Metal(triphenylphosphine)-atovaquone Complexes: Synthesis, Antimalarial Activity, and Suppression of Heme Detoxification.

To ascertain the bioinorganic chemistry of metals conjugated with quinones, the complexes [Ag(ATV)(PPh3)2] (1), [Au(ATV)(PPh3)]·2H2O (2), and [Cu(ATV)(PPh3)2] (3) were synthesized by the coordination of the antimalarial naphthoquinone atovaquone (ATV) to the starting materials [Ag(PPh3)2]NO3, [Au(PPh3)Cl], and [Cu(PPh3)2NO3], respectively. These complexes were characterized by analytical and spectroscopical techniques. X-ray diffraction of single crystals precisely confirmed the coordination mode of ATV to the metals, which was monodentate or bidentate, depending on the metal center. Both coordination modes showed high stability in the solid state and in solution. All three complexes showed negative log D values at pH 5, but at pH 7.4, while complex 2 continued to have a negative log D value, complexes 1 and 3 displayed positive values, indicating a more hydrophilic character. ATV and complexes 1-3 could bind to ferriprotoporphyrin IX (FePPIX); however, only complexes 1-3 could inhibit ß-hematin crystal formation. Phenotype-based activity revealed that all three metal complexes are able to inhibit the growth of P. falciparum with potency and selectivity comparable to those of ATV, while the starting materials lack this activity. The outcomes of this chemical design may provide significant insights into structure-activity relationships for the development of new antimalarial agents.

Alkoxy-Bridged Dicopper(II) Cores Meet Tetracyanonickelate Linkers: Structural, Magnetic, and Theoretical Investigation of Cu/Ni Coordination Polymers.

Two heterometallic Cu(II)/Ni(II) coordination polymers, [Cu2(Hbdea)2Ni(CN)4]n (1) and [Cu2(dmea)2Ni(CN)4]n·nH2O (2), were successfully self-assembled in water by reacting Cu(II) nitrate with H2bdea (N-butyldiethanolamine) and Hdmea (N,N-dimethylethanolamine) in the presence of sodium hydroxide and [Ni(CN)4]2-. These new coordination polymers were investigated by single-crystal and powder X-ray diffraction and fully characterized by FT-IR spectroscopy, thermogravimetry, elemental analysis, variable-temperature magnetic susceptibility measurements, and theoretical DFT and CASSCF calculations. Despite differences in crystal systems, in both compounds, each dinuclear building block [Cu2(µ-aminopolyalcoholate)2]2+ is bridged by diamagnetic [Ni(CN)4]2- linkers, resulting in 1D (1) or 2D (2) metal-organic architectures. Experimental magnetic studies show that both compounds display strong antiferromagnetic coupling (J = -602.1 cm-1 for 1 and -151 cm-1 for 2) between Cu(II) ions within the dimers mediated by the µ-O-alkoxo bridges. These results are corroborated by the broken symmetry DFT studies, which also provide further insight into the electronic structures of copper dimeric units. By reporting a facile self-assembly synthetic protocol, this study can be a model to widen a still limited family of heterometallic Cu/Ni coordination polymer materials with different functional properties.

Promising fluconazole based zinc(II) and copper(II) coordination polymers against Chagas disease.

A series of new transition metal coordination polymers, [Zn(Ac)2(FLZ)2]n (1), [Zn(FLZ)2(Cl)2]n (2), {[Zn(FLZ)2](NO3)2}n (3), [Cu(FLZ)2(CH3COO)4]n (4), {[Cu(FLZ)2Cl2]}n (5) and {[Cu(FLZ)2](NO3)2}n (6), were synthesized by the reaction of fluconazole (FLZ) with the respective zinc or copper salts under mild conditions. The molecular structure of these compounds was elucidated by several analytical and spectroscopy techniques such as elemental analyses, 1H and 13C{1H} nuclear magnetic resonance, electronic paramagnetic resonance, and infrared spectroscopy. Single-crystal X-ray diffraction confirmed the structure of the compounds 2, 4, 5 and 6 in solid state. The antichagasic activity of these compounds was evaluated against different forms of Trypanosoma cruzi. Compound 2 exhibited the highest activity against intracellular amastigotes. The ultrastructural changes in epimastigotes and intracellular amastigotes were investigated. These promising biological results demonstrated that the zinc or copper coordination polymers can form very active anti-parasitic compounds. The resulting compounds are more effective than the free azole drug and, consequently, great candidates for the treatment of Chagas disease.

Diverse 3-Methylthio-4-Substituted Maleimides through a Novel Rearrangement Reaction: Synthesis and Selective Cell Imaging.

A transition metal-free protocol for the preparation of fluorescent and non-fluoresent 3-methylthio-4-arylmaleimides in a single step through a new rearrangement from thiazolidine-2,4-diones is described. By employing the optimized reaction conditions, a broad scope of derivatives was prepared in ≤97% yield. The reaction tolerated several substituted aryl groups, including the challenging preparation of pyridyl-containing derivatives. A series of control experiments strongly suggested that the new rearrangement involves a key isocyanate intermediate and a further reaction with in situ-generated methylthiomethyl acetate. The photophysical properties of some of the synthesized derivatives as well as their use in live cell imaging were also investigated, revealing that some of the substituted maleimides are capable of selectively staining different regions of the cells.

Metal-Cocatalyst Interaction Governs the Catalytic Activity of MII-Porphyrazines for Chemical Fixation of CO2.

Chemical fixation of CO2 to produce cyclic carbonates can be a green and atomic efficient process. In this work, a series of porphyrazines (Pzs) containing electron-withdrawing groups and central MII ions (where M = Mg, Zn, Cu, and Co) were synthesized and investigated as catalysts for the cycloaddition of CO2 to epoxides. Then, the efficiency of the Pzs was tested by varying cocatalyst type and concentration, epoxide, temperature, and pressure. MgIIPz bearing trifluoromethyl groups (1) showed the best conversion, producing, selectively, 78% of propylene cyclic carbonate (PCC), indicating that a harder and stronger Lewis acid is more effective for epoxide activation. Moreover, cocatalyst variation showed a notable effect on the reaction yields. Spectrophotometric titrations, MALDI-TOF mass spectra, and theoretical calculations suggest poisoning of the catalyst when tetrabutylammonium chloride (TBAC) and large amounts of tetrabutylammonium bromide (TBAB) were used in the system. The same was not observed for tetrabutylammonium iodide (TBAI), indicating that the metal-cocatalyst interaction may govern the reaction rate. In addition, two rare examples of crystalline structures were obtained, proving the distorted square pyramidal geometry with water molecule as axial ligand. This is one of the first studies reporting Pzs as catalysts for the chemical fixation of CO2, and we believe that the intricate balance between cocatalyst concentration and conversion efficiency shown here may aid future studies in the area.

Multicomponent ionic crystals of diltiazem with dicarboxylic acids toward understanding the structural aspects driving the drug-release.

Diltiazem (DIL) is a calcium channel blocker antihypertensive drug commonly used in the treatment of cardiovascular disorders. Due to the high solubility and prompt dissolution of the commercial form hydrochloride (DIL-HCl) that is closely related to short elimination drug half-life, this API is known for exhibiting an unfitted pharmacokinetic profile. In an attempt to understand how engineered multicomponent ionic crystals of DIL with dicarboxylic acids can minimize these undesirable biopharmaceutical attributes, herein, we have focused on the development of less soluble and slower dissolving salt/cocrystal forms. By the traditional solvent evaporation method, two hydrated salts of DIL with succinic and oxalic acids (DIL-SUC-H2O and DIL-OXA-H2O), and one salt-cocrystal with fumaric acid (DIL-FUM-H2FUM) were successfully prepared. An in-depth crystallographic description of these new solid forms was conducted through single and powder X-ray diffraction (SCXRD, PXRD), Hirshfeld surface (HS) analysis, energy framework (EF) calculations, Fourier Transform Infrared (FT-IR) spectroscopy, and thermal analysis (TG, DSC, and HSM). Structurally, the inclusion of dicarboxylic acids in the crystal structures provided the formation of 2D-sheet assemblies, where ionic pairs (DIL+/anion-) are associated with each other via H-bonding. Consequently, a substantial lowering in both solubility (16.5-fold) and intrinsic dissolution rate (13.7-fold) of the API has been achieved compared to that of the hydrochloride salt. These findings demonstrate the enormous potential of these solid forms in preparing of novel modified-release pharmaceutical formulations of DIL.

Insights on a new sulfonamide chalcone with potential antineoplastic application.

Chalcones (E)-1,3-diphenyl-2-propene-1-ones, a class of biosynthetic precursor molecules of flavonoids, have a wide variety of biological applications. Besides the natural products, many synthetic derivatives and analogs became an object of continued interest in academia and industry. In this work, a synthesis and an extensive structural study were performed on a sulfonamide chalcone 1-Benzenesulfonyl-3-(4-bromobenzylidene)-2-(2-chlorophenyl)-2,3-dihydro-1H-quinolin-4-one with potential antineoplastic application. In addition, in silico experiments have shown that the sulfonamide chalcone fits well in the ligand-binding site of EGFR with seven µ-alkyl binding energy interactions on the ligand-binding site. Finally, the kinetic stability and the pharmacophoric analysis for EGFR indicated the necessary spatial characteristics for potential activity of sulfonamide chalcone as an antagonist.

Antifungal promising agents of zinc(II) and copper(II) derivatives based on azole drug.

A series of new metal complexes, [Zn(KTZ)2(Ac)2]·H2O (1), [Zn(KTZ)2Cl2]·0.4CH3OH (2), [Zn(KTZ)2(H2O)(NO3)](NO3) (3), [Cu(KTZ)2(Ac)2]·H2O (4), [Cu(KTZ)2Cl2]·3.2H2O (5), [Cu(KTZ)2(H2O)(NO3)](NO3)·H2O (6), were synthesized by a reaction of ketoconazole (KTZ) with their respective zinc or copper salts under mild conditions. Similarly, six corresponding metal-CTZ (clotrimazole) complexes [Zn(CTZ)2(Ac)2]·4H2O (7), [Zn(CTZ)2Cl2] (8), [Zn(CTZ)2(H2O)(NO3)](NO3)·4H2O (9), [Cu(CTZ)2(Ac)2]·H2O (10), [Cu(CTZ)2Cl2]·2H2O (11), [Cu(CTZ)2(H2O)(NO3)](NO3)·2H2O (12), were obtained. These metal complexes were characterized by elemental analyses, molar conductivity, 1H and 13C{1H} nuclear magnetic resonance, UV/Vis, and infrared spectroscopies. Further, the crystal structure for complexes 7 and 10 was determined by single-crystal X-ray diffraction. The antifungal activity of these metal complexes was evaluated against three fungal species of medical relevance: Candida albicans, Cryptococcus neoformans, and Sporothrix brasiliensis. Complexes 1 and 3 exhibited the greatest antifungal activity with a broad spectrum of action at low concentrations and high selectivity. Some morphological changes induced by these metal complexes in S. brasiliensis cells included yeast-hyphae conversion, an increase in cell size and cell wall damage. The strategy of coordination of clinic drugs (KTZ and CTZ) to zinc and copper was successful, since the corresponding metal complexes were more effective than the parent drug. Particularly, the promising antifungal activities displayed by Zn-KTZ complexes make them potential candidates for the development of an alternative drug to treat mycoses.

Significant increase in the secretion of extracellular vesicles and antibiotics resistance from methicillin-resistant Staphylococcus aureus induced by ampicillin stress.

Extracellular vesicles (EVs) containing specific cargo molecules from the cell of origin are naturally secreted from bacteria. EVs play significant roles in protecting the bacterium, which can contribute to their survival in the presence of antibiotics. Herein, we isolated EVs from methicillin-resistant Staphylococcus aureus (MRSA) in an environment with or without stressor by adding ampicillin at a lower concentration than the minimum inhibitory concentration (MIC). We investigated whether EVs from MRSA under stress condition or normal condition could defend susceptible bacteria in the presence of several ß-lactam antibiotics, and directly degrade the antibiotics. A comparative proteomic approach was carried out in both types of EVs to investigate ß-lactam resistant determinants. The secretion of EVs from MRSA under antibiotic stressed conditions was increased by 22.4-fold compared with that of EVs without stress. Proteins related to the degradation of ß-lactam antibiotics were abundant in EVs released from the stressed condition. Taken together, the present data reveal that EVs from MRSA play a crucial role in the survival of ß-lactam susceptible bacteria by acting as the first line of defense against ß-lactam antibiotics, and antibiotic stress leads to release EVs with high defense activity.

Crystal structure, vibrational frequencies and polarizability distribution in hydrogen-bonded salts of pyromellitic acid.

Structural features of moderate-to-strong O-H...O hydrogen bonds are related to the frequencies of O-H stretching vibrations and to the electric polarizability distribution in the donor and acceptor functional groups for crystals synthesized from the 1,2,4,5-benzenetetracarboxylic (pyromellitic) acid, namely: bis(3-aminopyridinium) dihydrogen pyromellitate tetrahydrate, (1); bis(3-carboxypyridinium) dihydrogen pyromellitate, (2); bis(3-carboxyphenylammonium) dihydrogen pyromellitate dihydrate, (3); and bis(4-carboxyphenylammonium) dihydrogen pyromellitate, (4). A combination of single-crystal X-ray diffraction, powder Raman spectroscopy and first-principle calculations in both crystalline and gaseous phases has shown that changes in the O-H...O hydrogen-bond geometry can be followed by changes in the corresponding spectral modes. Vibrational properties of moderate hydrogen bonds can be estimated from correlations based on statistical analysis of several compounds [Novak (1974). Struct. Bond. 18, 177-216]. However, frequencies related to very short O-H...O bonds can only be predicted by relationships built from a subset of structurally similar systems. Moreover, the way in which hydrogen bonds affect the polarizability of donor and acceptor groups depends on their strength. Moderate interactions enhance the polarizability and make it more anisotropic. Shorter hydrogen bonds may decrease the polarizability of a group as a consequence of the volume restraint implied by the neighbour molecule within a hydrogen-bonded aggregate. This is significant for evaluation of the electric susceptibility in crystals and, therefore, for estimation of refractive indices and birefringence.

Enhancing the solubility and permeability of the diuretic drug furosemide via multicomponent crystal forms.

Furosemide (FSM) is a biopharmaceutical classification system (BCS) class IV drug, being a potent loop diuretic used in the treatment of congestive heart failure and edema. Due to its low solubility and permeability, FSM is known for exhibiting poor oral bioavailability. In order to overcome or even minimize these undesirable biopharmaceutical attributes, in this work we have focused on the development of more soluble and permeable multicomponent solid forms of FSM. Using solvent evaporation as crystallization method, a salt and a cocrystal of FSM with imidazole (IMI) and 5-fluorocytosine (5FC) coformers, named FSM-IMI and FSM-5FC, respectively, were successfully prepared. A detailed structural study of these new solid forms was conducted using single and powder X-ray diffraction (SCXRD, PXRD), Fourier Transform Infrared (FT-IR) and proton Nuclear Magnetic Resonance (1H NMR) spectroscopy and thermal analysis (thermogravimetry, differential scanning calorimetry and hot-stage microscopy). Both FSM-IMI and FSM-5FC showed substantial enhancements in the solubility (up 118-fold), intrinsic dissolution (from 1.3 to 2.6-fold) and permeability (from 2.1 to 2.8-fold), when compared to the pure FSM. These results demonstrate the potential of these new solid forms to increase the limited bioavailability of FSM.

Binucleating Hydrazonic Ligands and Their µ-Hydroxodicopper(II) Complexes as Promising Structural Motifs for Enhanced Antitumor Activity.

Very few inorganic antineoplastic drugs have entered the clinic in the last decades, mainly because of toxicity issues. Because copper is an essential trace element of ubiquitous occurrence, decreased side effects could be expected in comparison with the widely used platinum anticancer compounds. In the present work, two novel hydrazonic binucleating ligands and their µ-hydroxo dicopper(II) complexes were prepared and fully characterized. They differ by the nature of the aromatic group present in their aroylhydrazone moieties: while H3L1 and its complex, 1, possess a thiophene ring, H3L2 and 2 contain the more polar furan heterocycle. X-ray diffraction indicates that both coordination compounds are very similar in structural terms and generate dimeric arrangements in the solid state. Positive-ion electrospray ionization mass spectrometry analyses confirmed that the main species present in a 10% dimethyl sulfoxide (DMSO)/water solution should be [Cu2(HL)(OH)]+ and the DMSO-substituted derivative [Cu2(L)(DMSO)]+. Scattering techniques [dynamic light scattering (DLS) and small-angle X-ray scattering] suggest that the complexes and their free ligands interact with bovine serum albumin (BSA) in a reversible manner. The binding constants to BSA were determined for the complexes through fluorescence spectroscopy. Moreover, to gain insight into the mechanism of action of the compounds, calf thymus DNA binding studies by UV-visible and DLS measurements using plasmid pBR322 DNA were also performed. For the complexes, DLS data seem to point to the occurrence of DNA cleavage to Form III (linear). Both ligands and their dicopper(II) complexes display potent antiproliferative activity in a panel of four cancer cell lines, occasionally even in the submicromolar range, with the complexes being more potent than the free ligands. Our data on cellular models correlate quite well with the DNA interaction experiments. The results presented herein show that aroylhydrazone-derived binucleating ligands, as well as their dinuclear µ-hydroxodicopper(II) complexes, may represent a promising structural starting point for the development of a new generation of highly active potential antitumor agents.

Synthesis, crystal structure and topological analysis of a three-dimensional polymeric network based on zinc(II), potassium and 5-sulfobenzene-1,3-dicarboxylate (SIP).

Aromatic polycarboxylate linkers provide structural rigidity and strong interactions among the metal centre and the carboxylate O atoms. A new three-dimensional coordination polymer namely, catena-poly[potassium [tetraaqua(µ-5-sulfobenzene-1,3-dicarboxylato)zinc(II)]], {K[Zn(C8H3O7S)(H2O)4]}n or {K[Zn(SIP)(H2O)4]}n, where SIP is 5-sulfobenzene-1,3-dicarboxylate or 5-sulfoisophthalate, was obtained and characterized by elemental analysis and IR vibrational spectroscopy, and the single-crystal structure was determined by X-ray diffraction analysis. The compound crystallizes in the monoclinic space group P21/n with Z = 4. Topological analysis revealed that K-O interactions form a two-dimensional network, which is uninodal 4-connected and can be described with a point symbol (44.62), and this plane network is classified as sql/Shubnikov. The layers are connected by Zn2+ ions coordinated to the SIP linker, forming a three-dimensional network. This net is a trinodal (3,5,6)-connected system with point symbol (3.44.52.62.73.83).(3.44.52.62.7).(3.72).

Synthesis of glycerol carbonate over a 2D coordination polymer built with Nd3+ ions and organic ligands.

In this work, a two-dimensional coordination polymer was synthesized and the structure was determined by single-crystal X-ray diffraction. The crystal structure belongs to the space group Pna21 and was characterized by Raman and FT-infrared spectroscopy, powder X-ray diffraction and Brunauer-Emmett-Teller surface area analysis. Catalyst activities were evaluated through the synthesis of glycerol carbonate from glycerol and urea using a batch reactor. After the optimization of both reaction and reaction conditions, the activity results showed that the coordination polymer used as a heterogeneous catalyst has good values of conversions and selectivity for the manufacturing of glycerol carbonate in a fine-chemical process. The analysis of powder X-ray diffraction and spectroscopy for the coordination polymer employed, before and after the reaction, shows that some changes have taken place in the crystal structure during the process, in spite of a recovery at the end of the reaction. The advantages and limitations of the coordination polymer were discussed and compared with those of the previous heterogeneous catalysts in the literature.

Novel gold(I) complexes with 5-phenyl-1,3,4-oxadiazole-2-thione and phosphine as potential anticancer and antileishmanial agents.

The current anticancer and antileishmanial drug arsenal presents several limitations concerning their specificity, efficacy, costs and the emergence of drug-resistant cells lines, which encourages the urgent need to search for new alternatives. Inspired by the fact that gold(I)-based compounds are promising antitumoral and antileishmanial drug candidates, we synthesized novel gold(I) complexes containing phosphine and 5-phenyl-1,3,4-oxadiazole-2-thione and evaluated their anticancer and antileishmanial activities. Synthesis was performed by reacting 5-phenyl-1,3,4-oxadiazole-2-thione derivatives with chloro(triphenylphosphine)gold(I) and chloro(triethylphosphine)gold(I). The novel compounds were characterized by infrared, Raman, 1H, 13C nuclear magnetic resonance, high-resolution mass spectra, and x-ray crystallography. The coordination of the ligands to gold(I) occurred through the exocyclic sulfur atom. All gold(I) complexes were active at low micromolar or nanomolar range with IC50 values ranging from <0.10 to 1.66 µM against cancer cell lines and from 0.9 to 4.2 µM for Leishmania infantum intracellular amastigotes. Compound (6-A) was very selective against murine melanoma B16F10, colon cancer CT26.WT cell lines and L. infantum intracellular amastigotes. Compound (7-B) presented the highest anticancer activity against both cancer cell lines while the promising antileishmanial lead was compound (6-A). Tiethylphosphine gold(I) complexes were more active than the conterparts triphenylphosphine derivatives for both anticancer and antileishmanial activities. Triethylphosphine gold(I) derivatives presented antimony cross-resistance in L. guyanensis demonstrating their potential to be used as chemical tools to better understand mechanisms of drug resistance and action. These findings revealed the anticancer and antileishmanial potential of gold(I) oxadiazole phosphine derivatives.

Structure elucidation of five novel isomeric saponins from the viscera of the sea cucumber Holothuria lessoni.

Sea cucumbers are prolific producers of a wide range of bioactive compounds. This study aimed to purify and characterize one class of compound, the saponins, from the viscera of the Australian sea cucumber Holothuria lessoni. The saponins were obtained by ethanolic extraction of the viscera and enriched by a liquid-liquid partition process and adsorption column chromatography. A high performance centrifugal partition chromatography (HPCPC) was applied to the saponin-enriched mixture to obtain saponins with high purity. The resultant purified saponins were profiled using MALDI-MS/MS and ESI-MS/MS which revealed the structure of isomeric saponins to contain multiple aglycones and/or sugar residues. We have elucidated the structure of five novel saponins, Holothurins D/E and Holothurinosides X/Y/Z, along with seven reported triterpene glycosides, including sulfated and non-sulfated saponins containing a range of aglycones and sugar moieties, from the viscera of H. lessoni. The abundance of novel compounds from this species holds promise for biotechnological applications.

Discovery of novel saponins from the viscera of the sea cucumber Holothuria lessoni.

Sea cucumbers, sometimes referred to as marine ginseng, produce numerous compounds with diverse functions and are potential sources of active ingredients for agricultural, nutraceutical, pharmaceutical and cosmeceutical products. We examined the viscera of an Australian sea cucumber Holothuria lessoni Massin et al. 2009, for novel bioactive compounds, with an emphasis on the triterpene glycosides, saponins. The viscera were extracted with 70% ethanol, and this extract was purified by a liquid-liquid partition process and column chromatography, followed by isobutanol extraction. The isobutanol saponin-enriched mixture was further purified by high performance centrifugal partition chromatography (HPCPC) with high purity and recovery. The resultant purified polar samples were analyzed using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS)/MS and electrospray ionization mass spectrometry (ESI-MS)/MS to identify saponins and characterize their molecular structures. As a result, at least 39 new saponins were identified in the viscera of H. lessoni with a high structural diversity, and another 36 reported triterpene glycosides, containing different aglycones and sugar moieties. Viscera samples have provided a higher diversity and yield of compounds than observed from the body wall. The high structural diversity and novelty of saponins from H. lessoni with potential functional activities presents a great opportunity to exploit their applications for industrial, agricultural and pharmaceutical use.

Treatment strategies for high resveratrol induction in Vitis vinifera L. cell suspension culture.

Bioprocesses capable of producing large scales of resveratrol at nutraceutical grade are in demand. This study herein investigated treatment strategies to induce the production of resveratrol in Vitis vinifera L. cell suspension cultures. Among seven investigated elicitors, jasmonic acid (JA), salicylic acid, ß-glucan (GLU), and chitosan enhanced the production of intracellular resveratrol manyfold. The combined treatment of JA and GLU increased extracellular resveratrol production by up to tenfold. The application of Amberlite XAD-7 resin for in situ removal and artificial storage of secreted resveratrol further increased resveratrol production by up to four orders of magnitude. The level of resveratrol produced in response to the combined treatment with 200 g/L XAD-7, 10 µM JA and 1 mg/mL GLU was approximately 2400 mg/L, allowing the production of resveratrol at an industrial scale. The high yield of resveratrol is due to the involvement of a number of mechanisms working in concert.

Characterization of anthocyanic vacuolar inclusions in Vitis vinifera L. cell suspension cultures.

Anthocyanic vacuolar inclusions (AVIs) are intra-vacuolar structures capable of concentrating anthocyanins and are present in over 50 of the highest anthocyanin-accumulating plant species. Presence of AVIs alters pigment intensity, total anthocyanin levels, pigment hue and causes bathochromic shifts in a spatio-temporal manner within various flowers, vegetables and fruits. A year-long study on Vitis vinifera cell suspension cultures found a strong correlation between AVI prevalence and anthocyanin content, but not the number of pigmented cells, growth rate or stilbene content. Furthermore, enhancement of the prevalence of AVIs and anthocyanins was achieved by treatment of V. vinifera cell suspension cultures with sucrose, jasmonic acid and white light. A unique autofluorescence of anthocyanins was used to demonstrate microscopically that AVIs proceed from the cytosol across the tonoplast and were able to coalesce intravacuolarly, with fewer, larger AVIs predominating as cells mature. Purification and characterisation of these bodies were performed, showing that they were dense, highly organic structures, with a lipid component indicative of membrane-encasement. These purified AVIs were also shown to comprise long-chain tannins and possessed an increased affinity for binding acylated anthocyanins, though no unique protein component was detected.

Purification, molecular cloning, and characterization of glutathione S-transferases (GSTs) from pigmented Vitis vinifera L. cell suspension cultures as putative anthocyanin transport proteins.

The ligandin activity of specific glutathione S-transferases (GSTs) is necessary for the transport of anthocyanins from the cytosol to the plant vacuole. Five GSTs were purified from Vitis vinifera L. cv. Gamay Fréaux cell suspension cultures by glutathione affinity chromatography. These proteins underwent Edman sequencing and mass spectrometry fingerprinting, with the resultant fragments aligned with predicted GSTs within public databases. The corresponding coding sequences were cloned, with heterologous expression in Escherichia coli used to confirm GST activity. Transcriptional profiling of these candidate GST genes and key anthocyanin biosynthetic pathway genes (PAL, CHS, DFR, and UFGT) in cell suspensions and grape berries against anthocyanin accumulation demonstrated strong positive correlation with two sequences, VvGST1 and VvGST4, respectively. The ability of VvGST1 and VvGST4 to transport anthocyanins was confirmed in the heterologous maize bronze-2 complementation model, providing further evidence for their function as anthocyanin transport proteins in grape cells. Furthermore, the differential induction of VvGST1 and VvGST4 in suspension cells and grape berries suggests functional differences between these two proteins. Further investigation of these candidate ligandins may identify a mechanism for manipulating anthocyanin accumulation in planta and in vitro suspension cells.