Polyamine derivatives from the bee pollen of Quercus mongolica with tyrosinase inhibitory activity.

Eighteen constituents, including nine new compounds, were isolated from the bee pollen of Quercus mongolica. The structures of the new compounds were established on the basis of combined spectroscopic analysis. Structurally, the nine new compounds are polyamine derivatives with phenolic moieties which were assigned as one putrescine derivative, mogolicine A (2), seven spermidine derivatives, mongolidines A-G (3-5, 8, 12, 14, 17) and one spermine derivative, mogoline A (18). Evaluation of the biological activity of isolated compounds revealed that the polyamine derivatives with coumaroyl and caffeoyl moieties showed tyrosinase inhibition with IC50 values of 19.5-85.8 µM; however, the addition of a methoxy group to phenolic derivatives reduced the inhibitory activity.

Identification of a novel acetylated form of branched-chain polyamine from a hyperthermophilic archaeon Thermococcus kodakarensis.

Long/branched-chain polyamines are unique polycations found in thermophiles. The hyperthermophilic archaeon Thermococcus kodakarensis contains spermidine and a branched-chain polyamine, N4-bis(aminopropyl)spermidine, as major polyamines. The metabolic pathways associated with branched-chain polyamines remain unknown. Here, we used gas chromatography and liquid chromatography-tandem mass spectrometry analyses to identify a new acetylated polyamine, N4-bis(aminopropyl)-N1-acetylspermidine, from T. kodakarensis; this polyamine was not found in other micro-organisms. The amounts of branched-chain polyamine and its acetylated form increased with temperature, indicating that branched-chain polyamines are important for growth at higher temperatures. The amount of quaternary acetylated polyamine produced was associated with the amount of N4-bis(aminopropyl)spermidine in the cell. The ratio of acetylated to non-acetylated forms was higher in the stationary phase than in the logarithmic growth phase under high-temperature stress condition.

ArgTX-636, a polyamine isolated from spider venom: A novel class of melanogenesis inhibitors.

To discover new molecules with an inhibitory activity of melanogenesis a hundred of scorpions, snakes, spiders and amphibians venoms were screened for their capacity to inhibit mushroom tyrosinase using 3,4-l-dihydroxyphenylalanine (l-DOPA) as substrate. The Argiope lobata spider venom proved to be the most active. HPLC fraction containing Argiotoxine-636 (ArgTX-636), a polyamine known for its numerous biological activities, was found to also show a good regulation activity of melanogenesis by inhibiting DOPA and 5,6-dihydroxyindole-2-carboxylic acid (DHICA) oxidases activities, wore by tyrosinase (TYR) and tyrosinase-related protein 1 (TRP-1), respectively. Our results demonstrate that ArgTX-636 reduced the mushroom tyrosinase activity in a dose-dependent way with a maximal half inhibitory concentration (IC50) value of 8.34µM, when l-DOPA is used as substrate. The Lineweaver-Burk study showed that ArgTX-636 is a mixed type inhibitor of the diphenolase activity. Moreover, ArgTX-636 inhibits DHICA oxydase activity of mushroom tyrosinase activity with IC50 at 41.3µM. ArgTX-636 has no cytotoxicity in B16F10 melanoma cells at concentrations up to 42.1µM. The effect of ArgTX-636 on melanogenesis showed that melanin production in B16F10 melanoma cell decreased by approximatively 70% compared to untreated cells. ArgTX-636 displayed no significant effect on the TYR expression while the protein level of TRP-1 decreased in B16F10 cells. Thus, ArgTX-636 could have particular interest for cosmetic and/or pharmaceutical use in order to reduce important dermatoses in black and mixed skins.

Fabclavines: bioactive peptide-polyketide-polyamino hybrids from Xenorhabdus.

The structure of the fabclavines-unique mixtures of nonribosomally derived peptide-polyketide hybrids connected to an unusual polyamino moiety-has been solved by detailed NMR and MS methods. These compounds have been identified in two different entomopathogenic Xenorhabdus strains, thereby leading also to the identification of the fabclavine biosynthesis gene cluster. Detailed analysis of these clusters and initial mutagenesis experiments allowed the prediction of a biosynthesis pathway in which the polyamino moiety is derived from an unusual type of fatty acid synthase that is normally involved in formation of polyunsaturated fatty acids. As fabclavines show broad-spectrum activity against bacteria, fungi, and other eukaryotic cells, they might act as "protection factors" against all kinds of food competitors during the complex life cycle of Xenorhabdus, its nematode host, and their insect prey.

Analysis of polyamidoamine dendrimers by isoelectric focusing.

Polyamidoamine dendrimers have been studied extensively for their potential applications in nanomedicine. Their uses as imaging, drug, and nucleic acid delivery agents are nearing clinical trials. As such, characterization of polyamidoamine dendrimers and their nano-devices is of immense importance for monitoring the efficiency of their synthesis, purity, and quality control of manufactured products as well as their in vivo behavior. We report here the analysis of polyamidoamine dendrimers possessing various cores and surface groups with a simple and inexpensive isoelectric focusing method. The isoelectric points of the dendrimers were readily determined from a calibration plot generated by running proteins with known pI values. The isoelectric points for various surface-modified polyamidoamine dendrimers ranged from 4 to 9. Polyamidoamine dendrimers possessing terminal hydroxyl groups gave a pI > 7, while those with terminal carboxyl groups exhibit a pI < 7. Generation number and cores of the dendrimers did not significantly affect their isoelectric points. Isoelectric focusing thus offers another important tool for characterizing these nanomolecules.

[Relationship between the anticoagulant activity of sulfated plant polysaccharides and the area of their precipitation with polycations during biospecific electrophoresis].

Polyanions (in an amount within 1.5 - 6.0 mg), including cellulose sulfates (excreted from Gossipium hirsutum L., molecular weight 22.0 kDa, degree of sulfation within 0.8 - 1.8), inulin sulfates (excreted from Helianthus tuberosus, molecular weight 8.0 kDa, degree of sulfation within 0.6 - 1.6), pectin sulfates (excreted from Abies sibirica L., molecular weight 24.0 kDa, degree of sulfation within 0.8 - 1.1), give rise to peaks of precipitation with polycations of protamine sulfate. Only cellulose sulfates (in amount within 0.38 - 6.00 mg) give the peaks of precipitation with chitosan polycations (molecular weight 10 kDa, degree of deacetylation 85%) during horizontal biospecific electrophoresis. The height of the peak of precipitation with protamin sulfate was found to grow with increasing antithrombin activity of cellulose sulfates and pectin sulfate (for polyanions in an amount within 1.5 - 6 mg). The size of the area of precipitation with chitosan was found to decrease with increasing antithrombin activity of cellulose sulfates.

JBIR-94 and JBIR-125, antioxidative phenolic compounds from Streptomyces sp. R56-07.

Two phenolic compounds, JBIR-94 (1) and JBIR-125 (2), were isolated from the fermentation broth of strain R56-07, which was identified by phylogenetic methods as a novel species of Streptomyces. The structures of 1 and 2 were assigned on the basis of 1D and 2D NMR spectroscopy and MS analyses. Compounds 1 and 2 exhibited 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity with an IC(50) value of 11.4 and 35.1 µM, respectively. These compounds are the first examples of hydroxycinnamic acid amides containing putrescine or spermidine produced by actinomycetes.

Solid-phase synthesis and biological evaluation of Joro spider toxin-4 from Nephila clavata.

Polyamine toxins from orb weaver spiders are attractive pharmacological tools particularly for studies of ionotropic glutamate (iGlu) receptors in the brain. These polyamine toxins are biosynthesized in a combinatorial manner, providing a plethora of related, but structurally complex toxins to be exploited in biological studies. Here, we have used solid-phase synthetic methodology for the efficient synthesis of Joro spider toxin-4 (JSTX-4) (1) from Nephila clavata, providing sufficient amounts of the toxin for biological evaluation at iGlu receptor subtypes using electrophysiology. Biological evaluation revealed that JSTX-4 inhibits iGlu receptors only in high µM concentrations, thereby being substantially less potent than structurally related polyamine toxins.

Removal of organic pollutants in model water and thermal wastewater using clay minerals.

Water treatment method was developed for the removal of different anionic dyes such as methyl orange and indigo carmine, and also for thymol applying sodium bentonite and cationic surfactant - hexadecyltrimethylammonium bromide (HTAB) - or polyelectrolytes (polydiallyldimethylammonium chloride, poly-DADMAC and poly-amines). The removal efficiency of these model substrates was examined in model water using UV-Vis spectrophotometry, HPLC and TOC analysis. The clay mineral and HTAB were added in one step to the polluted model water in Jar-test experiments. The influence of the cation exchange capacity (CEC) of the applied clay mineral and the presence of polyaluminium chloride coagulant (BOPAC) were also tested for the water treatment process. The structures of the in situ produced and pre-prepared organoclay composites were compared by XRD analysis. The rapid formation of organoclay adsorbents provided very efficient removal of the dyes (65-90 % in 3-10 mg/L TOC(0) range) with 200 mg/L sodium bentonite dose, however thymol was less efficiently separated. Adsorption efficiencies of the composites were compared at different levels of ion exchange such as at 40, 60 and 100 %. In the case of thymol, the elimination of inorganic carbon from the model water before the TOC analysis resulted in some loss of the analysed volatile compound therefore the HPLC analysis was found to be the most suitable tool for the evaluation of the process. This one-step adsorption method using in situ formed organoclay was better performing than the conventional process in which the montmorillonite-surfactant composite is pre-preapared and subsequently added to the polluted water. The purification performance of this method was also evaluated on raw and artificially polluted thermal wastewater samples containing added thymol.

High-throughput screening of salivary polyamine markers for discrimination of colorectal cancer by multisegment injection capillary electrophoresis tandem mass spectrometry.

Polyamine metabolites provide pathophysiological information on disease or therapeutic efficacy, yet rapid screening methods for these biomarkers are lacking. Here, we developed high-throughput polyamine metabolite profiling based on multisegment injection capillary electrophoresis triple quadrupole tandem mass spectrometry (MSI-CE-MS/MS), which allows sequential 40-sample injection followed by electrophoretic separation and specific mass detection. To achieve consecutive analysis of polyamine samples, 1 M formic acid was used as the background electrolyte (BGE). The BGE spacer volume had an apparent effect on peak resolution among samples, and 20 nL was selected as the optimal volume. The use of polyamine isotopomers as the internal standard enabled the correction of matrix effects in MS detection. This method is sensitive, selective and quantitative, and its utility was demonstrated by screening polyamines in 359 salivary samples within 360 min, resulting in discrimination of colorectal cancer patients from noncancer controls.

Identification and determination of urinary acetylpolyamines in cancer patients by electrospray ionization and time-of-flight mass spectrometry.

A method for the quantification of acetylpolyamines, N(1),N(12)-diacetylspermine (DiAcSpm), monoacetylspermidine (AcSpd), and N(1),N(8)-diacetylspermidine (DiAcSpd), identifying each compound simultaneously, was developed with the goal of evaluating these acetylpolyamines as potential biomarkers of cancer. The method consists of prepurification of acetylpolyamines in urine with commercially available cartridges and derivatization with heptafluorobutyric (HFB) anhydride. HFB derivatives of acetylpolyamines were determined simultaneously using (15)N-labeled acetylpolyamines as internal standards by electrospray ionization and time-of-flight mass spectrometry (ESI-TOF MS). After the method was validated, the urinary acetylpolyamines of 38 cancer patients were quantified with this method. A comparison of the concentrations of DiAcSpm with those measured by a colloidal gold aggregation method demonstrated a correlation coefficient of 0.996, showing that the two methods were equally satisfactory. Analysis of the correlation between DiAcSpd or AcSpd and DiAcSpm, performed for the first time, indicated the usefulness of DiAcSpm as a urinary biomarker of cancer. During the course of this work, two simple methods for the preparation of alpha,omega-diacetylpolyamines were developed, and a possibility to separate and determine the concentrations of the two isomers, N(1)-acetylspermidine and N(8)-acetylspermidine in AcSpd, was shown by tandem mass spectrometry (MS/MS).

Long-Term Cd Exposure Alters the Metabolite Profile in Stem Tissue of Medicago sativa.

As a common pollutant, cadmium (Cd) is one of the most toxic heavy metals accumulating in agricultural soils through anthropogenic activities. The uptake of Cd by plants is the main entry route into the human food chain, whilst in plants it elicits oxidative stress by unbalancing the cellular redox status. Medicago sativa was subjected to chronic Cd stress for five months. Targeted and untargeted metabolic analyses were performed. Long-term Cd exposure altered the amino acid composition with levels of asparagine, histidine and proline decreasing in stems but increasing in leaves. This suggests tissue-specific metabolic stress responses, which are often not considered in environmental studies focused on leaves. In stem tissue, profiles of secondary metabolites were clearly separated between control and Cd-exposed plants. Fifty-one secondary metabolites were identified that changed significantly upon Cd exposure, of which the majority are (iso)flavonoid conjugates. Cadmium exposure stimulated the phenylpropanoid pathway that led to the accumulation of secondary metabolites in stems rather than cell wall lignification. Those metabolites are antioxidants mitigating oxidative stress and preventing cellular damage. By an adequate adjustment of its metabolic composition, M. sativa reaches a new steady state, which enables the plant to acclimate under chronic Cd stress.

Combinatorial chemistry in insects: a library of defensive macrocyclic polyamines.

The pupal defensive secretion of the coccinellid beetle Epilachna borealis is composed principally of a combinatorial library of macrocyclic polyamines. These compounds constitute a previously unrecognized family of natural products, characterized by extremely large-ring lactonic structures derived from a small set of (2-hydroxyethylamino)alkanoic acids. The combinatorial assembly of these simple building blocks generates a high degree of structural diversity, which is further increased by slow, spontaneous intramolecular rearrangement of the macrocycles.

[The total large-scale synthesis of argiopine].

The total large-scale synthesis of a natural toxin argiopine, a polymethylenepolyamine derivative, was developed. It. consisted of 26 stages and included three key block schemes. Most of the stages proceeded quantitatively, which excluded the necessity of using the chromatographic separation of intermediates.

Cardiotoxic effects of pavetamine extracted from Pavetta harborii in the rat.

Previous studies have shown that crude extracts from Pavetta harborii as well as dried plant material have cardiotoxic effects on rats and sheep that can lead to heart failure. The active component has since been isolated and identified. This substance has been named pavetamine. The aim of this study was to determine whether pavetamine has cardiotoxic effects similar to those seen in previous reports, when administered to rats intraperitoneally. Sprague Dawley rats received two doses, initially 4 mg/kg and then 3 mg/kg pavetamine respectively and were monitored for 35 days before cardiodynamic parameters were measured by inserting a fluid-filled catheter into the left ventricle via the right carotid artery. These values were compared to those of control rats that had received only saline. Pavetamine significantly reduced systolic function and body mass in the treated rats, which indicates that it has the potential to induce heart failure in this animal model.

Modulation of oncogenic miRNA biogenesis using functionalized polyamines.

MicroRNAs are key factors in the regulation of gene expression and their deregulation has been directly linked to various pathologies such as cancer. The use of small molecules to tackle the overexpression of oncogenic miRNAs has proved its efficacy and holds the promise for therapeutic applications. Here we describe the screening of a 640-compound library and the identification of polyamine derivatives interfering with in vitro Dicer-mediated processing of the oncogenic miR-372 precursor (pre-miR-372). The most active inhibitor is a spermine-amidine conjugate that binds to the pre-miR-372 with a KD of 0.15 µM, and inhibits its in vitro processing with a IC50 of 1.06 µM. The inhibition of miR-372 biogenesis was confirmed in gastric cancer cells overexpressing miR-372 and a specific inhibition of proliferation through de-repression of the tumor suppressor LATS2 protein, a miR-372 target, was observed. This compound modifies the expression of a small set of miRNAs and its selective biological activity has been confirmed in patient-derived ex vivo cultures of gastric carcinoma. Polyamine derivatives are promising starting materials for future studies about the inhibition of oncogenic miRNAs and, to the best of our knowledge, this is the first report about the application of functionalized polyamines as miRNAs interfering agents.

Analysis of underivatized polyamines by reversed phase liquid chromatography with electrospray tandem mass spectrometry.

A reversed phase liquid chromatography-electrospray ionization-tandem mass spectrometric method (RP-LC-ESI-MS/MS) was developed to separate and detect polyamines with minimal sample pre-treatment and without any derivatization. Prior to MS/MS analysis, a complete chromatographic separation of polyamines was achieved by a linear gradient elution using heptafluorobutyric acid as a volatile ion-pair modifier, and signal suppression was prevented by post-column addition of 75% propionic acid in isopropanol to the column flow. The developed method was successfully applied to the identification of metabolites formed from N(1),N(12)-diethylspermine in the reaction catalyzed by recombinant human polyamine oxidase and to the detection of eight different polyamines in a standard mixture.

Analysis of guazatine mixture by LC and LC-MS and antimycotic activity determination of principal components.

Guazatine is a non-systemic contact fungicide, a mixture of reaction products from polyamines, comprising mainly octa-methylenediamine, iminodi(octamethylene)diamine, octamethylenebis(imino-octamethylene) diamine and carbamonitrile. In this work, the analysis of guazatine mixture by LC and LC-MS has been treated for the first time. In the guazatine mixture diamine derivatives account for 40% of the constituents of guazatine, triamines for 46%, tetramines for 11% and other amine derivatives for 3%. The most abundant individual components are the fully guanidated triamine (GGG, 30.6%) and the fully guanidated diamine (GG, 29.5%) followed by the monoguanidated diamine (GN, 9.8%) and a diguanidated triamine (GGN, 8.1%). The identification and separation of main components of commercial guazatine was performed through a new LC-MS method. The separation was performed on an Alltima C(18) column using linear gradient elution (formic acid in water and acetonitrile) with UV-detection at 200 nm and the identification was performed by ESI(+)-mass spectrometry analysis. The main components (GN, GG, GNG, GGN, GGG and GGGG) were then purified and separated from the mixture. Antimycotic activity of guazatine derivatives was determined on different species and strains belonging to genus Candida. The results obtained suggest that GNG and GGGG components can further be developed in new antifungal compounds with high potential for the treatment of Candida infections.

Separation of poly(amidoamine) (PAMAM) dendrimer generations by dynamic coating capillary electrophoresis.

The separation of compounds possessing amino groups (peptides, proteins, polyamino compounds) by capillary zone electrophoresis suffers from the interaction (sticking) of these solutes with the capillary wall. This sticking can result in the absence or incomplete separation of compounds or even in their retention in the capillary. Polyamidoamine (PAMAM) dendrimers are a class of spherical polymers with primary amino groups at the surface. These compounds can be separated reasonably well at acidic pH but not at neutral pH. A new method based on the dynamic coating of the capillary was developed for the separation of these compounds at pH 7.4. The method comprises separation in a fused-silica capillary (57 cm total length, 50 cm to the detector, ID 75 microm) and a background electrolyte consisting of a Tris-phosphate buffer (50 mmol/L, pH 7.4) and 0.05% (w/v) polyethyleneimine. This system is suitable for the separation of 7 generations of dendrimers (generations 0-6). The dynamic coating agent (polyethyleneimine) also improves the separation at acid pH.

Exploitation of the size-exclusion effect of reversed-phase high performance liquid chromatography for the direct analysis of diethylene triamine pentaacetic acid in therapeutic monoclonal antibody formulations.

Monoclonal antibodies (mAb) are being widely studied for the treatment of cancers and other diseases. The mAb is typically in a solution formulation and administered as an intravenous infusion. Ready-to-use solutions are favored for their clinical convenience but they can potentially suffer from a shorter shelf life due to accelerated rates of some forms of degradation such as oxidation, relative to lyophilized formulations. To improve stability, the chelating agent diethylene triamine pentaacetic acid (DTPA) is often used at very low concentrations in biologics formulations to prevent oxidation induced by metal ions. Because of its low concentration and susceptibility to changes in concentration during stability study or processing, the measurement of DTPA levels during formulation and process development is critical. In response to this need we developed a platform reversed-phase HPLC method that allows for the rapid and direct determination of DPTA concentrations which does not require the prior removal of mAbs in formulation samples. The method exploits the "size exclusion effect" of C18 columns with narrow pore sizes (90-120Å) to elute large mAb at the void volume, enabling direct injections of mAb samples for quantitation of DTPA. The method was found to be suitable for the analysis of DTPA in the range of 2-20µg/mL across multiple drug formulations containing different therapeutic mAb and antibody drug conjugates. The method was successfully validated for specificity, precision, accuracy, linearity, and robustness.