Low molecular weight linear polyethylenimine-b-poly(ethylene glycol)-b-polyethylenimine triblock copolymers: synthesis, characterization, and in vitro gene transfer properties

Novel ABA triblock copolymers consisting of low molecular weight linear polyethylenimine (PEI) as the A block and poly(ethylene glycol) (PEG) as the B block were prepared and evaluated as polymeric transfectant. The cationic polymerization of 2-methyl-2-oxazoline (MeOZO) using PEG-bis(tosylate) as a macroinitiator followed by acid hydrolysis afforded linear PEI-PEG-PEI triblock copolymers with controlled compositions. Two copolymers, PEI-PEG-PEI 2100-3400-2100 and 4000-3400-4000, were synthesized. Both copolymers were shown to interact with and condense plasmid DNA effectively to give polymer/DNA complexes (polyplexes) of small sizes (<100 nm) and moderate zeta-potentials (approximately +10 mV) at polymer/plasmid weight ratios > or =1.5/1. These polyplexes were able to efficiently transfect COS-7 cells and primary bovine endothelial cells (BAECs) in vitro. For example, PEI-PEG-PEI 4000-3400-4000 based polyplexes showed a transfection efficiency comparable to polyplexes of branched PEI 25000. The transfection activity of polyplexes of PEI-PEG-PEI 4000-3400-4000 in BAECs using luciferase as a reporter gene was 3-fold higher than that for linear PEI 25000/DNA formulations. Importantly, the presence of serum in the transfection medium had no inhibitive effect on the transfection activity of the PEI-PEG-PEI polyplexes. These PEI-PEG-PEI triblock copolymers displayed also an improved safety profile in comparison with high molecular weight PEIs, since the cytotoxicity of the polyplex formulations was very low under conditions where high transgene expression was found. Therefore, linear PEI-PEG-PEI triblock copolymers are an attractive novel class of nonviral gene delivery systems.     

Nonviral gene carriers based on diblock copolymers of poly(2-ethyl-2-oxazoline) and linear polyethylenimine

Diblock copolymers that consist of poly(2-ethyl-2-oxazoline) (PEOz) and linear polyethylenimine (LPEI) were prepared for use as nonviral gene carriers. The PEOz-b-LPEI copolymers were synthesized by coupling PEOz with LPEI in a thiol-disulfide exchange reaction between the sulfhydryl and pyridyl disulfide terminal groups. A polymer/DNA weight ratio (P/D) of over 12 was required to enable PEOz-b-LPEI to condense DNA completely. The DNA-condensing capability of the diblock copolymers was increased with increasing the hydrolytic degrees of the LPEI segment. The PEOz-b-LPEI polyplexes were stable in 150 mM NaCl aqueous solution and had a mean diameter around 190 nm, whereas BPEI and LPEI polyplexes formed large aggregates in the range 300-500 nm. In addition, these polyplexes exhibited the sensitivity to solution pH and were dissociated in the acidic buffers (pH < or = 5.5). The results of in vitro cell viability and luciferase assay indicated that PEOz-b-LPEI showed not only low cytotoxicity but also high transfection efficiency in gene expression.     

New amphiphilic poly(2-ethyl-2-oxazoline)/ poly(L-lactide) triblock copolymers

A new series of cationic, thermo-sensitive, and biodegradable poly(L-lactide)-poly(2-ethyl-2-oxazoline)-poly(L-lactide) (PLLA-PEOz-PLLA) triblock copolymers were synthesized by ring-opening polymerization. With increasing molecular weight and crystallinity of hydrophobic PLLA blocks, the critical micellization concentrations (CMC) occurred at lower concentration. The PLLA-PEOz-PLLA aqueous solution was transparent at room temperature. Heating the solution resulted in precipitations, which were caused by the combination of dehydration of water around PEOz and the aggregations of PLLA segments. Acid/base titration profiles indicated that PLLA-PEOz-PLLA were protonated at neutral and acidic conditions. Considerable buffering capacity was found over the entire pH range. The specific PLLA-PEOz-PLLA triblock copolymers with thermal- and pH-sensitive properties can be tailored by varying the compositions and can be applied as controlled release carries for biomedical applications.     

Synthesis and self-assembly of well-defined poly(amino acid) end-capped poly(ethylene glycol) and poly(2-methyl-2-oxazoline)

Poly(ethylene glycol) (PEG) and poly(2-methyl-2-oxazoline) (PMOx) are water-soluble, biocompatible polymers with stealth hemolytic activities. Poly(amino acid) (PAA) end-capped PEG and PMOx were prepared using amino-terminated derivatives of PEG and PMOx as macroinitiators for the ring-opening polymerization of γ-benzyl protected l-glutamate N-carboxyanhydride and S-benzyloxycarbonyl protected l-cysteine N-carboxyanhydride, respectively, in the presence of urea, at room temperature. The molecular weight of the PAA moiety was kept between M(n) = 2200 and 3000 g mol(-1). PMOx was polymerized by cationic ring-opening polymerization resulting in molecular weights of M(n) = 5000 and 10,000 g mol(-1), and PEG was a commercial product with M(n) = 5000 g mol(-1). Here, we investigate the self-assembly of the resulting amphiphilic block copolymers in water and the effect of the chemical structure of the block copolymers on the solution properties of self-assembled nanostructures. The PEG-block-poly(amino acid), PEG-b-PAA, and PMOx-block-poly(amino acid), PMOx-b-PAA, block copolymers have a narrow and monomodal molecular weight distribution (PDI < 1.3). Their self-assembly in water was studied by dynamic light scattering and fluorescence spectroscopy. In aqueous solution, the block copolymers associate into particles with hydrodynamic radii (R(H)) ranging in size from R(H) 70 to 130 nm, depending on the block copolymer architecture and the polymer molecular weight. Larger R(H) and critical association concentration values were obtained for copolymers containing poly(S-benzyloxycarbonyl-l-cysteine) compared to their poly(γ-benzyl-L-glutamate) analogue. FTIR investigations revealed that the poly(γ-benzyl-L-glutamate) block adopts a helical conformation, while the poly(S-benzyloxycarbonyl-L-cysteine) block exists as β-sheet.     

Derivatization of (+/-)-5-[(2-methylphenoxy)methyl]-2-amino-2-oxazoline,an imidazoline binding sites ligand,with (+)-(R)-alpha-methylbenzyl isocyanate for drug monitoring purposes

The derivatization of racemic 5-[(2-methylphenoxy)methyl]-2-amino-2-oxazoline, developed as an imidazoline binding sites ligand, with (+)-(R)-alpha-methylbenzyl isocyanate was performed in chloroform. The reaction led to two pairs of diastereomers, which were separated by RP-HPLC. A kinetic study of the derivatization reaction was achieved in order to establish conditions suitable for experimental drug monitoring.     

Estimation of the hydrophobicity of 2,4-diphenyl-1,3-oxazoline analogs and QSAR analysis of their ovicidal activity against Tetranychus [corrected] urticae

Partition coefficients of six 2-phenyl-1,3-oxazoline congeners containing 2-I, 2-NO2, 2-CF3, 2,6-(CH3)2, 2,6-F2, and 2-F-6-Cl substitutions on the phenyl moiety were measured in a 1-octanol/water system using the flask-shaking method. The effect on the hydrophobicity (LogP) of substituents on the phenyl moiety of 2-phenyl-1,3-oxazolines linearly correlated with that of benzamide congeners. logP values of other 2-(substituted phenyl)-1,3-oxazoline analogs were empirically estimated from the corresponding substituted benzamides. The ovicidal activity of 2-(substituted phenyl)-4-phenyl-1,3-oxazoline analogs against the two-spotted spider mite Tetranychus [corrected] urticae was quantitatively analyzed using the classical QSAR (Hansch-Fujita) method. Results showed that ovicidal activity increases with hydrophobicity. The introduction of inductive electron-withdrawing groups at ortho-positions increased ovicidal activity, but addition of steric bulk was unfavorable. Substitution at either the meta- or para-position was detrimental to the acaricidal activity.     

Stereochemistry of cobalt-catalyzed carbonylation of 2-oxazolines

The stereospecificity of the title reaction is studied using the (4R,5S)-4-methyl-2,5-diphenyl-2-oxazoline and (4R,5R)-4-methyl-2,5-diphenyl-2-oxazoline as the substrates. While the catalyst system containing BnCo(CO)₄ and BnCOCo(CO)₄ (1) was used initially, a convenient catalyst formulation generated from the commercially available Co₂(CO)₈ and AIBN (2) has been found more effective than catalyst system 1. The stereoselectivity in all cases favors inversion at the C(5)-position with diastereomeric excess up to >97%.     

Tailored poly(2-oxazoline) polymer brushes to control protein adsorption and cell adhesion

POx bottle-brush brushes (BBBs) are synthesized by SIPGP of 2-isopropenyl-2-oxazoline and consecutive LCROP of 2-oxazolines on 3-aminopropyltrimethoxysilane-modified silicon substrates. The side chain hydrophilicity and polarity are varied. The impact of the chemical composition and architecture of the BBB upon protein (fibronectin) adsorption and endothelial cell adhesion are investigated and prove extremely low protein adsorption and cell adhesion on BBBs with hydrophilic side chains such as poly(2-methyl-2-oxazoline) and poly(2-ethyl-2-oxazoline). The influence of the POx side chain terminal function upon adsorption and adhesion is minor but the side chain length has a significant effect on bioadsorption.     

Derivatization of (±)-5-[(2-Methylphenoxy)methyl]-2-amino-2-oxazoline,an Imidazoline Binding Sites Ligand,with (+)- (R) -α-Methylbenzyl Isocyanate for Drug Monitoring Purposes

The derivatization of racemic 5-[(2-methylphenoxy)methyl]-2-amino-2-oxazoline, developed as an imidazoline binding sites ligand, with (+)- (R) - α -methylbenzyl isocyanate was performed in chloroform. The reaction led to two pairs of diastereomers, which were separated by RP-HPLC. A kinetic study of the derivatization reaction was achieved in order to establish conditions suitable for experimental drug monitoring.     

A New Synthesis of 2-Methyl-6-methyleneocta-2,7-dien-4-ol,A Component of the Pheromane of California Five-spined Ips

A key intermediate, 2-isocyano-3-hydroxybutyrate (III) was isolated from a reaction of isocyanoacetate (I) with acetaldehyde (II) in the presence of Et₃N. It was found that III was readily converted into 2-isocyanocrotonate (V) and 2-isocyano-2-(1′-hydroxyethyl)-3-hydroxybutyrate (VI) which are undesirable compounds for the synthesis of threonine. However, by use of a metal catalyst (e.g. NiCl₂ or PdCl₂), the isocyano-hydroxy compound (III) was selectively converted into 5-methyl-4-alkoxycarbonyl-2-oxazoline (IV) which is an important precursor of threonine. Furthermore, chemical properties of IV were examined; the results suggested that cis-oxazoline was relatively sensitive to acid, base and heat.

On the basis of these results, the reaction of I with II was carried out using Et₃N-PdCl₂ as a catalyst to obtain threo-threonine (85% purity) in a good yield (85%).     

Amphiphilic model conetworks based on cross-linked star copolymers of benzyl methacrylate and 2-(dimethylamino)ethyl methacrylate: synthesis, characterization, and DNA adsorption studies

Six amphiphilic model conetworks of a new structure, that of cross-linked "in-out" star copolymers, were synthesized by the group transfer polymerization (GTP) of the hydrophobic monomer benzyl methacrylate (BzMA) and the ionizable hydrophilic monomer 2-(dimethylamino)ethyl methacrylate (DMAEMA) in a one-pot preparation. The synthesis took place in tetrahydrofuran (THF) using tetrabutylammonium bibenzoate (TBABB) as the catalyst, 1-methoxy-1-(trimethylsiloxy)-2-methyl-propene (MTS) as the initiator, and ethylene glycol dimethacrylate (EGDMA) as the cross-linker. Three heteroarm star-, two star block-, one statistical copolymer star-, and one homopolymer star-based networks were prepared. The synthesis of these star-based networks involved four to six steps, including the preparation of the linear (co)polymers, the "arm-first" and the "in-out" star copolymers, and finally the network. The precursors and the extractables were characterized using gel permeation chromatography (GPC) and proton nuclear magnetic resonance (1H NMR) spectroscopy. The degrees of swelling (DSs) of all the networks were measured in THF, while the aqueous DSs were measured as a function of pH. The DSs at low pH were higher than those at neutral or high pH because of the protonation of the DMAEMA units and were found to be dependent on the structure of the network. The DSs in THF were higher than those in neutral water and were independent of the structure. Finally, DNA adsorption studies onto the networks indicated that the DNA binding was governed by electrostatics.     

O-Benzylated thioglucoses, intermediates for the synthesis of (1→6)- and (1→4)-linked oligosaccharides: S-benzylation, coupling, and thioglycoside cleavage

The use of the chloroacetyl group as a protecting group has been studied for a 2-methylglyco-[2′,1′:4,5]-2-oxazoline. The reaction of chloroacetyl chloride or chloroacetic anhydride with 2-acetamido-1,3,4-tri-O-acetyl-2-deoxy-β-d-glucopyra-nose provided 2-acetamido-1,3,4-tri-O-acetyl-6-O-(chloroacetyl)-2-deoxy-β-d-glucopyranose which, on treatment with anhydrous ferric chloride in dichloromethane, produced the desired oxazoline. The glycosylating capability of the oxazoline has been investigated with aglycon hydroxides, to give the corresponding 2-acetamido-2-deoxy-β-d-glucopyranosides. The chloroacetyl group can be selectively removed by treatment with thiourea, and migration of O-acetyl groups was not observed under these conditions.     

Rapid deswelling response of poly(N-isopropylacrylamide)/poly(2-alkyl-2-oxazoline)/poly(2-hydroxyethyl methacrylate) hydrogels

Ternary poly( N-isopropylacrylamide)/poly(2-alkyl-2-oxazoline)/poly(2-hydroxyethyl methacrylate) (PNIPAAm/PROZO/PHEMA) hydrogels were prepared by the free-radical copolymerization of N-isopropylacrylamide (NIPAAm), 2-hydroxyethyl methacrylate (HEMA), and poly(2-alkyl-2-oxazoline) (PROZO) multifunctional macromonomers. The resulting polymeric materials were characterized by differential scanning calorimetry (DSC), thermogravimetric analysis (TGA), and scanning electron microscopy (SEM), as well as by equilibrium swelling experiments. All synthesized hydrogels display temperature sensitivity in the 28-38 degrees C range. A high rate of response was registered as compared to that of materials based only on PNIPAAm. The swelling-deswelling peculiar behavior was related to the chemical composition (hydrophile/hydrophobe balance), the length of the inserted PROZO sequence, and inner morphology, an aspect which points on its possible control by synthesis. It was evidenced that the architecture of the resulting porous materials has a high order degree, emerging from the self-assembling of the microgel particles, which provided numerous, nearly uniform, large water release channels.     

Palladium-catalysed asymmetric allylation and complex formation involving P,N-bidentate diamidophosphites with 1,3,2-diazaphospholidine cycles

New chiral P,N-bidentate 1,3,2-diazaphospholidine ligands were obtained by one-step phosphorylation of (2S,3S)-2-(ferrocenylideneamino)-3-methylpentan-1-ol and (4S,5S)-(−)-2-methyl-4-hydroxymethyl-5-phenyl-2-oxazoline. Complexation of the new ligands with [Rh(CO)₂Cl]₂ and [Pd(allyl)Cl]₂ was found to give chelate complexes [Rh(CO)Cl(η²-P,N)] and , correspondingly. With the new P,N-ligands, up to 70% ee was achieved in the asymmetric Pd-catalysed sulfonylation of 1,3-diphenyl-2-propenyl acetate with sodium p-toluenesulfinate. In the enantioselective alkylation of 1,3-diphenyl-2-propenyl acetate with dimethyl malonate, up to 91% enantioselectivity was achieved by using complexes as chiral catalysts.     

Preparation, characterization, and properties of fullerene-vinylpyrrolidone copolymers

Water-soluble fullerene (C(60))-N-vinylpyrrolidone copolymers were prepared by the radical polymerization method. The structures of the copolymers were characterized by Fourier transform infrared, UV-Vis, (1)H NMR, (13)C NMR, gel permeation chromatography, thermogravimetric analyses, and scanning electron microscopy (SEM). The results presented show that C(60) and vinylpyrrolidone (VP) can be copolymerized under different conditions. With a constant benzoyl peroxide amount, C(60) contents in the copolymers increase with increasing initial C(60):VP reactant ratio. The assembly behavior of water-soluble C(60)-N-vinylpyrrolidone copolymers was investigated by SEM. The results show that the copolymers create morphology that is sphere-like. Fullerene-containing micro-/nano-sized copolymer fibers were prepared, for the first time, by electrospinning. The cytotoxicity to cancer cell lines of the copolymers was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide and confocal laser scanning microscope. The results show that copolymers exhibit better cytotoxicity against HeLa cells and mouse osteogenic sarcoma cells (cytotoxicity of copolymers is better than that of fullerene complex). The mechanism of fullerene-VP copolymerization was investigated for the first time.     

Stereocontrolled routes to cis-hydroxyamino sugars, part VII: synthesis of daunosamine and ristosamine

The nitrogen of an allylic amine can serve as the fulcrum for stereocontrolled delivery of oxygen to an adjacent trigonal site, and cis-hydroxyamino sugars can thus be prepared. Methods for achieving the complementary procedure, namely, control of the delivery of nitrogen to an adjacent site by an allylic oxygen, are described. For example, treatment of methyl 2,3,6-trideoxy-- -erythro-hex-2-enopyranoside with trichloroacetonitrile gave an imidate ester which reacted with iodonium dicollidine perchlorate to give 2-trichloromethyl-(methyl 2,3,4,6-tetradeoxy-2-iodo-- -altropyranoside)-[3,4-d]-2-oxazoline. Exhaustive reductive dehalogenation of this product followed by hydrolysis led to methyl N-acetyl-- -ristosaminide. An analogous series of reactions was used to prepare the corresponding daunosaminide.     

Poly(2-oxazoline) hydrogels as next generation three-dimensional cell supports

Synthetic hydrogels selectively decorated with cell adhesion motifs are rapidly emerging as promising substrates for 3D cell culture. When cells are grown in 3D they experience potentially more physiologically relevant cell–cell interactions and physical cues compared with traditional 2D cell culture on stiff surfaces. A newly developed polymer based on poly(2-oxazoline)s has been used for the first time to control attachment of fibroblast cells and is discussed here for its potential use in 3D cell culture with particular focus on cancer cells toward the ultimate aim of high-throughput screening of anticancer therapies. Advantages and limitations of using poly(2-oxazoline) hydrogels are discussed and compared with more established polymers, especially polyethylene glycol (PEG).     

Poly(2-oxazoline) hydrogels as next generation three-dimensional cell supports

Synthetic hydrogels selectively decorated with cell adhesion motifs are rapidly emerging as promising substrates for 3D cell culture. When cells are grown in 3D they experience potentially more physiologically relevant cell–cell interactions and physical cues compared with traditional 2D cell culture on stiff surfaces. A newly developed polymer based on poly(2-oxazoline)s has been used for the first time to control attachment of fibroblast cells and is discussed here for its potential use in 3D cell culture with particular focus on cancer cells toward the ultimate aim of high-throughput screening of anticancer therapies. Advantages and limitations of using poly(2-oxazoline) hydrogels are discussed and compared with more established polymers, especially polyethylene glycol (PEG).     

Asymmetric carbon-carbon bond-forming reaction at the 2-position of a piperidine skeleton

An asymmetric carbon-carbon bond-forming reaction at the 2-position of a piperidine skeleton was exploited. This method consisted of a reaction between 1-(4-methoxybenzoyl)-3,4-didehydro-2-methoxypiperidines and dimethyl malonate catalyzed by Cu(II)-chiral 2,2'-isopropylidenebis(4-phenyl-2-oxazoline) to afford a 2-substituted piperidine skeleton with moderate enantioselectivity.