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Histone deacetylase 10 (HDAC10) is a zinc-dependent polyamine deacetylase enriched in the cytosol of eukaryotic cells. The active site of HDAC10 contains catalytic residues conserved in other HDAC isozymes that function as lysine deacetylases: Y307 assists the zinc ion in polarizing the substrate carbonyl for nucleophilic attack, and the H136-H137 dyad serves general base-general acid functions. As an inducer of autophagy, HDAC10 is an attractive target for the design of selective inhibitors that may be useful in cancer chemotherapy. Because detailed structural information regarding the catalytic mechanism of HDAC10 may inform new approaches to inhibitor design, we now report X-ray crystal structures of HDAC10 in which reaction intermediates with substrates N 8 -acetylspermidine and N -acetylputrescine are trapped in the active site. The Y307F substitution prevents activation of the substrate carbonyl for nucleophilic attack by the zinc-bound water molecule, thereby enabling crystallographic isolation of intact enzyme-substrate complexes. The H137A substitution removes the catalytically obligatory general acid, thereby enabling crystallographic isolation of oxyanionic tetrahedral intermediates. Finally, the acetate complex with the wild-type enzyme represents a product complex after dissociation of the polyamine coproduct. Taken together, these structures provide snapshots of the reaction coordinate of acetylpolyamine hydrolysis and are consistent with a mechanism in which tandem histidine residues H136 and H137 serve as general base and general acid catalysts, respectively. The function of the histidine dyad in the HDAC10 mechanism appears to be similar to that in HDAC6, but not HDAC8 in which both functions are served by the second histidine of the tandem pair.

A pair of new macrocyclic spermidine alkaloids, (+)-( S )-scocycamide and (-)-( R )-scocycamide, were isolated from the roots of Scopolia tangutica . Their structures were established by extensive spectroscopic data, electronic circular dichroism analyses, and chemical synthesis. They featured a unique 6/18 fused bicyclic framework with spermidine and catechol units, representing a new subtype of natural spermidine alkaloids. A plausible biosynthetic pathway was also proposed. They inhibited butyrylcholinesterase and exhibited antioxidant capacity, suggesting beneficial constituents against Alzheimer's disease and oxidation.

There is growing interest in the characterization of protein complexes and their interactions with ligands using native ion mobility mass spectrometry. A particular challenge, especially for membrane proteins, is preserving noncovalent interactions and maintaining native-like structures. Different approaches have been developed to minimize activation of protein complexes by manipulating charge on protein complexes in solution and the gas-phase. Here, we report the utility of polyamines that have exceptionally high charge-reducing potencies with some molecules requiring 5-fold less than trimethylamine oxide to elicit the same effect. The charge-reducing molecules do not adduct to membrane protein complexes and are also compatible with ion-mobility mass spectrometry, paving the way for improved methods of charge reduction.

Eukaryotic histone deacetylase 10 (HDAC10) is a Zn 2+ -dependent hydrolase that exhibits catalytic specificity for the hydrolysis of the polyamine N 8 -acetylspermidine. The recently determined crystal structure of HDAC10 from Danio rerio (zebrafish) reveals a narrow active site cleft and a negatively charged "gatekeeper" (E274) that favors the binding of the slender cationic substrate. Because HDAC10 expression is upregulated in advanced-stage neuroblastoma and induces autophagy, the selective inhibition of HDAC10 suppresses the autophagic response and renders cancer cells more susceptible to cytotoxic chemotherapeutic drugs. Here, we describe X-ray crystal structures of zebrafish HDAC10 complexed with eight different analogues of N 8 -acetylspermidine. These analogues contain different Zn 2+ -binding groups, such as hydroxamate, thiolate, and the tetrahedral gem-diolate resulting from the addition of a Zn 2+ -bound water molecule to a ketone carbonyl group. Notably, the chemistry that accompanies the binding of ketonic substrate analogues is identical to the chemistry involved in the first step of catalysis, i.e., nucleophilic attack of a Zn 2+ -bound water molecule at the scissile carbonyl group of N 8 -acetylspermidine. The most potent inhibitor studied contains a thiolate Zn 2+ -binding group. These structures reveal interesting geometric changes in the metal coordination polyhedron that accommodate inhibitor binding. Additional interactions in the active site highlight features contributing to substrate specificity. These interactions are likely to contribute to inhibitor binding selectivity and will inform the future design of compounds selective for HDAC10 inhibition.

In this paper, we demonstrate the ability to control and electrochemically monitor nucleic acid conformation by inducing collapse of short, surface-bound nucleotides (7-28 nucleotides). More specifically, we monitored changes in a 5'-electrode-bound DNA structure via changes in the faradaic current related to the reduction/oxidation of a 3'-terminal-appended redox molecule. Reversible DNA collapse was induced by cation condensation achieved by either reducing the dielectric permittivity of the interrogation solution or by the addition of multivalent cations such as the polyamine spermidine (3 + ). Additionally, we find that while the change in electrochemical signal associated with surface bound DNA collapse is dependent on nucleic acid length and surface packing density, the solution conditions (e.g., dielectric permittivity) required for collapse remain constant. As such, we find that collapse of the short DNA strands occurs when the effective charge of the DNA backbone is ∼73-89% neutralized by cations in solution/buffer, according to Manning's theory on cation condensation. This work provides new insight into the structure function relationship of surface-bound nucleic acids and how this is manifested in electrochemical signaling.

The aim of the bioassay-guided fractionation was the selection of the most potent group of compounds responsible for the protection of sunflower bee pollen grains. Synthesis of prospective antifungal polyamides of hydroxycinnamic acids was based on previous structural elucidation of ethanol soluble fraction by 1 H, 1 H-PFG-COSY, 1 H, 13 C-HSQC, FT-IR, FT-Raman, and LC-MS experiments. The main compounds found were tri- p-coumaroylspermidines accompanied by other HCAA of spermidine and putrescine. Several model HCAA derivatives were prepared to test their antifungal activity against widespread spoilage fungi ( A. niger 42 CCM 8189, F. culmorum DMF 0103, and P. verrucosum DMF 0023). A. niger CCM 8189 and F. culmorum DMF 0103 exhibited higher resistance to the antifungal effects of hydroxycinnamic acid amides, whereas P. verrucosum DMF 0023 was the most sensitive strain. It has been discovered the effect of HCAA polarity on the role of secondary metabolites in the microbial protection of pollen grains. The combination of bioassay-guided fractionation, structural elucidation, selection of prospective compounds, and their synthesis to determine their antifungal properties could be considered as an original approach.

The ubiquitous molecule spermidine is known for its pivotal roles in the contact mediation, fusion, and reorganization of biological membranes and DNA. In our model system, borosilicate beads were attached to atomic force microscopy cantilevers and used to probe mica surfaces to study the details of the spermidine-induced attractions. The negative surface charges of both materials were largely constant over the measured pH range of pH 7.8 to 12. The repulsion observed between the surfaces turned into attraction after the addition of spermidine. The attractive force was correlated with the degree of spermidine protonation, which changed from +3 to +1 over the measured pH range. The force was maximal at pH 7.8. To explain the observed pH and spermidine concentration dependence, two different theoretical approaches were used: a chemical model of the charge equilibrium of spermidine and Monte-Carlo simulations of the orientation of the rodlike spermidine molecules in the gap between the borosilicate and mica surfaces. Monte-Carlo simulations of the orientational ordering of the rodlike spermidine molecules suggested the induction of attractive interactions between the surfaces if the gap was bridged by the molecules. For larger gaps, the orientational distribution function of the spermidine molecules predicted a considerable degree of parallel attachment of the molecules to the surfaces, resulting in reduced effective surface charge densities of both surfaces, which reduced their electrostatic repulsion.

We have developed a one-step method to synthesize carbon quantum dots (CQD PAs ) from biogenic polyamines (PAs) as an antibacterial agent for topical treatment of bacterial keratitis (BK). CQDs synthesized by direct pyrolysis of spermidine (Spd) powder through a simple dry heating treatment exhibit a solubility and yield much higher than those from putrescine and spermine. We demonstrate that CQDs obtained from Spds (CQD Spds ) possess effective antibacterial activities against non-multidrug-resistant Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Salmonella enterica serovar Enteritidis bacteria and also against the multidrug-resistant bacteria, methicillin-resistant S. aureus. The minimal inhibitory concentration (MIC) of CQD Spds is ∼2500-fold lower than that of spermidine alone, demonstrating their strong antibacterial capabilities. Investigation of the possible mechanisms behind the antibacterial activities of the as-synthesized CQD Spds indicates that the super-cationic CQD Spds with small size (diameter ca. 6 nm) and highly positive charge (ζ-potential ca. +45 mV) cause severe disruption of the bacterial membrane. In vitro cytotoxicity, hemolysis, hemagglutination, genotoxicity, and oxidative stress and in vivo morphologic and physiologic cornea change evaluations show the good biocompatibility of CQD Spds . Furthermore, topical ocular administration of CQD Spds can induce the opening of the tight junction of corneal epithelial cells, thereby leading to great antibacterial treatment of S. aureus-induced BK in rabbits. Our results suggest that CQD Spds are a promising antibacterial candidate for clinical applications in treating eye-related bacterial infections and even persistent bacteria-induced infections.

Histatin 5 (Hst5) is a naturally occurring antimicrobial peptide that acts as the first line of defense against oral candidiasis. It has been shown that conjugation of the active Hst5 fragment, Hst5 4-15 , and the polyamine spermidine (Spd) improves the candidacidal effect. Knowledge about the structure of these conjugates is, however, very limited. Thus, the aim of this study was to characterize the structural properties of the Hst5 4-15 -Spd conjugates by performing atomistic molecular dynamics simulations in combination with small-angle X-ray scattering. It was shown that the Hst5 4-15 -Spd conjugates adopt extended and slightly rigid random coil conformations without any secondary structure in aqueous solution. It is hypothesized that the increased fungal killing potential of Hst5 4-15 -Spd, in comparison with the Spd-Hst5 4-15 conjugate, is due to the more extended conformations of the former, which cause the bonded Spd molecule to be more accessible for recognition by polyamine transporters in the cell.

N 1 -Acetylspermine oxidase (APAO) catalyzes the conversion of N 1 -acetylspermine or N 1 -acetylspermidine to spermidine or putrescine, respectively, with concomitant formation of N-acetyl-3-aminopropanal and hydrogen peroxide. Here we present the structure of murine APAO in its oxidized holo form and in complex with substrate. The structures provide a basis for understanding molecular details of substrate interaction in vertebrate APAO, highlighting a key role for an asparagine residue in coordinating the N 1 -acetyl group of the substrate. We applied computational methods to the crystal structures to rationalize previous observations with regard to the substrate charge state. The analysis suggests that APAO features an active site ideally suited for binding of charged polyamines. We also reveal the structure of APAO in complex with the irreversible inhibitor MDL72527. In addition to the covalent adduct, a second MDL72527 molecule is bound in the active site. Binding of MDL72527 is accompanied by altered conformations in the APAO backbone. On the basis of structures of APAO, we discuss the potential for development of specific inhibitors.

Liquid-liquid phase separation is responsible for formation of P granules, nucleoli, and other membraneless subcellular organelles composed of RNA and proteins. Efforts to understand the physical basis of liquid organelle formation have thus far focused on intrinsically disordered proteins (IDPs) as major components that dictate occurrence and properties. Here, we show that complex coacervates composed of low complexity RNA (polyuridylic acid, polyU) and short polyamines (spermine and spermidine) share many features of IDP-based coacervates. PolyU/polyamine coacervates compartmentalize biomolecules (peptides, oligonucleotides) in a sequence- and length-dependent manner. These solutes retain mobility within the coacervate droplets, as demonstrated by rapid recovery from photobleaching. Coacervation is reversible with changes in solution temperature due to changes in the polyU structure that impact its interactions with polyamines. We further demonstrate that lipid vesicles assemble at the droplet interface without impeding RNA entry/egress. These vesicles remain intact at the interface and can be released upon temperature-induced droplet dissolution.

The effect of spermidine on the kinetics, conformation, and dynamics of native trypsin was studied by steady-state thermal stability, intrinsic fluorescence, circular dichroism (CD), ultraviolet-visible (UV-vis) spectroscopy, and kinetic techniques, as well as molecular docking, at the temperatures of 298 and 308 K. The Stern-Volmer quenching constants (Ksv) for the trypsin-spermidine complex were obtained at two temperatures, revealing that spermidine quenched the intensity of trypsin through the static mode of the quenching mechanism. The corresponding thermodynamic parameters, Gibbs free-energy, enthalpy, and entropy changes, showed that the binding process was spontaneous. These values and the molecular docking technique revealed that the hydrogen bonding and van der Waals forces played a major role in stabilizing the complex. CD, absorption, and fluorescence results also indicated that spermidine binding had a partial effect on trypsin structure. Spermidine could also influence the activity of trypsin. Upon spermidine binding, the Vmax value of the enzyme was increased and the kcat/Km values were enhanced slightly. The Tm of the trypsin-spermidine complex was enhanced probably due to the higher H-bond formation and lower surface hydrophobicity after spermidine modification, as confirmed by UV-vis spectroscopy and fluorescence spectra. UV absorption and CD studies also indicated that the binding of spermidine to trypsin had induced microenvironmental changes around the enzyme, leading to changes in its secondary structure.

The bitterness in lulo (Solanum quitoense Lam.) fruit is increased during processing (juicing or drying). To identify the bitter-active compounds, the ethanolic fruit pulp extract was subjected to RP-18 solid-phase extraction, and then sensory-guided fractionated by HPLC. Two spermidine derivatives, N(1),N(4),N(8)-tris(dihydrocaffeoyl)spermidine and N(1),N(8)-bis(dihydrocaffeoyl)spermidine, were isolated and their structures confirmed by analysis of their HPLC-ESI/MS and (1)H and (13)C NMR data. The N(1),N(4),N(8)-tris(dihydrocaffeoyl)spermidine was synthesized and used as an authentic sample to unequivocally confirm the structure of this compound and to quantitate it in both fresh and dried fruit. In silico analyses demonstrated that spermidine derivatives identified in lulo pulp exhibited a strong ACE-I (angiotensin I-converting enzyme) inhibitory activity. Subsequently, these results were confirmed by in vitro analyses and showed the potential use of lulo fruit pulp as an ingredient of functional foods related to the prevention of blood hypertension.

A novel synthetic route to the chemoselectively protected N,S-ditritylglutathione monomethyl ester is described involving the chemical modification of the commercially available glutathione (GSH). The synthetic value of this building block in the facile preparation of GSH bioconjugates in a satisfying overall yield was exemplified by the case of trypanothione disulfide (TS2), a GSH-spermidine bioconjugate, involved in the antioxidative stress protection system of parasitic protozoa, such as trypanosoma and leishmania parasites.

The ubiquitous polyamine spermidine is indispensable for eukaryotic growth and cell proliferation. A conserved vital function of spermidine across eukaryotes is conferred by its aminobutyl group that is transferred to a single lysine in translation factor eIF5A to form the essential hypusine post-translational modification required for cellular translation. In direct contrast, although spermidine is absolutely essential for growth of α-proteobacterial plant pathogen Agrobacterium tumefaciens, we have found, by employing a suite of natural polyamines and synthetic methylated spermidine analogues together with spermidine biosynthetic mutants, that it is solely the 1,3-diaminopropane moiety of spermidine that is required for growth. Indeed, any polyamine containing an intact terminal 1,3-diaminopropane moiety can replace spermidine for growth, including the simple diamine 1,3-diaminopropane itself, a paradigm shift in understanding polyamine function in bacteria. We have identified for the first time a spermidine retroconversion activity in bacteria, producing diamine putrescine from triamine spermidine; however, exogenously supplied tetraamine spermine is resistant to retroconversion. When spermidine levels are pharmacologically decreased, synthesis of spermine from spermidine is induced via the same biosynthetic enzymes, carboxyspermidine dehydrogenase and carboxyspermidine decarboxylase that produce spermidine from putrescine, the first identification of a spermine biosynthetic pathway in bacteria. This also suggests that spermidine represses spermine biosynthesis, but when spermidine levels decrease, it is then converted by carboxyspermidine dehydrogenase and decarboxylase enzymes to spermine, which is resistant to retroconversion and constitutes a sequestered pool of protected 1,3-diaminopropane modules required for growth. We also identify an efficient N-acetylspermidine deacetylase activity, indicative of a sophisticated bacterial polyamine homeostasis system.

Kukoamines are a series of bioactive phytochemicals conjugated by a polyamine backbone and phenolic moieties. Understanding the structural diversity of kukoamine metabolites in plants is meaningful for drug discovery. In this study, an LC-MS/MS method was established for kukoamine profiling and characterization from lycii cortex (LyC) via a triple-quadrupole linear ion trap mass spectrometry (Q-TRAP). On the basis of the typical fragmentation of kukoamine, a diagnostic ion, which represents the features of the backbone and phenolic substitute, was chosen as the product ion for precursor ion scan, and then the screened precursor ions were applied to a successive multiple ion monitoring triggered enhanced product ion scan (MIM-EPI) to simultaneously present the profile survey and MS/MS acquisition. Because the MIM narrowed the ion scan range in Q1 and the ion trap enhanced the ion fragments passing through Q2, the qualitative capability of quadrupole MS can be greatly improved, especially for capture of the uncommon metabolites. There are 12 kukoamine metabolites identified from LyC, with either spermine or spermidine backbone and with conjugation of one to three dihydrocaffeoyls or other kinds of phenolic moieties. Except for kukoamines A and B, other metabolites were identified in LyC for the first time. This approach can be utilized for metabolite identification in other substrates.

The emergence of bacterial resistance and biofilm associated infections has created a challenging situation in global health. In this present state of affairs where conventional antibiotics are falling short of being able to provide a solution to these problems, development of novel antibacterial compounds possessing the twin prowess of antibacterial and antibiofilm efficacy is imperative. Herein, we report a library of amino acid tunable lipidated norspermidine conjugates that were prepared by conjugating both amino acids and fatty acids with the amine functionalities of norspermidine through amide bond formation. These lipidated conjugates displayed potent antibacterial activity against various planktonic Gram-positive and Gram-negative bacteria including drug-resistant superbugs such as methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus faecium, and β-lactam-resistant Klebsiella pneumoniae. This class of nontoxic and fast-acting antibacterial molecules (capable of killing bacteria within 15 min) did not allow bacteria to develop resistance against them after several passages. Most importantly, an optimized compound in the series was also capable of killing metabolically inactive persisters and stationary phase bacteria. Additionally, this compound was capable of disrupting the preformed biofilms of S. aureus and E. coli. Therefore, this class of antibacterial conjugates have potential in tackling the challenging situation posed by both bacterial resistance as well as drug tolerance due to biofilm formation.

We present a general coarse-grained model of sodium, magnesium, spermidine, and chlorine in implicit solvent. The effective potentials between ions are systematically parametrized using a relative entropy coarse-graining approach [Carmichael, S. P. and M. S. Shell, J. Phys. Chem. B, 116, 8383-93 (2012)] that maximizes the information retained in a coarse-grained model. We describe the local distribution of ions in the vicinity of a recently published coarse-grained DNA model and demonstrate a dependence of persistence length on ionic strength that differs from that predicted by Odijk-Skolnick-Fixman theory. Consistent with experimental observations, we show that spermidine induces DNA condensation whereas magnesium and sodium do not. This model can be used alongside any coarse-grained DNA model that has explicit charges and an accurate reproduction of the excluded volume of dsDNA.

A strategy is introduced for enhancing the cellular selectivity of Amphotericin B (AmB) and other classes of membrane-disrupting agents. This strategy involves attaching the agent to a molecular umbrella to minimize the disruptive power of aggregated forms. Based on this approach, AmB has been coupled to a molecular umbrella derived from one spermidine and two cholic acid molecules and found to have antifungal activities approaching that of the native drug. However, in sharp contrast to AmB, the hemolytic activity and the cytotoxcity of this conjugate toward HEK293 T cells have been dramatically reduced.

Natural and synthetic membrane active antibacterial agents offer hope as potential solutions to the problem of bacterial resistance as the membrane-active nature imparts low propensity for the development of resistance. In this report norspermidine based antibacterial molecules were developed that displayed excellent antibacterial activity against various wild-type bacteria (Gram-positive and Gram-negative) and drug-resistant bacteria (methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus faecium, and β-lactam-resistant Klebsiella pneumoniae). In a novel structure-activity relationship study it has been shown how incorporation of an aromatic amino acid drastically improves selective antibacterial activity. Additionally, the effect of stereochemistry on activity, toxicity, and plasma stability has also been studied. These rapidly bactericidal, membrane active antibacterial compounds do not trigger development of resistance in bacteria and hence bear immense potential as therapeutic agents to tackle multidrug resistant bacterial infections.

Biosensors, in which binding of ligands is detected through changes in the optical or electrochemical properties of a DNA layer confined to the sensor surface, are important tools for investigating DNA interactions. Here, we investigate if conformational changes induced in surface-attached DNA molecules upon ligand binding can be monitored by the quartz crystal microbalance with dissipation (QCM-D) technique. DNA duplexes containing 59-184 base pairs were formed on QCM-D crystals by stepwise assembly of synthetic oligonucleotides of designed base sequences. The DNA films were exposed to the cationic polyamines spermidine and spermine, known to condense DNA molecules in bulk experiments, or to the recombination protein Rad51, known to extend the DNA helix. The binding and dissociation of the ligands to the DNA films were monitored in real time by measurements of the shifts in resonance frequency (Δf) and in dissipation (ΔD). The QCM-D data were analyzed using a Voigt-based model for the viscoelastic properties of polymer films in order to evaluate how the ligands affect thickness and shear viscosity of the DNA layer. Binding of spermine shrinks all DNA layers and increases their viscosity in a reversible fashion, and so does spermidine, but to a smaller extent, in agreement with its lower positive charge. SPR was used to measure the amount of bound polyamines, and when combined with QCM-D, the data indicate that the layer condensation leads to a small release of water from the highly hydrated DNA films. The binding of Rad51 increases the effective layer thickness of a 59 bp film, more than expected from the know 50% DNA helix extension. The combined results provide guidelines for a QCM-D biosensor based on ligand-induced structural changes in DNA films. The QCM-D approach provides high discrimination between ligands affecting the thickness and the structural properties of the DNA layer differently. The reversibility of the film deformation allows comparative studies of two or more analytes using the same DNA layer as demonstrated here by spermine and spermidine.

RNA targeting is an evolving new approach to anticancer therapeutics that requires identification of small molecules to selectively target specific RNA structures. In this report, the interaction of biogenic polyamines spermine, spermidine and the synthetic analogue 1-naphthylacetyl spermine with three double-stranded RNA polynucleotides--poly(I)·poly(C), poly(C)·poly(G), and poly(A)·poly(U)--has been described to understand the structural and thermodynamic basis of the binding and the comparative efficacy of the analogue over the natural polyamines. Circular dichroism spectroscopy, thermal melting experiments, and ethidium bromide displacement assay were used to characterize the interaction. Microcalorimetry studies were performed to deduce the energetics of the interaction and atomic force microscopy experiments done to gain insight into the interaction at the molecular level. The experiments demonstrated structural perturbations in the polynucleotides on binding of the polyamines. Thermal melting studies showed enhanced stabilization of RNA-polyamine complexes with increase in the total standard molar enthalpy of transition. The binding affinity was strongest for poly(I)·poly(C) as revealed by microcalorimetry results and varied as poly(I)·poly(C) > poly(C)·poly(G) > poly(A)·poly(U). The order of affinity for the polyamines was spermine >1-naphthylacetyl spermine > spermidine. Total enthalpy-entropy compensation and high standard molar heat capacity values characterized the interactions. The results of the study on the binding of polyamines to dsRNAs presented here have been compared to those reported earlier with dsDNAs. The present findings advance our knowledge on the mechanism of interaction of polyamines with RNA and may help in the search for analogues that can interfere with biogenic polyamine metabolism and function.

The intrinsic ability of albumin to bind active substances in the physiological fluids has been explored to endow hydrogels with improved capability to regulate drug release. To develop such biomimetic-functional hydrogels, it is critical that albumin conformation is not altered and that the protein remains retained inside the hydrogel keeping its conformational freedom, i.e., it should be not chemically cross-linked. Thus, the hydrogels were prepared with various proportions of albumin by physical cross-linking of anionic polysaccharides (gellan gum and chondroitin sulfate) with the cationic endogen polyamine spermidine under mild conditions in order to prevent albumin denaturation. Texture and swelling properties of hydrogels with various compositions were recorded, and the effect of the preparation variables was evaluated applying neurofuzzy logic tools for hydrogels prepared with and without albumin and associating the antibiotic cloxacillin. Developed hydrogel systems were extensively analyzed by means of nuclear magnetic resonance (NMR) to determine weak-to-medium and strong binding modes and the equilibrium constants of the albumin-cloxacillin association. NMR techniques were also employed to demonstrate the successful modulation of the cloxacillin release from the albumin-containing hydrogels. In vitro microbiological tests carried out with Staphylococcus aureus and Staphylococcus epidermidis confirmed the interest of the albumin-containing hydrogels as efficient platforms for cloxacillin release in its bioactive form.

We investigated the collective conformational response of DNA polymer brushes to condensation induced by the trivalent cation spermidine. DNA brushes, a few kilobase-pairs long, undergo a striking transition into macroscopic domains of collapsed chains with fractal dendritic morphology. Condensation is initiated by focal nucleation of a towerlike bundle, which laterally expands in a chain-reaction cascade of structural chain-to-chain collapse onto the surface. The transition exhibits the hallmarks of a first-order phase transition with grain boundaries, hysteresis, and coexistence between condensed and uncondensed phases. We found that an extended DNA conformation is maintained throughout the transition and is a prerequisite for the formation of large-scale dendritic domains. We identified a critical DNA density above which the nucleation propensity and growth rate sharply increase. We hypothesize that the ability of DNA-scaffolding proteins to modify the local DNA density within a genome may act as a dynamic and sensitive mechanism for spatial regulation of DNA transactions in vivo by selective condensation of chromosomal territories. By assembling a DNA brush along a patterned line narrower than twice the DNA contour length and tuning the local surface densities, we were able to initiate nucleation at a predefined location and induced growth of a single condensed nanowire over a distance 2 orders of magnitude longer than the single-chain contour. Our results demonstrate spatial control of condensation as a new tool for constructing DNA-based synthetic systems with important implications for regulation of DNA transactions on surfaces.

LC-UV-MS analyses of inflorescence extracts of Sambucus nigra L. (elder, Adoxaceae) revealed the presence of numerous acyl spermidines, with isomers of N,N-diferuloylspermidine and N-acetyl-N,N-diferuloylspermidine being most abundant. Pollen was the main source of the acyl spermidines in the inflorescence. Three of the major acyl spermidines were isolated and their structures determined by NMR spectroscopy as N⁵,N¹⁰-di-(E,E)-feruloylspermidine and the new compounds N¹-acetyl-N⁵,N¹⁰-di-(Z,E)-feruloylspermidine and N¹-acetyl-N⁵,N¹⁰-di-(E,E)-feruloylspermidine. An isomer of N,N,N-triferuloylspermidine was also obtained and identified as N¹,N⁵,N¹⁰-tri-(E,E,E)-feruloylspermidine. In addition to stereoisomers of the isolated acyl spermidines, other acyl spermidines detected by the positive ion LC-UV-MS were isomers of N-caffeoyl-N,N-diferuloylspermidine, N-coumaroyl-N,N-diferuloylspermidine, N-caffeoyl-N-feruloylspermidine, N-coumaroyl-N-feruloylspermidine, N-acetyl-N-caffeoyl-N-feruloylspermidine, and N-acetyl-N-coumaroyl-N-feruloylspermidine. Analysis of commercial elderflower drinks showed that acyl spermidines were persistent in these processed elderflower products. Examination of inflorescence extracts from Sambucus canadensis L. (American elder) revealed the presence of acyl spermidines that were different from those of S. nigra.

The cellular polyamines spermine, spermidine, and their metabolic precursor putrescine, have long been associated with cell-growth, tumor-related gene regulations, and Alzheimer's disease. Here, we show by in vitro spectroscopy and AFM imaging, that these molecules promote aggregation of amyloid-beta (Aβ) peptides into fibrils and modulate the aggregation pathways. NMR measurements showed that the three polyamines share a similar binding mode to monomeric Aβ(1-40) peptide. Kinetic ThT studies showed that already very low polyamine concentrations promote amyloid formation: addition of 10 μM spermine (normal intracellular concentration is ~1 mM) significantly decreased the lag and transition times of the aggregation process. Spermidine and putrescine additions yielded similar but weaker effects. CD measurements demonstrated that the three polyamines induce different aggregation pathways, involving different forms of induced secondary structure. This is supported by AFM images showing that the three polyamines induce Aβ(1-40) aggregates with different morphologies. The results reinforce the notion that designing suitable ligands which modulate the aggregation of Aβ peptides toward minimally toxic pathways may be a possible therapeutic strategy for Alzheimer's disease.

In this study, stable supramolecular hydrogels have been obtained from the assembly of graphene oxide (GO) in presence of polyamines including tris(aminoethyl)amine, spermine, and spermidine [biologically active molecule]. One of these hydrogels has been well characterized by various techniques including field-emission scanning electron microscopy (FE-SEM), transmission electron microscopy (TEM), atomic force microscopy (AFM), X-ray diffraction (XRD) study, and Raman spectroscopy. TEM and AFM studies of one of these hydrogels have revealed the presence of a network structure of cross-linked nanosheets. This suggests the supramolecular assembly of GO in the presence of polyamines using the acid-base type electrostatic interaction. In presence of a mild reducing agent (vitamin C), one of these GO hydrogels has been transformed into a reduced graphene oxide (RGO)-based hydrogel by a simple in situ reduction of GO sheets within the hydrogel matrix. Moreover, noble metal nanoparticle containing RGO based hybrid hydrogels have been obtained using in situ and simultaneous co-reduction of GO and noble metal precursors within the GO gel matrix. The elegance of this method is in situ, "green chemical" and simultaneous reduction of GO and metal salts within the hydrogel matrix to form RGO-based hybrid gel and concomitant stabilization of metal nanoparticles (MNPs) within the gel system. The nascently formed MNPs are homogeneously and uniformly distributed on the surface of the RGO nanosheets within the hybrid gel. Interestingly, this MNP containing RGO-based hybrid hydrogel matrix acts as a potential catalyst for the reduction of aromatic nitro to amino group. The catalyst (hybrid gel matrix) can be separated easily after the reaction and reused several times.

Acrolein (Acr) is a major component in cigarette smoke and a ubiquitous environmental pollutant. It is also formed as a product of lipid peroxidation. Following ring closure via the Michael addition, Acr modifies deoxyguanosine (dG) in DNA by forming cyclic 1,N(2)-propanodeoxyguanosine adducts (OHPdG). The reactions of Acr with dG yield, depending on the direction of ring closure, two regioisomers, α- and γ-OHPdG, in approximately equal amounts. However, previous (32)P-postlabeling studies showed that the γ isomers were detected predominantly in the DNA of rodent and human tissues. Because of the potential differential biological activity of the isomeric OHPdG adducts, it is important to confirm and study the chemical basis of the regioselective formation of γ isomers in vivo. In this study, it is confirmed that γ-OHPdG adducts are indeed the major isomers formed in vivo as evidenced by a LC-MS/MS method specifically developed for Acr-derived dG adducts. Furthermore, we have shown that the formation of γ-isomers is increased in the presence of amino-containing compounds, including amino acids, proteins, and cell lysates. A product of Acr and arginine that appears to mediate the regioselective formation of γ isomers was identified, but its structure was not fully characterized due to its instability. This study demonstrates that intracellular amino-containing compounds may influence the regiochemistry of the formation of OHPdG adducts and reveals a mechanism for the preferential formation of γ-OHPdG by Acr in vivo.

Cluster expansion techniques are used to obtain microconstants and microenthalpies of protonation reactions. The approach relies on the analysis of macroscopic protonation constants and protonation enthalpies within a homologous series. Various linear aliphatic polyamines are considered, including 3,4-tri (spermidine), 3,4,3-tet (spermine), and 2,2,2,2-pent. Besides the full resolution of the microscopic protonation equilibria, one obtains information on the temperature dependence of the microstate probabilities. We find that the concentrations of the dominant microspecies increase with increasing temperature. Due to the large negative protonation enthalpies that are typical for amines, higher temperatures generally favor the less protonated species.

Furan is toxic and carcinogenic in rodents. Because of the large potential for human exposure, furan is classified as a possible human carcinogen. The detailed mechanism by which furan causes toxicity and cancer is not yet known. Since furan toxicity requires cytochrome P450-catalyzed oxidation of furan, we have characterized the urinary and hepatocyte metabolites of furan to gain insight into the chemical nature of the reactive intermediate. Previous studies in hepatocytes indicated that furan is oxidized to the reactive α,β-unsaturated dialdehyde, cis-2-butene-1,4-dial (BDA), which reacts with glutathione (GSH) to form 2-(S-glutathionyl)succinaldehyde (GSH-BDA). This intermediate forms pyrrole cross-links with cellular amines such as lysine and glutamine. In this article, we demonstrate that GSH-BDA also forms cross-links with ornithine, putrescine, and spermidine when furan is incubated with rat hepatocytes. The relative levels of these metabolites are not completely explained by hepatocellular levels of the amines or by their reactivity with GSH-BDA. Mercapturic acid derivatives of the spermidine cross-links were detected in the urine of furan-treated rats, which indicates that this metabolic pathway occurs in vivo. Their detection in furan-treated hepatocytes and in urine from furan-treated rats indicates that polyamines may play an important role in the toxicity of furan.

The polyamines are organic polycations present at millimolar concentrations in eukaryotic cells where they participate in the regulation of vital cellular functions including proliferation and differentiation. Biological evaluation of rationally designed polyamine analogs is one of the cornerstones of polyamine research. Here we have synthesized and characterized novel C-methylated spermidine analogs, that is, 2-methylspermidine, 3-methylspermidine, and 8-methylspermidine. 3-Methylspermidine was found to be metabolically stable in DU145 cells, while 8-methylspermidine was a substrate for spermidine/spermine N(1)-acetyltransferase (SSAT) and 2-methylspermidine was a substrate for both SSAT and acetylpolyamine oxidase. All the analogs induced the splicing of the productive mRNA splice variant of SSAT, overcame growth arrest induced by 72-h treatment with ornithine decarboxylase (ODC) inhibitor α-difluoromethylornithine, and were transported via the polyamine transporter. Surprisingly, 2-methylspermidine was a weak downregulator of ODC activity in DU145 cells. Our data demonstrates that it is possible to radically alter the biochemical properties of a polyamine analog by changing the position of the methyl group.

The reentrant condensation of 21-bp oligonucleotide in the presence of spermidine was investigated by laser light scattering and capillary electrophoresis. 21-bp oligonucleotide showed a bimodal distribution in 1 × TE buffer, with the slow mode being the characteristic diffusion of polyelectrolyte in solution without enough salt. At the fixed spermidine concentration, the reentry of oligonucleotide underwent aggregation, phase separation, and disassociation in sequence with time, and the kinetics was extremely slow. For example, it took more than 1200 h (50 days) for the reentry to complete at 21 mM spermidine. Higher spermidine concentration led to faster kinetics. After reentry, the slow mode disappeared, and the charges of oligonucleotide were at least partially neutralized. No prominent charge inversion was observed. The kinetics of oligonucleotide reentry in the presence of spermidine gained insight in the interactions of polyelectrolyte in aqueous solution.

High-resolution melting (HRM) analysis exploits the reduced thermal stability of DNA fragments that contain base mismatches to detect single nucleotide polymorphisms (SNPs). However, the capacity of HRM to reveal other features of DNA chemistry remains unexplored. DNA methylation plays a key role in regulating gene expression and is essential for normal development in many higher organisms. The presence of methylated bases perturbs the double-stranded DNA structure, although its effect on DNA thermal stability is largely unknown. Here, we reveal that methylated DNA has enhanced thermal stability and is sufficiently divergent from nonmethylated DNA to allow detection and quantification by HRM analysis. This approach reliably distinguishes between sequence-identical DNA differing only in the methylation of one base. The method also provides accurate discrimination between mixes of methylated and nonmethylated DNAs, allowing discrimination between DNA that is 1% and 0% methylated and also between 97.5% and 100% methylated. Thus, the method provides a new means of adjusting thermal optima for DNA hybridization and PCR-based techniques and to empirically measure the impact of DNA methylation marks on the thermostability of regulatory regions. In the longer term, it could enable the development of new techniques to quantify methylated DNA.

We describe here the syntheses and the biological properties of new alkylaminooxysterols. Compounds were synthesized through the trans-diaxial aminolysis of 5,6-alpha-epoxysterols with various natural amines including histamine, putrescine, spermidine, or spermine. The regioselective synthesis of these 16 new 5alpha-hydroxyl-6beta-aminoalkylsterols is presented. Compounds were first screened for dendrite outgrowth and cytotoxicity in vitro, and two leads were selected and further characterized. 5alpha-Hydroxy-6beta-[2-(1H-imidazol-4-yl)ethylamino]cholestan-3beta-ol, called dendrogenin A, induced growth control, differentiation, and the death of tumor cell lines representative of various cancers including metastatic melanoma and breast cancer. 5alpha-Hydroxy-6beta-[3-(4-aminobutylamino)propylamino]cholest-7-en-3beta-ol, called dendrogenin B, induced neurite outgrowth on various cell lines, neuronal differentiation in pluripotent cells, and survival of normal neurones at nanomolar concentrations. In summary, we report that two new alkylaminooxysterols, dendrogenin A and dendrogenin B, are the first members of a class of compounds that induce cell differentiation at nanomolar concentrations and represent promising new leads for the treatment of cancer or neurodegenerative diseases.

Molecular umbrella provided a promising avenue for the design of the intracellular delivery of hydrophilic therapeutic agents. However, the limited understanding of its cellular uptake would be a roadblock to its effective application. Herein, we investigate the ability and mechanism of cellular entry of a fluorescently labeled diwalled molecular umbrella, which was synthesized from cholic acid, spermine, and 5-carboxyfluorescein, into Hela cells, with the extent of uptake analyzed by confocal fluorescence microscopy and flow cytometry. It is found that the as-synthesized diwalled molecular umbrella can greatly facilitate cellular uptake of hydrophilic agent, 5-carboxyfluorescein. In vitro experiments with diffuse marker, endocytic marker, and inhibitors suggested that several distinct uptake pathways (e.g., passive diffuse, clathrin-mediated endocytosis, and caveolae/lipid-raft-dependent endocytosis) are involved in the internalization of diwalled molecular umbrella. These results, together with its low toxicity and good biocompatibility, thus demonstrate the suitability of molecular umbrella for application as vectors in drug delivery systems.

The Kir2.1 potassium channel owes its inward-rectifying behavior to blocking by multivalent ions, e.g., magnesium and spermine, which access the channel from the cytoplasm and are thought to bind within the pore. To investigate the pathway followed by these ions from the cytoplasm through the pore, we have used multiscale modeling (via continuum electrostatics calculations, docking, and molecular dynamics simulations) to identify possible binding sites en route. On its way to eventually binding in the cavity, magnesium interacts extensively with Glu299, which lines the pore in the center of the intracellular domain. Interaction sites for spermine are formed by Asp255, Glu299, and Glu224. Entropic factors seem to favor interactions of spermine within the center of the cytoplasmic domain.

The enzyme spermidine/spermine N (1)-acetyltransferase (SSAT) catalyzes the transfer of acetyl groups from acetylcoenzyme A to spermidine and spermine, as part of a polyamine degradation pathway. This work describes the crystal structure of SSAT in complex with coenzyme A, with and without bound spermine. The complex with spermine provides a direct view of substrate binding by an SSAT and demonstrates structural plasticity near the active site of the enzyme. Associated water molecules bridge several of the intermolecular contacts between spermine and the enzyme and form a "proton wire" between the side chain of Glu92 and the N1 amine of spermine. A single water molecule can also be seen forming hydrogen bonds with the side chains of Glu92, Asp93, and the N4 amine of spermine. Site-directed mutation of Glu92 to glutamine had a detrimental effect on both substrate binding and catalysis and shifted the optimal pH for enzyme activity further into alkaline solution conditions, while mutation of Asp93 to asparagine affected both substrate binding and catalysis without changing the pH dependence of the enzyme. Considered together, the structural and kinetic data suggest that Glu92 functions as a catalytic base to drive an otherwise unfavorable deprotonation step at physiological pH.

DNA condensates were formed by treating linear pUC19 plasmids ligated to an AQ-containing oligomer with spermidine. The condensates are toroid-shaped objects having a radius of 70 to 100 nm. Irradiation of the condensates with UV light (absorbed by the anthraquinone) causes the one-electron oxidation of the DNA and concomitant reaction at GG steps of the oligomer. Analysis of the distance dependence of the reaction at guanine in these condensates reveals a dependency on the position of the AQ. This observation is attributed to a reduction in the rate for trapping of the radical cation in the relatively dehydrated interior of the condensate.

A new spermidine triamide derivative has been isolated from peanut flowers and identified as N (1)-acetyl- N (5), N (10)-di- p-( EE)-coumaroylspermidine on the basis of detailed analysis of NMR, MS, and UV data. Two other spermidine conjugates, N (1), N (5), N (10)-tri- p-( EEE)-coumaroylspermidine and di- p-( EE)-coumaroylspermidine, as well as four flavonoid conjugates (quercetin-3-glucoside, quercetin-3-glucuronide, isorhamnetin-3-glucoside, and isorhamnetin-3-glucuronide) that have been previously reported in organs of other plants, have been found in this study in peanut ( Arachis hypogaea L.), a representative of the Leguminosae family, for the first time. The dynamics of photoisomerization in the spermidine conjugates have been investigated.

The chemistry and biology of acetyl-protected spermidine-bridged dinuclear platinum complexes [{ trans-PtCl(NH 3) 2] 2-mu-NH 2(CH 2) 3N(COR)(CH 2) 4NH 2]X 2 (R = H, X = Cl (1,1/t,t-spermidine, BBR3571); R = CH 3 , X = Cl ( 2); R = CH 2 Cl, X = ClO 4 ( 3); R = CF 3 , X = Cl ( 4)) are compared with their carbamate analogues. The compounds are potential prodrugs for the parent compound 1, a highly potent antitumor agent. At pH 6-8 hydrolysis of the blocking group with the release of the "parent" protonated species follows the order 4 > 3 >> 2. For 4, rate constants for the deprotection increase in this pH range. The DNA binding profile of 4 is similar to the Boc derivative, confirming the central influence of charge on DNA binding properties. The differences in cytotoxicity for the protected compounds in ovarian carcinoma cell lines sensitive and resistant to cisplatin cannot completely be explained by spontaneous release of 1,1/t,t-spermidine at physiological pH. Inherent cytotoxicity and cell line specificity may contribute to the observed behavior. The properties of the compounds present them also as possible "second-generation" analogues of the clinically relevant trinuclear complex [{ trans-PtCl(NH 3) 2} 2-mu- trans-Pt(NH 3) 2(NH 2(CH 2) 6NH 2) 2](NO 3) 4, ( 8, BBR3464).

The synthesis of bifunctional dinuclear platinum complexes, [{PtCl(dach)}(2)-mu-Y](n+)Cl(n) (1-3; Y = H(2)N(CH(2))(3)NH(2)(CH(2))(4)NH(2), H(2)N(CH(2))(6)NH(2)(CH(2))(6)NH(2), and H(2)N(CH(2))(6)NH(2)(CH(2))(2)NH(2)(CH(2))(6)NH(2), respectively; Figure 1) is reported. There was no labilization of the polyamine linker groups of the cis-1,2-diaminocyclohexane complexes in the presence of sulfur-containing species at physiological pH, in contrast to previous studies preformed on trans complexes. Metabolism reactions are somewhat dependent on the nature of the polyamine: at physiological pH, the spermidine complex 1 forms an inert (tetraamine)platinum species in which one platinum is chelated by a central and terminal amino group. The stability of cis-geometry complexes may make them viable second-generation polynuclear platinum clinical candidates.

Trypanothione reductase (TR) catalyzes the NADPH-dependent reduction of trypanothione disulfide (1). TR plays a central role in the trypanosomatid parasite's defense against oxidative stress and has emerged as a promising target for antitrypanosomal drugs. We describe the synthesis and activity of dethiotrypanothione and analogues (2-4) as inhibitors of Trypanosoma cruzi TR. The syntheses of these macrocycles feature ring-closing olefin metathesis (RCM) reactions catalyzed by ruthenium catalyst 17. Derivative 4 is our most potent inhibitor with a Ki=16 microM.

Naturally occurring polyamines putrescine, cadaverine, spermidine, and spermine are analogues of the species-specific long-chain polyamines found in diatoms. Scanning electron microscopy and energy-dispersive spectroscopy show that the reactions of a soluble Ti(IV) precursor with spermidine and spermine, but not putrescine or cadaverine, produce nanostructured irregular polyhedra of titanium oxide. At 25 degrees C, the average size of the particles formed with spermidine is 400 +/- 150 nm, and with spermine, 140 +/- 50 nm. Although the particles are X-ray amorphous at room temperature, annealing studies reveal that the particles adopt crystallinity at higher temperatures characteristic of anatase (TiO2). The major portion of the biopolyamines is not coprecipitated with the solid but is left in solution. Kinetic measurements reveal an initial fast step followed by two slower phases of reaction. At 25 degrees C, k(1obs) and k(2obs) for the reaction with spermidine are 5 x 10(-3) s(-1) and 3.6 x 10(-4) s(-1), respectively, and for spermine, 4.8 x 10(-3) s(-1) and 4.2 x 10(-4) s(-1), respectively. Taken together, the data suggest spermidine and spermine are biocatalysts for the precipitation of nanostructured titanium oxide.

The biocoordination chemistry of antimony and bismuth has been extensively investigated due to the historical use of these metals in medicine. Structures of bismuth antiulcer agents and interactions of Bi3+ with proteins and enzymes, such as transferrin and lactoferrin, the histidine-rich protein Hpn, and urease, have been characterized. Sb5+ is a prodrug and is bioreduced or activated to its active form Sb3+ intracellularly. Antimony binds to biomolecules, such as glutathione, trypanothione, and nucleotides, and forms binary and ternary complexes, which may allow it to be trafficked in cells. These studies have improved our understanding of the mechanism of action of bismuth and antimony drugs, which in turn allows the future design of drugs.

A complete analysis of the transverse acoustic modes (TAMs) for the homologous series of alpha,omega-diamines (H2N(CH2)nNH2) (n = 2-10, n = 12) as well as for the biogenic polyamines spermidine and spermine was undertaken, by Raman and inelastic neutron scattering (INS) spectroscopies combined with density functional theory (DFT) calculations. A complete assignment of the whole set of TAMs was carried out, for both the undeuterated and N-deuterated species. 1,2-Diaminoethane was found to display exceptional behavior, probably due to the formation of dimers in the solid state. An n-even/n-odd dependence of the low frequency INS pattern was observed for these polyamines. The very good accordance between their INS experimental TAMs and the ones previously reported for the corresponding n-alkanes suggest a close conformational similarity between these systems.

Nitric oxide (NO) and hyaluronic acid (HA), two species widely different in terms of molecular complexity and biological competence, are both known to play an important role in the wound healing process. To combine the properties of HA and NO, we synthesized new NO-donors based on hyaluronic acid derivatives exhibiting a controlled NO-release under physiological conditions (in vitro tests). Since two molecules of NO can form a covalent bond with secondary amines to yield structures, named NONO-ates, able to release NO in solution, we used spermidine bound to HA as the NO-linker. The HA-spermidine derivative was obtained by controlled HA amidation in aqueous media, activating the biopolymer carboxylate groups with a water soluble carbodiimide. The resulting derivative, soluble in water, was fully characterized by high field 1H and 13C NMR spectroscopy. The amount of grafting of spermidine on HA was determined by integration of suitable 1H NMR signals. In addition, cross-linked derivatives of HA were synthesized by the Ugi's four-component reaction using formaldehyde, cyclohexylisocyanide, and spermidine. The HA-spermidine networks were characterized by 13C CP-MAS NMR spectroscopy. The degree of cross-linking of the networks was also determined. Finally, the release of NO from the swollen hydrogels freshly saturated with NO, in contact with aqueous media, was monitored by means of UV spectrophotometric measurements.

Efficient syntheses of metabolically stable alpha-methylspermidine 1, alpha-methylspermine 2, and bis-alpha,alpha'-methylated spermine 3 starting from ethyl 3-aminobutyrate are described. The biological tolerance for these compounds was tested in wild-type mice and transgenic mice carrying the metallothionein promoter-driven spermidine/spermine N(1)-acetyltransferase gene (MT-SSAT). The efficient substitution of natural polyamines by their derivatives was confirmed in vivo with the rats harboring the same MT-SSAT transgene and in vitro with the immortalized fibroblasts derived from these animals. Enantiomers of previously unknown 1-amino-8-acetamido-5-azanonane dihydrochloride 4 were synthesized starting from enantiomerically pure (R)- and (S)-alaninols. The studies with recombinant human polyamine oxidase (PAO) showed that PAO (usually splits achiral substrates) strongly favors the (R)-isomer of 4 that demonstrates for the first time that the enzyme has hidden potency for stereospecificity.

A series of molecular umbrella conjugates, derived from cholic acid, deoxycholic acid, spermidine, lysine, and 5-mercapto-2-nitrobenzoic acid, have been synthesized and found capable of transporting an attached 16-mer oligonucleotide (S-dT16) across liposomal membranes made from 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidyldglycerol (POPG), and cholesterol [POPC/POPG/cholesterol (65/5/30; mol/mol/mol, v/v/v)] at 37 degrees C. Those molecular umbrellas containing four choloyl (or deoxycholoyl) groups resulted in significantly faster rates of transport as compared to those containing only two such moieties. A model that accounts for these membrane transport processes is proposed.

The utility of polyamines as vectors for the intracellular transport of iron chelators is further described. Consistent with earlier results with polyamine analogues, these studies underscore the importance of charge in the design of polyamine-vectored chelators. Four polyamine conjugates are synthesized, two of terephthalic acid [N(1)-(4-carboxy)benzoylspermine (7) and its methyl ester (6)] and two of (S)-2-(2,4-dihydroxyphenyl)-4,5-dihydro-4-methyl-4-thiazolecarboxylic acid [(S)-4'-(HO)-DADFT] [(S)-4,5-dihydro-2-[2-hydroxy-4-(12-amino-5,9-diazadodecyl-oxy)phenyl]-4-methyl-4-thiazolecarboxylic acid (10) and its ethyl ester (9)]. These four molecules were evaluated in murine leukemia L1210 cells for their impact on cell proliferation (48- and 96-h IC(50) values), their ability to compete with spermidine for the polyamine transport apparatus (K(i)), and their intracellular accumulation. The data revealed that when neutral molecules (cargo fragments) were fixed to the polyamine vector, the conjugates competed well with spermidine for transport and were accumulated intracellularly to millimolar levels. However, this was not the case when the cargo fragments were negatively charged. Metabolic studies of the polyamine-vectored (S)-4'-(HO)-DADFTs in rodents indicated that not only did the expected deaminopropylation step occur, but also a surprisingly high level of oxidative deamination at the terminal primary nitrogens took place. Finally, the iron-clearing efficiency of the (S)-4'-(HO)-DADFT conjugates was determined in a bile-duct-cannulated rodent model. Attaching the ligand to a polyamine vector had a profound effect on increasing the iron-clearing efficiency of this chelator relative to its parent drug.

Dihydromotuporamine C (4) and its 4,4-triamine analogue (5) were synthesized in good yield using ring-closing metathesis (RCM) methods. Comparison of their biological activities (Ki determinations in L1210 cells and IC50 determinations in L1210, CHO, and CHO-MG cells) revealed that the motuporamine derivatives do not use the polyamine transporter (PAT) for cellular entry. Bioevaluation of a N1-(anthracen-9-ylmethyl)-N1-(ethyl)homospermidine control (7) revealed that the presence of a N1 tertiary amine center imparted a significant reduction in the PAT affinity of the polyamine conjugate and abolished its PAT-targeting selectivity.