Preparation and properties of immobilized pectinase onto the amphiphilic PS-b-PAA diblock copolymers

Well-defined amphiphilic block copolymers poly(styrene-b-acrylic acid) (PS-b-PAA) with controlled block length were synthesized using atom transfer radical polymerization (ATRP). Pectinase enzyme was immobilized on the well-defined amphiphilic block copolymers PS-b-PAA. The carboxyl groups on the amphiphilic PS-b-PAA diblock copolymers present a very simple, mild, and time-saving process for enzyme immobilization. Various characteristics of immobilized pectinase such as the pH and temperature stability, thermal stability, and storage stability were valuated. Among them the pH optimum and temperature optimum of free and immobilized pectinase were found to be pH 6.0 and 65 degrees C.     

Amphiphilic block copolymers as bile acid sorbents: 2. Polystyrene-b-poly(N,N,N-trimethylammoniumethylene acrylamide chloride): self-assembly and application to serum cholesterol reduction

This paper presents morphological studies and preliminary bile salt binding properties of the new amphiphilic diblock copolymer polystyrene-b-poly(N,N,N-trimethylammoniumethylene acrylamide chloride) (PS-b-PTMEACl)(1) (see Figure 1), a derivative of PS-b-poly(tert-butylacrylate) (PS-b-PtBuA). In an aqueous environment, PS-b-PTMEACl forms simple spheres (approximately 20 nm diameter), large compound micelles (>100 nm diameter), and larger, more complex architectures as presented and discussed below. The colloidal stability with respect to sodium chloride and as a function of particle concentration is also considered. Finally, PS-b-PTMEACl aggregates were prepared and tested as an alternative to the commercially available bile salt sequestrant resins that target coronary heart disease due to elevated cholesterol levels. Electron micrographs were employed to visualize the colloid-based polyelectrolyteminus signbiosurfactant interaction and chromatographic separation analytical methods were used to quantify the sequestration. The results indicate that although at this preliminary stage they require laborious preparation, self-assembled aggregates may present an interesting alternative to the clinically used bile salt sequestrants.     

Poly(N,N,N-trimethylammoniumalkyl acrylamide chloride) based hydrogels for serum cholesterol reduction

Hydrogels present an attractive alternative to nanoscale block copolymer aggregates and microscale resin beads as potential asystemic serum cholesterol reduction materials. Not only would the oral delivery of these materials be more pleasant than the sand-like bile salt anion sequestrant beads but also the hydrogel preparation is much simpler than the copolymer aggregate analogues [Cameron, N. S.; Eisenberg, A.; Brown, G. R. Biomacromolecules 2002, 3, 116-123. Cameron, N. S.; Eisenberg, A.; Brown, G. R. Biomacromolecules 2002, 3, 124-132]. Our goal was to explore these materials building on our experience with bulk resins and self-assembled copolymers. In this paper, following a brief introduction to hydrogels and their application to hypercholesterolemia, the synthesis, characterization, and preliminary glycocholate binding properties of poly(N,N,N-trimethylammoniumalkyl acrylamide chloride)gel are presented [Cameron, N. S.; Eisenberg, A.; Brown, G. R. Polym. Preprints 2002, 43, 771-772].     

Synthesis of beta- and gamma-fluorenylmethyl esters of respectively N alpha-Boc-L-aspartic acid and N alpha-Boc-L-glutamic acid

The orthogonal synthesis of N alpha-Boc-L-aspartic acid-gamma-fluorenylmethyl ester and N alpha-Boc-L-glutamic acid-delta-fluorenylmethyl ester is reported. This is a four-step synthesis that relies on the selective esterification of the side-chain carboxyl groups on N alpha-CBZ-L-aspartic acid and N alpha-CBZ-L-glutamic acid. Such selectivity is accomplished by initially protecting the alpha-carboxyl group through the formation of the corresponding 5-oxo-4-oxazolidinone ring. Following side-chain esterification, the alpha-carboxyl and alpha-amino groups are deprotected with acidolysis. Finally, the alpha-amino group is reprotected with the t-butyl-oxycarbonyl (Boc) group. Thus aspartic acid and glutamic acid have their side-chain carboxyl groups protected with the base-labile fluorenylmethyl ester (OFm) and their alpha-amino groups protected with the acid-labile Boc group. These residues, when used in conjunction with N alpha-Boc-N epsilon-Fmoc-L-lysine, are important in the formation of side-chain to side-chain cyclizations, via an amide bridge, during solid-phase peptide synthesis.     

Synthesis and erythropoietin receptor binding affinities of N,N-disubstituted amino acids

N,N-Dicinnamyl, N-benzyl-N-cinnamyl, and N,N-dibenzyl amino acids were prepared and evaluated in an EPO binding assay. Several derivatives of aspartic acid, glutamic acid, and lysine exhibited moderate (10-50 microM) affinity for EBP; 'dimerization' of the most potent analogues by coupling with linear diamines led to EPO competitors having 1-2 microM binding affinities.     

Orthogonal protecting groups for N(alpha)-amino and C-terminal carboxyl functions in solid-phase peptide synthesis

For the controlled synthesis of even the simplest dipeptide, the N(alpha)-amino group of one of the amino acids and the C-terminal carboxyl group of the other should both be blocked with suitable protecting groups. Formation of the desired amide bond can now occur upon activation of the free carboxyl group. After coupling, peptide synthesis can be continued by removal of either of the two protecting groups and coupling with the free C-terminus or N(alpha)-amino group of another protected amino acid. When three functional amino acids are present in the sequence, the side chain of these residues also has to be protected. It is important that there is a high degree of compatibility between the different types of protecting groups such that one type may be removed selectively in the presence of the others. At the end of the synthesis, the protecting groups must be removed to give the desired peptide. Thus, it is clear that the protection scheme adopted is of the utmost importance and makes the difference between success and failure in a given synthesis. Since R. B. Merrifield introduced the solid-phase strategy for the synthesis of peptides, this prerequisite has been readily accepted. This strategy is usually carried out using two main protection schemes: the tert-butoxycarbonyl/benzyl and the 9-flourenylmethoxycarbonyl/tert-butyl methods. However, for the solid-phase preparation of complex or fragile peptides, as well as for the construction of libraries of peptides or small molecules using a combinatorial approach, a range of other protecting groups is also needed. This review summarizes other protecting groups for both the N(alpha)-amino and C-terminal carboxyl functions.     

Efficient solid phase peptide synthesis. Use of methanesulfonic acid alpha-amino deprotecting procedure and new coupling reagent, 2-(benzotriazol-1-yl)oxy-1,3-dimethylimidazolidinium hexafluorophosphate (BOI).

An efficient method for solid phase peptide synthesis was developed, which consists of N alpha-selective deprotection by dilute methanesulfonic acid, in situ neutralization and rapid coupling reaction using benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP) or 2-(benzotriazol-1-yl)oxy-1,3- dimethylimidazolidinium hexafluorophosphate (BOI) reagent. Selective removal of the N alpha-Boc group by dilute methanesulfonic acid was of more advantage than removal by TFA in terms of stability of semipermanent protecting groups and suppression of undesired side reactions. The use of in situ neutralization and rapid coupling method reduced intramolecular aminolytic cyclization by shortening exposure of the deprotected nucleophilic amino group. A successful synthesis of porcine brain natriuretic peptide (pBNP) has been achieved using this efficient solid phase peptide synthesis scheme.     

Synthesis of tritium-labelled isopenicillin N, penicillin N and 6-aminopenicillanic acid.

1. Phenoxymethylpenicillin sulphoxide 4-methoxybenzyl ester was labelled with 3H in its 2-beta-methyl group. Its specific radioactivity was 362 mCi/mmol. 2. Removal of the side chain of this compound yielded the corresponding ester of 6-aminopenicillanic acid sulphoxide and coupling of the latter with the appropriate protected alpha-aminoadipic acid gave 4-methoxybenzyloxycarbonylisopenicillin N sulphoxide di-4-methoxybenzyl ester or the corresponding derivative of penicillin N. 3. Removal of the protective groups by hydrogenolysis and reduction of the sulphoxide group yielded 3H-labelled isopenicillin N or penicillin N. 4. 3H-labelled phenoxymethylpenicillin sulphoxide was obtained by hydrogenolysis from its 4-methoxybenzyl ester. Reduction of its sulphoxide group and subsequent removal of the side chain gave 3H-labelled 6-aminopenicillanic acid.     

Macroporous polyacrylamide monolithic gels with immobilized metal affinity ligands: the effect of porous structure and ligand coupling chemistry on protein binding

Macroporous polyacrylamide gels (MPAAG) with iminodiacetic acid (IDA) functionality were prepared by (i) chemical modification of polyacrylamide gel, (ii) co-polymerization of acrylamide with allyl glycidyl ether (AGE) and N,N'metylene-bis(acrylamide) (MBAAm) followed by coupling IDA ligand or (iii) by copolymerization of acrylamide and MBAAm with functional monomer carrying IDA-functionality (1-(N,N-bis(carboxymethyl)amino-3-allylglycerol). Screening for optimized conditions for the production of the MPAAG with required porous properties was performed in a 96-well chromatographic format that allowed parallel production and analysis of the MPAAG prepared from reaction mixtures with different compositions. Scanning electron microscopy of the fabricated MPAAG revealed two different types of the porous structures: monomodal macroporous structure with large interconnected pores separated by dense non-porous pore walls in case of plain gels or gels produced via copolymerization with AGE. The other type of the MPAAG (gel produced via co-polymerization with functional monomer carrying IDA-functionality) had bimodal pore structure with large interconnected pores separated by the pore walls pierced through with micropores. The effect of different modifications of MPAAG monoliths and of porous structure of the MPAAG (monomodal and bimodal porous structure) on protein binding has been evaluated.     

Stability of 100 homo and heterotypic coiled-coil a-a' pairs for ten amino acids (A, L, I, V, N, K, S, T, E, and R)

We present the thermal stability monitored by circular dichroism (CD) spectroscopy at 222 nm of 100 heterodimers that contain all possible coiled-coil a-a' pairs for 10 amino acids (I, V, L, N, A, K S, T, E, and R). This includes the stability of 36 heterodimers for 6 amino acids (I, V, L, N, A, and K) previously described and 64 new heterodimers including the 4 amino acids (S, T, E, and R). We have calculated a double mutant alanine thermodynamic cycle to determine a-a' pair coupling energies to evaluate which a-a' pairs encourage specific dimerization partners. The four new homotypic a-a' pairs (T-T, S-S, R-R, E-E) are repulsive relative to A-A and have destabilizing coupling energies. Among the 90 heterotypic a-a' pairs, the stabilizing coupling energies contain lysine or arginine paired with either an aliphatic or a polar amino acid. The range in coupling energies for each amino acid reveals its potential to regulate dimerization specificity. The a-a' pairs containing isoleucine and asparagine have the greatest range in coupling energies and thus contribute dramatically to dimerization specificity, which is to encourage homodimerization. In contrast, the a-a' pairs containing charged amino acids (K, R, and E) show the least range in coupling energies and promiscuously encourage heterodimerization.     

Radioactive analogs of neurotensin. II. Preparation of tritiated neurotensin from a synthetic multi-unsaturated derivative

In this second paper on the synthesis of neurotensin analogues as precursors for radiolabelling, solid phase synthesis of two polyunsaturated peptides, [Dah6, delta Pro7,10]-neurotensin and acetyl-[delta Pro10]-neurotensin-(8-13), are described. The first one contains one triple bond and two double bonds susceptible to tritiation in the same molecule, the second one contains one double bond in the shortest sequence having neurotensin activity. The C-terminal residue, Boc-Leu, was esterified on the chloromethyl-resin by its cesium salt. For the other amino acids a double coupling was carried out, the first one with dicyclohexylcarbodimide and the second one with the amino acid hydroxybenzotriazole ester. Acylation of the second amino acid, on the resin, presented some difficulties to achieve completeness and several acetylations and benzoylations had to be performed in order to block the last 4 per cent of free amines. It seems that these difficulties are related to some batches of chloromethyl-resin. Incorporation of both acetylenic lysine, N alpha-Boc-N epsilon-Z-L-2,6-diamino-4-hexynoic acid, whose synthesis is described, and N alpha-Boc-L-3,4-dehydroproline was without problems in this synthesis. After cleavage by hydrofluoric acid the crude peptides were purified by gel filtration on Bio-Gel P2 and ion exchange chromatography on carboxymethylcellulose (CM 52). [Dah6, delta Pro7,10]-neurotensin so obtained (51 per cent compared to starting Boc-Leu-resin) was in homogeneous form as characterized by amino acid analysis, thin layer chromatography in different systems and high performance liquid chromatography. The hydrogenation or tritiation product was identical with native neurotensin. Unsaturated derivative and neurotensin obtained after catalytic hydrogenation were as active as native neurotensin in inhibition of 125I-[Trp11]-neurotensin binding to rat brain synaptic membranes and in guinea pig ileum contractility test. Substitution of proline and lysine by their dehydro-derivatives did not affect the biological properties of neurotensin. The tritiated neurotensin (160-180 Ci/mmol) should be a good agent for biological characterization of neurotensin receptors and for investigation of the peptide metabolism.     

Synthesis of N4-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-L-asparagine analogues: succinamide, L-2-hydroxysuccinamide, and L-2-hydroxysuccinamic acid hydrazide analogues

The syntheses of three analogues of N4-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-L-asparagine are described. N-(2-Acetamido-2-deoxy-beta-D-glucopyranosyl)succinamide was synthesized by the reaction of pentafluorophenyl succinamate with 2-acetamido-2-deoxy-beta-D-glucopyranosylamine. 2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-beta-D-glucopyranosylamine was synthesized, and the complete assignment of the 1H NMR spectrum is given. Reaction of the protected beta-D-glycosylamine with L-malic acid chloralid in the presence of a coupling agent (EEDQ) gave N4-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-beta-D-glucopyranosyl)-L-malamic acid chloralid that was deprotected two ways: (1) using ammonia, which gave N4-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-L-2-hydroxysuccinamide, and (2) using hydrazine, which gave N4-(2-acetamido-2-deoxy-1-D-glucopyranosyl)-L-2-hydroxysuccinamic acid hydrazide.     

4'-alpha-C-Branched N,O-nucleosides: synthesis and biological properties

The synthesis of 4'-alpha-C-branched N,O-nucleosides has been described, based on the 1,3-dipolar cycloaddition of nitrones with vinyl acetate followed by coupling with silylated nucleobases, The obtained compounds have been evaluated for their activity against HSV-1, HSV-2, HTLV-1. Cytotoxicity and apoptotic activity have been also investigated: compound 10c shows moderate apoptotic activity in Molt-3 cells.     

衍生化UPLC-MS法测定酸性植物激素

为有效利用超高效液相-高分辨率飞行时间质谱(UPLC-TOF-MS)对酸性植物激素进行定性定量分析,选取几种有机酸衍生化试剂:2-溴苯乙酮(BP)、2-二甲氨基乙胺(DMED)、1-(3-二甲基氨基丙基)-3-乙基碳二亚胺(EDC)和溴代丙酮基三甲基溴化铵(BTA),分别与脱落酸(ABA)、赤霉素(GA3)、吲哚乙酸(IAA)、茉莉酸(JA)和水杨酸(SA)进行衍生化反应。通过比较上述各植物激素的质谱响应值,判断衍生化试剂的效果。将植物激素标准品进行浓度梯度稀释,并与衍生化试剂反应,测定它们的定量限。用衍生效果最佳的试剂与植物粗提液反应,检验其应用效果。结果显示,几种衍生化试剂均可大幅度提高酸性植物激素的质谱响应值,其中DMED衍生后植物激素的质谱灵敏度提高幅度最大,且反应稳定、重复性好。DMED衍生后, ABA、GA3、IAA、JA和SA的定量限分别为0.05、0.2、0.1、0.1和0.5 ng·mL^–1,与未衍生化相比,分别降低100、25、50、50和10倍,即DMED衍生化使几种植物激素的质谱灵敏度提高10–100倍,显著高于目前报道的灵敏度。将该方法应...     

Solid-phase peptide synthesis of somatostatin using mild base cleavage of N alpha-9-fluorenylmethyloxycarbonylamino acids

Experimental details for the "Fmoc solid phase peptide synthesis" of somatostatin are described. The 9-fluorenylmethyloxycarbonyl group was rapidly and quantitatively cleaved by 55% piperidine in dimethylformamide and monitored (u.v.) manually. For a kinetic study, a centrifugal reactor with a photometric control system and reference cell was used at each stage. The symmetrical anhydride coupling reaction was rapid and either acetic anhydride or fluorescamine termination was incorporated to minimize formation of deletion peptides. Anchor-bond cleavage was effected with trifluoroacetic acid which simultaneously removed all the acid labile tert.-butyl side chain protecting groups. N alpha-9-fluorenylmethyloxycarbonyl peptides may be obtained by omitting the piperidine deprotection step after the last cycle of synthesis. From several syntheses, analytically pure di-S-protected somatostatin 14-peptide was obtained in 55-60% overall yield. The S-protecting groups were removed and the product was purified by gel filtration to give homogeneous dihydrosomatostatin (91%) yield. Oxidation of dihydrosomatostatin with potassium ferricyanide and purification by countercurrent distribution provided analytically pure homogeneous somatostatin.     

Nucleic acid-specific photoactivation of oligodeoxyribonucleotides labeled with deuterated dihydro-N,N,N′,N′-tetramethylrhodamine using green light

We developed a simple protocol for high-yielding synthesis of conjugates of a deuterated dihydro-N,N,N′,N′-tetramethylrhodamine (F*) with oligodeoxyribonucleotides and a 2′-OMe RNA (a representative nuclease-resistant, chemically modified oligonucleotide) using easily accessible starting materials including NaBD₄ and conjugates of oligonucleotides with N,N,N′,N′-tetramethylrhodamine (F). These compounds were found to be stable in air and insensitive to light at 525, 635 and 650 nm, whereas slow activation occurs upon their exposure to 470 nm light. However, at the conditions of the templated reaction, in the presence of a target nucleic acid and a photocatalyst based on the eosin structure, the F* is oxidized forming fluorescent F. This reaction is >30-fold faster than the background reaction in the absence of the template. Moreover, the presence of a single mismatch in the target nucleic acid slows down the templated reaction by eightfold. These activatable dyes can potentially find applications as nucleic acid-specific probes for super-resolution imaging in live cells.     

Acrylamide and Glycidamide Impair Neurite Outgrowth in Differentiating N1E.115 Neuroblastoma Without Disturbing Rapid Bidirectional Transport of Organelles Observed by Video Microscopy

Abstract: The nature of the pathogenic insult in acrylamide neuropathy is unknown, but axonal transport disturbances are suspected. Using N1E.115 neuroblastoma in vitro, we examined acrylamide and related compounds in terms of general cytotoxicity, ability to block neurite outgrowth, and effects on neurite integrity and fast axonal transport. Acrylamide, glycidamide, and methylene-bisacrylamide were weakly cytotoxic in a ⁵¹Cr-release assay, but only at ≥10 m M (order of efficacy: methylene-bis-acrylamide > glycidamide > acrylamide). Neurite outgrowth by differentiating cells was inhibited at 100-fold lower concentrations, with similar EC₅₀ values for all three toxicants, i.e., acrylamide, 70 ± 15 μ M ; methylene-bis-acrylamide, 92 ± 31 μ M ; glycidamide, 120 ± 30 μ M . Only glycidamide (1 m M ) caused degeneration of established neurites within a period of 48 h. Video-enhanced contrast differential interference contrast microscopy was used to test the effect of acrylamide and glycidamide on organelle transport in the neurites. In exposures of ≤48 h at 1 m M , neither toxicant altered bidirectional organelle flux, measured as organelles transported per minute per micrometer of neurite diameter. Anterograde and retrograde organelle speeds were also undisturbed. These results suggest that mechanisms other than direct inhibition of organellar motility are responsible for acrylamide's neurotoxicity in vivo.     

Biological and chemical characteristics of allergen preparations from Neisseria (N. meningitidis, N. gonorrhoeae, N. perflava)]

Various methods of isolating allergen fractions from N. meningitidis, N. gonorrhoeae and N. perflava were tested. The biological activity of the preparation was found to depend on the method of its production, which determined its chemical composition. When gonococcal and meningococcal allergens and N. perflava allergen were used in skin tests, cross reactions were observed. Nevertheless, as the intensity and size of skin reaction was much greater when a homologous preparation was administered, it was possible to differentiate the presence of sensitization to a definite microbial species. Electrophoresis in acrylamide gel revealed the heterogeneity of allergen preparations. The ability of the preparation to induce skin reaction was not connected with its serological properties.     

A synthesis of N6,N6,N6-trimethyl-L-lysine dioxalate in gram amounts

A procedure is described for the synthesis of crystalline N6,N6,N6-trimethyl-L-lysine dioxalate in gram amounts starting from the commercially available N2-tert-butoxycarbonyl-N6-benzyloxycarbonyl-L-lysine, which is reacted with methyl iodide in methanol in the presence of potassium hydrogen carbonate after deprotection of the side-chain amino group by catalytic hydrogenation. The work-up involves only filtrations and evaporations.     

Effects of expander-treating a barley-based concentrate on ruminal fermentation, bacterial N synthesis, escape of dietary N, and performance of dairy cows

Three primiparous dairy cows in early lactation with cannulas in rumen, duodenum and ileum were used in a 3×3 Latin square design to study effects of expander treatment of a barley-based concentrate. The concentrate was either pelleted at 75–80°C or expander treated at 125–130°C prior to pelleting. The diets consisted of 6.7 kg DM of grass silage and 10 kg DM of (1) 100% pelleted, (2) 50% pelleted and 50% expanded or (3) 100% expanded concentrate. The diets were offered as a mixed ration in four equal meals daily. Ruminal fermentation, bacterial N synthesis, duodenal, ileal and faecal flow of nutrients, and animal performance were monitored. Expander treatment numerically increased ruminal digestion of starch, which explained the observed increase in ruminal VFA concentration and the lowered ruminal pH (P<0.05). The proportion of butyrate in rumen liquid increased, whereas the proportion of propionate decreased in the expanded compared to the pelleted treatment (P<0.05). Expander treatment tended to increase rumen volume and rumen NDF pool size. Ruminal digestion of NDF was numerically lower in the expanded than in the pelleted treatment. No differences in bacterial N synthesis or efficiency of synthesis were observed among treatments. Expander treatment numerically increased the duodenal flow of non-ammonia N (NAN) and amino acid N (AAN), and seemed to increase the flow of non-ammonia non-bacterial N (NANBN) to the duodenum to a similar extent as was indicated by nylon bag studies. Milk production and milk fat and protein content were increased by the expander treatment (P<0.05), indicating that expander treatment increased the supply of nutrients for milk production.