Reversal in helix sense of copoly(beta-alkyl-L-aspartate-beta-benzyl-L-aspartate)

The conformation of copoly(beta-alkyl-L-aspartate-beta-benzyl-L-aspartate), in which the alkyl group is ethyl, propyl, butyl, hexyl, nonyl, dodecyl, or stearyl, was studied in solution and the solid state by optical rotatory dispersion and circular dichroism methods. The helix sense of the copolyaspartate studied here is transformed from a left-handed to right-handed alpha-helix as the degree of alkylation increases. Reversal in helix sense occurs, i.e., the left-handed alpha-helix based on the handedness of poly(beta-benzyl-L-aspartate) is transformed into a right-handed alpha-helix with increase in alkyl groups with right-handed nature. Reversal in helix sense is also observed for copolyaspartates with an intermediate or high degree of alkylation as temperature rises. Copolyaspartates with hexyl, nonyl, or dodecyl groups exhibit an induced circular dichroism around 230-238 nm and can form an ordered side chain structure which is broken down at high temperature. One has to consider the conformation of the omega-helix and beta-form of the copolyaspartates in the solid state in addition to the reversal in helix sense. Copolyaspartates with a low degree of alkylation are in the alpha-helical conformation over the low temperature range and adopt the omega-helical conformation in the high temperature range, indicative of a thermal alpha-omega transition. A small number of alkyl groups can be incorporated into the benzene ring stacking of the omega-helix, but not a large number. All the copolyaspartates can assume the beta-form at high temperatures. The helix conformation is not significantly affected by the formation of side chain crystals of the copolyaspartate with a large number of stearyl groups, in contrast to copolyglutamate.     

Synthesis of 3-[4-(dimethylamino)phenyl]alkyl-2-oxindole derivatives and their effects on neuronal cell death

Novel 3-[4-(dimethylamino)phenyl]alkyl-2-oxindole analogs were synthesized by either of the following two pathways: (1) a sequence of Knoevenagel condensation of oxindole with (4-dimethylamino)cinnamaldehyde–hydrogenation, or (2) alkylation of oxindole dianion with [(4-dimethylamino)phenyl]alkyl halides. Subsequent alkylation at C-3 and/or N-1 of the oxindole skeleton by anion-based methods provided additional substituted derivatives for structure-activity relationship studies. Their effects on neuronal cell death induced by oxidative stress were evaluated by lactate dehydrogenase assay. Compounds with the alkyl chain length of 2–4 significantly suppressed the neuronal cell death. No significant change occurred in the activity by substitution with less-polar groups. The stereochemistry at C-3 of the oxindole core was also irrelevant for the neuroprotective effects of these compounds.     

Self-assembled nanoparticles of dextrin substituted with hexadecanethiol

The amphiphilic molecule dextrin-VA-SC16 (dexC16) was synthesized and studied in this work. DexC16 has a hydrophilic dextrin backbone with grafted acrylate groups (VA) substituted with hydrophobic 1-hexadecanethiol (C16). A versatile synthetic method was developed allowing control of the dextrin degree of substitution with the hydrophobic chains (DSC16, number of alkyl chains per 100 dextrin glucopyranoside residues). Materials with different DSC16 were prepared and characterized using 1H NMR. DexC16 self-assembles in water through association of the hydrophobic alkyl chains, originating nanoparticles. The nanoparticles properties were studied by dynamic light scattering (DLS), fluorescence spectroscopy, and atomic force microscopy (AFM).     

Amphiphilic biopolymers (amphibiopols) as new surfactants for membrane protein solubilization

The aim of this study was to develop new surfactants for membrane protein solubilization, from a natural, biodegradable polymer: the polysaccharide pullulan. A set of amphiphilic pullulans (HMCMPs), differing in hydrophobic modification ratio, charge ratio, and the nature of the hydrophobic chains introduced, were synthesized and tested in solubilization experiments with outer membranes of Pseudomonas fluorescens. The membrane proteins were precipitated, and then resolubilized with various HMCMPs. The decyl alkyl chain (C(10)) was the hydrophobic graft that gave the highest level of solubilization. Decyl alkyl chain-bearing HMCMPs were also able to extract integral membrane proteins from their lipid environment. The best results were obtained with an amphiphilic pullulan bearing 18% decyl groups (18C(10)). Circular dichroism spectroscopy and membrane reconstitution experiments were used to test the structural and functional integrity of 18C(10)-solubilized proteins (OmpF from Escherichia coli and bacteriorhodopsin from Halobacterium halobium). Whatever their structure type (alpha or beta), 18C(10) did not alter either the structure or the function of the proteins analyzed. Thus, HMCMPs appear to constitute a promising new class of polymeric surfactants for membrane protein studies.     

Isolation and identification of products from alkylation of nucleic acids: ethyl- and isopropyl-purines.

Ethylation and isopropylation of guanine in alkaline solution, or of adenine in formic acid, by alkyl methanesulphonates gave the following products: 1-, N2-, 3-, O6-, 7- and 9-alkylguanines; 1-, 3-, 7- and 9-alkyladenines. The products were identified from their characteristic u.v-absorption spectra, by comparison with either known ethyladenines or with the corresponding known methyladenines, and were also characterized by mass spectrometry. Their chromatographic properties on paper, t.l.c. and various columns were determined. DNA was alkylated in neutral solution with 14C-labelled alkyl methanesulphonates and the ratios of the alkylpurines formed were obtained, and compared for alkylation by methyl, ethyl and isopropyl methanesulphonates and by N-methyl-N-nitrosourea. The extents of alkylation at O-6 of guanine relative to those at N-7 of guanine varied with the reactivity of the methylating agents according to the predictions of Swain & Scott (1953) relating nucleophilicity of the groups alkylated with the substrate constants of the alkylating agents. The relative extents of alkylation at N-3 of adenine did not follow this correlation.     

Synthesis and characterization of amphiphilic monodisperse compounds and poly(ethylene imine)s: influence of their microstructures on the antimicrobial properties

Amphiphilic monodisperse compounds (series B-I and B-II) and poly(ethylene imine)s (PEI-I, PEI-II, and PEI-III) with different microstructures were prepared from primary amines or poly(ethylene imine) with functional carbonates bearing cationic, hydrophobic, or amphiphilic groups. Their inhibition potential against proliferation of E. coli , S. aureus , and B. subtilis was investigated and their hemolytic activities were determined. The influence of the microstructures, the alkyl chain length and the distribution of cationic and hydrophobic groups, on their antimicrobial efficacy was studied. Amphiphilic compounds with long alkyl chains (C14-C18) directly linked to the cationic groups (series B-I) are more effective against both Gram-positive and Gram-negative bacteria than amphiphilic compounds in which the hydrophobic and cationic groups (series B-II) are connected by a spacer. Poly(ethylene imine)s with amphiphilic grafts (B-I) called PEI-II are more effective than amphiphilic PEIs with the same alkyl chain but with randomly linked cationic and hydrophobic graft called PEI-I or with the amphiphilic grafts (B-II) called PEI-III. The influence of the inoculum size on the MIC value was investigated exemplarily with compounds of series B-I against S. aureus .     

Inhibition of classical pathway of complement activation with negative charged derivatives of bisphenol A and bisphenol disulphates

In order to obtain strong inhibitors of classical pathway of complement activation the low weight negative charged compounds have been investigated. On the basis of bisphenol A anionic derivatives with one or two carboxylic, sulphate and phosphate groups the critical role of negative charged groups for complement-inhibiting activity has been established. It was determined that two sulphate or phosphate groups in the molecule provide the most inhibiting effect. At the next stage a set of bisphenol disulphates of varying structures has been synthesized and investigated. Bulky hydrophobic groups (cyclohexyliden, fluorenyliden, anthronyliden) at the central part of the bisphenol molecule it was found to increase complement-inhibiting activity markedly. The replacement of the ortho-positions to the charged group by halogens or alkyl groups (allyl, propyl) increases the inhibiting effect. It was showed by ELISA that several compounds studied interact with C1q, C1r /C1s components of complement. For the set of bisphenol disulphates the QSAR equation with hydrophobic coefficient and electronic parameters has been formulated. Both hydrophobic and electrostatic interactions it was established to have a great significance for the inhibition of classical pathway of complement activation.     

A biologically active hydrophobic T-1-conotoxin from the venom of Conus spurius

A major, very hydrophobic peptide, sr5a, was purified from the venom duct of Conus spurius specimens collected in the Yucatan Channel, Mexico. Its amino acid sequence (IINWCCLIFYQCC; calculated monoisotopic mass assuming two disulfide bridges 1616.68 Da) was determined by automatic Edman degradation after reduction and alkylation, and confirmed by mass spectrometry (ESI monoisotopic mass, 1616.60; MALDI monoisotopic mass 1616.42 Da). The primary structure of sr5a showed the pattern that characterizes the family of the T-1-conotoxins, which belong to the T-superfamily of conotoxins. The disulfide bonds were determined by partial reduction and alkylation with N-ethylmaleimide, followed by total reduction and alkylation with 4-vinylpyridine, and automatic Edman sequencing. The connectivity of the Cys residues (I-III, II-IV) is the same as that found in the T-1-conotoxin family. When injected intracranially (2.0 nmol) into mice, peptide sr5a caused depressed behavioral activity.     

Cytotoxicity of alkylating agents towards sensitive and resistant strains of Escherichia coli in relation to extent and mode of alkylation of cellular macromolecules and repair of alkylation lesions in deoxyribonucleic acids

1. A quantitative study was made of the relationship between survival of colony-forming ability in Escherichia coli strains B/r and B(s-1) and the extents of alkylation of cellular DNA, RNA and protein after treatment with mono- or di-functional sulphur mustards, methyl methanesulphonate or iodoacetamide. 2. The mustards and methyl methanesulphonate react with nucleic acids in the cells, in the same way as found previously from chemical studies in vitro, and with proteins. Iodoacetamide reacts only with protein, principally with the thiol groups of cysteine residues. 3. The extents of alkylation of cellular constituents required to prevent cell division vary widely according to the strain of bacteria and the nature of the alkylating agent. 4. The extents of alkylation of the sensitive and resistant strains at a given dose of alkylating agent do not differ significantly. 5. Removal of alkyl groups from DNA of cells of the resistant strains B/r and 15T(-) after alkylation with difunctional sulphur mustard was demonstrated; the product di(guanin-7-ylethyl) sulphide, characteristic of di- as opposed to mono-functional alkylation, was selectively removed; the time-scale of this effect suggests an enzymic rather than a chemical mechanism. 6. The sensitive strain B(s-1) removed alkyl groups from DNA in this way only at very low extents of alkylation. When sensitized to mustard action by treatment with iodoacetamide, acriflavine or caffeine, the extent of alkylation of cellular DNA corresponding to a mean lethal dose was decreased to approximately 3 molecules of di(guanin-7-ylethyl) sulphide in the genome of this strain. 7. Relatively large numbers of monofunctional alkylations per genome can be withstood by this sensitive strain. Iodoacetamide had the weakest cytotoxic action of the agents investigated; methyl methanesulphonate was significantly weaker in effect than the monofunctional sulphur mustard, which was in turn weaker than the difunctional sulphur mustard. 8. Effects of the sulphur mustards on nucleic acid synthesis in sensitive and resistant strains were studied. DNA synthesis was inhibited in both strains at low doses in a dose-dependent manner, but RNA and protein synthesis were not affected in this way. 9. DNA synthesis in E. coli B(s-1) was permanently inhibited by low doses of mustards. In the resistant strains 15T(-) and B/r a characteristic recovery in DNA synthesis was observed after a dose-dependent time-lag. This effect could be shown at low doses in the region of the mean lethal dose. 10. Cellular DNA was isotopically prelabelled and the effect of mustards on stability of DNA was investigated. With resistant strains a dose-dependent release of DNA nucleotide material into acid-soluble form was found; this was much more extensive with the difunctional mustard (about 400 nucleotides released per DNA alkylation) than with the monofunctional mustard (about 10 nucleotides per alkylation). With the sensitive strain no dose-dependent release was found, though the DNA was less stable independent of cellular alkylation. 11. The results are discussed in terms of the concepts that alkylation of cellular DNA induces lesions which interfere with DNA replication, but which can be enzymically ;repaired'. The possible nature of these lesions is discussed in terms of the known reactions of the alkylating agents with DNA.     

Use of self-assembled monolayers of different wettabilities to study surface selection and primary adhesion processes of green algal (Enteromorpha) zoospores

We investigated surface selection and adhesion of motile zoospores of a green, macrofouling alga (Enteromorpha) to self-assembled monolayers (SAMs) having a range of wettabilities. The SAMs were formed from alkyl thiols terminated with methyl (CH(3)) or hydroxyl (OH) groups or mixtures of CH(3)- and OH-terminated alkyl thiols and were characterized by measuring the advancing contact angles and by X-ray photoelectron spectroscopy. There was a positive correlation between the number of spores that attached to the SAMs and increasing contact angle (hydrophobicity). Moreover, the sizes of the spore groups (adjacent spores touching) were larger on the hydrophobic SAMs. Video microscopy of a patterned arrangement of SAMs showed that more zoospores were engaged in swimming and "searching" above the hydrophobic sectors than above the hydrophilic sectors, suggesting that the cells were able to "sense" that the hydrophobic surfaces were more favorable for settlement. The results are discussed in relation to the attachment of microorganisms to substrata having different wettabilities.     

A novel surface-active peptide derivative exhibits in vitro inhibition of platelet aggregation

A tetrapeptide corresponding to a region of the N-terminal portion of lactotransferrin with hydrophobic alkyl groups at the terminal ends was synthesized and its physicochemical properties as well as its effect on thrombin-stimulated platelet aggregation were examined. The tetrapeptide derivative, in the aggregated state, produced inhibitory effect on platelet aggregation. The concentration dependent activity of the peptide was analyzed in the light of micelle formation, with the micellar aggregate comprising four tetrapeptide units. The unique action of this peptide derivative on the inhibition of platelet aggregation might be useful in the development of potent antithrombotic drugs.     

Change in membrane fluidity induced by polyphenols is highly dependent on the position and number of galloyl groups

The cell membrane fluidity was very important in adipogenesis and galloyl groups on polyphenolic structures could enhance their antiadipogenic activity. However, the effect of polyphenols on membrane fluidity and the role of galloyl groups in fluidity changes remain unclear. Therefore, the present study chose structurally different polyphenols to compare their effects on the membrane morphology and fluidity of 3T3-L1 preadipocytes, and then the reasons behind the changes of membrane fluidity induced by galloylated polyphenols were explored from structural and molecular insights using liposome model and molecular dynamic simulation technology. Our results indicated that galloylated polyphenols could significantly change 3T3-L1 cell membrane morphology and decrease membrane fluidity, while non-galloylated ones could not. The membrane interference effect of polyphenols was enhanced as the number of galloyl groups increased. Morever, the decrease in membrane fluidity induced by galloylated polyphenols was due to the disturbance of polyphenols on lipid alkyl chains in the cell membrane. Galloylated polyphenols could not only locate in the polar head, but also insert into hydrophobic center of lipid bilayer to interfere with the lipid alkyl chains arrangement, thus decreasing the membrane fluidity and showing strong affinity for the membrane. In addition, differences in position of galloyl groups in polyphenols induced distinct effect on cell membranes interactions, thus affecting the binding manner and bioactivity. The results expanded the understanding on the strong antiadipogenic activity of galloylated polyphenols through the aspect of their effects on cell membrane by both experimental and theoretically simulated ways.     

Polymer-bound alkyltriazenes for mild racemization-free esterification of amino acid and peptide derivatives.

A novel tool for polymer-assisted solution phase (PASP) esterification of amino acid and peptide derivatives has been developed. When treated with carboxylic acids, polymer-bound alkyltriazenes react with a loss of nitrogen and transfer of the alkyl moiety to the carboxylate anion to form the corresponding alkyl esters. There are no limitations with regard to either the protecting groups or the nature of the amino acid. Furthermore no racemization occurs at the chiral centers of the amino acids as demonstrated by chiral GC-MS analyses. Alkyltriazene-resins were also applied successfully to the esterification of peptide acids and other peptidic structures, such as tripalmitoyl-S-glyceryl-cysteine (Pam3Cys). The triazene-mediated esterification reaction is exceptionally mild, and there is no need for prior activation of the carboxy groups. This method is therefore particularly suitable for the alkylation of complex peptidomimetic structures prone to racemization and for acid-sensitive structures.     

DNA-directed alkylating ligands as potential antitumor agents: sequence specificity of alkylation by intercalating aniline mustards

The sequence preferences for alkylation of a series of novel parasubstituted aniline mustards linked to the DNA-intercalating chromophore 9-aminoacridine by an alkyl chain of variable length were studied by using procedures analogous to Maxam-Gilbert reactions. The compounds alkylate DNA at both guanine and adenine sites. For mustards linked to the acridine by a short alkyl chain through a para O- or S-link group, 5'-GT sequences are the most preferred sites at which N7-guanine alkylation occurs. For analogues with longer chain lengths, the preference of 5'-GT sequences diminishes in favor of N7-adenine alkylation at the complementary 5'-AC sequence. Magnesium ions are shown to selectively inhibit alkylation at the N7 of adenine (in the major groove) by these compounds but not the alkylation at the N3 of adenine (in the minor groove) by the antitumor antibiotic CC-1065. Effects of chromophore variation were also studied by using aniline mustards linked to quinazoline and sterically hindered tert-butyl-9-aminoacridine chromophores. The results demonstrate that in this series of DNA-directed mustards the noncovalent interactions of the carrier chromophores with DNA significantly modify the sequence selectivity of alkylation by the mustard. Relationships between the DNA alkylation patterns of these compounds and their biological activities are discussed.     

Structure-based design and synthesis of phosphinate isosteres of phosphotyrosine for incorporation in Grb2-SH2 domain inhibitors. Part 2

A series of novel phosphinates, derived from 4-phosphonomethylphenylalanine, are described as isosteres of phosphotyrosine. Benzyl (or alkyl) phosphinomethylphenylalanine derivatives were prepared by alkylation of an amino acid P-H phosphinate.     

N-alkylated chitosan as a potential nonviral vector for gene transfection

Alkylated chitosans (ACSs) were prepared by modifying chitosan (CS) with alkyl bromide. The self-aggregation of ACSs in acetic acid solution was characterized by fluorescence spectroscopy and dynamic light scattering method. The results indicate that introducing alkyl side chains leads to the self-aggregation of ACSs, and CS with a 99% deacetylation degree shows no aggregation due to the electrostatic repulsion. The electrophoresis experiment demonstrates that the complex between CS and DNA was formed at a charge ratio (+/-) of 1/1; ACS/DNA complexes were formed at a lower charge ratio (+/-) of 1/4. A small amount of alkylated chitosans play the same shielding role as chitosan in protecting DNA from DNase hydrolysis. Differential scanning calorimetry (DSC) and atomic force microscopy (AFM) were employed separately to investigate the thermodynamic behavior of dipalmitoyl-sn-glycero-3-phosphocholine (DPPC)/CS and DPPC/ACS mixtures and the variation in topological structure of DPPC membrane induced by CS and ACS. It is shown that CS and ACS can cause the fusion of DPPC multilamellar vesicles as well as membrane destabilization. In contrast, the perturbation effect induced by ACS is more evident due to the hydrophobic interaction. CS and ACS were used to transfer plasmid-encoding CAT into C(2)C(12) cell lines. Upon elongating the alkyl side chain, the transfection efficiency is increased and levels off after the number of carbons in the side chain exceeds 8. It is proposed that the higher transfection efficiency of ACS is attributed to the increasing entry into cells facilitated by hydrophobic interactions and easier unpacking of DNA from ACS carriers due to the weakening of electrostatic attractions between DNA and ACS.     

Regioselective synthesis of long-chain ethers and their sulfates derived from methyl beta-D-galactopyranoside and derivatives via dibutylstannylene acetal intermediates

A number of different conditions were investigated for the alkylation of the dibutylstannylene acetals of methyl beta-d-galactopyranoside with long-chain primary alkyl bromides, decyl, dodecyl, and tetradecyl bromide. The best yields of the major products, the 3-O-alkyl ethers, were obtained by reaction of the alkyl bromide with the monodibutylstannylene acetal in DMF in the presence of cesium fluoride for extended periods of time at moderate temperatures (65 degrees C). These products were always accompanied by minor amounts of the 3,6-di-O-alkyl derivative. Performing the reaction with excess alkyl halide on the bis(dibutylstannylene) acetal resulted in more of the 3,6-di-O-alkyl derivative, particularly for the shorter alkyl bromides, but this product was never predominant. Sulfation of the dibutylstannylene acetal of methyl 3-O-tetradecyl-beta-D-galactopyranoside resulted in the 6-sulfate in 96% yield.     

Identification of bis-quindolines as new antiinfective agents

Several N-substituted quindolines were made to further evaluate the role of N-alkylation on the activity of indoloquinolines as antifungal agents. While N-5 substitution is required for these activities, N-10 alkylation alone leads to inactive products but is tolerated in the presence of N-5 alkyl groups. It was also discovered that bis-quindolines appear to have a more expanded antimicrobial spectrum and lower cytotoxicity than their monomeric counterparts.     

The effect of hydrophobic interaction on endotoxin adsorption by polymeric affinity matrix

Endotoxin, a major pyrogen of concern to the biological industry, is a lipopolysaccharide containing a highly hydrophobic region, lipid A, in its structure. The effect of hydrophobic interaction on endotoxin adsorption from an aqueous solution was studied by covalently bonding aminoalkyl groups with varying hydrocarbon lengths to a cellulose and acrylic composite matrix. The amount of endotoxin adsorbed on the matrix increased with the increasing length of alkyl groups, demonstrating the contribution of hydrophobic interaction between endotoxin and the solid matrix. Both the hydrophobic and the charge interaction prove to be effective for endotoxin adsorption, and a synergistic effect from the dual chemical forces is achievable under specified conditions. The effect of solvent, pH and salts on endotoxin adsorption provides further evidence for the importance of hydrophobic force as a means of removing endotoxin from aqueous solutions.     

Hydrophobic displacement chromatography of proteins

Displacement chromatography of proteins was successfully carried out in both hydrophobic interaction and reversed-phase chromatographic systems using low-molecular weight displacers. The displacers employed for hydrophobic displacement chromatography were water soluble, charged molecules containing several short alkyl and/or aryl groups. Spectroscopy was employed to verify the absence of structural changes to the proteins displaced on these hydrophobic supports. Displacement chromatography on a reversed-phase material was employed to purify a growth factor protein from its closely related variants, demonstrating the high resolutions that can be achieved by hydrophobic displacement chromatography. This process combines the high-resolution/high-throughput characteristics of displacement chromatography with the unique selectivity of these hydrophobic supports and offers the chromatographic engineer a powerful tool for the preparative purification of proteins.