An efficient and versatile synthesis of bisPNA-peptide conjugates based on chemoselective oxime formation

Oligomers with two identical peptide nucleic acid sequences joined by a flexible hairpin linker (bisPNA) can stably bind to specific DNA sequences without altering plasmid supercoiling, thus offering a unique opportunity to attach various functional entities to high molecular weight DNA. Current synthetic approaches, however, severely limit the possibility to link peptides or other chemical moieties (i.e., sugars, oligonucleotides, etc.) to bisPNA. Here we report a novel strategy for the synthesis of bisPNA-peptide conjugates in which chemoselective ligation of bisPNA to peptides was accomplished through oxime formation between an oxy-amine-containing peptide and a bisPNA-methyl ketone (complementary modifications can also be used). The described synthesis is highly efficient, does not require a protection strategy, and is carried out under mild aqueous conditions. Through this methodology long peptide sequences in either C to N or N to C polarity can be linked to bisPNA. In addition, this protocol makes the conjugation of cysteine-containing peptides feasible and allows disulfide bond formation to be controlled. This same approach can be exploited to link oligonucleotides, sugars, or other chemical entities to bisPNA.     

The spider acylpolyamine Mygalin is a potent modulator of innate immune responses

Mygalin is an antibacterial molecule isolated from the hemocytes of the spider Acanthoscurria gomesiana. It was identified as bis-acylpolyamine spermidine. We evaluated the modulator effects of synthetic Mygalin in the innate immune response. We demonstrate that Mygalin induces IFN-γ synthesis by splenocytes increasing the nitrite secretion by splenocytes and macrophages. A specific inhibitor of iNOS abrogated Mygalin-induced nitrite production in macrophages independent of IFN-γ activation. In addition, Mygalin-activated macrophages produced TNF-α but not IL-1β, demonstrating that Mygalin does not act directly on the inflammasome. Furthermore, this compound did not affect spontaneous or Concanavalin A-induced proliferative responses by murine splenocytes and did not induce IL-5 or apoptosis of splenocytes or bone marrow-derived macrophages. These data provide evidence that Mygalin modulates the innate immune response by inducing IFN-γ and NO synthesis. The combined immune regulatory and antibacterial qualities of Mygalin should be explored as a strategy to enhance immune responses in infection.     

Structural and biological characterization of one antibacterial acylpolyamine isolated from the hemocytes of the spider Acanthocurria gomesiana

We have isolated a 417Da antibacterial molecule, named mygalin, from the hemocytes of the spider Acanthoscurria gomesiana. The structure of mygalin was elucidated by tandem mass spectrometry (MS/MS) and by two spectroscopic techniques, nuclear magnetic resonance (NMR) and ultraviolet (UV) spectroscopy. Mygalin was identified as bis-acylpolyamine N1,N8-bis(2,5-dihydroxybenzoyl)spermidine, in which the primary amino groups of the spermidine are acylated with the carboxyl group of the 2,5-dihydroxybenzoic acid. Mygalin was active against Escherichia coli at 85muM, being this activity inhibited completely by catalase. Therefore, the antibacterial activity of mygalin was attributed to its production of hydrogen peroxide (H(2)O(2)). The putative mechanisms of formation of H(2)O(2) from mygalin are discussed. To our knowledge this is the first report of one bis-acylpolyamine with antibacterial activity purified from animal source.     

An efficient and versatile approach for the construction of oligonucleotide microarrays

A new immobilization chemistry for covalent attachment of phosphorylated oligonucleotides on epoxy-activated glass surface via opening of oxirane ring is described. The proposed strategy results in excellent immobilization efficiency, spot homogeneity, and morphology. The constructed microarray was successfully demonstrated for discrimination of nucleotide mismatches.     

蜘蛛毒素的研究进展及其相关应用

蜘蛛毒素含有多种化学成分,除毒性作用外这些物质对机体产生多重影响并具有多种活性。近年来,大量新的毒性组分不断被分离纯化,其结构和功能性作用被广泛深入研究,蜘蛛毒素成为了生物毒素领域新的研究热点。本文对蜘蛛毒素在离子通道作用机制方面的最新研究进展进行了阐述,同时还探讨了蜘蛛毒素在医学实践中新的应用和发展。     

An efficient synthesis of phosphatidylcholines

This article deals with two of the major steps involved in phospholipid synthesis: the preparation of the optically pure precursors, sn-glycero-3-phosphocholine (GPC) and -ethanolamine, from a convenient lipid source such as egg yolk, and acylation of hydroxyl groups present in those precursors involving an acid to yield the corresponding phospholipid. Phosphatidylcholines and phosphatidylethanolamines were separated from lipids extracted from egg yolk using low-pressure column chromatography. The advantages of this method include the use of smaller volumes of solvents and silica gel and reuse of adsorbent. Acylation of GPC is aided by ultrasound from a common laboratory bath cleaner. Ultrasound-assisted base-catalyzed esterification of GPC is accomplished between 2-6 hours providing a phospholipid in more than 80% yield. This scheme is particularly valuable in the synthesis of polymerizable phospholipids.     

Genes and evolution of two-domain toxins from lynx spider venom

Spiderines are comparatively long polypeptide toxins (∼110 residues) from lynx spiders (genus Oxyopes). They are built of an N-terminal linear cationic domain (∼40 residues) and a C-terminal knottin domain (∼60 residues). The linear domain empowers spiderines with strong cytolytic activity. In the present work we report 16 novel spiderine sequences from Oxyopes takobius and Oxyopes lineatus classified into two subfamilies. Strikingly, negative selection acts on both linear and knottin domains. Genes encoding Oxyopes two-domain toxins were sequenced and found to be intronless. We further discuss a possible scenario of lynx spider modular toxin evolution.     

Syntheses and biological activities of fluorescent-labeled analogs of acylpolyamine toxin NPTX-594 isolated from the venom of Madagascar Joro spider

Acylpolyamine-type spider toxins are known to be potent and specific blockers against glutamate receptors (GluRs). The present study describes the syntheses and biological activities of several fluorescent-labeled analogs related to a Madagascar Joro spider toxin NPTX-594 to analyze visually the unknown interaction between spider toxins and GluRs.     

An efficient enzymatic synthesis of thiamin pyrophosphate

Thiamin pyrophosphate was synthesized in 71% yield, on a multi-milligram scale, using overexpressed thiazole kinase, pyrimidine kinase, thiamin phosphate synthase, and thiamin phosphate kinase. This provides a facile route to isotopically labeled thiamin pyrophosphate from its readily available pyrimidine and thiazole precursors.     

An efficient synthesis of methyl 2,3-anhydro-alpha-D-ribofuranoside

An efficient synthesis of methyl 2,3-anhydro-alpha-D-ribofuranoside is reported. Its preparation is achieved via a four-step sequence from methyl 2,3,5-tri-O-benzoyl-alpha-D-arabinofuranoside in 74% overall yield.     

Modulation of P2X3 receptors by spider toxins

Recently, the novel peptide named purotoxin-1 (PT1) has been identified in the venom of the spider Geolycosa sp. and shown to exert marked modulatory effects on P2X3 receptors in rat sensory neurons. Here we studied another polypeptide from the same spider venom, purotoxin-2 (PT2), and demonstrated that it also affected activity of mammalian P2X3 receptors. The murine and human P2X3 receptors were heterologously expressed in cells of the CHO line, and nucleotide-gated currents were stimulated by CTP and ATP, respectively. Both PT1 and PT2 negligibly affected P2X3-mediated currents elicited by brief pulses of the particular nucleotide. When subthreshold CTP or ATP was added to the bath to exert the high-affinity desensitization of P2X3 receptors, both spider toxins strongly enhanced the desensitizing action of the ambient nucleotides. At the concentration of 50nM, PT1 and PT2 elicited 3-4-fold decrease in the IC(50) dose of ambient CTP or ATP. In contrast, 100nM PT1 and PT2 negligibly affected nucleotide-gated currents mediated by mP2X2 receptors or mP2X2/mP2X3 heteromers. Altogether, our data point out that the PT1 and PT2 toxins specifically target the fast-desensitizing P2X3 receptor, thus representing a unique tool to manipulate its activity.     

Two classes of channel-specific toxins from funnel web spider venom

1. The paralytic effects and neuromuscular actions of Agelenopsis aperta venom on insects were analyzed biochemically and electrophysiologically. 2. Paralysis caused by Agelenopsis venom is correlated with two effects on neuromuscular transmission: postsynaptic inhibition and presynaptic excitation. These effects are explained by the actions of two classes of toxins purified by RPLC, the alpha- and mu-agatoxins. 3. The alpha-agatoxins are low molecular weight, acylpolyamines which cause rapid, reversible paralysis correlated with use-dependent postsynaptic block of EPSPs and ionophoretic glutamate potentials. The mu-agatoxins are cysteine-rich polypeptides which cause irreversible paralysis and repetitive action potentials originating in presynaptic axons or nerve terminals. 4. The joint actions of the alpha- and mu-agatoxins lead to significantly higher rates of paralysis than are obtained by either toxin class alone, and this may relate to enhancement by excitatory mu-agatoxins of use-dependent block caused by alpha-agatoxins.     

An efficient synthesis and biological activity of substituted p-benzoquinones

An efficient synthesis of 2,5-diarylamino-3,6-dichloro-1,4-benzoquinone derivatives has been achieved by condensing mono substituted anilines with tetrachloro-p-benzoquinone in presence of fused sodium acetate as condensing agent under microwave irradiation without any solvent. All the synthesized compounds were tested for their antibacterial and antitumour activity using standard drugs.     

An efficient and scalable synthesis of 2,6-diaminopurine riboside

An efficient process to synthesize 2,6-diaminopurine riboside in high yield and quality is described. This inexpensive approach was scaled up to multi-hundred kilogram quantities for use in oligonucleotide therapeutics.     

An efficient, practical synthesis of 2-methoxyestradiol

An efficient and practical scheme to synthesize 2-methoxyestradiol has been developed. The key step was the copper-mediated methoxylation using ethyl acetate as a co-catalyst to introduce a methoxyl group. These synthetic procedures of four steps from 17β-estradiol as starting material gave 2-methoxyestradiol with a 61% overall yield.     

An efficient enzymatic synthesis of 5-aminovaleric acid

The title compound was prepared enzymatically from l-lysine in an excellent yield and under buffer-free conditions. l-Lysine was oxidized by the action of l-lysine α-oxidase from Trichoderma viride followed by spontaneous oxidative decarboxylation of the intermediate 6-amino-2-oxocaproic acid in the reaction medium. l-Lysine α-oxidase was immobilized on an epoxy-activated solid support (Sepabeads EC-EP) and the activity of both solution-based and immobilized enzyme in this reaction was determined.     

An efficient synthesis of a heterobifunctional coupling agent

An efficient synthesis of 4-[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl]-N-(6-[[6-([6-[(2,5-dioxopyrrolidin-1-yl)oxy]-6-oxohexyl]amino)-6-oxohexyl]amino]-6-oxohexyl)cyclohexanecarboxamide (12), a heterobifunctional coupling agent, was developed, which is critical for chemoselective conjugation of proteins and enzymes. The synthesis involved seven steps starting from 6-{[(benzyloxy)carbonyl]amino}hexanoic acid (1), and multigram quantities of coupling agent (12) were prepared using this protocol in excellent overall yield and 99.6% purity by reversed phase HPLC. The new method is suitable for the synthesis of coupling agent 12, consistently with purity >99%, and is useful for the preparation of other analogous coupling agents.