Synthesis and anthelmintic activity of substituted (R)-phenyllactic acid containing cyclohexadepsipeptides

The substituted (R)-phenyllactic acid containing cyclohexadepsipeptides (CHDPs) represent novel enniatin derivatives with strong in vivo activities against the parasitic nematode Haemonchus contortus Rudolphi in sheep. 2D NMR spectroscopic analysis revealed for the substituted (R)-phenyllactic acid containing CHDPs one major conformer with an unsymmetrically folded conformation lacking a cis-amide bond. A correlation between the substitution pattern and its anthelmintic activity was found. Here we report on a simple total synthetic pathway of the precursor for this particular type of CHDPs and an efficient modification of the benzylic side chain (R-PhLac(2)).     

Effects of side chain modification on the activity of propyl cyclohexaneacetate as an attractant to the German cockroach

Propyl cyclohexaneacetate, a synthetic attractant to the German cockroach, Blattella germanica, appears to consist of a head (cyclohexane ring) and a tail (ester linkage). The tail was modified as regards a number of structural parameters, and the change in activity was interpreted in terms of the corresponding receptor site.Irrespective of the position and direction of the ester linkage, six atoms were optimum for the side chain. The activity increased when the terminal methyl group was replaced with chlorine, and decreased when changed into methylene. The methyl branch in the alcohol unit depressed the activity. The order of attraction among the esters with six atom side chain was as follows: propyl cyclohexaneacetate > cyclohexylmethyl butanoate ≧ ethyl 3-cyclohexylpropanoate ≧ cyclohexyl pentanoate > butyl cyclohexanecarboxylate = 2-cyclohexylethyl propanoate.Ethers, ketones and hydrocarbons which were derived from the esters with six atom side chains by replacing either or both of the carbonyl groups and ether oxygen with methylene(s) were inferior to the parent esters. Their relative activities were in the following order: pentyl cyclohexyl ether > propyl 2-cyclohexylethyl ether > butyl cyclohexyl-methyl ether = pentyl cyclohexyl ketone = butyl cyclohexylmethyl ketone = 6-cyclohexylhexane. The SAR in respect of the ester group resembled that in the muscarinic activity of acetylcholine.     

Synthesis and anthelmintic activity of cyclohexadepsipeptides with (S,S,S,R,S,R)-configuration

The (S,S,S,R,S,R)-configurated cyclohexadepsipeptides (CHDPs) represent novel enniatin derivatives with strong in vivo activity against the parasitic nematode Haemonchus contortus Rudolphi in sheep. 2D NMR spectroscopic analysis revealed for the major conformation the asymmetric conformer, containing a cis-amide bond between C(alpha) protons of neighbouring 2-hydroxy-(S)-carboxylic acid and N-methyl-(S)-amino acid. The absolute configuration of the novel CHDPs was determined by X-ray crystallography. A correlation between the major conformer and its anthelmintic activity was found. Here, we report on a simple total synthetic pathway for this particular type of CHDPs.     

5-Alkyl-2-alkylamino-6-(2,6-difluorophenylalkyl)-3,4-dihydropyrimidin-4(3H)-ones, a new series of potent, broad-spectrum non-nucleoside reverse transcriptase inhibitors belonging to the DABO family

2-Alkylamino-6-[1-(2,6-difluorophenyl)alkyl]-3,4-dihydro-5-alkylpyrimidin-4(3H)-ones (F(2)-NH-DABOs) 4, 5 belonging to the dihydro-alkoxy-benzyl-oxopyrimidine (DABO) family and bearing different alkyl- and arylamino side chains at the C(2)-position of the pyrimidine ring were designed as active against wild type (wt) human immunodeficiency virus type 1 (HIV-1) and some relevant HIV-1 mutants. Biological evaluation indicated the importance of the further anchor point of compounds 4, 5 into the non-nucleoside binding site (NNBS): newly synthesized compounds were highly active against both wild type and the Y181C HIV-1 strains. In anti-wt HIV-1 assay the potency of amino derivatives did not depend on the size or shape of the C(2)-amino side chain, but it associated with the presence of one or two methyl groups (one at the pyrimidine C(5)-position and the other at the benzylic carbon), being thymine, alpha-methyluracil or alpha-methylthymine derivatives almost equally active in reducing wt HIV-1-induced cytopathogenicity in MT-4 cells. Against the Y181C mutant strain, 2,6-difluorobenzyl-alpha-methylthymine derivatives 4d, 5h'-n' showed the highest potency and selectivity among tested compounds, both a properly sized C(2)-NH side chain and the presence of two methyl groups (at C(5) and benzylic positions) being crucial for high antiviral action.     

Discovery of S-444823, a potent CB1/CB2 dual agonist as an antipruritic agent

The optimization of a series of 3-carbamoyl 2-pyridone derivatives as CB agonists is reported. These efforts resulted in the discovery of 3-(2-(1-(cyclohexylmethyl)-2-oxo-1,2,5,6,7,8,9,10-octahydrocycloocta[b]pyridine-3-carboxamido)thiazol-4-yl)propanoic acid (21), a potent dual CB1/CB2 agonist without CNS side effects induced by CB1 receptor activation. It exhibited strong inhibition of scratching as a 1.0% acetone solution in the pruritic model.     

Differential degradation of nonylphenol isomers by Sphingomonas xenophaga Bayram

Sphingomonas xenophaga Bayram, isolated from the activated sludge of a municipal wastewater treatment plant, was able to utilize 4-(1-ethyl-1,4-dimethylpentyl)phenol, one of the main isomers of technical nonylphenol mixtures, as a sole carbon and energy source. The isolate degraded 1 mg of 4-(1-ethyl-1,4-dimethylpentyl)phenol/ml in minimal medium within 1 week. Growth experiments with five nonylphenol isomers showed that the three isomers with quaternary benzylic carbon atoms [(1,1,2,4-tetramethylpentyl)phenol, 4-(1-ethyl-1,4-dimethylpentyl)phenol, and 4-(1,1-dimethylheptyl)phenol] served as growth substrates, whereas the isomers containing one or two hydrogen atoms in the benzylic position [4-(1-methyloctyl)phenol and 4-n-nonylphenol] did not. However, when the isomers were incubated as a mixture, all were degraded to a certain degree. Differential degradation was clearly evident, as isomers with more highly branched alkyl side chains were degraded much faster than the others. Furthermore, the C9 alcohols 2,3,5-trimethylhexan-2-ol, 3,6-dimethylheptan-3-ol, and 2-methyloctan-2-ol, derived from the three nonylphenol isomers with quaternary benzylic carbon atoms, were detected in the culture fluid by gas chromatography-mass spectrometry, but no analogous metabolites could be found originating from 4-(1-methyloctyl)phenol and 4-n-nonylphenol. We propose that 4-(1-methyloctyl)phenol and 4-n-nonylphenol were cometabolically transformed in the growth experiments with the mixture but that, unlike the other isomers, they did not participate in the reactions leading to the detachment of the alkyl moiety. This hypothesis was corroborated by the observed accumulation in the culture fluid of an as yet unidentified metabolite derived from 4-(1-methyloctyl)phenol.     

Differential Degradation of Nonylphenol Isomers by Sphingomonas xenophaga Bayram

Sphingomonas xenophaga Bayram, isolated from the activated sludge of a municipal wastewater treatment plant, was able to utilize 4-(1-ethyl-1,4-dimethylpentyl)phenol, one of the main isomers of technical nonylphenol mixtures, as a sole carbon and energy source. The isolate degraded 1 mg of 4-(1-ethyl-1,4-dimethylpentyl)phenol/ml in minimal medium within 1 week. Growth experiments with five nonylphenol isomers showed that the three isomers with quaternary benzylic carbon atoms [(1,1,2,4-tetramethylpentyl)phenol, 4-(1-ethyl-1,4-dimethylpentyl)phenol, and 4-(1,1-dimethylheptyl)phenol] served as growth substrates, whereas the isomers containing one or two hydrogen atoms in the benzylic position [4-(1-methyloctyl)phenol and 4-n-nonylphenol] did not. However, when the isomers were incubated as a mixture, all were degraded to a certain degree. Differential degradation was clearly evident, as isomers with more highly branched alkyl side chains were degraded much faster than the others. Furthermore, the C₉ alcohols 2,3,5-trimethylhexan-2-ol, 3,6-dimethylheptan-3-ol, and 2-methyloctan-2-ol, derived from the three nonylphenol isomers with quaternary benzylic carbon atoms, were detected in the culture fluid by gas chromatography-mass spectrometry, but no analogous metabolites could be found originating from 4-(1-methyloctyl)phenol and 4-n-nonylphenol. We propose that 4-(1-methyloctyl)phenol and 4-n-nonylphenol were cometabolically transformed in the growth experiments with the mixture but that, unlike the other isomers, they did not participate in the reactions leading to the detachment of the alkyl moiety. This hypothesis was corroborated by the observed accumulation in the culture fluid of an as yet unidentified metabolite derived from 4-(1-methyloctyl)phenol.     

Molecular dynamics simulation on structural conformation of conjugated polymer-functionalised films for optimal fluorescent performance

The sensitive structure-related fluorescent properties of p-phenyleneethynylene (PPE)-functionalised fluorescent films with side chains and without side chains in different chemical environments are studied by molecular dynamics simulations. The calculations demonstrate that the structural change plays a crucial role in the fluorescent performance of PPE films, in which a major contribution is from the conformation of side chains. The PPE molecules with sides chains immobilised on SiO₂ substrate prefer to aggregate together in a dry state, but are more likely to stay in a monomolecular state in THF solvent. To an optimal concentration of the solvent molecules, the side chains are even perpendicular to the backbones of the molecules. The aggregation and separation of the PPE molecules with side chains are found to be controlled by the contraction and extension of the side chains connected to PPE. The van der Waals' force between the side chains is mainly responsible for these changes, which leads to a spreading out of the side chains in the presence of THF. All the results from the simulation studies can successfully explain the experimental observations.     

Structure-activity relationships of 1,5-biaryl pyrroles as EP1 receptor antagonists

The preliminary SAR of a series of novel 1,5-biaryl pyrrole EP1 receptor antagonists derived from compound 1 is described. Replacement of the benzyl group of 1 with isosteric groups was investigated. The most effective replacement was found to be the isobutyl group. The cyclopentylmethyl and cyclohexylmethyl groups were also effective benzyl replacements. The cyclohexylmethyl derivative 19 demonstrated the lowest metabolic clearance within this series. In addition, several high affinity substituted benzyl analogues were also identified. Compound 39 was found to have good bioavailability in rats and demonstrated efficacy in the established FCA preclinical model of inflammatory pain with a calculated ED50 of 9.2mg/kg.     

Original preparation of conjugates for antibody production against Amicoumacin-related anti-microbial agents

Amicoumacins are natural products with potent anti-ulcerogenic and anti-bacterial activities, and have been isolated from different Bacillus genera. They belong to a family of 3,4-dihydroisocoumarin derivatives bearing hydroxylated amino acid side chains. The 3,4-dihydroisocoumarin moiety of Amicoumacins has been obtained in two steps from 2-methoxybenzoic acid by combining directed and benzylic metalation strategies. The use of s-BuLi in both steps gave satisfactory and reproducible yields. For the development of an immunoassay (ELISA) of Amicoumacin-related compounds in biological media, the deprotected 3,4-dihydroisocoumarin moiety has been coupled to the BSA carrier protein via a homobifunctional linker deriving from d-tartaric acid. This approach enabled to introduce the hydroxylated portion of Amicoumacin directly during the preparation of hapten-protein conjugates. The coupling ratio was evaluated by mass spectrometry. The hapten-protein conjugate showing the best coupling ratio was used to generate polyclonal immunosera in rabbits. After immunoserum titration, ELISA conditions were set up and specificity tests were performed on solutions of pure parent compounds, semi-purified Amicoumacin B as well as on culture supernatants of strains known for their Amicoumacin production. This immunoassay is suitable for a rapid and simple screening test for the production of Amicoumacins and its related compounds by bacterial strains.     

The carbohydrate side chains of the major plasma serpins of horse and wallaby: analyses of enzymatic and chemically treated (including 'Smith degradation') protein blots by lectin binding

The carbohydrate side chains of the major plasma serpins of the horse and wallaby have been characterized by lectin analyses of protein blots from two-dimensional gels using the major human plasma serpin, alpha 1-protease inhibitor, as a control. Eight lectins were used in the characterization in conjunction with enzymatic deglycosylation of complex and high mannose side chains, chemical desialylation and defucosylation, and one round of 'Smith degradation', all being performed on the nitrocellulose blots. Assuming a standard complex side chain structure, the results of the 21 lectin/treatment combinations were interpreted as indicating that the equine proteins consist of partially sialylated biantennary side chains except for the most acidic proteins which have triantennary side chains. The wallaby proteins have bi- and triantennary side chains with a bisecting N-acetylglucosamine and fucose residue present.     

Pseudomonas aeruginosa lipopolysaccharide: evidence that the O side chains and common antigens are on the same molecule.

We investigated whether Pseudomonas aeruginosa produces two distinct lipopolysaccharides (LPS) containing either serologically variable O side chains or a neutral polysaccharide common antigen, designated A bands, that reacts with monoclonal antibody (MAb) E87. Immunoprecipitation of LPS and free O side chains with O-side-chain-specific antibodies or MAb E87 resulted in coprecipitation of both polysaccharides when antibody of either specificity was employed. Chromatography of LPS and free O side chains in a disaggregating deoxycholate buffer indicated the two polysaccharide antigens cochromatograph when eluates were analyzed by sensitive and specific enzyme-linked immunosorbent assay inhibitions. The LPS from a mutant of strain PAO1 that lacks polymerized O side chains but retains the common antigen eluted in fractions containing smaller LPS molecules, indicating the necessity of polymerized O side chains for elution in early fractions containing large LPS monomers. A phosphomannomutase mutant of P. aeruginosa PAO1 makes a rough LPS lacking both O side chains and common antigen but still produces a small (< 6-kDa) common antigen component detectable in cell lysates. Introduction of the cloned pmm gene into this strain restored production of a smooth LPS expressing large MAb E87-reactive common antigen. Destruction with NaOH of O side chains on recombinant LPS molecules eluting early from the molecular sieve column resulted in a shift of the MAb E87-reactive antigen to the late-eluting fractions. These results indicate that on most P. aeruginosa LPS molecules, O side chains and neutral polysaccharide common antigens are both present.     

Statistical and conformational analysis of the electron density of protein side chains

Protein side chains make most of the specific contacts between proteins and other molecules, and their conformational properties have been studied for many years. These properties have been analyzed primarily in the form of rotamer libraries, which cluster the observed conformations into groups and provide frequencies and average dihedral angles for these groups. In recent years, these libraries have improved with higher resolution structures and using various criteria such as high thermal factors to eliminate side chains that may be misplaced within the crystallographic model coordinates. Many of these side chains have highly non-rotameric dihedral angles. The origin of side chains with high B-factors and/or with non-rotameric dihedral angles is of interest in the determination of protein structures and in assessing the prediction of side chain conformations. In this paper, using a statistical analysis of the electron density of a large set of proteins, it is shown that: (1) most non-rotameric side chains have low electron density compared to rotameric side chains; (2) up to 15% of chi1 non-rotameric side chains in PDB models can clearly be fit to density at a single rotameric conformation and in some cases multiple rotameric conformations; (3) a further 47% of non-rotameric side chains have highly dispersed electron density, indicating potentially interconverting rotameric conformations; (4) the entropy of these side chains is close to that of side chains annotated as having more than one chi(1) rotamer in the crystallographic model; (5) many rotameric side chains with high entropy clearly show multiple conformations that are not annotated in the crystallographic model. These results indicate that modeling of side chains alternating between rotamers in the electron density is important and needs further improvement, both in structure determination and in structure prediction.     

Design,synthesis and evaluation of 4,7-diamino-1,10-phenanthroline G-quadruplex ligands

A series of 4,7-diamino-1,10-phenanthroline derivatives carrying positively charged side chains has been synthesized, and their G-quadruplex interaction evaluated by circular dichroism (CD) and surface plasmon resonance (SPR). In absence of side chains, 4,7-diamino-1,10-phenanthroline exhibits a weak but significant G-quadruplex stabilizing effect, compared to no stabilization by 1,10-phenanthroline. We hypothesize that this effect is due to increased basicity of the phenanthroline nitrogens and protonation or ion chelation to form a central positive charge which stack on the G-tetrad above the central ionic column. Introduction of positively charged side chains results in compounds with appreciable G-quadruplex stabilizing properties and high aqueous solubility, with the longer side chains giving more potent compounds. Ligands carrying guanidine side chains in general show higher quadruplex stabilizing activity and distinctly slower kinetic properties than their amino and dimethylamino analogues, possibly due to specific hydrogen bond interactions with the G-quadruplex loops.     

Synthesis and structural studies of N-p-toluensulfonyl cyclodipeptides

In view of the chemical and structural interest of cyclopeptides bearing an electron withdrawing substituent directly bonded at the amide nitrogen atom, the two N-p-toluensulfonyl (N-tosyl) derivatives cyclo[-Phe(Tos)-D-Phe-] (I) and cyclo[-Phe(Tos)-D-Pro-] (II) have been synthesized and their stereochemistry defined. The molecular structure of I, as determined by x-ray diffraction analysis, is reported together with 1H-nmr parameters indicating the preferred rotameric conformation in chloroform solution. The N-tosyl group alters the geometry of the cyclodipeptide ring by lengthening both the N-C bonds departing from the tosylated nitrogen and reducing the corresponding ring angle. The 6-membered peptide ring adopts an unusual "sofa" conformation with the Tos-Phe C alpha atom deviating 0.230(3) A out of the mean plane of the other five ring atoms. One of the two S-O bonds forms a planar system that involves the tosylated nitrogen and the corresponding amide carbonyl. In the crystal, both the benzylic side chains are folded over the heterocyclic ring, whereas in chloroform solution, the benzylic side chain of the D-Phe prefers an extended conformation.     

Phe and Asn side chains in DNA double strands

The contribution of amino acid side chains to the recognition of DNA by peptides or proteins is evaluated by substituting single nucleobases of a DNA double strand by amino acid side chains. C-nucleosides with the side chains of phenylalanine and asparagine were synthesized and incorporated in DNA. This modification should allow to keep the double strand conformation. Hydrogen bonds, pi-pi-interactions and solvation have an influence on the double strand stability.     

Organization of pectic arabinan and galactan side chains in association with cellulose microfibrils in primary cell walls and related models envisaged

The structure of arabinan and galactan domains in association with cellulose microfibrils was investigated using enzymatic and alkali degradation procedures. Sugar beet and potato cell wall residues (called 'natural' composites), rich in pectic neutral sugar side chains and cellulose, as well as 'artificial' composites, created by in vitro adsorption of arabinan and galactan side chains onto primary cell wall cellulose, were studied. These composites were sequentially treated with enzymes specific for pectic side chains and hot alkali. The degradation approach used showed that most of the arabinan and galactan side chains are in strong interaction with cellulose and are not hydrolysed by pectic side chain-degrading enzymes. It seems unlikely that isolated arabinan and galactan chains are able to tether adjacent microfibrils. However, cellulose microfibrils may be tethered by different pectic side chains belonging to the same pectic macromolecule.     

Optimum interaction of sterol side chains with phosphatidylcholine.

The specificity of the interaction between the cholesterol side chain and egg phosphatidylcholine was precisely defined by examining the effect of three new analogues of cholesterol with modified side chains on the ordering of two steroid spin labels in liposomes. The complete side chain of cholesterol was shown to be required for maximum ordering. Sterols with side chains shorter or longer than cholesterol caused significantly less ordering.     

The role of solvent polarity in the free energy of transfer of amino acid side chains from water to organic solvents

The transfer free energies of amino acid side chains from water to N-methylacetamide have been determined and compared with those obtained from other model systems. Although the process of transfer from water to N-methylacetamide represents transfer from a lower dielectric phase to a higher dielectric phase, the transfer free energies of most of the amino acid side chains are nearly the same as those obtained from the water to ethanol system. Among the apolar side chains studied, only the transfer free energies of methionine and the aromatic side chains are apparently influenced to some extent by the polarity of the organic solvent phase. The transfer free energies of the neutral polar side chains also exhibit significant dependence on solvent polarity. The van't Hoff plots for most of the apolar side chains exhibit nonlinear curves, indicating that the enthalpy of transfer from water to N-methylacetamide is temperature-dependent. It is suggested that to assess the contribution of the hydrophobic free energy to the stability of globular proteins, it is probably not necessary to account for variation in the internal environment of the protein.     

Optimum interaction of sterol side chains with phosphatidylcholine

The specificity of the interaction between the cholesterol side chain and egg phosphatidylcholine was precisely defined by examining the effect of three new analogues of cholesterol with modified side chains on the ordering of two steroid spin labels in liposomes. The complete side chain of cholesterol was shown to be required for maximum ordering. Sterols with side chains shorter or longer than cholesterol caused significantly less ordering.