The development and application of a novel safety-catch linker for BOC-based assembly of libraries of cyclic peptides

Cyclic peptides are appealing targets in the drug-discovery process. Unfortunately, there currently exist no robust solid-phase strategies that allow the synthesis of large arrays of discrete cyclic peptides. Existing strategies are complicated, when synthesizing large libraries, by the extensive workup that is required to extract the cyclic product from the deprotection/cleavage mixture. To overcome this, we have developed a new safety-catch linker. The safety-catch concept described here involves the use of a protected catechol derivative in which one of the hydroxyls is masked with a benzyl group during peptide synthesis, thus making the linker deactivated to aminolysis. This masked derivative of the linker allows BOC solid-phase peptide assembly of the linear precursor. Prior to cyclization, the linker is activated and the linear peptide deprotected using conditions commonly employed (TFMSA), resulting in deprotected peptide attached to the activated form of the linker. Scavengers and deprotection adducts are removed by simple washing and filtration. Upon neutralization of the N-terminal amine, cyclization with concomitant cleavage from the resin yields the cyclic peptide in DMF solution. Workup is simple solvent removal. To exemplify this strategy, several cyclic peptides were synthesized targeted toward the somatostatin and integrin receptors. From this initial study and to show the strength of this method, we were able to synthesize a cyclic-peptide library containing over 400 members. This linker technology provides a new solid-phase avenue to access large arrays of cyclic peptides.

An efficient Fmoc strategy for the rapid synthesis of peptide para-nitroanilides

A new strategy has been developed for the rapid synthesis of peptide para-nitroanilides (pNA). The method involves derivatization of commercially available tritylchloride resin (TCP-resin) with 1,4-phenylenediamine, subsequent coupling with desired amino acids by the standard Fmoc protocol, and oxidation of the intermediate para-aminoanilides (pAA) with Oxone(R). This procedure allows easy assembly of the desired para-aminoanilides (pAA) on standard resin and efficient oxidation and purification of the corresponding para-nitroanilides (pNA). The method allows easy access to any desired peptide para-nitroanilides, which are useful substrates for the characterization and study of proteolytic enzymes.

Exploring privileged structures: the combinatorial synthesis of cyclic peptides

Head-to-tail cyclic peptides have been reported to bind to multiple, unrelated classes of receptor with high affinity. They may therefore be considered to be privileged structures. This review outlines the strategies by which both macrocyclic cyclic peptides and cyclic dipeptides or diketopiperazines have been synthesised in combinatorial libraries. It also briefly outlines some of the biological applications of these molecules, thereby justifying their inclusion as privileged structures.

Synthesis and structural analysis of the N-terminal domain of the thyroid hormone-binding protein transthyretin

Transthyretin (TTR) is a 55 kDa protein responsible for the transport of thyroid hormones and retinol in human serum. Misfolded forms of the protein are implicated in the amyloid diseases familial amyloidotic polyneuropathy and senile systemic amyloidosis. Its folding properties and stabilization by ligands are of current interest due to their importance in understanding and combating these diseases. To assist in such studies we developed a method for the solid phase synthesis of the monomeric unit of a TTR analogue and its folding to form a functional 55 kDa tetramer. The monomeric unit of the protein was chemically synthesized in three parts, comprising amino acid residues 151, 5499 and 102127, and ligated using chemoselective thioether ligation chemistry. The synthetic protein was folded and assembled to a tetrameric structure in the presence of the TTRs native ligand, thyroxine, as shown by gel filtration chromatography, native gel electrophoresis, TTR antibody recognition and thyroid hormone binding. In the current study the solution structure of the first of these fragment peptides, TTR(151) is examined to determine its intrinsic propensity to form beta-sheet structure, potentially involved in amyloid fibril formation by TTR. Despite the presence of extensive beta-structure in the native form of the protein, the Nterminal fragment adopts an essentially random coil conformation in solution.

Synthesis and characterization of delta-atracotoxin-ar1a, the lethal neurotoxin from venom of the Sydney funnel-web spider (Atrax robustus)

delta-Atracotoxin-Ar1a (delta-ACTX-Ar1a) is the major polypeptide neurotoxin isolated from the venom of the male Sydney funnel-web spider, Atrax robustus. This neurotoxin targets both insect and mammalian voltage-gated sodium channels, where it competes with scorpion alpha-toxins for neurotoxin receptor site-3 to slow sodium-channel inactivation. Progress in characterizing the structure and mechanism of action of this toxin has been hampered by the limited supply of pure toxin from natural sources. In this paper, we describe the first successful chemical synthesis and oxidative refolding of the four-disulfide bond containing delta-ACTX-Ar1a. This synthesis involved solid-phase Boc chemistry using double coupling, followed by oxidative folding of purified peptide using a buffer of 2 M GdnHCl and glutathione/glutathiol in a 1:1 mixture of 2-propanol (pH 8.5). Successful oxidation and refolding was confirmed using both chemical and pharmacological characterization. Ion spray mass spectrometry was employed to confirm the molecular weight. H-1 NMR analysis showed identical chemical shifts for native and synthetic toxins, indicating that the synthetic toxin adopts the native fold. Pharmacological studies employing whole-cell patch clamp recordings from rat dorsal root ganglion neurons confirmed that synthetic delta-ACTX-Ar1a produced a slowing of the sodium current inactivation and hyperpolarizing shifts in the voltage-dependence of activation and inactivation similar to native toxin. Under current clamp conditions, we show for the first time that delta-ACTX-Ar1a produces spontaneous repetitive plateau potentials underlying the clinical symptoms seen during envenomation. This successful oxidative refolding of synthetic delta-ACTX-Ar1a paves the way for future structure-activity studies to determine the toxin pharmacophore.

Soil remediation time to achieve clean-up goals II: influence of natural organic matter and water contents

This work reports a relatively rapid procedure for the forecasting of the remediation time (RT) of sandy soils contaminated with cyclohexane using vapour extraction. The RT estimated through the mathematical fitting of experimental results was compared with that of real soils. The main objectives were: (i) to predict the RT of soils with natural organic matter (NOM) and water contents different from those used in experiments; and (ii) to analyse the time and efficiency of remediation, and the distribution of contaminants into the soil matrix after the remediation process, according to the soil contents of: (ii1) NOM; and (ii2) water. For sandy soils with negligible clay contents, artificially contaminated with cyclohexane before vapour extraction, it was concluded that: (i) if the NOM and water contents belonged to the range of the prepared soils, the RT of real soils could be predicted with relative differences not higher than 12%; (ii1) the increase of NOM content from 0% to 7.5% increased the RT (1.8–13 h) and decreased the remediation efficiency (RE) (99–90%) and (ii2) the increase of soil water content from 0% to 6% increased the RT (1.8–4.9 h) and decreased the RE (99–97%). NOM increases the monolayer capacity leading to a higher sorption into the solid phase. Increasing of soil water content reduces the mass transfer coefficient between phases. Concluding, NOM and water contents influence negatively the remediation process, turning it less efficient and more time consuming, and consequently more expensive.

Organochlorine pesticide residues in strawberries from integrated pest management and organic farming

A rapid, specific, and sensitive method based on theQuick Easy Cheap Effective Rugged and Safe (QuEChERS) method and a cleanup using dispersive solid-phase extraction with MgSO4, PSA, and C18 sorbents has been developed for the routine analysis of 14 pesticides in strawberries. The analyses were performed by three different analytical methodologies: gas chromatography (GC) with electron capture detection (ECD), mass spectrometry (MS), and tandem mass spectrometry (MS/MS). The recoveries for all the pesticides studied were from 46 to 128%, with relative standard deviation of <15% in the concentration range of 0.005-0.250 mg/kg. The limit of detection (LOD) for all compoundsmetmaximumresidue limits (MRL) accepted in Portugal for organochlorine pesticides (OCP). A survey study of strawberries produced in Portugal in the years 2009-2010 obtained from organic farming (OF) and integrated pest management (IPM) was developed. Lindane and β-endosulfan were detected above the MRL in OF and IPM. Other OCP (aldrin, o,p0-DDT and their metabolites, and methoxychlor) were found below the MRL. The OCP residues detected decreased from 2009 to 2010. The QuEChERS method was successfully applied to the analysis of strawberry samples.

A Comparative Study of the Organic Acid Content of the Hemolymph of Schistosoma mansoni-Resistant and Susceptible Strains of Biomphalaria glabrata

The freshwater snail Biomphalaria glabrata is an intermediate host of the trematode Schistosoma mansoni. However, some strains of B. glabrata are resistant to successful infection by S. mansoni larvae. The present work examines the profile of organic acids present in S. mansoni-resistant and -susceptible strains of B. glabrata, in order to determine whether the type of organic acid present is related to susceptibility. The organic acids were extracted from the hemolymph of two susceptible B. glabrata strains (PR, Puerto Rico and Ba, Jacobina-Bahia from Brazil), and from the resistant strains 13-16-R1 and 10R2, using solid phase extraction procedures followed by high performance liquid chromatography. The organic acids obtained were analyzed and identified by comparison with known standards. Pyruvate, lactate, succinate, malate, fumarate, acetate, propionate, ß-hydroxybutyrate and acetoacetate were detected in all hemolymph samples. Under standard conditions, the concentration of each of these substances varied among the strains tested and appeared to be specific for each strain. An interesting variation was the low concentration of pyruvate in the hemolymph of PR-snails. Only the concentration of fumarate was consistently different (p&pound; 0.05) between resistant and susceptible strains

Direct and indirect root defences of milkweed (Asclepias syriaca): trophic cascades, trade-offs and novel methods for studying subterranean herbivory

P>1. Entomopathogenic nematodes can function as indirect defence for plants that are attacked by root herbivores. By releasing volatile organic compounds (VOCs), plants signal the presence of host insects and thereby attract nematodes.2. Nonetheless, how roots deploy indirect defences, how indirect defences relate to direct defences, and the ecological consequences of root defence allocation for herbivores and plant biomass are essentially unknown.3. We investigate a natural below-ground tritrophic system, involving common milkweed, a specialist root-boring beetle and entomopathogenic nematodes, and asked whether there is a negative genetic correlation between direct defences (root cardenolides) and indirect defences (emission of volatiles in the roots and nematode attraction), and between constitutive and inducible defences.4. Volatiles of roots were analysed using two distinct sampling methods. First, we collected emissions from living Asclepias syriaca roots by dynamic headspace sampling. This method showed that attacked A. syriaca plants emit five times higher levels of volatiles than control plants. Secondly, we used a solid phase micro-extraction (SPME) method to sample the full pool of volatiles in roots for genetic correlations of volatile biosynthesis.5. Field experiments showed that entomopathogenic nematodes prevent the loss of biomass to root herbivory. Additionally, suppression of root herbivores was mediated directly by cardenolides and indirectly by the attraction of nematodes. Genetic families of plants with high cardenolides benefited less from nematodes compared to low-cardenolide families, suggesting that direct and indirect defences may be redundant. Although constitutive and induced root defences traded off within each strategy (for both direct and indirect defence, cardenolides and VOCs, respectively), we found no trade-off between the two strategies.6. Synthesis. Constitutive expression and inducibility of defences may trade off because of resource limitation or because they are redundant. Direct and indirect defences do not trade off, likely because they may not share a limiting resource and because independently they may promote defence across the patchiness of herbivore attack and nematode presence in the field. Indeed, some redundancy in strategies may be necessary to increase effective defence, but for each strategy, an economy of deployment reduces overall costs.

Synthesis and immunological characterization of 104-mer and 102-mer peptides corresponding to the N- and C-terminal regions of the Plasmodium falciparum CS protein.

We investigated the immunogenicity and the conformational properties of the non-repetitive sequences of the Plasmodium falciparum circumsporozoite (CS) protein. Two polypeptides of 104 and 102 amino acids long, covering, respectively, the N- and C-terminal regions of the CS protein, were synthesized using solid phase Fmoc chemistry. The crude polypeptides were purified by a combination of size exclusion chromatography and RP-HPLC. Sera of mice immunized with the free polypeptides emulsified in incomplete Freund's adjuvant strongly reacted with the synthetic polypeptides as well as with native CS protein as judged by ELISA and IFAT assays. Most importantly, these antisera inhibited the sporozoite invasion of hepatoma cells. In addition, sera derived from donors living in a malaria endemic area recognized the CS 104- and 102-mers. Conformational studies of the CS polypeptides were also performed by circular dichroism spectroscopy showing the presence of a weakly ordered structure that can be increased by addition of trifluoroethanol. The obtained results indicate that the synthetic CS polypeptides and the natural CS protein share some common antigenic determinants and probably have similar conformation. The approach used in this study might be useful for the development of a synthetic malaria vaccine.

Convergent Approaches for the Synthesis of the Anti-tumoral Peptide, Kahalalide F. Study of Orthogonal Protecting Groups

Kahalalide compounds are peptides that are isolated from a Hawaiian herbivorous marine species of mollusc, Elysia rufescens, and its diet, the green alga Bryopsis sp. Kahalalide F and its synthetic analogues are the most promising compounds of the Kahalalide family because they show anti-tumoral activity. Linear solid-phase syntheses of Kahalalide F have been reported. Here we describe several new improved synthetic routes based on convergent approaches with distinct orthogonal protection schemes for the preparation of Kahaladide analogues. These strategies allow a better control and characterization of the intermediates because more reactions are performed in solution. Five derivatives of Kahalalide F were synthesized using several convergent approaches.

Characterization of volatile organic gunshot residues in fired handgun cartridges by headspace sorptive extraction

In forensic investigation of firearm-related cases, determination of the residual amount of volatile compounds remaining inside a cartridge could be useful in estimating the time since its discharge. Published approaches are based on following the decrease of selected target compounds as a function of time by using solid phase micro-extraction (SPME). Naphthalene, as well as an unidentified decomposition product of nitrocellulose (referred to as "TEA2"), are usually employed for this purpose. However, reliability can be brought into question given their high volatility and the low reproducibility of their extracted quantities. In order to identify alternatives and therefore develop improved dating methods, an extensive study on the composition and variability of volatile residues in nine different types of cartridges was carried out. Analysis was performed using headspace sorptive extraction (HSSE), which is a more exhaustive technique compared to SPME. 166 compounds were identified (several of which for the first time), and it was observed that the final compositional characteristics of each residue were strongly dependent on its source. Variability of single identified compounds within and between different types of cartridge, as well as their evolution over time, was also studied. Many explosion products containing up to 4 aromatic rings were found to be globally present in high proportions amongst residues. 27 of them (excluding naphthalene) also presented detectable decreases during the first 24 h. Therefore, they could be used as complementary target analytes in future dating methods.

Synthesis of Janus compounds for the recognition of G-U mismatched nucleobase pairs

The design and synthesis of two Janus-type heterocycles with the capacity to simultaneously recognize guanine and uracyl in G-U mismatched pairs through complementary hydrogen bond pairing is described. Both compounds were conveniently functionalized with a carboxylic function and efficiently attached to a tripeptide sequence by using solid-phase methodologies. Ligands based on the derivatization of such Janus compounds with a small aminoglycoside, neamine, and its guanidinylated analogue have been synthesized, and their interaction with Tau RNA has been investigated by using several biophysical techniques, including UV-monitored melting curves, fluorescence titration experiments, and 1H NMR. The overall results indicated that Janus-neamine/guanidinoneamine showed some preference for the +3 mutated RNA sequence associated with the development of some tauopathies, although preliminary NMR studies have not confirmed binding to G-U pairs. Moreover, a good correlation has been found between the RNA binding affinity of such Janus-containing ligands and their ability to stabilize this secondary structure upon complexation.

Convergent Approaches for the Synthesis of the Anti-tumoral Peptide, Kahalalide F. Study of Orthogonal Protecting Groups

Kahalalide compounds are peptides that are isolated from a Hawaiian herbivorous marine species of mollusc, Elysia rufescens, and its diet, the green alga Bryopsis sp. Kahalalide F and its synthetic analogues are the most promising compounds of the Kahalalide family because they show anti-tumoral activity. Linear solid-phase syntheses of Kahalalide F have been reported. Here we describe several new improved synthetic routes based on convergent approaches with distinct orthogonal protection schemes for the preparation of Kahaladide analogues. These strategies allow a better control and characterization of the intermediates because more reactions are performed in solution. Five derivatives of Kahalalide F were synthesized using several convergent approaches.

Efficient synthesis of sulfonamide derivatives on solid supports catalyzed using solvent-free and microwave-assisted methods

In this work we report the synthesis of sulfonamide derivatives using a conventional procedure and with solid supports, such as silica gel, florisil, alumina, 4Å molecular sieves, montmorillonite KSF, and montmorillonite K10 using solvent-free and microwave-assisted methods. Our results show that solid supports have a catalytic activity in the formation of sulfonamide derivatives. We found that florisil, montmorillonite KSF, and K10 could be used as inexpensive alternative catalysts that are easily separated from the reaction media. Additionally, solvent-free and microwave-assisted methods were more efficient in reducing reaction time and in increasing yield.