Stereoselective solvolyses of amino acid p-nitrophenyl esters by optically active imidazole-containing polymers

Solvolytic reactions of N-methoxycarbonyl-D- and L-phenylalanine p-nitrophenyl ester (D- 2 and L- 2 ) by poly(N-methacryloyl-L-histidine) and poly(N-methacryloyl-L-histidine methyl ester) were carried out. However, no significant difference between the solvolytic rates of the two enantiomeric substrates was observed. To investigate the effect of hydrophobic interaction, N-acetyl-, N-pentanoyl- and N-octanoyl-L-histidine were prepared and their effect on the ester solvolyses was studied. The rates were generally enhanced as the alkanoyl group became larger. Furthermore N-octanoyl-L-histidine showed some degree of stereoselective catalysis. Accordingly, when polymers with increased hydrophobicity were prepared by copolymerizing N-methacryloyl-L-histidine and its methyl ester with dodecyl methacrylate and were subjected to catalyze the solvolyses of optically active esters, the apparent catalytic rate constant for L- 2 was observed to be 1,3 times larger than that for the D- 2 . The enantio-selectivity became more pronounced as the hydrophobic character of polymer catalysts was enhanced.     

Hydrolysis of carboxylic acid p-nitrophenyl esters by cooperative imidazole functions flanked by hydrophobic groups in a polymer

N^α-Acryloyl-L -histidine was synthesized and copolymerized with N-vinylpyrrolidone using a radical initiator. The resultant copolymers having a variety of compositions were used as catalysts for the hydrolysis of p-nitrophenyl esters of carboxylic acids in 20% aqueous dioxane, and the second-order rate constant was determined. Because of the hydrophobic character of these copolymer catalysts, p-nitrophenyl laurate that carries a long acyl group was condensed along the copolymer chain by hydrophobic interactions, hence the copolymer catalysts were almost five times as reactive as N^α-acryloyl-L -histidine for the hydrolysis at pH 7,92. Increasing the pH to 10,07, the rate constant ratio increased to almost 15. This was explained in terms of the enhanced catalytic activity due to the increased cooperations between neutral imidazole functions. Since the rate increase attained was much smaller than that observed in the enzyme, the contribution to the enzyme efficiency of fixing the functional groups in a specific arrangement was relieved.     

Synthesis of optically active polymers by asymmetric catalysts. VIII. Asymmetric polyaddition of dimercaptan to dimethacrylate by optically active amine as catalyst

Optically active polymers could be obtained by the asymmetric polyaddition of tetramethylene dimercaptan to ethylene dimethacrylate using S-isobutylethylenimine or its polymer as catalyst. Polymer catalysts showed an enhanced activity in the asymmetric polyaddition.     

Enhancement of the mutagenicity of amino acid pyrolysates by phthalate esters

The ability of phthalic acid, phthalic acid anhydride, and various phthalate esters to enhance the mutagenicity of many amino acid pyrolysates was observed with the Ames test (Salmonella typhimurium TA98), but not the SOS Chromotest. Phthalate enhancement of the mutagenicity of 4-nitroquinoline-1-oxide, 2-nitrofluorene, and benzo[a]pyrene was not observed with either test. The mutagenicity-enhancing ability may be related to the induction of enzymes such as P450IIB, that metabolize amino acid pyrolysates. By quantitative structure activity relationship (QSAR) analysis, a good correlation was observed between the mutagenicity-enhancing activity of phthalates and their octanol-water partition coefficients. © 1994 Wiley-Liss, Inc.     

Gene transfection of hyperbranched PEI grafted by hydrophobic amino acid segment PBLG

The complex copolymer of hyperbranched polyethylenimine (PEI) with hydrophobic poly(gamma-benzyl L-glutamate) segment (PBLG) at their chain ends was synthesized. This water-soluble copolymer PEI-PBLG (PP) was characterized for DNA complexation (gel retardation assay, particle size, DNA release and DNase I protection), cell viability and in vitro transfection efficiency. The experiments showed that PP can effectively condense pDNA into particles. Size measurement of the complexes particles indicated that PP/DNA tended to form smaller nanoparticles than those of PEI/DNA, which was caused by the hydrophobic PBLG segments compressing the PP/DNA complex particles in aqueous solution. The representative average size of PP/DNA complex prepared using plasmid DNA (pEGFP-N1, pDNA) was about 96 nm. The condensed pDNA in the PP/pDNA complexes was significantly protected from enzymatic degradation by DNase I. Cytotoxicity studies by MTT colorimetric assays suggested that the PP had much lower toxicity than PEI. The in vitro transfection efficiency of PP/pDNA complexes improved a lot in HeLa cells, Vero cells and 293T cells as compared to that of PEI-25K by the expression of Green Fluorescent Protein (GFP) as determined by flow cytometry. Thus, the water-soluble PP copolymer showed considerable potential as carriers for gene delivery.     

Synthesis of optically active polymers by asymmetric catalysts. VII. Stereoselective polymerization of propylene oxide catalyzed by organomagnesium compounds

The reaction of organomagnesium compounds with propylene oxidene was studied to clarify the mechanism of the dialkylmagnesium catalyzed isotactic polymerization of propylene oxide. Dialkylmagnesium reacts with the monomer first to alkylmagnesium alkoxide and then to magnesium dialkoxide. In the reaction of alkylmagnesium alkoxide with propylene oxide in polar solvents preferentially the alkyl group reacts. Asymmetric selection in the D,L -copolymerization of the monomer was observed with dialkylmagnesium as catalyst, but not with magnesium dialkoxide. This observation is discussed in terms of the “catalyst control” mechanism of stereoregulation. The retained optical activity of the polymer is in accordance with the CH₂? O scission of the monomer ring found in the above reactions.     

Role of non-protein amino acid L-canavanine in autoimmunity

Association of SLE and alfalfa was first reported in a volunteer who developed lupus-like autoimmunity while ingesting alfalfa seed for a hypercholesterolemia study. This was corroborated with studies in monkeys fed with alfalfa sprout that developed SLE. Re-challenge with L-canavanine relapsed the disease. Arginine homologue L-canavanine, present in alfalfa, was suspected as a cause. L-canavanine can be charged by arginyl tRNA synthetase to replace L-arginine during protein synthesis. Aberrant canavanyl proteins have disrupted structure and functions. Induction or exacerbation of SLE by alfalfa tablets reported in a few cases remains controversial. Epidemiological studies on the relationship between alfalfa and SLE are sparse. In mice, NZB/W F1, NZB, and DBA/2 mice fed with L-canavanine show exacerbation/triggering of the SLE, however, BALB/c studies were negative. L-canavanine incorporation may be more efficient in the presence of inflammation or other conditions that can cause arginine deficiency. The L-canavanine induced apoptotic cells can be phagocytosed and a source of autoantigens processed by endosomal proteases. Endogenous canavanyl proteins are ubiquitinated and processed via proteasome. Incorporation of L-canavanine into proteasome or endosome can also cause disruption of antigen processing. Alfalfa/L-canavanine-induced lupus will be an interesting model of autoimmunity induced by the modification of self-proteins at the translational level.     

Aminolysis of α-hydroxy acid esters with α-amino acid salts; first step in the synthesis of optically active 2,5-morpholinediones

A process for preparing optically active 2,5-morpholinediones 1 is described starting from optically active α-amino acids and optically active α-hydroxy esters. The process involves three steps: In the first step the sodium salt of an α-amino acid is condensed with an α-hydroxy ester to yield an optically active N-(α-hydroxy acyl)-α-amino acid 4 . In the second step the hydroxy acid obtained is subjected to esterification with ethanol. In the third step the N-(α-hydroxy acyl)-α-amino acid ethyl ester 5 is subjected to cyclization with an acidic catalyst. For some examples the cyclization was also performed directly from the hydroxy acid 4 . Morpholinediones 1 are useful as precursors for the preparation of poly(depsipeptides) 2 and copolymers containing depsipeptide repeating units.     

The role of hydrophobic amino acid grafts in the enhancement of membrane-disruptive activity of pH-responsive pseudo-peptides

pH-responsive polymers have been synthesised by grafting l-valine (PV-75), l-leucine (PL-75) and l-phenylalanine (PP-75) onto the pendant carboxylic acid moieties of a pseudo-peptide, poly(l-lysine iso-phthalamide), at a stoichiometric degree of substitution of 75 mol%. The effect of such modification on the pH-, concentration- and time-dependent cell membrane-disruptive activity of the grafted polymers has been investigated using a haemolysis model. At 0.025 mg mL^?1, the grafted polymers were almost non-haemolytic at pH 7.4, but mediated considerable membrane lysis after 60 min in the pH range characteristic of early endosomes, which ranked in the order: PP-75 > PL-75 > PV-75 > poly(l-lysine iso-phthalamide). PP-75 was 35-fold more lytic on a molar basis than the membrane-lytic peptide melittin. With increasing concentration, the grafted polymers showed an increased ability to lyse cell membranes and caused noticeable membrane disruption at physiological pH. The mechanism of the polymer-mediated membrane destabilisation has been investigated. The in-vitro cytotoxicity of the grafted polymers has been assessed using a propidium iodide fluorescence assay. It has been demonstrated by confocal microscopy that the grafted polymers can induce a significant release of endocytosed materials into the cytoplasm of HeLa cells, which is a feature critical for drug delivery applications.     

Enantioselective hydrolysis of dipeptide amino acid esters by di- and tripeptide-type L-histidine derivatives in a bilayer vesicular system

The enantioselective hydrolysis of dipeptide-type amino acid esters (Z-(L )-Ala-(L or D )-Ala-PNP ( 5a ), Z-(L )-Ala-(L or D )-Leu-PNP ( 5b ), and Z-(L )-Ala-(L or D )-Phe-PNP ( 5c )) by di- or tri-peptide nucleophiles (Z-(L )-Leu-(L )-His ( 2a ), Z-(L )-Phe-(L )-His ( 2b ), and Z-(L )-Leu-(L )-His ( 3 )) in the bilayer vesicular aggregates of didodecyldimethylammonium bromide ( 6 ) resulted in the enantiomer rate ratio of LL /LD = 1, 1 to 18, the value of which was considerably higher than that (L /D = 1,0 to 4,6) in the hydrolysis of Z-(L or D )-Ala-PNP ( 4a ), Z-(L or D )-Leu-PNP ( 4b ), and Z-(L or D )-Phe-PNP ( 4c ) by the identical vesicular system and that (L /D = 0,7 to 3,1) in the hydrolysis of the dipeptide substrates 5a – c by Z-(L )-His ( 1 ) and 6 . The high enantioselectivity (LL /LD = 18) in the hydrolysis of 5c by the system of 2a and 6 was enhanced to be LL /LD = 36 by lowering the temperature from 25 to 10°C.     

Role of excitatory amino acid transporter 1 in neonatal rat neuronal damage induced by hypoxia-ischemia

The role of excitatory amino acid transporter 1 in neonatal rat neuronal damage was studied following hypoxia-ischemia. To induce hypoxia-ischemia injury, rats on postnatal day 7 were exposed to 8 % oxygen for 2 h following unilateral common carotid artery ligation. According to brain damage scoring based on Cresyl Violet staining, the neuronal damage time-dependently changed in the ischemic regions following hypoxia-ischemia. Immunohistochemical studies showed that excitatory amino acid transporter 1 expression was mainly observed in the cerebral cortex ipsilateral to common carotid artery ligation and markedly increased at 24 h and 48 h following hypoxia-ischemia. Combined with confocal laser scanning microscopic analysis, double staining showed that excitatory amino acid transporter 1 positive staining appeared in neurons as well as astrocytes after hypoxia-ischemia. Most excitatory amino acid transporter 1 positive staining cells exhibited regular morphological characteristics and only a few were double-stained by terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphosphate nick-end labeling. Down-regulation of excitatory amino acid transporter 1 expression by intraventricular administration of specific antisense oligonucleotide exacerbated neuronal damage in hypoxia-ischemia brain. These results suggest that the increase of excitatory amino acid transporter 1 expression may be involved in a pathophysiological process of hypoxia-ischemia brain damage and may reflect a self-compensative mechanism for protecting neurons from further injury.     

Investigation of the aminolysis of p-nitrophenyl esters of (meth)acryloylaminophenoxyacetic acids and their copolymers with N-(2-hydroxypropyl)methacrylamide by 6-aminopenicillanic acid

Copolymers of N-(2-hydroxypropyl)methacrylamide with p-nitrophenyl esters of (meth)acryloylaminophenoxyacetic acids were prepared and characterized. The aminolysis of monomeric and polymeric p-nitrophenyl esters by 6-aminopenicillanic acid was studied. The aminolysis rate was structure-dependent. Acryloyl derivatives were more reactive than methacryloyl derivatives. In both cases, the ortho-derivatives were more reactive than the para-derivatives. This phenomenon can be explained by the formation of an intramolecular hydrogen bond in the ortho-derivatives. The synthetic methods studied are suitable for the binding of biologically active compounds containing an aliphatic amino group to polymeric carriers.     

Synthesis of optically active polymers by asymmetric catalysts. IV. Dialkylzinc – alcohol system as catalyst for asymmetric-selective polymerization of propylene oxide

The system diethylzinc/optically active alcohol was examined as catalyst for asymmetric-selective polymerization of propylene oxide. Optically active alcohols with rigid structure are effective for the asymmetric selection. D (–)-1-Methoxypropanol-2 as well as poly(D -propylene oxide) of low molecular weight with hydroxyl end groups select L (–)-propylene oxide. ?Catalyst control”? mechanism of the stereoselection in the polymerization is suggested on this and other bases.