Effects of temperature on the polymerization of γ-ethyl DL-glutamate N-carboxy anhydride in DMF initiated by tri-n-propylamine

Polymerization of γ-ethyl DL -glutamate N-carboxy anhydride (NCA) in DMF has been carried out at various temperatures and with the use of tri-n-propylamine as the initiator. It was found that on decreasing the polymerization temperature the reaction rate is lowered but the molecular weight and the helix content of the final polymer are enhanced. An overall activation energy of ～4 kcal/mole has been found for the polymerization process. Preliminary experiments carried out on the polymerization of γ-benzyl D -glutamate NCA in DMF and with tri-n-propylamine as the initiator showed a strong depressing effect of the CO₂ evolved during the polymerization, both on the reaction rate and on the molecular weight of the polymer. All data are interpreted in terms of the Bamford-Swarc mechanism.     

Solvent effects in N-carboxyanhydride (NCA) polymerization initiated by strong base type initiators

The γ-benzyl-L -glutamate N-carboxyanhydride (NCA) polymerization initialed by diisopropylamine was studied in dimethylformamide (DMF)-dioxane mixtures of different compositions. It was found that the shape of the conversion versus time plots and the molecular weights of the polymers depend on the solvent composition. Auto-catalysis is present only when dioxane predominates in the solvent mixtures. Moreover, the molecular weight of the final polymer depends strongly on the precipitation conditions when the polymerization is carried out in DMF.     

Candida antarctica lipase B-catalyzed synthesis of poly(butylene succinate): shorter chain building blocks also work

Lipase catalysis was successfully employed to synthesize high molecular weight poly(butylene succinate) (PBS). Attempts to copolymerize succinic acid with 1,4-butanediol were unsuccessful due to phase separation of the reactants. To circumvent this problem, monophasic reaction mixtures were prepared from diethyl succinate and 1,4-butanediol. The reactions were studied in bulk as well as in solution. Of the organic solvents evaluated, diphenyl ether was preferred, giving higher molecular weight products. After 24 h in diphenyl ether, polymerizations at 60, 70, 80, and 90 degrees C yielded PBS with M(n) of 2000, 4000, 8000, and 7000, respectively. Further increase in reaction time to 72 h resulted in little or no further increase in M(n). However, increasing the reaction time produced PBS with extraordinarily low M(w)/M(n) due to the diffusion and reaction between low-molecular weight oligomers and chains that occurs at a greater frequency than interchain transesterification. Time-course studies and visual observation of polymerizations at 80 degrees C revealed PBS precipitates at 5 to 10 h, limiting the growth of chains. To maintain a monophasic reaction mixture, the polymerization temperature was increased from 80 to 95 degrees C after 21 h. The result was an increase in the PBS molecular weight to M(w) = 38 000 (M(w)/M(n) = 1.39). This work paves the way for the synthesis of PBS macromers and polymers that contain variable quantities of monomers with chemically sensitive moieties (e.g., silicone, epoxy, vinyl). Furthermore, this study established the feasibility of using lipase catalysis to prepare polyesters from alpha,omega-linear aliphatic diethyl ester/diol monomers with less than six carbons.     

Structure of branched poly-α-amino acids in dimethylformamide. I. Light scattering

Light scattering of multichain poly-α-amino acids was studied in dimethylformamide (DMF). The polymers consisted of a backbone of poly-L -lysine of degree of polymerization n with side chains of benzyl L -glutamate and benzyl L -aspartate of degree of polymerization, m, on each ε-amino group. The backbone length n is known and m is obtained by amino acid analysis. The results on a series of such materials confirm this structure and show that the molecules are dissolved in highly compacted conformations. It was found that DMF is a poor solvent for these polymers. In the case of the higher molecular weight polymers, the solutions initially were not molecularly disperse. The aggregates were resistant to dilution in the experimental range. Mild heat treatment, however, disaggregated the solutions irreversibly, and the light-scattering data indicated that a structural rearrangement of the molecules had occurred.     

Poly( -p-nitrobenzyl-L-glutamate): synthetic aspects, sedimentation, and viscosity studies

A series of poly(γ-p-nitrobenzyl-L -glutamates), PNBG, has been synthesized by the polymerization of N-carboxyanhydride (NCA) derivatives of γ-p-nitrobenzyl-L -glutamate, NBG, using triethylamine as an initiator. We studied the influence of (a) the solvents dioxane, nitrobenzene, dimethylformamide (DMF), and DMF–1,2-dichloroethane mixture and (b) the anhydride–initiator ratio (A/I) for the polymerization in nitrobenzene (A/I varying from 50 to 750) on the properties of the polymers obtained. In order to improve its synthesis, NBG, was prepared by three different methods. Ten samples of PNBG, ranging in M_w from 10,000 to 50,000, were examined viscometrically in DMF and dichloroacetic acid (DCA) and by ultracentrifugation in DMF. The data for [η] and S_o (limiting sedimentation coefficient) as functions of M_w for PNBG in DMF were utilized, applying theories of Kuhn and Kuhn,¹³ Schachman,¹⁴ and Perrin, ¹⁵ for the estimation of the length per monomeric residue h. Viscosity data gave a h value of about 2.3 Å, Whereas sedimentation yielded 1.5 Å. Treating viscosity and sedimentation data for poly(γ-benzyl-L -glutamate), PBLG, in the same way leads to somewhat higher hvalues (2.4 Å and 1.7 Å, respectively). Although a nitroaromatic effect was shown to be absent for PNBG in DMF, it can be concluded that in this medium PNBG has a somewhat more compact structure than PBLG.     

A radioimmuno—chromatographic scanning method for the analysis of testosterone conjugates in urine and serum

A method for the analysis of testosterone (and 5α-dihydrotestosterone) conjugates in human serum and urine samples is described. The samples were brought to pH 1 and extracted with a diethyl ether—methanol mixture. After evaporation the residues were run in a thin-layer chromatography system, individual samples' paths were cut into 1-cm long pieces and eluted with methanol. The methanol was evaporated and the residue subjected to acid hydrolysis. The released steroid was extracted by diethyl ether and measured by radioimmunoassay. The methodology described represents a new approach to the qualitative and quantitative study of steroid conjugates in serum and urine, and can easily be applied to the study of steroid conjugates in other biological material.     

Aggregation of poly(γ-benzyl-α,L-glutamate)

The aggregation of poly(γ-benzyl-α,L -glutamate) and its enantiomer in toluene has been investigated by following the viscosity as a function of temperature, concentration, molecular weight, molecular-weight distribution, helix chirality, and shear rate. The temperature and concentration data for a 138,000-molecular-weight sample was fitted to an open, reversible end-to-end aggregation model. The aggregation numbers resulting from this fit were consistent with the sudden onset in non-Newtonian flow resulting from only a 0.2-wt% increase in concentration. The association equilibrium constant was then used to predict viscosity for comparison with other data, in particular, the effect of molecular weight and molecular-weight distribution. A mixture of right-and left-handed helices showed the aggregation was not chiral selective. The stiffness of end-to-end aggregated (hydrogen-bonded) molecules differed little from their covalent counterparts, at least below a molecular weight of ～10⁶. We conclude that polybenzylglutamate aggregation in toluene can be described by an open end-to-end aggregation model.     

A chemiluminometric method for the determination of urea in serum using a three-enzyme bioreactor

A flow injection chemiluminometric assay for urea has been developed based on a minicolumn bioreactor packed with immobilized enzyme-bearing glass beads. The reactor contains immobilized urease, L -glutamate dehydrogenase and L -glutamate oxidase, aligned in this order (upstream to the downstream). When the sample is introduced into the bioreactor, urea is first hydrolysed by urease to produce ammonia, which is then converted into L -glutamate by L -glutamate dehydrogenase. L -Glutamate is finally oxidized by L -glutamate oxidase to produce hydrogen peroxide, which is quantified by measuring chemiluminescence emitted upon admixing with luminol and potassium ferricyanide. One assay cycle is completed within 1 minute. The method is sensitive (detection limit 0.5 nmol) and is linear in the range 0–30 mmol/l. It can be readily applied to the determination of urea in human serum, and requires no blank corrections for ammonia and/or L -glutamate present in serum samples.     

Alternate Procedure for the Preparation of the Coenzyme A-Synthesizing Protein Complex of Bakers' Yeast

Abstract

The coenzyme A-synthesizing protein complex (CoA-SPC) of Bakers' yeast synthesizes coenzyme A in an in vitro system from the precursors ATP, D-pantothenic acid and L-cysteine. CoA-SPC has been produced on a small scale by freezing Bakers' yeast cells in a mixture of diethyl ether and solid CO₂. followed by a thawing period, and subsequent removal of the diethyl ether by vacuum. The resulting yeast lysate was then stirred for 18 h in the presence of t-Factor to solubilize CoA-SPC. When a greater quantity of CoA-SPC was needed, the danger associated with the use of a large volume of diethyl ether was apparent. Therefore, the freezing step involving diethyl ether and solid CO₂ has been replaced by a process of slowly drying fresh Bakers' yeast until approximately 34% of the initial weight of the yeast remained as dry solids. The yeast solids were ground to further disrupt the cell wall and membrane structure. The grinding step was followed by rehydration of the dry yeast solids with deionized H₂O and stirring the rehydrated yeast for 18 h to solubilize CoA-SPC.     

Linear copolymeric poly(thia-alkanedioates) by lipase-catalyzed esterification and transesterification of 3,3'-thiodipropionic acid and its dimethyl ester with alpha,omega-alkanediols

Linear copolymeric polyesters (polyoxoesters) containing thioether functions [poly(3,3'-thiodipropionic acid-co-alpha,omega-alkanediols)] were formed in good yield by esterification of an equimolar mixture of 3,3'-thiodipropionic acid (4-thiaheptane-1,7-dioic acid) and 1,6-hexanediol (weight average molecular mass, M(W) >600 Da: approximately 81% after 6 h) or 1,12-dodecanediol (M(W) > 900 Da: approximately 90% after 6 h) catalyzed by immobilized lipase B from Candida antarctica (Novozym 435) for up to 336 h in moderate vacuo without a solvent or drying reagent in the reaction mixture. Poly (3,3'-thiodipropionic acid-co-1,6-hexanediol) and poly (3,3'-thiodipropionic acid-co-1,12-dodecanediol) were extracted from the reaction mixtures using tetrahydrofurane and precipitated from tetrahydrofurane-iso-hexane (1:1, v/v) at approximately 0 degrees C. The precipitate of poly(3,3'-thiodipropionic acid-co-1,6-hexanediol) showed a maximum molecular weight of 6 x 10(5) Da corresponding to a M(W) of approximately 24,200 Da and a degree of polymerization of up to 2,150 monomer units. The precipitated poly(3,3'-thiodipropionic acid-co-1,12-dodecanediol) showed a maximum molecular weight of 8 x 10(5) Da corresponding to a M(W) of approximately 27,200 Da and a maximum degree of polymerization of up to 2,200 monomer units. The chemical structures of both polyesters containing thioether functions were confirmed by chemical derivatization and NMR spectrometry. The chemical structures of various low-molecular weight reaction intermediates of the esterification of 3,3'-thiodipropionic acid with 1,6-hexanediol were elucidated by GC-MS.     

Purification and initial characterization of lipase from the scutella of corn seedlings

The lipase from the scutella of corn (Zea mays) MO-17 seedlings was purified 272-fold to apparent homogeneity as evidenced by sodium dodecyl sulfate polyacrylamide gel electrophoresis and double immunodiffusion. The procedure involved isolation of the lipid bodies, extraction with diethyl ether, DE-52 ion exchange chromatography, and sucrose density gradient centrifugation. The enzyme had an approximate molecular weight of 270,000 daltons after sucrose density gradient centrifugation, and 65,000 daltons after sodium dodecyl sulfate polyacrylamide gel electrophoresis. The lipase contained no cysteine and its molecular weight in sodium dodecyl sulfate was not reduced by β-mercaptoethanol. The amino acid composition as well as a biphasic partition using Triton X-114 revealed the enzyme to be a hydrophobic protein. Rabbit γ-globulin containing antibodies raised against the purified lipase formed one precipitin line with the lipase in a double diffusion test, and precipitated all the lipase activity from a solution.     

Antiradical activity of hydrolyzed and non-hydrolyzed extracts from Helichrysi inflorescentia and its phenolic contents

A methanol extract was obtained from defatted (petroleum ether) inflorescence of Helichrysum arenarium (L.) Moench (perennial herb native to Middle and Southeast Europe). The extract was evaporated under reduced pressure and the dry residue was dissolved in hot water. The aqueous solution was stored for 6 d at 4 degrees C and the precipitate discarded. The remaining solution was divided into three aliquots a, b and c. Part a was extracted with ethyl acetate to obtain extract (A), part b was extracted with diethyl ether to obtain extract (B) and part c was subjected to alkaline hydrolysis and then extracted with diethyl ether to obtain extract (C). Extracts (A), (B) and (C) were evaporated under reduced pressure to obtain the dry residues A, B and C which were further investigated for phenolic compound content by TLC and HPLC and for antiradical activity with 2,2-diphenyl-1-picrylhydrazyl radical (DPPH*) as a substrate. Residue C exhibited stronger antiradical properties than non-hydrolysed residues A and B. HPLC analysis showed a great increase of caffeic acid in residue C. We concluded that the hydrolysis process led to a significant increase of free caffeic acid (strong antioxidant) concentration resulting in increased antiradical activity of residue C.     

Quantitative analysis of 5-fluorouracil and 5,6-dihydrofluorouracil in plasma by gas chromatography mass spectrometry

5-Fluorouracil and 5,6-dihydro-5-fluorouracil were analysed in the plasma of patients by combined gas chromatography mass spectrometry. 5-Bromouracil was the internal standard. After extraction from plasma with an isopropanol-diethyl ether mixture (20/80) the components were pentylated and the derivatives produced extracted into diethyl ether. Electron impact mass spectrometry was used for the simultaneous quantitative determinations of 5-fluorouracil and 5,6-dihydro-5-fluorouracil (detection limit 10 ng ml-1 5-fluorouracil, 80 ng ml-1 5,6-dihydro-5-fluorouracil). Chemical ionization was utilized to measure 5,6-dihydro-5-fluorouracil concentrations less than 80 ng ml-1 (sensitivity 10 ng ml-1). The biological applicability of these two techniques was demonstrated by analysing plasma samples from patients after administration of 5-fluorouracil or 5'-deoxyfluorouridine by intravenous injections and infusions.     

Simultaneous determination of tolmetin and its metabolite in biological fluids by high-performance liquid chromatography

A highly sensitive and selective high-performance liquid chromatographic assay has been developed for the separation and quantitation of tolmetin and its major metabolite in human biological fluids, viz. plasma, urine and synovial fluid. Analysis of plasma and synovial fluid required only 0.5 ml of the sample. The sample was washed with diethyl ether and extracted with diethyl ether—chloroform (2:1). The extracted compounds were injected onto a reversed-phase column (RP-2) and absorbance was measured at 313 nm. The standard curves in plasma were found to be linear for both tolmetin and the metabolite at concentrations from 0.04 to 10.0 μg/ml. Urine samples (0.5 ml) were diluted (1:1) with methanol containing the internal standard and were directly injected onto the reversed-phase (RP-2) column. Standard curves of tolmetin and metabolite in urine were linear in the range 5–300 μg/ml. Serum and synovial fluid concentrations of tolmetin and its metabolite in patients receiving multiple doses of tolmetin sodium were determined using the assay procedure.     

Extraction and quantitation of dolichol and dolichyl phosphate in soybean embryo tissue

A procedure for the quantitative extraction of both dolichol and dolichyl phosphate (Dol-P) in plant tissue (soybean embryos) into diethyl ether from an alkaline saponification mixture is described. A complete and quantitative separation of total dolichol and total Dol-P is then obtained based on their respective solubilities in diethyl ether and water. After separation dolichol and Dol-P can both be analyzed and quantitated directly by reverse-phase HPLC on C18 columns without additional purification. The two major homologs of dolichol and Dol-P are those with 17 and 18 isoprene units. The total dolichol and total Dol-P contents of dry embryos were 96.3 +/- 0.8 and 5.3 +/- 0.1 micrograms/g, respectively. The post-HPLC recoveries for dolichol and Dol-P were 101 +/- 2 and 84 +/- 3% respectively, using [1-14C]dolichol and Dol-P containing 20 isoprene units as recovery standards. Dol-P estimations could be carried out on material equivalent to as little as 65 mg embryo tissue.     

Highly sensitive high-performance liquid chromatographic determination method for a new erythromycin derivative, EM523, and its major metabolites in human plasma and urine using post-column tris(2,2′-bipyridine) ruthenium(III) chemiluminescence detection

A method for the simultaneous determination of de(N-methyl)-N-ethyl-8,9-anhydroerythromycin A 6,9-hemiacetal (EM523, I) and its three metabolites in human plasma and urine has been developed using high-performance liquid chromatography (HPLC) with chemiluminescence (CL) detection. Plasma and urine samples spiked with erythromycin as an internal standard were extracted with a mixture of dichloromethane and diethyl ether under alkaline conditions. The ortanic layer was evaporated under a stream of nitrogen gas. The reconstituted sample was injected into an HPLC apparatus and separated on an ODS column using a gradient elution method. The elute was reacted on-line with a mixture of tris(2,2′-bipyridine) ruthenium(II) and peroxodisulfate, and the generated CL intensity was detected. Optimization of the CL reaction conditions resulted in a sensitive and stable CL intensity for the determination of I and its metabolites. The recovery of each compound from human plasma and urine, and the sensitivity, linearity, accuracy and precision of the method were satisfactory. The lower limits of quantitation for each compound using 0.2 ml of plasma and 0.1 ml of urine were 1 and 00 ng/ml, respectively. This method has been used for the determination of I in samples from clinical trials.     

PEGA supports for combinatorial peptide synthesis and solid-phase enzymatic library assays

Permeable resins cross-linked with long PEG chains were synthesized for use in solid-phase enzyme library assays. High molecular weight bis-amino-polyethylene glycol (PEG) 4000, 6000, 8000 were synthesized by a three-step reaction starting from PEG-bis-OH. Macromonomers were synthesized by partial or di-acryloylation of bis-amino-PEG derivatives. Bis/mono-acrylamido–PEG were copolymerized along with acrylamide by inverse suspension copolymerization to yield a less cross-linked resin (Type I, compounds 6–9 ). Furthermore, acryloyl–sarcosin ethyl ester was co-polymerized along with bis-acrylamido PEG to obtain more crosslinked capacity resin (Type II, compounds 13–19 ). N,N-Dimethylacrylamide was used as a co-monomer in some cases. The polymer was usually obtained in a well-defined beaded form and was easy to handle under both wet and dry conditions. The supports showed good mechanical properties and were characterized by studying the swelling properties, size distribution of beads, and by estimating the amino group capacity. Depending on the PEG chain length, the monomer composition and the degree of cross-linking the PEGA supports showed a high degree of swelling in a broad range of solvents, including water, dichloromethane, DMF, acetonitril, THF and toluene; no swelling was observed in diethyl ether. The PEGA resins (Type I ) with an amino acid group capacity between 0.07 and 1.0 mmol/g could be obtained by variation of the monomer composition in the polymerization mixture. Fluorescent quenched peptide libraries were synthesized on the new polymer using a multiple column library synthesizer and incubated with the matrix metalloproteinase MMP-9 after it had been activated by 4-aminophenyl mercuric acetate resulting in 67/83 kDa active enzyme. The bright beads were separated manually under a fluorescence microscope and sequenced to obtain peptide substrates for MMP-9. After treatment with ethylene diamine, high-loaded resins (Type II ) have been employed in continuous flow peptide synthesis to yield peptides in excellent yield and purity. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

Extraction and identification of different prostaglandins in Allium cepa

Green onions (Allium cepa) were homogenized in a blender and extracted by normal extraction methods except that diethyl ether was used as the first extracting solvent. Different analytical procedures were used for the identification of the prostaglandins separated. TLC was applied using silica gel 60 F254 plates and a mixture of benzene, dioxane and acetic acid (20:10:1) as eluent, and the Rf values were compared with those of authentic samples. GC analysis on an SE 30 packed column and FID was applied; relative retention times of the onion extract components were measured and matched with authentic prostaglandin samples using cholesterol as an internal standard. GC-MS analyses using the same conditions adopted for GC analysis were conducted on a Finnigan MAT 112S instrument. Four peaks were identified. The prostaglandins identified were F1 alpha, E1, B1 and A2.     

Inactivation of viable Ascaris eggs by reagents during enumeration.

Various reagents commonly used to enumerate viable helminth eggs from wastewater and sludge were evaluated for their potential to inactivate Ascaris eggs under typical laboratory conditions. Two methods were used to enumerate indigenous Ascaris eggs from sludge samples. All steps in the methods were the same except that in method I a phase extraction step with acid-alcohol (35% ethanol in 0.1 N H(2)SO(4)) and diethyl ether was used whereas in method II the extraction step was avoided by pouring the sample through a 38-microm-mesh stainless steel sieve that retained the eggs. The concentration of eggs and their viability were lower in the samples processed by method I than in the samples processed by method II by an average of 48 and 70%, respectively. A second set of experiments was performed using pure solutions of Ascaris suum eggs to elucidate the effect of the individual reagents and relevant combination of reagents on the eggs. The percentages of viable eggs in samples treated with acid-alcohol alone and in combination with diethyl ether or ethyl acetate were 52, 27, and 4%, respectively, whereas in the rest of the samples the viability was about 80%. Neither the acid nor the diethyl ether alone caused any decrease in egg viability. Thus, the observed inactivation was attributed primarily to the 35% ethanol content of the acid-alcohol solution. Inactivation of the eggs was prevented by limiting the direct exposure to the extraction reagents to 30 min and diluting the residual concentration of acid-alcohol in the sample by a factor of 100 before incubation. Also, the viability of the eggs was maintained if the acid-alcohol solution was replaced with an acetoacetic buffer. None of the reagents used for the flotation step of the sample cleaning procedure (ZnSO(4), MgSO(4), and NaCl) or during incubation (0.1 N H(2)SO(4) and 0.5% formalin) inactivated the Ascaris eggs under the conditions studied.     

From oligomers to molecular giants of soybean oil in supercritical carbon dioxide medium: 1. Preparation of polymers with lower molecular weight from soybean oil

Polymers with a low molecular weight derived from soybean oil have been prepared in a supercritical carbon dioxide medium by cationic polymerization. Boron trifluoride diethyl etherate was used as an initiator. Influences of polymerization temperature, amount of initiator, and carbon dioxide pressure on the molecular weight were investigated. It is shown that the higher polymerization temperature favors polymers with relatively higher molecular weights. Larger amounts of initiator also provide polymers with higher molecular weights. Higher pressure favors polymers with relatively higher molecular weights. The applications of these soy-based materials will be in the lubrication and hydraulic fluid areas.