Solubilization mechanism of α-glycosylated naringin based on self-assembled nanostructures and its application to skin formulation.

We previously reported that α-glycosylated naringin (naringin-G), synthesized by enzyme-catalyzed transglycosylation, can enhance the solubility of poorly water-soluble compounds without surface-active property. However, the solubilization mechanism has not been fully elucidated. In this study, the solubilization mechanism of naringin-G was investigated using nuclear magnetic resonance (NMR) spectroscopy, and its application in skin formulations was further investigated. 1H NMR and dynamic light scattering measurements at various concentrations confirmed the self-assembled nanostructures of naringin-G above a critical aggregation concentration of approximately 2.2 mg/mL. Two-dimensional 1H-1H nuclear Overhauser effect spectroscopy and solubility tests revealed that flavone with poor water solubility, could be solubilized in its self-assembled structure with a stoichiometric relationship with naringin-G. When naringin-G was included in the skin formulation, the permeated amount and permeability coefficient (Papp) of flavones improved up to four times with increasing amounts of naringin-G. However, flavone solubilization by adding an excessive amount of naringin-G resulted in a decreased permeated amount and Papp of flavones, indicating the interplay between the apparent solubility and skin permeability of flavones. Naringin-G, which forms a nanoaggregate structure without exhibiting surface-active properties, has the potential to enhance the solubility and skin permeation of poorly water-soluble compounds.

Electronic substituent effect on the conformation of a phenylalanine-incorporated cyclic peptide.

The Phe-incorporated cyclic peptide [cyclo(-Phe1-oxazoline2-d-Val3-thiazole4-Ile5-oxazoline6-d-Val7-thiazole8-)] is in a conformational equilibrium between square and folded forms in solution. In the folded form, a CH⋯π interaction between the Phe1 aromatic ring and the Oxz2 methyl group is observed. We endeavored to control the local conformation and thus modulate the CH⋯π interaction and flexibility of the Phe1 side chain by controlling the electronic substituent effects at the 4-position of the aromatic ring of the Phe1 residue. The effect of the 4-substituent on the global conformation was indicated by the linear relationship between the conformational free energies (ΔGo) determined through NMR-based quantification and the Hammett constants (σ). Electron-donating substituents, which had relatively strong CH⋯π interactions, promoted peptide folding by restraining the loss in entropy. Local control by the 4-substituent effects suggested that the Phe side chain exerts an entropic influence on the folding of these cyclic peptides.

Unveiling the flavone-solubilizing effects of α-glucosyl rutin and hesperidin: probing structural differences through NMR and SAXS analyses.

Flavonoids often exhibit broad bioactivity but low solubility and bioavailability, limiting their practical applications. The transglycosylated materials α-glucosyl rutin (Rutin-G) and α-glucosyl hesperidin (Hsp-G) are known to enhance the dissolution of hydrophobic compounds, such as flavonoids and other polyphenols. In this study, the effects of these materials on flavone solubilization were investigated by probing their interactions with flavone in aqueous solutions. Rutin-G and Hsp-G prepared via solvent evaporation and spray-drying methods were evaluated for their ability to dissolve flavones. Rutin-G had a stronger flavone-solubilizing effect than Hsp-G in both types of composite particles. The origin of this difference in behavior was elucidated by small-angle X-ray scattering (SAXS) and nuclear magnetic resonance analyses. The different self-association structures of Rutin-G and Hsp-G were supported by SAXS analysis, which proved that Rutin-G formed polydisperse aggregates, whereas Hsp-G formed core-shell micelles. The observation of nuclear Overhauser effects (NOEs) between flavone and α-glucosyl materials suggested the existence of intermolecular hydrophobic interactions. However, flavone interacted with different regions of Rutin-G and Hsp-G. In particular, NOE correlations were observed between the protons of flavone and the α-glucosyl protons of Rutin-G. The different molecular association states of Rutin-G or Hsp-G could be responsible for their different effects on the solubility of flavone. A better understanding of the mechanism of flavone solubility enhancement via association with α-glucosyl materials would permit the application of α-glucosyl materials to the solubilization of other hydrophobic compounds including polyphenols such as flavonoids.

Experimental evidence for CH⋯π interaction-mediated stabilization of the square form in phenylglycine-incorporated ascidiacyclamide.

Ascidiacyclamide [cyclo(-Ile-oxazoline-D-Val-thiazole-)2] is a cytotoxic cyclic peptide from ascidian. We examined the potential of the CH⋯π interaction at the diagonal position of ascidiacyclamide by comparing the interactions of Ile, Val, Abu (2-aminobutyric acid) or Ala with Ile, Chg (cyclohexylglycine) or Phg (phenylglycine). In solution, ascidiacyclamides are in a conformational equilibrium between square and folded forms. The CH⋯π interaction is expected to contribute to stabilization of the square form, which enhances the peptides' cytotoxicity. The distances between the alkyl side chain of Xaa and the π-plane of Phg were estimated from the crystal structures. The conformational free energies (ΔG°) determined through NMR-based quantitation indicated remarkable stabilization of the square form upon incorporation of Phg. These observations were consistent with the circular dichroism (CD) spectral measurements. Chemical shift perturbation studies suggested that stabilization of the square form of Phg-incorporated peptides was due to the CH⋯π interaction with the alkyl side chain of Xaa. Greater enthalpic losses were caused during the folding process of Phg-incorporated peptides than Ile- or Chg-incorporated peptides. It is suggested that these enthalpic losses are relevant to the CH⋯π interaction energies, which must be disrupted during folding. In addition, the CH⋯π interactions in the Phg-incorporated peptides increased cytotoxicity.

Structural Investigation of Hesperetin-7-O-Glucoside Inclusion Complex with ß-Cyclodextrin: A Spectroscopic Assessment.

Flavonoids are biologically active natural products of great interest for their potential applications in functional foods and pharmaceuticals. A hesperetin-7-O-glucoside inclusion complex with ß-cyclodextrin (HEPT7G/ßCD; SunActive® HCD) was formulated via the controlled enzymatic hydrolysis of hesperidin with naringinase enzyme. The conversion rate was nearly 98%, estimated using high-performance liquid chromatography analysis. The objective of this study was to investigate the stability, solubility, and spectroscopic features of the HEPT7G/ßCD inclusion complex using Fourier-transform infrared (FTIR), Raman, ultraviolet-visible absorption (UV-vis), 1H- and 13C- nuclear magnetic resonance (NMR), differential scanning calorimetry (DSC), liquid chromatography/mass spectroscopy (LC-MS), scanning electron microscopy (SEM), and powdered X-ray diffraction (PXRD) spectroscopic techniques including zeta potential, Job's plot, and phase solubility measurements. The effects of complexation on the profiles of supramolecular interactions in analytic features, especially the chemical shifts of ß-CD protons in the presence of the HEPT7G moiety, were evaluated. The stoichiometric ratio, stability, and solubility constants (binding affinity) describe the extent of complexation of a soluble complex in 1:1 stoichiometry that exhibits a greater affinity and fits better into the ß-CD inner cavity. The NMR spectroscopy results identified two different configurations of the HEPT7G moiety and revealed that the HEPT7G/ßCD inclusion complex has both -2S and -2R stereoisomers of hesperetin-7-O-glucoside possibly in the -2S/-2R epimeric ratio of 1/1.43 (i.e., -2S: 41.1% and -2R: 58.9%). The study indicated that encapsulation of the HEPT7G moiety in ß-CD is complete inclusion, wherein both ends of HEPT7G are included in the ß-CD inner hydrophobic cavity. The results showed that the water solubility and thermal stability of HEPT7G were apparently increased in the inclusion complex with ß-CD. This could potentially lead to increased bioavailability of HEPT7G and enhanced health benefits of this flavonoid.

Enhancement of Acinetobacter baumannii biofilm growth by cephem antibiotics via enrichment of protein and extracellular DNA in the biofilm matrices.

AIMS: The aims were to determine the effects of subinhibitory concentrations of eight cephem and carbapenem antibiotics on the biofilm formation of Acinetobacter baumannii cells and examine their effects on pre-established biofilms. METHODS AND RESULTS: Effects of antibiotics on biofilm formation were assayed using microtitre plates with polystyrene peg-lids. Cefmetazole, ceftriaxone, ceftazidime and cefpirome increased the biomass of pre-established biofilms on pegs in the range of their sub-minimum inhibitory concentrations (MICs), whereas none increased biofilm formation by planktonic cells. Carbapenems had a negative effect. The constituents of antibiotic-induced biofilms were analysed. Ceftriaxone or ceftazidime treatment markedly increased the matrix constituent amounts in the biofilms (carbohydrate, 2.7-fold; protein, 8.9-12.7-fold; lipid, 3.3-3.6-fold; DNA, 9.1-12.2-fold; outer membrane vesicles, 2.7-3.8-fold and viable cells, 6.8-10.1-fold). The antibiotic-enhanced biofilms had increased outer membrane protein A and were resistant to the anti-biofilm effect of azithromycin. CONCLUSIONS: Some cephems increased the biomass of pre-established biofilms in the ranges of their sub-MICs. The antibiotic-enhanced biofilms possessed more virulent characteristics than normal biofilms. SIGNIFICANCE AND IMPACT OF THE STUDY: Incomplete administration of certain cephems following biofilm-related Ac. baumannii infections could adversely cause exacerbated and chronic clinical results.

Structural study of the recognition mechanism of tau antibody Tau2r3 with the key sequence (VQIINK) in tau aggregation.

One of the histopathological features of Alzheimer's disease (AD) is higher order neurofibrillary tangles formed by abnormally aggregated tau protein. The sequence 275VQIINK280 in the microtubule-binding domain of tau plays a key role in tau aggregation. Therefore, an aggregation inhibitor targeting the VQIINK region in tau may be an effective therapeutic agent for AD. We have previously shown that the Fab domain (Fab2r3) of a tau antibody that recognizes the VQIINK sequence can inhibit tau aggregation, and we have determined the tertiary structure of the Fab2r3-VQIINK complex. In this report, we determined the tertiary structure of apo Fab2r3 and analyzed differences in the structures of apo Fab2r3 and Fab2r3-VQIINK to examine the ligand recognition mechanism of Fab2r3. In comparison with the Fab2r3-VQIINK structure, there were large differences in the arrangement of the constant and variable domains in apo Fab2r3. Remarkable structural changes were especially observed in the H3 and L3 loop regions of the complementarity determining regions (CDRs) in apo Fab2r3 and the Fab2r3-VQIINK complex. These structural differences in CDRs suggest that formation of hydrophobic pockets suitable for the antigen is important for antigen recognition by tau antibodies.

Effect of the powerful plasticity of the tert-butyl side chain on the conformational equilibrium of ascidiacyclamides.

Ascidiacyclamide [cyclo(-Ile1,5 -oxazoline2,6 -d-Val3,7 -thiazole4,8 -)2 ] is a cytotoxic cyclic peptide from ascidian. Through structural analyses using monosubstituted analogues (Xaa1 : Ala, 2-aminobutyric acid, Val, cyclohexylglycine, and phenylglycine), we previously demonstrated the conformational equilibrium between its square and folded forms. As the bulkiness of the Xaa1 residue side chain was reduced, spontaneous folding was promoted, and the cytotoxicity decreased accordingly. In the present study, five disubstituted analogues in which a tert-leucine residue (Tle) was incorporated at the 5-position of the abovementioned monosubstituted analogues were synthesized, after which their structures were analyzed using X-ray diffraction, circular dichroism (CD) spectral measurements, and 1 H NMR-based quantitative analysis. The side chains of the Tle and Ile residues are structural isomers of one another, and the Tle residue bearing the tert-butyl group can be expected to play a role as a building block. In fact, peptides incorporating Tle5 exhibited much less spontaneous folding than their Ile5 counterparts in both crystal and solution. Increases in enthalpy and entropy due to the tert-butyl group during the folding process resulted in increased conformational free energy (ΔG°). The powerful plasticity of the tert-butyl group would stabilize the square form relating with cytotoxicity.

Crystal structure of the human tau PHF core domain VQIINK complexed with the Fab domain of monoclonal antibody Tau2r3.

Neurofibrillary tangles formed by abnormally aggregated tau protein are a histopathological feature of tauopathies. A tau aggregation inhibitor is a potential therapeutic agent for tauopathies. In this study, we prepared a monoclonal antibody for tau, monoclonal antibody to tau protein (Tau2r3), using as epitope the 272 GGKVQIINKKLD283 peptide in the microtubule-binding domain of tau, the key region mediating tau aggregation. We show that Tau2r3 clearly inhibits tau aggregation. To analyze the inhibition mechanism of Tau2r3, we solved the crystal structure of the Fab domain of Tau2r3 (Fab2r3) in complex with the VQIINK peptide. In the Fab2r3-VQIINK structure, the second and sixth polar residues and the fourth hydrophobic residue of VQIINK are crucial for binding to Fab2r3. The structural data for the Fab2r3-VQIINK complex could contribute to the design of new tau aggregation inhibitors.

NMR-based quantitative studies of the conformational equilibrium between their square and folded forms of ascidiacyclamide and its analogues.

Ascidiacyclamide [cyclo(-Ile1,5-oxazoline2,6-D-Val3,7-thiazole4,8-)] (1) is a cytotoxic cyclic peptide from the ascidian, or sea squirt. Through structural analyses using asymmetric analogues [Xxx1: Ala (2), Val (3), Leu (4), Phe (5), cyclohexylalanine (6) and phenylglycine (7)], we previously showed 1 to exist in a conformational equilibrium between square and folded forms. In the present study, five new asymmetric analogues [Xxx1: 2-aminobutyric acid (8), 2-aminopentyric acid (9), tert-butylalanine (10), cyclohexylglycine (11) and tert-leucine (12)] were synthesized, and their structures were analyzed with X-ray diffraction and CD spectral measurements. Variable temperature 1H NMR measurements were performed to determine their equilibrium constants and their thermodynamic parameters. The use of two reference peptides made these quantitative studies possible. T3ASC, which contains three thiazole rings as a result of replacing oxazoline2 with thiazole, and dASC, in which the two oxazoline rings were deleted, were respectively used as square and folded reference peptides. The estimated parameters enabled more detailed discussion of the relationship between the bulkiness of substituents and the conformational free energies (ΔG°) of the peptides as well as the relationship between structure and cytotoxicity. The ΔG° values for peptides 1, 2, 3, 8, 9 and 11 decreased with decreases in the bulkiness of their substituents. We suggest that spontaneous folding is promoted as the bulkiness of substituents decreases. Peptides 7 and 12, which have large positive ΔG° values independently of temperature, did not exhibit spontaneous folding at any temperature; that is, their conformations were very stable in the square form. Peptides 4, 5, 6 and 10 had negative ΔG° values, despite their bulky substituents. Peptides with a positive ΔG° value showed cytotoxicity, and peptides with a negative ΔG° value showed reduced or no cytotoxicity. However, peptides 5 and 6 showed cytotoxicity equal to or stronger than 1. Those ten peptides except for 5 and 6 showed a clear structure-cytotoxicity relationship based on ΔG° values.

Conformational properties of ascydiacyclamide analogues with cyclic α-amino acids instead of oxazoline residues.

Ascidiacyclamide [ASC, cyclo(-Ile-oxazoline-d-Val-thiazole-)2] is a cyclic octapeptide isolated from tunicates. We designed ASC analogues [cyclo(-Ile-Xxx-d-Val-thiazole-)2] in which Pro or a homologue was substituted for oxazoline: [Pro]ASC (Xxx: proline), [Aze]ASC (Xxx: (S)-Azetidine-2-carboxylic acid), [Pip]ASC (Xxx: (S)-Piperidine-2-carboxylic acid) and [ΔPro]ASC (Xxx: (S)-3-pyrroline-2-carboxylic acid) to explore their potential to serve as substitutes for the oxazoline ring. The conformations of these analogues were examined using X-ray diffraction, 1H NMR and CD spectroscopy. In both the crystal and solution states, the conformations of [Pro]ASC, [Aze]ASC and [ΔPro]ASC were novel square structures having two trans imide bonds and stabilized by two intramolecular hydrogen bonds. The crystal structure of [Pip]ASC was a folded conformation with cis and trans imide bonds. Three isomers (cc, ct and tt) were present in a solution of [Pip]ASC. From crystal structures, the degree of puckering in the side chains of Pro and its homologues was estimated to be in the order Pip > Pro > Aze > ΔPro. [Pro]ASC and [Pip]ASC showed strong cytotoxicity, but [Aze]ASC and [ΔPro]ASC showed no cytotoxicity. Among the four analogues, there is consistency between the prolyl ring puckering and cytotoxicity, but not between the peptide backbone structure and cytotoxicity.

Carapanosins A-C from Seeds of Andiroba (Carapa guianensis, Meliaceae) and Their Effects on LPS-Activated NO Production.

Two new phragmalin-type limonoids, Carapanosins A and B (1 and 2), and a new gedunin-type limonoid, Carapansin C (3), together with five known limonoids (4-8) were isolated from the oil of Carapa guianensis AUBLET (Meliaceae) seeds, a traditional medicine in Brazil and Latin American countries. Their structures were elucidated on the basis of spectroscopic analyses using 1D and 2D NMR techniques and HRFABMS. Compounds 1-8 were evaluated for their effects on the production of NO in LPS-activated mouse peritoneal macrophages. The NO inhibitory assay suggested that Compounds 3, 6, and 8 may be valuable as potential inhibitors of macrophage activation.

Naphthablins B and C, Meroterpenoids Identified from the Marine Sediment-Derived Streptomyces sp. CP26-58 Using HeLa Cell-Based Cytological Profiling.

HeLa cell-based cytological profiling (CP) was applied to an extract library of marine sediment-derived actinomycetes to discover new cytotoxic secondary metabolites. Among the hit strains, Streptomyces sp. CP26-58 was selected for further investigation to identify its cytotoxic metabolites. CP revealed that the known ionophore tetronasin (1) was responsible for the cytotoxic effect found in the extract. Furthermore, three naphthoquinone meroterpenoids, naphthablin A (2) and two new derivatives designated as naphthablins B (3) and C (4), were isolated from other cytotoxic fractions. The structures of the new compounds were elucidated based on analysis of their HRESIMS and comprehensive NMR data. The absolute configurations of the new compounds were deduced by simulating ECD spectra and calculating potential energies for the model compounds using density function theory (DFT) calculations. Compound 1 showed a significant cytotoxic effect against HeLa cells with an IC50 value of 0.23 µM, and CP successfully clustered 1 with calcium ionophores.

Conformational transformation of ascidiacyclamide analogues induced by incorporating enantiomers of phenylalanine, 1-naphthylalanine or 2-naphthylalanine.

We designed five ascidiacyclamide analogues [cyclo(-Xxx(1) -oxazoline(2) -d-Val(3) -thiazole(4) -l-Ile(5) -oxazoline(6) -d-Val(7) -thiazole(8) -)] incorporating l-1-naphthylalanine (l-1Nal), l-2-naphthylalanine (l-2Nal), d-phenylalanine (d-Phe), d-1-naphthylalanine (d-1Nal) or d-2-naphthylalanine (d-2Nal) into the Xxx(1) position of the peptide. The conformations of these analogues were then examined using (1) H NMR, CD spectroscopy, and X-ray diffraction. These analyses suggested that d-enantiomer-incorporated ASCs [(d-Phe), (d-1Nal), and (d-2Nal)ASC] transformed from the folded to the open structure in solution more easily than l-enantiomer-incorporated ASCs [(l-Phe), (l-1Nal), and (l-2Nal)ASC]. Structural comparison of the two analogues containing isomeric naphthyl groups showed that the 1-naphthyl isomer induced a more stable open structure than the 2-naphthyl isomer. In particular, [d-1Nal]ASC showed the most significant transformation from the folded to the open structure in solution, and exhibited the strongest cytotoxicity toward HL-60 cells.

Carapanolides T-X from Carapa guianensis (Andiroba) Seeds.

Two new mexicanolide-type limonoids, carapanolides T-U (1-2), and three new phragmalin-type limonoids, carapanolides V-X (3-5), were isolated from the seeds of Carapa guianensis (andiroba). Their structures were determined on the basis of 1D- and 2D-NMR spectroscopy.

CH-π interaction in VQIVYK sequence elucidated by NMR spectroscopy is essential for PHF formation of tau.

One of the histopathological features of Alzheimer's disease (AD) is higher order neurofibrillary tangles formed by abnormally aggregated tau protein. Investigation of the mechanism of tau aggregation is important for the clarifying the cause of AD and the development of therapeutic drugs. The microtubule-binding domain, which consists of repeats of similar amino acids (R1-R4) is thought to form the core component of paired helical filament (PHF). The hexapeptide(306) VQIVYK(311) of R3 has been shown to take a key role of promoting tau aggregation and assumed that its CH-π interaction between the side chains of Ile308 and Tyr310 would contribute in stabilizing the filament. In this work, we investigated a short isoform of tau (4RTau), R3, VQIVYK peptide and their mutants by thioflavin S (ThS) fluorescence, and NMR measurements, and proved for the first time that this CH-π interaction stabilizes the filament at the atomic level. In addition, by molecular modeling, we revealed that this interaction further supports an extended amphipathic structure for molecular self-association during the process of PHF formation of tau protein. The present work indicates new approach that inhibits the CH-π interaction for developing a therapeutic agent for AD.

Creation of an HDAC-based yeast screening method for evaluation of marine-derived actinomycetes: discovery of streptosetin A.

A histone deacetylase (HDAC)-based yeast assay employing a URA3 reporter gene was applied as a primary screen to evaluate a marine-derived actinomycete extract library and identify human class III HDAC (SIRT) inhibitors. On the basis of the bioassay-guided purification, a new compound designated as streptosetin A (1) was obtained from one of the active strains identified through the yeast assay. The gross structure of the new compound was elucidated from the 1D and 2D NMR data. The absolute stereostructure of 1 was determined based on X-ray crystal structure analysis and simulation of ECD spectra using time-dependent density functional theory calculations. This compound showed weak inhibitory activity against yeast Sir2p and human SIRT1 and SIRT2.

C-H ... π interplay between Ile308 and Tyr310 residues in the third repeat of microtubule binding domain is indispensable for self-assembly of three- and four-repeat tau.

Information on the structural scaffold for tau aggregation is important in developing a method of preventing Alzheimer's disease (AD). Tau contains a microtubule binding domain (MBD) consisting of three or four repeats of 31 and 32 similar residues in its C-terminal half. Although the key event in tau aggregation has been considered to be the formation of ß-sheet structures from a short hexapeptide (306)VQIVYK(311) in the third repeat of MBD, its aggregation pathway to filament formation differs between the three- and four-repeated MBDs, owing to the intermolecular and intramolecular disulphide bond formations, respectively. Therefore, the elucidation of a common structural element necessary for the self-assembly of three-/four-repeated full-length tau is an important research subject. Expanding the previous results on the aggregation mechanism of MBD, in this paper, we report that the C-H … π interaction between the Ile308 and Tyr310 side chains in the third repeat of MBD is indispensable for the self-assembly of three-/four-repeated full-length tau, where the interaction provides a conformational seed for triggering the molecular association. On the basis of the aggregation behaviours of a series of MBD and full-length tau mutants, a possible self-association model of tau is proposed and the relationship between the aggregation form (filament or granule) and the association pathway is discussed.

A new class of rhodamine luminophores: design, syntheses and aggregation-induced emission enhancement.

A new class of rhodamine luminophores, 3',3''-bis(oxospiroisobenzofuran)-3,7-bis(dialkylamino)benzopyrano-xanthene derivatives (ABPX), have been successfully developed. The emission behavior of ABPX series is directly opposite to the concentration quenching of conventional rhodamine dyes. ABPX series exhibit aggregation-induced emission enhancement (AIEE).

Interplay between I308 and Y310 residues in the third repeat of microtubule-binding domain is essential for tau filament formation.

Investigation of the mechanism of tau polymerization is indispensable for finding inhibitory conditions or identifying compounds preventing the formation of paired helical filament or oligomers. Tau contains a microtubule-binding domain consisting of three or four repeats in its C-terminal half. It has been considered that the key event in tau polymerization is the formation of a ß-sheet structure arising from a short hexapeptide (306)VQIVYK(311) in the third repeat of tau. In this paper, we report for the first time that the C-H⋯π interaction between Ile308 and Tyr310 is the elemental structural scaffold essential for forming a dry "steric zipper" structure in tau amyloid fibrils.