Aminoethyl Group Research Articles

Poly(β-aminoester)s with Pendant Primary Amines for Efficient Gene Delivery

Three hydrolytically degradable poly(beta-aminoester)s containing ester bonds in the main chain and primary amines in the side chain, synthesized by Michael polyaddition, were applied to deliver foreign DNA into cells in vitro. These linear polycations can condense DNA into small-sized particles with positive surface charge at high N/P ratios. Their high buffer capacity at pH 5-7 facilitated the escape of DNA from the endosome and resulted in efficient gene expression. Under the optimal conditions, poly(beta-aminoester)s with a pendant aminoethyl group showed higher transfection efficiencies than branched poly(ethylenimine) (PEI) 25KDa in 293T cells. The effect of side chain structure of the poly(beta-aminoester) on transfection efficiency has been investigated, which indicated that the poly(beta-aminoester) containing the pendant aminoethyl group was the most efficient carrier for both of 293T cells and COS-7 cells. The combination of hydrolytical degradation, high buffer capacity, relatively low cytotoxicity, and high transfection efficiency suggested that this kind of poly(beta-aminoester)s are novel promising nonviral gene carriers.

Acetylcholinesterase inhibitory activity of novel chitooligosaccharide derivatives

In this study, three kinds of chitooligosaccharides (COS) with different substitution groups, including aminoethyl (AE), dimethylaminoethyl (DMEM) and diethylaminoethyl (DEAE) groups, were evaluated for their inhibitory activities against cholinesterase (acetylcholinesterase and butyrylcholinesterase). These COS derivatives were synthesized and their structures were characterized as AE-COS, DMAE-COS and DEAE-COS, respectively; the structures were elucidated via spectroscopic techniques ( 1H NMR). Of these, DMAE- and DEAE-COS were synthesized for the first time in this work. These COS derivatives evidenced potent acetylcholinesterase (AChE) inhibitory activities with IC 50 values of 56.5 ± 0.26, 24.1 ± 0.39 and 9.2 ± 0.33 μg/ml, respectively; however, these compounds exhibited no activity against butyrylcholinesterase. These were further analyzed for their inhibitory pattern on AChE via Lineweaver–Burk plots. AE-COS showed non-competition type inhibition, and DMAE-COS and DEAE-COS exhibited competition type inhibition against AChE. In this study, we suggested that COS derivatives have potential as AChE inhibitors for preventing Alzheimer’s disease.

Electronic Spectra and Nonlinear Optical Properties of Thiazole Chromophores

Electronic spectra and second-order nonlinear optical (NLO) properties of eight thiazole chromophores were calculated by density functional theory (DFT) at B3LYP/6-31+G level. Results showed that thiazole chromophore molecules with active hydroxy-ethyl, alkoxy-ethyl, fluoro-ethyl, and amino-ethyl groups have high β values of about 1.6×10-28 esu which are in good agreement with the available experimental results. Maximum absorption wavelengths λmax of the compounds in the gas phase ranged from 480 to 488 nm. The λmax was red-shifted as the solvent polarity increased for the thiazole chromophore molecules with an active hydroxy-ethyl group.

The conformational landscape of 5-methoxytryptamine studied by rotationally resolved fluorescence spectroscopy and resonant ionization spectroscopy

Rotationally resolved electronic spectra of three different conformers of 5-methoxytryptamine were recorded in a molecular beam. 5-Methoxy substitution reduces the number of observed conformers to three compared to seven that have been reported for tryptamine. Quantum chemical calculations indicate that anti-rotamers of the methoxy-group are more stable relative to the syn-forms. Two gauche structures (Gpy(in) and Gph(in)) of the ethylamino group with respect to the indole chromophore were found to be less stable than the other seven. The lowest electronically excited state has been identified as the (1)L(b) one for all observed conformers which was confirmed by quantum-chemical calculations. Based on the comparison of rotational constants obtained from fits using evolutionary algorithms with those from calculations, the three observed conformers were determined to be the Gpy(up), Gph(up), Gpy(out) ethylamino side-chain conformations.

Electrochemical reduction of atrazine: NMR evidence for reduction of the triazine ring

Over the last decade, researchers have investigated the electrochemical reduction pathways of a popular triazine herbicide known as atrazine (2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine) using controlled potential electrolysis and a mercury pool electrode. There is general agreement that a two-electron dechlorination process is a likely reduction pathway. However, Faraday’s law calculations indicated an overall four-electron reduction process. Additional pathways including the elimination of the ethyl group, elimination of the ethylamino group, and reduction of the triazine ring have been proposed. We believe to have NMR evidence for the reduction of the triazine ring using controlled potential electrolysis and a mercury pool electrode. We would like to add our results to the ongoing studies of this important triazine herbicide.

Effects of structure and modification on sustained release properties of starches

Starches of different sources and compositions were investigated to determine the effect of structure and chemical modification on the sustained release properties of the resultant modified starches. Starches were cross-linked with epichlorohydrin and substituted with carboxymethyl or aminoethyl groups at different levels. Substitution efficiency was overall higher for waxy corn and potato starches than for Hylon VII, and was higher for starches at low cross-linking levels than those at high cross-linking ones. Waxy corn starch displayed better sustained release properties when cross-linked to a lower level, whereas Hylon VII showed better performances when cross-linked to a higher level. Matrices substituted with carboxymethyl and aminoethyl groups at the high level showed better sustained release properties than those substituted at the low level. The proportion and structure of amylose and amylopectin in starches from different botanical sources strongly influenced the level of modification required to produce a satisfactory sustained release matrix.

Preparation of Zwitterionic Ribonucleoside Phosphoramidites for Solid-Phase siRNA Synthesis

RNA oligomers, carrying 2'-O-modified nucleosides, proved to be extremely useful in different antisense strategies, including RNAi. The 2'-O-alkyl modification, carrying an amino functionality, deserves special attention due to its ability to neutralize the negatively charged phosphate backbone, leading to improved physicochemical and pharmaceutical properties of antisense agents. Here, we report a very short, convenient, and straightforward synthesis of phosphoramidites for all four 2'-aminoethyl-modified natural ribonucleosides, where the aminoethyl group is introduced in a single alkylation step.

1,3-Bis(ethylamino)-2-nitrobenzene, 1,3-bis(n-octylamino)-2-nitrobenzene and 4-ethylamino-2-methyl-1H-benzimidazole

1,3-Bis(ethylamino)-2-nitrobenzene, C(10)H(15)N(3)O(2), (I), and 1,3-bis(n-octylamino)-2-nitrobenzene, C(22)H(39)N(3)O(2), (II), are the first structurally characterized 1,3-bis(n-alkylamino)-2-nitrobenzenes. Both molecules are bisected though the nitro N atom and the 2-C and 5-C atoms of the ring by twofold rotation axes. Both display intramolecular N-H...O hydrogen bonds between the amine and nitro groups, but no intermolecular hydrogen bonding. The nearly planar molecules pack into flat layers ca 3.4 A apart that interact by hydrophobic interactions involving the n-alkyl groups rather than by pi-pi interactions between the rings. The intra- and intermolecular interactions in these molecules are of interest in understanding the physical properties of polymers made from them. Upon heating in the presence of anhydrous potassium carbonate in dimethylacetamide, (I) and (II) cyclize with formal loss of hydrogen peroxide to form substituted benzimidazoles. Thus, 4-ethylamino-2-methyl-1H-benzimidazole, C(10)H(13)N(3), (III), was obtained from (I) under these reaction conditions. Compound (III) contains two independent molecules with no imposed internal symmetry. The molecules are linked into chains via N-H...N hydrogen bonds involving the imidazole rings, while the ethylamino groups do not participate in any hydrogen bonding. This is the first reported structure of a benzimidazole derivative with 4-amino and 2-alkyl substituents.

Pd-catalyzed coupling of arylamines and 2-bromo-3,3,3-trifluoropropene

Palladium-catalyzed coupling of arylamines and 2-bromo-3,3,3-trifluoropropene (BTP) was investigated. When a toluene solution of aniline and BTP was heated at 110 °C in the presence of Pd 2(dba) 3·CHCl 3, 1,1′-diphenylphosphinoferrocene and Cs 2CO 3 under an argon atmosphere, N-(1,1,1-trifluoro-2-propylidene)aniline was obtained in excellent GC and 19F NMR yields (99%) and isolated yield (92%). Cs 2CO 3 was exclusively effective in the coupling reaction among the bases tested. The coupling using 2-aminobenzonitriles as substrates provided not only 2- N-(1,1,1-trifluoro-2-propylidene)aminobenzonitriles but also 4-amino-2-trifluoromethylquinolines and 2-trifluoromethyl-4- N-(1,1,1-trifluoro-2-propylidene)aminoquinolines. The strong electron-withdrawing character of CF 3 will enhance the acidity of the methyl proton in the N-(1,1,1-trifluoromethyl-2-propylidene)amino group, resulting in the attack of the methyl proton to the carbon in the cyano group to give 4-amino-2-trifluoromethylquinolines. Moreover, the one-pot synthesis of 2-trifluoromethylindoles with 2-bromoanilines and BTP was achieved by use of Pd(OAc) 2, 2-dicyclohexylphosphino-2′,4′,6′-triisopropylbipenyl and Cs 2CO 3. The Pd-catalyzed intramolecular Heck coupling of the vinyl group in 2-bromo- N-(1-trifluoromethyl)vinylanilines, which is the tautmeric isomer of 2-bromo- N-(1,1,1-trifluoro-2-propylidene)anilines, and the C Br bond presumably furnished indole rings. The C N double bond of N-(1,1,1-trifluoro-2-propylidene)amino group obtained here was smoothly hydrogenated to N-(1-methyl-2,2,2-trifluoro)ethylamino group using LiAlH 4 or H 2 with Pd/C.

5-(Diethylamino)-2-[(Z)-(1-naphthylimino)methyl]phenol

In the title molecule, C21H22N2O, the angle between the mean planes of the 1-naphthylimino and 2-methylyphenyl groups is 63.3 (2)degrees. The two diethyl extensions from the 5-diethylamino group are twisted in a + and - antiperiplanar conformation. One of the ethyl arms is disordered over two conformations with occupancies of 0.644 (3) and 0.356 (3). The crystal packing is stabilized by intermolecular C-H center dot center dot center dot pi interactions between pi orbitals of the 1-naphthyl (I/II) rings and H atoms from a nearby benzene ring as well as from an ethyl C atom of an ethylamino group. The molecules are stacked along the b axis in alternate inverted chains with the 1-naphthyl rings obliquely parallel to the ab face of the unit cell. Intramolecular interactions between the hydroxyl H atom and the imino N atom provide additional conformational stability.

Aminoethyl chitooligosaccharides inhibit the activity of angiotensin converting enzyme

In the present research, chitooligosaccharides (COS) with molecular weight 800–3000 Da and 90% of deacetylation were chemically modified by grafting 2-chloroethylamino hydrochloride at C-6 position to synthesize angiotensin I converting enzyme (ACE) inhibitory chitin derivatives based on the properties of ACE inhibitors. The synthetic product was designated as aminoethyl chitooligosaccharide (AE-COS) with molecular weight 800.79–4765 Da. Its IC 50 value on ACE was 0.8017 μg/mL. Furthermore, Lineweaver-Burk plots revealed that the inhibition was competitive via obligatory binding site of ACE. Therefore, these results exhibited that substitution of the hydrogen atom at the C-6 position of pyranose residue by the aminoethyl group promotes ACE inhibitory effects of COS.

Synthesis of Aminated Poly(1-vinylimidazole) for a New pH-Sensitive DNA Carrier

The poly(1-vinylimidazole) (PVIm) with aminoethyl groups has been synthesized as a new pH-sensitive DNA carrier. The resulting aminated PVIm (PVIm-NH2) was water-soluble in spite of the deprotonation of the imidazole groups at physiological pH, as determined by acid-base titration and solution turbidity measurement. Hemolysis assay showed the PVIm-NH2 enhanced membrane disruptive ability at endosomal pH, owing to the protonation of the imidazole groups with a pKa value around 6.0. Agarose gel retardation assay proved that the introduced aminoethyl groups worked as an anchor groups to retain DNA. The resulting PVIm-NH2 formed the ternary complex with DNA and lactosylated poly(L-lysine), leading to the significant gene expression in human hepatoma HepG2 cells.

Design of Aminated Poly(1-vinylimidazole) for a New pH-Sensitive Polycation To Enhance Cell-Specific Gene Delivery

Poly(1-vinylimidazole) (PVIm) with aminoethyl groups has been synthesized as a new pH-sensitive polycation to enhance cell-specific gene delivery. The resulting aminated PVIm (PVIm-NH2) was water-soluble despite deprotonation of the imidazole groups at physiological pH, as determined by acid-base titration and solution turbidity measurement. Hemolysis assay showed that PVIm-NH2 enhanced membrane disruptive ability at endosomal pH, owing to the protonation of the imidazole groups with a pKa value around 6.0. Agarose gel retardation assay proved that the introduced aminoethyl groups worked as anchor groups to retain DNA. Furthermore, the ternary complex of DNA, PVIm-NH2, and a poly(L-lysine) conjugated with lactose molecules, PLL-Lac, at pH 7.4 dissociated the PLL-Lac polycation by protonation of the imidazole groups of PVIm-NH2 at pH 6.0. The resulting PVIm-NH2/DNA binary complexes easily released DNA, as compared with the PLL-Lac/PVIm-NH2/DNA ternary complex, which was examined by competitive exchange with dextran sulfate. By using PVIm-NH2 as a pH-sensitive DNA carrier, as well as PLL-Lac as a cell-targeting DNA carrier, the resulting ternary complex specifically mediated the gene expression, which depended on the protonation of the imidazole groups, on human hepatoma HepG2 cells with asialoglycoprotein receptors. These results suggest that the cell-specific gene delivery mediated by PLL-Lac was enhanced by PVIm-NH2 as a new pH-sensitive polycation.

Gene delivery with biocompatible cationic polymer: Pharmacogenomic analysis on cell bioactivity

The availability of non-viral gene delivery systems is determined by their capacity and safety during gene introduction. In this study, the safety issues of polyplex were analyzed from the standpoint of the biomolecular mechanisms. P[Asp(DET)], a newly developed polymer, polyasparagine carrying the N-(2-aminoethyl)aminoethyl group as the side chain which was recently revealed to show good transfection efficiency to primary cells, was compared to conventional linear poly(ethylenimine) (LPEI). After transfection toward a bioluminescent cell line, P[Asp(DET)] maintained the expression level of stably expressing luciferase. In contrast, LPEI showed a decrease in the luciferase expression, while the similar expression of exogenous reporter gene was obtained. Evaluation of the housekeeping genes expression as well as the profiles of pDNA uptake after transfection suggested the time-dependent toxicity of LPEI that perturbs cellular homeostasis. Consistently, the induction of osteogenic differentiation by functional gene introduction was achieved only by P[Asp(DET)], even though appreciable expression of the gene was achieved by LPEI. It is crucial that this aspect of safety be taken into account, especially when the gene introduction is applied to primary cells to regulate such cell function as differentiation. This biomolecular analysis focusing on cellular homeostasis is beneficial for assessing the practicability of the gene delivery systems for clinical application.

Bacterial adhesion to and viability on positively charged polymer surfaces

Secondary and tertiary amino groups were introduced into polymer chains grafted onto a polyethylene flat-sheet membrane to evaluate the effects of surface properties on the adhesion and viability of a strain of the Gram-negative bacterium Escherichia coli and a strain of the Gram-positive bacterium Bacillus subtilis. The characterization of the surfaces containing amino groups, i.e. ethylamino (EA) and diethylamino (DEA) groups, revealed that the membrane potentials are proportional to amino-group densities and contact angle hysteresis. A high bacterial adhesion rate constant k was observed at high membrane potential, which indicates that membrane potential could be used as an indicator for estimating bacterial adhesion to the EA and DEA sheets, especially in B. subtilis. The bacterial adhesion rate constant of E. coli markedly increased at a membrane potential higher than -7.8 mV, whereas that of B. subtilis increased at a membrane potential higher than -8.3 mV, at which the dominant effect on bacterial adhesion is expected to change. The viability experiments revealed that approximately 80% of E. coli cells adhering to the sheets with high membrane potential were inactivated after a contact time of 8 h, whereas 60% of B. subtilis cells were inactivated. Furthermore, E. coli viability significantly decreased at a membrane potential higher than -8 mV, whereas B. subtilis viability decreased as membrane potential increased, which reflects differences in cell wall structure between E. coli and B. subtilis.

Low frequency backbone vibrations of individual conformational isomers: Tryptamine

The low frequency vibrations of the ethylamino backbone of six conformers of tryptamine have been studied in the ground and excited states using dispersed fluorescence spectroscopy, rotationally resolved laser induced fluorescence, and ab initio calculations. Four low frequency vibrational modes of the backbone, which involve torsional and librational motions of the ethylamino group, have been identified. The three anti conformers show a substantially different vibrational pattern than the four conformers in which the amino group is in gauche position with respect to the pyrrole and the phenyl ring, respectively.

An Enzyme-Linked Immunosorbent Assay (ELISA) to Determine the Specificity of the Sugar-Binding Protein NgcE, a Component of the ABC Transporter forN-Acetylglucosamine inStreptomyces olivaceoviridis

The ATP-binding cassette (ABC) transporter Ngc for N-acetylglucosamine (GlcNAc) of the chitin-degrader Streptomyces olivaceoviridis comprises the solute-binding protein NgcE, which has highest affinity for GlcNAc and N,N'-diacetylchitobiose {(GlcNAc)2} and reduced affinity for longer chitooligomers. NgcE was used to develop a generally applicable enzyme-linked immunosorbent assay (ELISA) system. As a prerequisite, the reducing end of (GlcNAc)2 was coupled with the ethylamino group of 2-(4-aminophenyl)ethylamine. The resulting conjugate was linked with amino groups of bovine serum albumin (BSA) to gain the neoglycoprotein BSA-APEA-(GlcNAc)2, which was fixed to wells in microtitre-plates. The NgcE protein was shown to bind efficiently to the immobilized BSA-APEA-(GlcNAc)2. In competition assays, the affinity of NgcE was 1,000-fold higher for GlcNAc and (GlcNAc)2 than for (GlcNAc)3 and (GlcNAc)4. These results are consistent with those previously obtained by surface plasmon resonance. Since the ELISA method can be performed very rapidly at low cost, it should be an efficient general tool to determine the affinity of a ligand to its cognate solute-binding protein.

PH-Dependent Dissociation of Carbohydrate Ligand Polycations from DNA Ternary Complexes

Abstract Here, we have demonstrated that carbohydrate ligand polycations are pH-dependently dissociated from DNA polyion complexes. In response to pH, DNA ternary complexes have dissociated the ligand polycations, resulting in DNA binary complexes. Namely, the ternary complexes of DNA, a poly(l-histidine) modified with aminoethyl groups, PLH-NH2, and a poly(l-lysine) conjugated with lactose molecules, PLL-Lac, at pH 7.5 have dissociated the PLL-Lac polycations by the protonation of imidazole groups of PLH-NH2 at pH 6.0. The pH-dependent dissociation of the ligand polycations from DNA polyion complexes at endosomal pH is expected to offer unique design for DNA delivery systems.

Design and Development of Thermolytic DNA Oligonucleotide Prodrugs

Deoxyribonucleoside phosphoramidites functionalized with the thermolytic 2-(N-formyl-N-methyl)aminoethyl group for phosphorus protection (1a-d) have been prepared and employed in the solid-phase synthesis of CpG ODN fma1555. Given that this modified oligonucleotide can be converted to the immunomodulatory CpG ODN 1555 under neutral conditions at 37 degrees C, its biologic activity was demonstrated in vivo by studies showing that intraperitoneal administration of CpG ODN fma1555 in mice resulted in the activation of cytokine-secreting splenocytes. Furthermore, administration of CpG ODN fma1555 to mice that were challenged intradermally in the ear with live L. major metacyclic promastigotes, reduced the severity of Leishmania skin lesions over time to an extent similar to that obtained with CpG ODN 1555. In another infectious model experiment, CpG ODN fma1555 protected newborn mice from death (65% survival) when administered 3 days before infection with the aggressive Tacaribe (TCRV) virus. A comparable immunoprotection was obtained by treatment of TCRV-infected mice with CpG ODN 1555 administered on the same day of infection (45% survival). However, when TCRV-infected mice were treated with CpG ODN fma1555 on the day of infection, they died as a consequence of the relatively slow conversion of the oligonucleotide prodrug to the bioactive CpG ODN 1555. Co-administration of both CpG ODN 1555 and CpG ODN fma1555 to mice 3 days prior to TCRV infection or on the day of infection provided protection from death (45-65% survival) and thus widened the immunoprotection window against TCRV-infection.

Development of an immunobiosensor assay for the beta-adrenergic compound zilpaterol*

A surface plasmon resonance biosensor method was developed to measure zilpaterol residues in sheep urine. A CM-5 sensor chip previously reacted with ethylenediamine to produce an aminoethyl group was coupled with 4-carboxybutyl zilpaterol activated using EDC/NHS. Five polyclonal and four monoclonal antibodies were screened for their suitability to detect low levels of zilpaterol using the biosensor technology. Total binding was greater for polyclonal than monoclonal antibodies, but a less diluted antibody solution was required for polyclonal antibodies. A fixed antibody concentration and various concentrations of zilpaterol were injected to obtain a standard curve for each antibody to allow for B0 and IC50 determination. The stability of the assay was assessed by the consistency of B0 in repeated experiments extending at least six hours. A measure of non-specific binding allowed the assessment of the specificity of the antibody-immobilized ligand interaction. The effect of varying concentrations of urine on B0 and IC50 was evaluated to assess the degree of “matrix” effect that would be present in an assay. Based on these criteria the most promising antibody (2E10, a monoclonal antibody) was selected for further evaluation. This antibody had good sensitivity with IC50=4.47±0.41 ng/ml (n=11) in buffer). Both intra- and inter-assay variation studies showed excellent recovery and reproducibility for concentrations between 2 ng/ml and 8 ng/ml. A comparison of the biosensor method with a previously developed ELISA demonstrated that both methods give equivalent results (slope of the correlation plot = 1.02) with a high correlation (r2=0.91) between them.