loss in experimental process. We analyzed the adsorption isotherm by fitting it with the Freundlich and the Langmuir models and the fitting curves are shown in Fig. 2b. The Langmuir model was fitting better than the Freundlich model, and the correlation coefficients R² for the Langmuir and the Freundlich models are 0.97 and 0.93, respectively. The better Langmuir model fitting indicates that the adsorption of MB on TONS surface is homogenous.¹¹ The maximum adsorption capacity fitted by the Langmuir model is 3937 mg g⁻¹. This adsorption capacity is, to the best of our knowledge, the largest for the MB adsorption among all the reports (see Table S1 in the ESI^†).^1,15–17,35 However, the dye removal efficiency is low even at the low initial concentration. Only half even less dye can be adsorbed as shown in Fig. 2a. Fig. 2c shows the morphology of the TONS after the MB adsorption at initial concentration of 1000 mg l⁻¹. The 2D feature of the TONS is clearly presented, indicating its good structural stability. In contrast to the large adsorption capacity of the TONS, its layered counterparts KLTO and HTO show limited adsorption capacity as shown Fig. 2d (see the adsorption data for KLTO in Fig. S2^†). Our data show that the adsorption capacities of the KLTO and HTO are 3.45 and 2.63 mg g⁻¹, respectively.Open in a separate windowFig. 2(a) The adsorption capacity and adsorption efficiency of titanate nanosheets at different initial concentrations; (b) the adsorption isotherm fitting with the Freundlich and the Langmuir models; (c) the typical morphology of TONS after MB adsorption (the initial concentration at 1000 mg l⁻¹); (d) the UV-Vis spectra of MB solution with concentration of 10 mg l⁻¹ before and after HTO adsorption. Fig. 3a presents the UV-Vis spectra of the MB (10 mg l⁻¹) and the TONS solution centrifuged at different time. The TONS shows rapid adsorption of the MB molecules. The concentration of the MB monomers dropped to a negligible concentration within 1 min, and a new peak appeared at ∼575 nm (purple by naked eyes) which can be assigned to the trimer form of the MB.^36–38 It is noted that the adsorption of MB monomers onto the TONS is much faster than other material systems, such as clay and graphene oxide.^14,39 The reason beyond this phenomenon is probably due to the large surface area and high charge density. It has been reported that the external adsorption of the MB is a fast process comparing with the internal adsorption in the clay system.³⁹ In this work, the TONS were dispersed in aqueous solution, so the external adsorption was dominating in the adsorption process. Another interesting feature is that the concentration of the MB trimers increased as the time proceeded. Because the MB monomers had been adsorbed onto the TONS within 1 min, the increase of the MB trimer concentration after 1 min suggests that the MB trimers came from desorption of the MB from the TONS. We analyzed the kinetic of trimer formation by assuming that the MB⁺ ions participating formation of trimer are irrelevant to the kinetic of desorption of MB–TONS complex, and we found the third order kinetic model is the best fitting to the experimental data among first, second and third order kinetic models, see Fig. 3b. The third order kinetic model yielded the correlation coefficient value of 0.965, while the first and second order kinetic models yielded the correlation coefficient values of 0.909 and 0.948, respectively. The data suggest that recombinative molecular desorption occurred, that is, multiple MB molecules desorbed and formed trimers. The desorption of counterions on the TONS was also observed with the TBA⁺ ions because the TBA–TONS complex went through an ionization process in the course of time.⁴⁰ The data here suggest the MB–TONS complex also went through an ionization process in the course of time after the rapid combination.Open in a separate windowFig. 3(a) UV-Vis spectra of MB and titanate nanosheets solution centrifuged at different time. (b) The trimer adsorption intensity vs. t for describing the desorption kinetics of methylene blue (10 mg l⁻¹) on the TONS.We also investigated the effect of the initial concentration of the MB on adsorption. Fig. 4a shows the UV-Vis spectra of residual (equilibrium) solution at different initial concentrations. The data show that the trimers were the main product after desorption at low concentrations (<100 mg l⁻¹), while the dimers (peak position at ∼615 nm) appeared and became more and more pronounced at high concentrations (>300 mg l⁻¹). We plotted the intensity change of monomers and dimers in reference to trimers in Fig. 4b. It is clear that the intensity of monomers didn''t change significantly in reference to trimers, but the intensity of the dimers did when the initial concentration of the MB solution increased, especially at 500 mg l⁻¹. It is noted that one should not compare the absolute values instead of the trend in the curves. The right vertical axis in the Fig. 4b presents the ratio of active surface area of adsorbed MB : TONS in the function of initial MB concentration in our study. We calculated the values assuming the MB molecules have only one side active to occupy the surface of the TONS. Fig. 4b shows that the number of the adsorbed MB⁺ ions was sufficient to cover the entire TONS surface and the second layer of the MB⁺ formed on the TONS surface when the initial concentration of MB solution reached 300 mg l⁻¹. Taking into consideration that the dimer started forming at the initial concentration of 300 mg l⁻¹, (see Fig. 4a) and the dimer formation enhanced further as the MB⁺ concentration increased, these data suggest that the formation of first layer MB⁺ on TONS surfaces suppresses the formation of trimers and promotes the formation of dimers. This is reasonable because the formation of first layer MB⁺ reduces the charge density of the MB–TONS complex. Bujdák et al. report that the high charge density of clay induces high order agglomeration of MB dye and low charge density suppresses the agglomeration.⁴¹ In our study, the charge density of the TONS remains constant, but the surface charge density of nanosheet complex changes with different amount of MB adsorption. The charge density of the MB–TONS complex determines the yield either trimer or dimer.Open in a separate windowFig. 4(a) UV-Vis spectra of residual (equilibrium) solution, (b) the intensity change of the monomer and dimer in the reference with trimer and the ratio of active surface area of adsorbed MB : TONS (the red dash line marks the position of left vertical axis at 1).In these colloidal systems, the TONS complex is governed by electrostatic force.³¹ According to the double layer theory, the TONS should be tightly bounded with the counterions such as MB⁺, TBA⁺ and H⁺ in first layer. In our solutions which are very basic, the concentration of these ions is in following order, TBA⁺ (0.0129 mol l⁻¹) > MB⁺ (0.00313 mol l⁻¹ for 1000 mg l⁻¹) ≫ H⁺. So, the TBA⁺ and MB⁺ ions are the main contributors to build the first layer. Given the size of the MB⁺ molecules 1.7 × 0.76 × 0.33 nm, the charge density is calculated to be 1.78q_e/nm² (where q_e is the electron charge) in the form of perpendicular to the TONS.⁴² In reality, the charge density should be smaller than this value because MB molecules are preferable to bound with TONS at certain angle. For example, the angle was found to be 65–70 °C on mica surface.⁴¹ The charge density of the TBA⁺ and NSTO is 1.63 and 4.72q_e/nm², respectively.²⁸ Therefore, one layer of the TBA⁺ and MB⁺ ions is insufficient to compensate the charge on the TONS surface. The TBA⁺ and MB⁺ ions in second and outer layers are necessary, but are loosely attracted to the TONS by the Coulomb force. The large dye adsorption capacity is probably due to this multilayer dye molecule adsorption behavior and the large surface area of the TONS. The mechanism of the ultra-high adsorption is to be verified.In summary, we studied the charged TONS for the MB adsorption treatment. We found that the TONS possess an ultra-high dye adsorption, and the adsorption capacity of titanate nanosheets is found up to 3937 mg g⁻¹. However, our data show that the dye removal efficiency of titanate nanosheets is low as less than 50%. Based on our analysis, we conclude that the large dye adsorption capacity is due to the large surface area of the TONS and the multilayer dye molecule adsorption behavior because of the presence of the surface charge. Furthermore, our study shows that the adsorption is a rapid process, and the loosely adsorbed MB molecules go through desorption to form high order agglomerates in the course of time. Our analysis indicates the desorption process a recombinative molecular desorption. What''s more, the product after desorption varies with the initial concentration of MB. Low concentration yields trimers, and high concentration promotes formation of dimers.It is well known that MB is a well-used redox indicator for photocatalysis study, at the end, we would like to highlight that one should use MB as the indicator more carefully because the color change may not be always due to the catalytic effect.     

Photoswitchable probe with distinctive characteristics for selective fluorescence imaging and long-term tracing

A photo-switchable and high-contrast bio-imaging indicator 4,4′-(1E,1′E)-(4,4′-(cyclopentene-1,2-diyl)bis(5-methylthiophene-4,2-diyl))bis(methan-1-yl-1-ylidene)bis(azan-1-yl-1-ylidene)bis(2-(benzo[d]thiazol-2-yl)phenol) (BMBT) has been demonstrated, by integrating photochromophore with excited-state intramolecular proton transfer (ESIPT) moiety. The ability of reversible emission switching enables arbitrarily selective labeling or concealing of cells simply by controlling light irradiation. Besides, when the emission was switched on, BMBT is demonstrated to exhibit unique characteristics of aggregation induced emission (AIE), providing a high on–off ratio for favorable bio-imaging. Thus, the non-labeling and easily-controlled selective imaging, as well as good biocompatibility indicates BMBT to be a favorable cell probe with great potentials for functional bio-imaging fluorophore.

In this work, a photoswitchable probe was synthesized by integrating a photochromophore with an excited-state intramolecular proton transfer (ESIPT) moiety. It was explored to be a favorable fluorophore for selective fluorescence imaging and long-term tracing.

Although there are many conventional fluorescent probes used for fluorescent imaging in the past few years,^1–5 such as rhodamine,⁶ cyanine dye,⁷ quantum dots,^8,9 and lanthanide probes,¹⁰ these fluorescent probes can only respond irreversibly to one event.^11,12 In comparison, photochromophores,¹³ which can reversibly response with UV and visible light,¹⁴ are more valuable fluorescent probes for regional optical marking of interested cells.^15,16 Because of their favourable characteristics, such as excellent thermal stability, good fatigue resistance and fast response time, diarylethenes derivatives have drawn wide spread concern of researchers.^17–20 Furthermore, the special optical properties of diarylethenes enable them to be suitable for long time and real time monitoring in bioimaging.The significance of targeted imaging of fluorescence probe for bio-samples in vitro or in vivo is well-known for their high sensitivity, non-invasiveness, real-time detection and especially selectivity.^21–24 However, the synthesis of these targeting materials is usually complicated. Moreover, for the same type of cells, no selectivity is demonstrated by these complex targeting agents.^21–24 Therefore, easy-prepared and easy-controlled non-labeling fluorescence imaging agents have always been pursued by both the industry and scientific communities. Among the above-mentioned photochromophores, diarylethenes are expected to be promising photochromic imaging candidates due to their favorable characteristics described in preceding paragraph. Prospectively, they can be used for selective long-term tracing if the photostability can be ensured.In our previous work, a series of AIE-active excited-state intramolecular proton transfer (ESIPT) complexes have been demonstrated to be good bio-imaging candidate with many advantages such as simple preparation, good biocompatibility, high quantum yields, fast cell staining as well as long-term anti-photobleaching.^25–27 Sometimes, ESIPT compounds exhibit dual emission, originated from keto and enol state, respectively. This caused extremely fast four-level photophysical cycle (E–E*–K*–K–E), mediated by intramolecular H-bonds immediately after photoexcitation, enables two emissions.^28,29 Herein, we have demonstrated a new type of multifunctional bio-imaging materials based on facile synthesis design concept, by introducing a photochromic diarylethene moiety to enable regional emission turn-on, and introducing an ESIPT moiety to allow good photo stability for long-term tracing. To the best of our knowledge, this is the first example with integrating abilities of non-labeling selectively long-term regional tracing.The new diarylethene derivative (BMBT, see the molecular structure in Scheme 1) has been synthesized (Fig. S1^†) via condensation reaction of 5-amino-2-(benzo[d]thiazol-2-yl)phenol with 4,4-(cyclopentene-1,2-yl)-bis(5-methyl-thiophene-2-formaldehyde), according to a previous reported procedure.^30,31 The molecular structures and purities were confirmed by ¹H NMR spectroscopy and mass spectroscopy (Fig. S6 and S7,^† see the synthesis details and full molecular characterizations in the ESI^†).Open in a separate windowScheme 1Structure and photochromic process of BMBT.The photoirradiation-induced changes in absorption and fluorescence spectra at room temperature are investigated in THF/DMEM mixture (1.0 × 10⁻⁵ mol L⁻¹). The open-ring isomer mainly exhibits two absorption peaked at 304 and 380 nm, respectively (Fig. S2^†). This is ascribed to the internal charge transfer and π–π transition of 2-(2′-hydroxy-phenyl)benzothiazole (HBT),^10,30 coupled with the CT inside the HBT unit from the hydroxyphenyl ring to the benzothiazole ring (Scheme 1). BMBT exhibits two emissions; one blue emission peak around 458 nm and a red emission peak around 600 nm, corresponding to the enol and keto emission, respectively (Fig. 1). Upon irradiation with ultraviolet light at 365 nm, the absorption band at longer wavelength region centered at 595 nm increased obviously with irradiation time (Fig. S2^†). This is caused by the formation of the closed-ring isomer (see the photochromic reaction in Scheme 1). Correspondingly, due to spectroscopic overlap between this longer-wavelength absorbance with the red emission ranged from 560–700 nm, the relative intensity of the red emission substantially decreases with the UV irradiation (Fig. 1), because of efficient energy transfer. Upon further visible light (λ = 520 nm) irradiation, the closed-ring isomer transfers back to the initial open-ring isomer, and thus the longer-wavelength absorption decreases and the red emission restores. This indicates good reversibility of the photochromic reaction.Open in a separate windowFig. 1Fluorescence emission changes of BMBT in THF/DMEM (1 : 200, vol : vol) mixture upon irradiation with 365 nm light (λ_ex = 385 nm) and visible light (λ_ex = 520 nm).Excitingly, BMBT exhibit prominent characteristics of Aggregation-Induced Emission (AIE).^31–33 The fluorescence intensity of BMBT in THF solution was relatively weak, while the powder or nanoparticles of the material dispersed in DMEM buffer exhibited a significantly enhanced emission (Fig. 2). When the DMEM buffer fraction was increased gradually from 0% to 20%, the fluorescence intensity only slightly enhanced. When the DMEM fraction was further increased to 40%, the emission exhibits a significant enhancement. The total intensity at the blue enol emission increased more than 10 times at 100% DMEM fraction as compared with that in THF solution. This AIE property enables high signal-to-noise ratios for favorable bioimaging (Fig. S3^†).Open in a separate windowFig. 2Fluorescence spectra of 1 × 10⁻⁵ M BMBT in the THF/DMEM mixture at different water fractions (λ_ex = 385 nm).Based on the good reversibility of light response and AIE characteristics, the practical application of the BMBT as bioprobe was further investigated. The biological imaging of BMBT was observed by using confocal laser scanning microscopy (CLSM). Blue luminescence in the cytoplasm of HeLa cells was observed after incubation with a THF/DMEM (1 : 200, vol : vol) solution of BMBT (20 μM) for 30 min at 37 °C (Fig. 2 inset). The overlay of luminescent images and bright-field images confirmed that BMBT was located mainly in the cytoplasm of cells rather than the membrane and nucleus (Fig. S3^†). Intense intracellular luminescence with a high signal-to-noise ratio (I₁/I₂ > 7) was detected between the cytoplasm (regions 3 and 1) and nucleus (region 2), also implying weak even few nuclear uptake of BMBT (Fig. S4^†). Besides, BMBT has a low cytotoxicity with the cellular viabilities estimated to be greater than 85% after 24 h incubation with the highest cultural concentration of 50 μM BMBT (Fig. S5^†).The luminescence switching of BMBT can also be achieved while alternating UV and visible light illumination in fixed HeLa cells. Cells (shown in red circle, Fig. 3a) were irradiated with 488 nm light (0.5 mW) for 3 min, the blue fluorescence of the irradiated cells was lamped off while the surrounding cells remained almost unchanged. Such fluorescence quenching is likely ascribed to the intramolecular fluorescence resonance energy transfer of BMBT, due to the intensified short-wavelength absorption band (270–450 nm) of the closed-open form of BMBT with the blue emission band (420–560 nm).³⁴ Upon irradiation with 405 nm light (1.25 mW) the fluorescence of all the selected cell was rapidly recovered within 1 min, caused by decrease of the relative intensity of the 270–450 nm absorption. The fluorescence can be repeatedly erased and recovered many rounds without significant fluorescence quenching, which was hardly achieved by conventional fluorophores (Fig. 3b).^35,36Open in a separate windowFig. 3(a) CLSM image (above) and the overlay image (bottom) of fixed HeLa cells incubated with 20 μM BMBT for 30 min at 37 °C (1) and (5) in original state; (2) and (6) irradiated by 488 nm light (0.5 mW) for a single cell; (3) and (7) all cells, and; (4) and (8) recovered by 405 nm light (1.25 mW). (b) Fluorescence switching of fixed HeLa cells by alternating UV (405 nm, 1.25 mW, 10 s/time) and visible (488 nm, 0.5 mW, 3 min/time) light illumination (λ_ex = 405 nm).This characteristic of selectively opto-marking or de-marking of cells may be used for non-invasive and dynamic tracing of the interested objects in vitro. That is, BMBT can arbitrarily opto-label or de-label interested cells without affecting cell proliferation. As shown in Fig. 4, the cell marked in the red circle was treated with visible light illumination (488 nm, 0.5 mW, 3 min), and its fluorescence was effectively quenched. Cells division of the remaining “bright” cells was observed under the microscope field of vision for a long-term tracing with time up to 36 h. Multiple new cells were produced, as indicated by upper white arrow. Even, as indicated by bottom white arrow, the tacked cells were observed to be doubled. The high brightness of the fluorescence in the proliferated cells indicated the good photo stability of BMBT. This indicated that BMBT can be used as a cell marker for arbitrarily selective erasing the fluorescence of the designated cell. It can also be used for selective lighting their emission by making them as the remaining bright cells after selective photo-erasing or selective photo-recovering after full erasing. This long-term tracking with non-labeling and selective optically marking or de-marking is seldom reported by other photochromophore-based bioimaging agents.³⁷ Herein, the excellent anti-photo bleaching characteristic in the long-term tracing is attributed to high photo stability of the HBT moiety.^26,27Open in a separate windowFig. 4Cells was treated with visible (488 nm, 0.5 mW, 3 min) light illumination for erasing the fluorescence. The remaining bright cells were incubated for another 36 hours, which were observed to be amplified normally (λ_ex = 405 nm).     

Manipulation of light transmission from stable magnetic microrods formed by the alignment of magnetic nanoparticles

Due to the increasing energy consumption, smart technologies have been considered to automatically control energy loss. Smart windows, which can use external signals to modulate their transparency, can regulate solar energy by reflecting excess energy and retaining the required energy in a building without using additional energy to cool or heat the interiors of the building. Although many technologies have been developed for smart windows, they still need to be economically optimised. Here, we propose a facile method to synthesise magnetic microrods from magnetic nanoparticles by alignment using a magnetic field. To maximise the transparency difference in the ON and OFF states, we controlled the nanoparticle concentration in a dispersion liquid, magnetic field application time, and viscosity of the dispersant. Interestingly, the magnetic microrods remained stable when we mixed short-chain polymers (polyethylene glycol) with a liquid dispersant (isopropyl alcohol). Furthermore, the Fe₂O₃ microrods maintained their shape for more than a week, while the Fe₃O₄ microrods clustered after a day because they became permanent magnets. The anisotropic features of the magnetic rods were used as a light valve to control the transparency of the smart window.

Magnetic microrods were synthesised from magnetic nanoparticles by alignment using a magnetic field. The transparency difference was maximised and the anisotropic features of the rods were used as a light valve to control the transparency of a smart window.

Buildings, factories, and houses consume a considerable amount of energy resulting in energy shortages. In addition, the depletion of fossil fuels is threatening the current energy supply. Therefore, the smart window technology is considered for reducing the current energy consumption, because a “SMART” window can regulate solar energy.^1,2 When it is too sunny outside, for example, the window can block the sunlight to remove the need for operating cooling systems which consume electrical energy. In addition, the smart technology can be used to artificially control the transparency of windows, which enhances the comfort in automobiles and aircrafts. Many researchers have developed optimised materials that use external signals, such as temperature, electricity, or magnetic field to control their transmittance.^1–11 For example, electrochromic and thermochromic materials, such as nanocrystal and amorphous metal oxide composites, monolithic-phased vanadium dioxide (VO₂), and hydrogel microparticles, have been recently studied for use in smart windows. To adjust optical transmittance, nanocrystals embedded in metal oxide exploit their electrochemical charging and discharging,³ vanadium dioxide uses a reversible metal-to-insulator transition,⁴ and hydrogel microparticles control their diameter to modulate the light scattering by changing the temperature.⁵ Mechanoresponsive smart windows, which use the light scattering effects on particle-embedded films or micropillars, are also potential candidates for these applications.^6–8 In addition, the use of mechanoresponsive and electrically driven wrinkles on a surface has been proposed as a smart window to scatter the light.^9,10 Functional materials such as liquid crystals (LCs) and particles have been used to manipulate transmittance. Polymer-dispersed liquid crystals (PDLCs) modulate the alignment direction of LCs by applying electrical signals.^1,2,11 Suspended particle (SP)-based smart windows that utilise the alignment of microparticles to change their transmittance have also been used.^1,2 In addition, magnetic materials have been used to modulate transmittance.^12–14 The use of magnetically responsive elastomeric micropillar arrays has also been demonstrated for controlling the light.¹³ Previously, our group reported a smart window inspired by a squid skin that uses the movement of magnetic nanoparticles within a tapered structure to control its transparency.¹⁴ However, the switching time based on this movement was long for practical utilisation. Although many materials have been developed for smart windows, these materials still need to be economically optimised.Here, we introduce a facile method for preparing stable magnetic microrods that can be utilised for smart windows by changing the direction of the magnetic field. Therefore, we applied a magnetic field to align magnetic Fe₂O₃ nanoparticles to form magnetic microrods. These rods have high aspect ratios; therefore, they can be used as light valves to control light transparency. Furthermore, we examined the effect of the nanoparticle concentration, magnetic field application time, and dispersant fluid viscosity on the formation of these magnetic rods. We also assessed their stability via mechanical agitation. Finally, we compared the stability of the Fe₂O₃ rods with that of microrods formed from Fe₃O₄ nanoparticles. Fig. 1(a) shows a schematic illustration of the formation of the magnetic microrods by applying a magnetic field. Initially, we prepared the magnetic nanoparticles and dispersion liquid mixture; thereafter, we applied a magnetic field using a neodymium magnet for 10 to 30 minutes to obtain magnetic rods dispersed in liquid. The Fe₂O₃ nanoparticles used were ∼50 nm in diameter. Meanwhile, an isopropyl alcohol (IPA) and polyethylene glycol (PEG) (molecular weight = 300) mixture was used as the dispersion liquid. Once the rods were formed, the applied magnetic field was removed. The dispersion liquid was filled in a transparent cavity (4 cm × 4 cm × 3 mm); thereafter, the direction of the magnetic rods was switched by controlling the magnetic field. A detailed procedure of the transparent cavity preparation is described in the experimental section and ESI (Fig. S1^†). The smart window filled with magnetic rods was placed on a display device (Samsung Galaxy J7), and then, the device screen was examined through the window. When the magnetic rods were vertically oriented (Fig. 1(b)), the university logo shown on the screen could be seen through the transparent cavity filled with magnetic rods (Fig. 1(c)). As shown in the microscopic image in Fig. 1(d), the transparent region could be seen. The black region is the top view of the vertically oriented magnetic rods. When we switched the orientation of the magnetic rods (Fig. 1(e)), the display device screen could not be seen (Fig. 1(f)) because the magnetic rods were parallel to the substrate, making the whole region dark (Fig. 1(g)). Since the transmittance change originated from the rotation of the magnetic rods, a faster response to the magnetic field was achieved here than in our previous study. In the previous study, we used the movement of nanoparticles within a confined microstructure filled with a dispersant to induce the transmittance change.¹⁴ The real time switch of the transmittance is found in the ESI (Movie S1^†). To adjust the anisotropy features of the magnetic rods, we controlled the magnetic nanoparticle concentration, magnetic field application time, and dispersion liquid viscosity. Fig. 2(a) shows the display device screen through the smart window (thickness = 3 mm) filled with microrods formed by applying the magnetic field for 30 min at different magnetic nanoparticle concentrations. To control the concentration of nanoparticles, the mixing ratio of PEG and IPA was fixed at 4 : 6. At a concentration of 0.1 wt%, the screen was visible through the smart window in the OFF state; however, it darkened as the concentration increased. In contrast, at a concentration of 2 wt%, the screen in the ON state was dark. To measure the length of the magnetic rods, we placed one drop of a liquid on a glass slide and measured its length with a microscope (Fig. 2(b)). When the concentration increased, the length of the rod increased until the concentration reached 1 wt%. The rods clustered at a concentration of 2 wt%. Fig. 2(c) shows that the rod length is proportional to the concentration. The length of the rod at a concentration of 2 wt% was excluded because it could not be measured due to clustering. Fig. 2(d) shows the transmittance measured by a window tint metre (AT-173, Guangzhou Amittari Instruments Co., Ltd.) at different nanoparticle concentrations. The transmittance in the ON and OFF states decreased with increasing nanoparticle concentration. At a concentration of 0.5 wt%, the transmittances were 4.5% and 62% in the OFF and ON states, respectively.Open in a separate windowFig. 1(a) Schematic illustration of the formation of the magnetic microrods by applying a magnetic field. (b) Schematic illustration of magnetic rods vertically oriented by controlling the magnetic field. (c) Smart phone screen showing the university logo through the transparent cavity filled with magnetic rods. (d) Microscopic image of the vertically oriented magnetic rods. (e) Schematic illustration of magnetic rods oriented parallel to the surface by controlling the magnetic field. (f) Smart phone screen showing the university logo through the transparent cavity filled with magnetic rods when the rods are in parallel to the surface. (g) Microscopic image of the parallel oriented magnetic rods. The scale bars represent 100 μm.Open in a separate windowFig. 2(a) Pictures of the display screen through the smart window for different concentrations of magnetic nanoparticles. (b) Microscopic images of the microrods in different concentration. The scale bars represent 100 μm. (c) Graph of the magnetic rod lengths versus the magnetic nanoparticles concentration. (d) Graph of the transmittance under different concentration conditions. Fig. 3(a) depicts a graph of the length of the magnetic rods with respect to the magnetic field application time (the fixed concentration of magnetic particles = 0.5%, and the viscosity of dispersion liquids = 4.4 cPs). The rod length was proportional to the application time and saturated after approximately 30 min. The transmittance had a similar trend as that shown by the rod length, as shown in Fig. 3(b). To control the dispersion liquid viscosity, in this study, a mixture of low viscosity IPA and high viscosity PEG was used. We measured the liquid mixture viscosities at different ratios (see the ESI Fig. S2^†) using a rheometer (Rheometer R/S plus, Brookfield). The measured viscosities of IPA and PEG were 0.78 and 91 cPs, respectively. When we increased the fraction of the relatively viscous PEG, the viscosity increased. Fig. 3(c) shows the rod length under different liquid viscosity conditions (the concentration was fixed at 0.5 wt% and time at 30 min). When the viscosity was higher, the rod length decreased. As shown in Fig. 3(d), the transmittance had the same trend as that shown by the lengths of the magnetic rods. We note the experimental data in Fig. 1, ​,4,4, ​,5,5, and ​and66 were obtained in the optimized condition for forming microrods (concentration = 0.5 wt%, magnetic field applying time = 30 min, and the viscosity of dispersion liquids = 4.4 cPs).Open in a separate windowFig. 3(a) Graph showing the length of the magnetic rods with respect to the magnetic field application time. (b) Graph of the transmittance with respect to the magnetic field application time. (c) Graph of the rod length under different liquid viscosity conditions. (d) Graph of the transmittance under different liquid viscosity conditions.Open in a separate windowFig. 4(a) SEM image of the magnetic rods. (b) Graph showing the relation between the length of the magnetic rods and transmittance at a fixed concentration.Open in a separate windowFig. 5Photovoltaic measurement results in ON and OFF states in a smart window.Open in a separate windowFig. 6(a) Transmittance in the ON and OFF states before and after agitation with a vortex mixer for 30 s when IPA only and a mixture of IPA and PEG are used. (b) Microscopic image of the disassembled microrods formed in IPA only after mechanical agitation. (c) Microscopic image of the stable microrods formed in a liquid mixture after mechanical agitation. The scale bars represent 100 μm. (d) Schematic illustration of the PEG coating on the magnetic rods to stabilise them. (e) Transmittance in the ON and OFF states after making the magnetic rods and after 24 h when using Fe₂O₃ and Fe₃O₄. (f) Pictures in the ON and OFF states of a smart window filled with Fe₃O₄ microrods after leaving the sample for a day. (g) Microscopic image of the clustered Fe₃O₄ magnetic rods after a day. The scale bars in (b), (c), and (g) represent 100 μm. Fig. 4(a) shows a scanning electron microscope (SEM) image of the magnetic rods. The rod length and width were 50 and 6 μm, respectively, which implies that the aligned magnetic nanoparticles were parallel to each other to be grown in both directions to the magnetic field. The anisotropy of the microfeatures showed a transmittance change as the orientation changed, as shown in Fig. 2 and ​and3.3. When the number of nanoparticles is N, the number of rods is inversely proportional to the rod length l. When the magnetic rods are vertically oriented, the transmittance (T) can be derived using eqn (1) below.T ∼1 − αN/l1where α is the parameter incorporating the compactness of the nanoparticles, density, and the relationship between the vertical and parallel growths of the magnetic rods. Fig. 4(b) shows the experimental data and a graph, based on eqn (1), of the relationship between the length of the magnetic rods and the transmittance at a fixed concentration (0.5 wt%). At a longer rod length, the transmittance in the ON state was higher, as expected from eqn (1).To emphasize the possibility of regulation of solar energy which has a broad wavelength spectrum, we measured J–V curves with a photovoltaic cell in ON and OFF states of the smart window. We used a calibrated reference photovoltaic cell (91150-KG5) and placed the smart window on it. First, we measured the efficiency of the solar cell as a reference (4.92%), which used a window without nanoparticles. And then, we measured the efficiency of a solar cell covered by the smart window in the ON state (3.24%) and OFF state (0.43%), respectively (Fig. 5). The ON/OFF ratio of the efficiency of the solar cells was about 7.5 which could modulate the solar energy with a wide spectrum. It is also noted that we assumed that the regulation of thermal radiation is dominant in our smart windows because the concentration of magnetic nanoparticles is less than 2 wt%, which can be a small effect on conduction and convection of heat transfer between the windows.The formation of the linear magnetic chains or rods has been studied for decades by applying a magnetic field on the nanoparticles.^15–21 The aligned magnetic microfeatures can be incorporated into polymers to enhance their mechanical properties or promote their suitability for special uses or serve as nanometre-sized stir bars for mixing. The lengths of the magnetic chains were controlled by the magnetic field, nanoparticle concentration, and magnetic field application time. Other groups have shown that the chain length is proportional to the field strength, concentration, and magnetic field application time, which agrees with our experimental results.^15,16,21 Another important aspect in the formation of the magnetic chains is their stability. When formed in a polymer, the chains are cannot be moved after the polymer is cured.^17,18 However, when the magnetic chains are formed in a liquid, the chains can be disassembled after the magnetic field is removed. Another challenge is clustering when the microchains become permanent magnets. To maintain the aligned features of the magnetic chains, polymeric surfactants bonded to a colloid¹⁹ or silica encapsulating the chain are used after forming the magnetic chains.²⁰ The Velev group demonstrated flexible microfilaments by assembling lipid-coated iron oxide particles.²¹ These researchers explained that the filaments were formed by a combination of the dipole–dipole interparticle attraction and magnetophoretic attractions of the particles. In addition, these filaments were stable due to nanocapillary lipid binding. Their work can provide explanation to our experimental observations.In this study, we used viscous PEG (40 wt%) as one of the dispersion liquids in the mixture, which can hinder interparticle attraction for obtaining longer microrods. Therefore, the rod length was shorter when the PEG fraction was higher (Fig. 3(c–d)). PEG could have adhered to the surfaces of the nanoparticles and coated the entire surface of the microrods, making the magnetic rods stable even after the magnetic field was removed. To prove this, the magnetic rods formed in IPA only and those formed from the mixture were compared. After the formation of the magnetic rods, the transmittance values in the ON/OFF states of the two samples were measured. Thereafter, a vortex mixer (Genie2, Neolab) was used to mechanically vibrate (1380 rpm for 30 s) the magnetic rods. Fig. 6(a) shows the transmittance in the ON and OFF states before and after agitation. For the magnetic rods formed in IPA, the transmittances in the ON and OFF states were 45.8% and 4.7%, respectively. However, when we agitated them mechanically, the transmittance in the ON state reduced to 1.6%. On the other hand, for the magnetic rods formed in the liquid mixture (40% PEG), the transmittance did not change in the ON state even after mechanical agitation. Fig. 6(b and c) shows the microscopic images of the magnetic rods after 30 s of agitation. When we used IPA only, the magnetic rods disassembled after agitation (Fig. 6(b)) However, the rods maintained their shape when a mixture of PEG and IPA was used (Fig. 6(c)). Fig. 6(d) shows a schematic illustration of the formation of the stable magnetic rods. PEG plays a role as a stabiliser by coating the magnetic rods after they have been formed, thereby stabilizing them.For the ageing test, we prepared smart windows with magnetic rods formed from the Fe₂O₃ and Fe₃O₄ nanoparticles. After applying the magnetic field, the magnetic rods were formed in both cases. When the Fe₂O₃ nanoparticles were used, the transmittances in the ON/OFF states did not change after the sample was allowed to remain for 24 h (Fig. 6(e)). When Fe₃O₄ nanoparticles were used, however, the transmittances in the OFF states changed significantly (from 21.6% to 52.5%) after 24 h. Fig. 6(f) shows the display screen through the smart window filled with Fe₃O₄ microrods after 24 h in the ON and OFF states. The microrods were clustered and moved to the boundary. When we examined these magnetic rods in the dark region, as shown in Fig. 6(e), using a microscope, we found that the magnetic rods had clustered (Fig. 6(g)). Meanwhile, the magnetic rods formed from the Fe₂O₃ nanoparticles were stable even after one week. Liu et al. reported the magnetization (M) - magnetizing field (H) curves of Fe₂O₃ and Fe₃O₄ nanoparticles and they demonstrated that Fe₂O₃ showed superparamagnetism whereas Fe₃O₄ exhibited ferromagnetic behaviour.²² It can explain Fe₂O₃ microrods can be rotated by the magnetic field without clustering and Fe₃O₄ microrods are clustered because they became permanent magnets.     

A solid-phase approach for the synthesis of α-aminoboronic acid peptides

A solid-phase synthesis of α-aminoboronic acid peptides using a 1-glycerol polystyrene resin is described. Standard Fmoc solid-phase peptide chemistry is carried out to construct bortezomib and ixazomib. This approach eliminates the need for liquid–liquid extractions, silica gel column chromatography, and HPLC purifications, as products are isolated in high purity after direct cleavage from the resin.

A solid-phase synthesis of α-aminoboronic acid peptides using a 1-glycerol polystyrene resin is described.

α-Aminoboronic acids are currently being investigated for their utility as reversible covalent inhibitors in a diverse range of therapeutic applications (Fig. 1).¹ These compounds'' Lewis acidity enables the formation of stable tetrahedral adducts with nucleophilic residues in biological targets (Fig. 2). In 2003, the first boronic acid drug, bortezomib, was approved for the treatment of multiple myeloma.² Ixazomib, a related α-aminoboronic acid inhibitor, was later approved in 2015 for the same indication.³Open in a separate windowFig. 1α-Aminoboronic acids featured in various drug discovery programs. Flaviviral protease inhibitor (1), HCV protease inhibitor (2), LepB inhibitor (3).Open in a separate windowFig. 2Yeast 20S proteasome in complex with bortezomib (PDB ID 2F16). The boronic acid forms a stable tetrahedral adduct with the N-terminal threonine (Thr1).Peptidic α-aminoboronic acids, such as bortezomib and ixazomib, have traditionally been assembled using standard peptide coupling techniques,⁴ wherein an α-aminoboronic ester is introduced onto a pre-constructed peptide and is subsequently deprotected to unmask the boronic acid. Metal-catalyzed decarboxylative borylation strategies have also been reported for the preparation of α-aminoboronic acid peptides.⁵ This approach provides direct access to these compounds from their parent peptide constructs but sacrifices stereochemical integrity.Regardless of the method, α-aminoboronic acid/ester peptides are difficult to prepare for a number of reasons.⁶ First, the C–B bond can be oxidatively labile.⁷ Second, α-aminoboronic acids and esters containing an unsubstituted α-amino group can undergo a spontaneous 1,3-rearrangement (Scheme 1, A); this process can be minimized or suppressed entirely if the amino group is rapidly acylated or protonated.⁸ Third, boronic esters can be hydrolytically labile, especially at low pH (Scheme 1, B).^6,9 Therefore, any multistep approach must entail careful extractive workups and purifications to ensure that the ester remains intact.Open in a separate windowScheme 1General considerations for preparing α-aminoboronic acid peptides.While solid-phase peptide synthesis (SPPS) has become a standard method for the construction of peptides,¹⁰ this technology has remained underexplored for the preparation of α-aminoboronic acid peptides.¹¹ An approach of this type could eliminate liquid–liquid extractions and HPLC purifications and could enable high-throughput access to this class of compounds. To the best of our knowledge, there has only been one report of C-terminal SPPS to generate α-aminoboronic acid peptides (Scheme 2).¹² Although this study provides a critical conceptual foundation, the approach it describes lacks the simplicity of a traditional solid-phase approach, requiring a complex 8-step synthesis to prepare resin-bound α-aminoboronic ester 6 for SPPS. This limitation may preclude its use as a general strategy for the preparation of α-aminoboronic acid peptides.Open in a separate windowScheme 28-Step preparation of a resin-bound α-aminoboronic acid for C-terminal SPPS.We sought to identify an approach that could enable access to resin-bound α-aminoboronic acids for SPPS in a limited number of steps using the emerging supply of commercially available α-aminoboronic acid building blocks. The Klein group recently described the use of a 1-glycerol polystyrene resin that could be used for Fmoc SPPS to construct boronic acid-containing peptides.^13–15 These results prompted us to explore the use of this resin for preparation of α-aminoboronic acid peptides, specifically bortezomib and ixazomib.Considering the unique reactivity of α-aminoboronic acids, we needed to devise a concise loading strategy that would suppress the potential for C to N boron migration. This required the amine to remain protonated or acylated throughout the loading process. These considerations lead to the design of a two-step loading protocol (Scheme 3). Commercially available boroleucine pinanediol ester 7 was hydrolysed with aqueous HCl. The boroleucine salt (8) was isolated in quantitative yield, free of pinanediol impurities, after a simple liquid–liquid extraction. The crude boroleucine salt was then shaken with the 1-glycerol polystyrene resin (loading capacity 0.6 mmol g⁻¹),¹³ Fmoc chloride, and N,N-diisopropylethylamine to provide resin-bound Fmoc-protected boroleucine 9.Open in a separate windowScheme 32-Step protocol for loading boroleucine onto a 1-glycerol polystyrene resin.With the C-terminal α-aminoboronic acid resin in hand, we used standard Fmoc SPPS coupling techniques¹⁶ to synthesize bortezomib (Scheme 4). Fmoc deprotection (piperidine, DMF) and amide coupling (Fmoc-Phe-OH, TBTU, N,N-diisopropylethylamine, DMF) delivered intermediate 10. A subsequent Fmoc deprotection/coupling sequence with pyrazinecarboxylic acid produced resin-bound bortezomib 11. Hydrolysis of the resin bound peptide was accomplished with gentle shaking in a THF/water mixture.¹³ Filtration and concentration delivered bortezomib (7-steps from boroleucine pinanediol ester 7) in 54% yield and in >95% purity.Open in a separate windowScheme 4Solid-phase synthesis of bortezomib.The synthesis of ixazomib (Scheme 5) was accomplished in an analogous manner. Fmoc deprotection (piperidine, DMF) and amide coupling (Fmoc-Gly-OH, TBTU, N,N-diisopropylethylamine, DMF) delivered intermediate 12. The deprotection/coupling sequence was repeated with 2,5-dichlorobenzoic acid to generate resin-bound ixazomib 13. Finally, boronic ester hydrolysis (THF/water) provided ixazomib in 49% yield and in >95% purity.Open in a separate windowScheme 5Solid-phase synthesis of ixazomib.     

Improved antibacterial performance using hydrogel-immobilized lysozyme as a catalyst in water

Silver nanoparticle-based catalysts are used extensively to kill bacteria in drinking water treatment. However secondary contamination and their high cost require scientists to seek alternatives with non-toxicity, high activity and low cost. In this article, we develop a new hydrogel-immobilized lysozyme (h-lysozyme) that shows excellent antibacterial performance, including high activity duration of up to 55 days, inhibition efficiency as high as 99.4%, good recycling capability of up to 11 cycles, a wide temperature window and extremely low concentration. The immobilized lysozyme displayed greatly improved bacterial inhibition with both Gram-negative E. coli and Gram-positive B. subtilis, which enables broad antibacterial applications in various water systems. In parallel, the non-toxic structure and high stability of the h-lysozyme without additional contamination make it a promising alternative to nanoparticle catalysts fur use in drinking water purification.

Hydrogel-immobilized lysozyme for antibacterial membrane modification.

Conventional disinfection technologies for drinking water include physical treatments, such as microfiltration, ultrafiltration and UV irradiation, and chemical methods using chlorine, bromine, silver, copper, zinc and ozone.^1–3 However, most technologies generate by-products and heavy metal residues along with high cost, which largely limit their further applications.^4,5 Recently lysozyme has been attracting attention in the preservation of foods and the inhibition of bacterial and virus growth.^6–8 Lysozyme is an antimicrobial enzyme produced by animals that forms part of the innate immune system,^9–11 and which exists in secretions, including tears, saliva, human milk, and mucus. In addition to its antimicrobial activity,^12,13 lysozyme also shows potential in anti-biofouling and water disinfection.^13–15Most isolated enzymes pose a challenge in terms of stability, sensitivity to environmental change and recycling.^16,17 In particular, applying lysozyme onto a membrane using chemical methods drastically reduces its activity.^18–20 Scientists thus attempt to immobilize enzymes to improve their performance and expand their applications.^21,22 Saeki et al. immobilized lysozyme on a reverse osmosis (RO) membrane via an amine coupling reaction with a reduced water flux.¹³ Forming a robust chemical cross-linking configuration using glutaraldehyde molecules¹⁷ and Lentikats® polymers¹⁹ can improve the stability, but it will deform the initial structure and thus lose some activity. Immobilizing lysozyme into a polymer network can enhance the interaction area with biological contaminants and thus improve the removal efficiency at a low dosage.^23–25Here, we developed a new hydrogel-immobilized lysozyme (h-lysozyme) to inhibit both Gram-negative (E. coli) and Gram-positive (B. subtilis) bacterial growth in water. The immobilized lysozyme showed greatly improved activity, efficiency and recycling capability at room temperature.^26–28 Owing to the strong bonding to the hydrogel and the porous structure of the cross-linked network, the antibacterial activity can be retained for a long period, e.g., months to years.^28,29 The large surface area of the hydrogel also increases the bacterial molecular capture ability and thus increases the reaction opportunities with lysozyme. The excellent antibacterial performance along with the non-toxicity and low cost of the h-lysozyme make it a promising alternative to replace the current silver particles in various water environments.³⁰As illustrated in Fig. 1a, a cross-linked porous hydrogel network is formed after the polarization of poly(ethylene glycol)methyl ether acrylate (PEGMA) monomer via UV irradiation. During this process, the lysozyme molecules are immobilized into the matrix. The largely exposed surface increases the interaction with bacteria while the porous structure provides sufficient space to capture bacteria (Fig. 1b). Then the presence of lysozyme introduces a catalytic reaction of the hydrolysis of peptidoglycan, which is the major component of the cell wall of most bacteria, as well as counteracting the osmotic pressure of the cytoplasm and binary fission during bacterial cell reproduction. Upon the reaction, the 1,4-beta-linkages between N-acetyl-d-glucosamine (NAG) and N-acetylmuramic acid (NAM) in peptidoglycan are broken (Fig. 1c). As a result, the hydrolyzed NAG and NAM molecules lead to the lysis of the bacteria.^31,32Open in a separate windowFig. 1Schematic diagram of hydrogel-immobilized lysozyme for antibacterial membrane modification. (a) The synthesis process, (b) the antibacterial process and (c) mechanism for killing bacteria.A lysozyme with high activity and long stability is critical in antibacterial applications. It was found that the activity of lysozyme powder can remain for as long as two years.^24,32 However, the activity of freestanding lysozyme will begin to be mitigated after being dispersed in water solution over a long time.^33,34 In order to compare their activity, pure lysozyme powder, lysozyme in deionized (DI) water and h-lysozyme samples were checked by an ultraviolet (UV) spectrophotometer. In parallel, the samples under −20 °C, −4 °C, room temperature (RT) and high temperatures of 60 °C, 65 °C, 70 °C, 80 °C were also measured.Lysozyme activity was checked via a typical process using whole cells of Micrococcus lysodeikticus (ML) as substrate.^35,36 The ML was dissolved in 0.1 M phosphate buffer saline (PBS) solution (pH = 6.24) at RT. Then the concentration was adjusted until the measured UV density at 450 nm reached approximately 1.3. The optical density (OA) evolution vs. time was recorded to calculate the activity:Activity (U mg⁻¹) = ΔOD₄₅₀ × 1000/m1Here ΔOD₄₅₀ is the difference in optical density between 15 s and 75 s, and m is the mass (mg) of lysozyme in 0.5 mL solution. The relative activity (R%) is obtained:R% = (A_i/A_f) × 1002A_i stands for the measured activity of samples (U mg⁻¹), and A_f represents the activity of freestanding lysozyme under same conditions.It was found that h-lysozyme exhibits a 120–250% higher relative activity compared to lysozyme powder or lysozyme in water solution (less than 100%) at RT (Fig. 2a), −4 °C (Fig. 2b) and −20 °C (Fig. 2c), respectively. Activity retention of up to 55 days was also recorded, indicating long-term stability. The fluctuation in the relative activity of the immobilized lysozyme is due to variations during sample preparation and UV measurement. At high temperature, 60 °C, the h-lysozyme displayed similar activity to lysozyme powder while it was higher than that of lysozyme in water (Fig. 2d). A relatively high activity at higher temperatures of 65, 70 and even 80 °C was still maintained, which demonstrates that h-lysozyme has good temperature tolerance. The hydrophilicity and large surface area of the PEG polymer based hydrogel attract plenty of bacteria, thus increasing the contact interface between the lysozyme and the ML molecules. As a result, a clearly improved activity under varying environments and temperatures was achieved.Open in a separate windowFig. 2The activity of h-lysozyme at (a) room temperature, (b) −4 °C, (c) −20 °C and (d) high temperatures (60 °C, 65 °C, 70 °C, 80 °C).The activity of the lysozyme in water at different concentrations was investigated.^37,38 It was found that the lysozyme has high activity at 0.04 mg mL⁻¹ (Fig. 3a, black). The activity of killing bacteria was decreased until an extremely low concentration of 0.0004 mg mL⁻¹ (Fig. 3a, blue). The recycling activity of the pure lysozyme was studied, as shown in Fig. 3b. It can be observed that most activity was lost after two cycles, which was caused by the decreased contact between lysozyme and ML due to the accumulation of debris on the surface. As a comparison, the hydrogel network with large spaces enhances the accommodation of debris and dead bacteria, resulting in a large exposed interface remaining. The recycling activity of both lysozyme and h-lysozyme is shown in Fig. 3c. It can clearly be seen that the immobilized lysozyme showed a high activity of about 30% even after 11 cycles, while the activity of the lysozyme without immobilization dropped to less than 10% after 8 cycles. These results further confirm that the porous hydrogel framework delivers sufficient contact interface between the lysozyme and bacteria to drastically improve the activity. In addition, the cross-linked net matrix also expands the acceptance capacity of the killed bacteria, avoiding surface coverage causing a loss of activity, which is a generic problem in the traditional silver particle killing process.Open in a separate windowFig. 3The recycling activity test of lysozyme. (a) The activity of lysozyme with different concentrations at OD₄₅₀. (b) The activity test of lysozyme in solution for two cycles. (c) The comparison of activity of pure lysozyme and h-lysozyme as function of cycles.The antibacterial capability was first tested by exposing the h-lysozyme to the Gram-negative bacterial module E. coli. In order to fast check the bacterial regrowth, a lysogeny broth media (LB) bacterial culture solution was used.³⁹Fig. 4a illustrates that UV-lysozyme (red) has the highest inhibition compared to hydrogel (blue), lysozyme (green) or control (black) samples. Specifically the E. coli was completely inhibited by lysozyme in the first 2 hours and showed a negligible increase even after 8 hours, which indicates a remarkable inhibition of E. coli. It should be noted that UV-irradiated lysozyme showed slightly improved inhibition due to the activation of lysozyme under irradiation. Interestingly, the hydrogel also displayed certain inhibition to E. coli growth (blue). Large amounts of bacteria were trapped inside the matrix and thus further growth was prevented due to the change of environment.Open in a separate windowFig. 4The antibacterial results with different samples. (a) OD₆₀₀ regrowth curves of E. coli bacteria exposed to different samples (blank as control, 3.2 mg mL⁻¹ UV irradiated lysozyme, blank hydrogel and 3.2 mg mL⁻¹ lysozyme) at 37 °C for 8 h. (b) The concentration of E. coli in DI water (10⁵ CFU initial concentration) after treatment with 3.2 mg mL⁻¹ lysozyme, blank hydrogel and h-lysozyme, the natural E. coli as control. (c) The calculated removal efficiency of E. coli after treatment with 3.2 mg mL⁻¹ lysozyme, blank hydrogel and h-lysozyme. (d) The concentration of E. coli in DI water (10⁵ CFU initial concentration) after treatment with 1 mg mL⁻¹ lysozyme, blank hydrogel and h-lysozyme, the E. coli as control. (e) The calculated removal efficiency of E. coli after treatment with 1 mg mL⁻¹ lysozyme, blank hydrogel and h-lysozyme.In order to mimic the real environment in drinking water, a solution with an initial E. coli concentration of 10⁵ CFU mL⁻¹ was prepared and exposed to different samples of control (E. coli), lysozyme, h-lysozyme and hydrogel, respectively. It was observed that h-lysozyme showed excellent E. coli inhibition similar to that of pure lysozyme (Fig. 4b). The E. coli inhibition efficiency was calculated based on the measured concentration vs. the control concentration (Fig. 4c). The inhibition efficiencies of h-lysozyme and lysozyme were 99.4% and 99.0% in the first hour and they were still 96.3% and 98.8% after 30 hours, respectively. The E. coli inhibition with a low lysozyme concentration of 1 mg mL⁻¹ was also studied, as shown in Fig. 4d. As can be seen from the figure, h-lysozyme shows the best inhibition performance in contrast to pure lysozyme, hydrogel or control samples. The inhibition efficiency of h-lysozyme after 26 hours was as high as 97.3%, which is higher than that of pure lysozyme of 96.4% (Fig. 4e), indicating promising potential in long-term antibacterial capability.In order to expand the antibacterial applications, the Gram-positive bacteria Bacillus subtilis (B. subtilis) in water were also tested.^30,40 As shown in Fig. 5a, h-lysozyme exhibited the lowest concentration, 1.6 × 10⁷ CFU mL⁻¹, of bacteria after 30 hours (blue). A corresponding inhibition efficiency of >92% after 26 hours was achieved (Fig. 5b), enabling better antibacterial durability.Open in a separate windowFig. 5(a) Concentration evolution of B. subtilis in DI water (10⁵ CFU initial concentration) after treatment with 3.2 mg mL⁻¹ lysozyme, blank hydrogel and h-lysozyme, the natural B. subtilis as control. (b) The removal efficiency of B. subtilis after treatment with 3.2 mg mL⁻¹ lysozyme, blank hydrogel and h-lysozyme and B. subtilis as control.In this work, we successfully developed a hydrogel-immobilized lysozyme that showed greatly improved antibacterial capability with high inhibition activity, wide concentration and temperature ranges and long recycling performance in drinking water. The increased interfaces between the lysozyme and bacteria molecules due to the large surface area from the porous structure of the hydrogel improve the inhibition of both Gram-negative and Gram-positive bacteria such as E. coli and B. subtilis. In addition, the industrial availability and non-toxicity of the lysozyme with hydrogel provide a promising alternative to the existing silver-based antibacterial catalysts for drinking water treatment.