Exploring the substrate specificity of OxyB, a phenol coupling P450 enzyme involved in vancomycin biosynthesis

OxyB is a cytochrome P450 enzyme that catalyzes the first oxidative phenol coupling reaction during vancomycin biosynthesis. The preferred substrate is a linear peptide linked as a C-terminal thioester to a peptide carrier protein (PCP) domain of the glycopeptide antibiotic non-ribosomal peptide synthetase. Previous studies have shown that OxyB can efficiently oxidize a model hexapeptide-PCP conjugate (R-Leu(1)-R-Tyr(2)-S-Asn(3)-R-Hpg(4)-R-Hpg(5)-S-Tyr(6)-S-PCP) (Hpg = 4-hydroxyphenylglycine) into a macrocyclic product by phenolic coupling of the aromatic rings in residues-4 and -6. In this work, the substrate specificity of OxyB has been explored using a series of N-terminally truncated peptides related in sequence to this model hexapeptide-PCP conjugate. Deletion of one or three residues from the N-terminus afforded a penta- (Ac-Tyr-Asn-Hpg-Hpg-Tyr-S-PCP) and a tri- (Ac-Hpg-Hpg-Tyr-S-PCP) peptide that were also efficiently transformed into the corresponding macrocyclic cross-linked product by OxyB. The tripeptide, representing the core of the macrocycle in vancomycin created by OxyB, is thus sufficient, as a thioester with the PCP domain, for phenol coupling to occur. The related tetrapeptide-PCP thioester was not cyclized by OxyB, neither was a related model hexapeptide containing tryptophan in place of tyrosine-6, nor were tripeptides (related to the natural product K-13) with the sequence Ac-Tyr-Tyr-Tyr-S-PCP cross-linked by OxyB.     

The formation of conjugates with PEG–chitosan improves the biocatalytic efficiency and antitumor activity of L-asparaginase from <Emphasis Type="Italic">Erwinia carotovora</Emphasis>

The ChitoPEGylation method, which is a novel approach to regulating the catalytic properties of enzymes that is based on the formation of a covalent conjugate of an enzyme with branched copolymers of chitosan, has been developed. The efficiency of this method has been demonstrated using a new recombinant preparation of L-asparaginase from Erwinia carotovora (EwA) as a model. The molecular architecture and composition of EwA conjugates with PEG–chitosans have been optimized. It has been shown that the decisive factors that affect the activity of the EwA conjugates are the molecular weight of and PEGylation degree of chitosan. It has been found that the EwA conjugation with PEG–chitosan increases, its cytostatic activity against human chronic myeloid leukemia K562 cells, Burkitt’s lymphoma Raji cells, and acute lymphoblastic leukemia Jurkat cells. These data provide new approaches to the synthesis of L-asparaginase preparations with improved biocatalytic properties.     

Grafting of 18β-Glycyrrhetinic Acid and Sialic Acid onto Chitosan to Produce a New Amphipathic Chitosan Derivative: Synthesis,Characterization, and Cytotoxicity

Chitosan is the only cationic polysaccharide found in nature. It has broad application prospects in biomaterials, but its application is limited due to its poor solubility in water. A novel chitosan derivative was synthesized by amidation of chitosan with 18β-glycyrrhetinic acid and sialic acid. The chitosan derivatives were characterized by Fourier transform infrared spectroscopy, thermogravimetric analysis, and measurement of the zeta potential. We also investigated the solubility, cytotoxicity, and blood compatibility of chitosan derivatives. 18β-glycyrrhetinic acid and sialic acid could be grafted onto chitosan molecular chains. The thermal stability of the synthesized chitosan derivatives was decreased and the surface was positively charged in water and phosphate-buffered saline. After chitosan had been modified by 18 β-glycyrrhetinic acid and sialic acid, the solubility of chitosan was improved greatly in water and phosphate-buffered saline, and percent hemolysis was <5%. Novel amphiphilic chitosan derivatives could be suitable polymers for biomedical purposes.     

Chromium (VI) ion adsorption features of chitosan film and its chitosan/zeolite conjugate 13X film

This research evaluated the importance of the adsorption properties of chitosan a chitosan/zeolite conjugate film for the removal of Cr(VI) ions from solutions in the 5-260 mg/L concentration range, when the pH was adjusted to 4.0 and 6.0. The uptake capacities of the films formed by chitosan and by the chitosan/zeolite conjugate were calculated by mass balance. The equilibrium isotherms were fitted to the Langmuir, Freundlich and Redlich-Peterson models. The chitosan film seems to be a good sorbent for Cr(VI) at pH 4, but its physical instability suggests the need for a more resilient support. Due to this fact zeolite was added to the chitosan matrix in solution and a chitosan/zeolite (CS/Zeo) film was thus formed. The solubility of the film and the characterization of the different matrices by FTIR, TGA and X-Ray showed that a cross-linked structure was formed between the chitosan and zeolite and the solubility of the film increased. In this study, the low manufacturing cost of the CS/Zeo matrix, the good uptake of Cr(VI) at acidic pH (17.28 mg/g) and the non desorption of Cr(VI) from the film in water suggests this combination should be tested in industrial environment.     

Mechanisms and kinetics of trisodium 2-hydroxy-1,1'-azonaphthalene-3,4',6-trisulfonate adsorption onto chitosan

Chitosan, a naturally abundant biopolymer, has widely been studied for metal adsorption from various solutions, but the extension of chitosan as an adsorbent to remove organic substances from water and wastewater has seldom been explored. In this study, the adsorption of an azo dye, trisodium 2-hydroxy-1,1'-azonaphthalene-3,4',6-trisulfonate (1), from aqueous solution onto the various degrees of deacetylated chitosan has been investigated. Equilibrium studies have been carried out to determine the capacity of chitosan for dye. The experimental data were analyzed using two isotherm correlations, namely, Langmuir and Freundlich equations. The linear correlation coefficients were determined for each isotherm and the Langmuir provided the best fit. The experimental adsorption isotherms were perfectly reproduced in the simulated data obtained from numerical analysis on the basis of the Langmuir model and the isotherm constants. Adsorption of (1) onto the chitosan flakes was found to be strongly depending on degrees of deacetylation in chitosan and temperatures. Significant amounts of (1) were adsorbed by chitosan 8B (higher degree of deacetylated chitosan), but the adsorption capacity was reduced remarkably with increasing solution temperatures. Thermodynamic parameters such as change in free energy (DeltaG), enthalpy (DeltaH), and entropy (DeltaS) were also determined. In addition, kinetic study indicated that the adsorption process mechanisms were both transport- and attachment-limited.     

Chitosan-supported imine palladacycle complex and its catalytic performance for heck reaction

Summary A chitosan-supported imine palladacycle complex has been synthesized by salicyladehyde via grafting on chitosan to form chitosan-Schiff base, followed by reacting with Li₂PdCl₄ in methanol. The catalyst has high activity and stereoselectivity for arylation of conjugate alkenes with aryl halides. This chitosan-supported imine palladium catalyst can be recovered and reused.     

Studies on asterosaponins—V : A novel steroid conjugate, 5α-pregn-9(11)-ene-3β,6α-diol-20-one-3-sulfate,from a starfish saponin,asterosaponin A

A new crystalline steroid conjugate was obtained by partial acid hydrolysis of asterosaponin A, a steroidal saponin from the starfish, Asterias amurensis. The structure of the conjugate was established as 5α-pregn-9(11)-ene-3β,6α-diol-20-one-3-sulfate (2) on the basis of elemental analysis, IR and PMR spectra measurement and chemical reaction. Solvolysis of compound 2 yielded 5α-pregn-9(11)-ene-3β,6α-diol-20-one. Oxidation with chromium trioxide-pyridine complex followed by solvolysis afforded a new steroid, 5α-pregn-9(11)-ene-3β-ol-6,20-dione, whose structure was deduced by the measurement of ORD curve and PMR spectra. Thus, the location of carbohydrate moiety in asterosaponin A has been assigned to 6α-hydroxy group of the steroid conjugate.     

Vesicle formed by amphiphilc cucurbit[6]uril: versatile, noncovalent modification of the vesicle surface, and multivalent binding of sugar-decorated vesicles to lectin

We report a novel vesicle formed by an amphiphilic CB[6] derivative, the surface of which can be easily modified via host-guest interactions by taking advantage of molecular cavities, readily accessible at the vesicle surface, and their strong affinity toward polyamines. Amphiphilic CB[6] derivative 1 synthesized by reaction between (allyloxy)12CB[6] and 2-[2-(2-methoxyethoxy)ethoxy]ethanethiol affords a vesicle that has been characterized by TEM, light scattering, and fluorescent dye entrapment experiments. Treatment of vesicle 1 with FITC (fluorescein isothiocyanate)-spermine conjugate ligand 2, in which spermine serves as a binding motif to CB[6] and FITC as a fluorescent tag, produced a surface-modified vesicle, which can be easily visualized by a confocal microscope. This result provides us with a new noncovalent, modular approach to the modification of vesicle surfaces. By treating the vesicle derived from the amphiphilic CB[6] with a tag-attached polyamine, we can easily decorate the surface of the vesicle with the tag. Sugar-decorated vesicles were prepared by this noncovalent method, and their interactions with concanavalin A (ConA) were studied. The binding constant of the vesicle decorated with mannose-spermidine conjugate 3 to ConA was measured to be approximately 3 x 104 M-1, which is almost 3 orders of magnitude higher than that of free ligand 3 to ConA (K = approximately 50 M-1). On the other hand, the binding constant of the vesicle coated with galactose-spermidine conjugate 4 to ConA was too small to be measured. These results illustrate the specific and multivalent interactions between the mannose-decorated vesicle and ConA. The ability for facile surface modification suggests many practical applications, including its use in targeted drug delivery and immunization.     

A fullerene-paclitaxel chemotherapeutic: synthesis, characterization, and study of biological activity in tissue culture

A fullerene-paclitaxel conjugate has been synthesized as a slow-release drug for aerosol liposome delivery of paclitaxel for lung cancer therapy. The conjugate was designed to release paclitaxel via enzymatic hydrolysis and subsequently has shown a half-life of release of 80 min in bovine plasma. A liposome formulation of the conjugate has been prepared using dilauroylphosphatidylcholine (DLPC), and its IC50 is virtually identical to the IC50 for a paclitaxel-DLPC formulation in human epithelial lung carcinoma A549 cells. With both clinically relevant kinetics of hydrolysis and significant cytotoxicity in tissue culture, the conjugate holds promise for enhanced therapeutic efficacy of paclitaxel in vivo.     

Synthesis and Characterization of a Modified Chitosan

Chitosan has been modified by incorporating a cationic moiety N- (3-chloro 2-hydroxypropyl) trimethyl ammonium chloride (CHPTAC) onto its primary amine group in aqueous alkali medium. A series of modified chitosan copolymers (Chito-cat-1 to Chito-cat-5) was synthesized by varying the CHPTAC to chitosan ratio. The modified chitosan copolymers have been characterized by viscometry, elemental analysis, infrared spectroscopy (IR), thermal analysis and X-ray diffractometry. From the above investigations, it has been confirmed that the cationic moiety was successfully incorporated onto chitosan.     

Identification of by-products from an orthogonal peptide ligation by oxime bonds using mass spectrometry and tandem mass spectrometry

Synthetic proteins with unusual architecture are obtained through chemoselective ligation, a method based on the condensation of unprotected peptides under mild aqueous conditions. The last step of a new procedure leading to a tri-branched conjugate consists of the chemoselective ligation reaction between an (aminooxy)acetyl peptide and a peptide aldehyde resulting from a first ligation via an oxime bond. In order to optimize the reaction conditions, electrospray ionization mass spectrometry combined with liquid chromatography and tandem mass spectrometry has been used. In addition to the target tri-branched conjugate, two other conjugates were characterized allowing documentation of transoximation reactions in peptide chemistry. A fourth conjugate was identified as a side product appearing after the first ligation. Data obtained by low-energy collision-induced dissociation led to a rapid and reliable identification of impurities observed in the (aminooxy)acetyl peptide despite a previous high performance liquid chromatography (HPLC) purification. This highlights the great reactivity of the aminooxy group towards carbonyl-containing compounds.     

Synthesis and Characterization of a Chitosan-bearing Binucleating Ligand and Its pH Dependency on Copper(II) Adsorption

A novel chitosan derivative has been synthesized by the condensation of a binucleating ligand, 2,6-bis[(N-methylpiperazine-1-yl)methyl]-4-formylphenol (BNL), with chitosan (CTS). The resulting material (CTS-BNL) was characterized and its adsorption properties towards copper(II) ions in aqueous solutions of various pH have been studied, the adsorption capacity (Q(e)) is about 0.94 mmol g(-1) at pH 6 and 1.45 mmol g(-1) at pH 8.5. This higher adsorption capacity may be due to the binucleating ligand anchored to chitosan.     

Radioimmunoassay for the determination of 2-methoxyestriol concentration in plasma of pregnant women.

A specific assay method has been developed for the determination of 2-methoxyestriol in plasma of pregnant women. The quantitation was achieved by radioimmunoassay after extraction of the plasma with ethyl acetate and purification on a Sephadex LH-20 column. In order to obtain the immunogen for 2-methoxyestriol, 6-oxo-2-methoxyestriol was converted to its 6-(O-carboxymethyl)oxime derivative. The derivative was then coupled to bovine serum albumin by the mixed anhydride method, and rabbits were immunized with this conjugate. The antiserum obtained was partially purified by affinity chromatography on estrone 17-(O-carboxymethyl)oxime-aminohexyl Sepharose conjugate to eliminate the cross-reactive antibodies. Plasma 2-methoxyestriol concentrations in pregnancy were estimated to be mean values of 41.1 pg/ml (12th-14th week), 85.3 pg/ml (27th-29th week), and 97.5 pg/ml (37th-41st week).     

探索利用无机结合肽PT2(DRTSTWR)制备纳米铂晶体

实验中首先利用固定化于PET(聚对苯二甲酸己二醇酯)膜上无机结合肽PT2 (DRTSTWR)制备出铂微晶体. 然后用游离的PT2与PtCl₄在室温和pH中性环境中反应24 h, 所得产物用TEM观察, 其多数为1～2 nm呈方形和球形晶体, 经EDX分析显示晶体的元素组成中Pt远大于Cl的含量, 显然晶体不可能是PtCl₄或PtCl₂; 样品的XPS谱图中出现了结合能值为71.0 eV峰(Pt_4f标准值71.1 eV), 确认晶体为铂纳晶, 由此推断无机结合肽PT2在无细胞状态下能够作为模板仿生合成铂晶体. 继而在反应体系中加入不同浓度的壳聚糖和聚丙烯酸钠, 观察到纳米粒子的粒径和形貌随表面活性剂的加入发生了改变. 加入5倍的壳聚糖制备的铂晶体的直径在12～15.5 nm之间, 晶体排布似乎有成线状的趋势. 加入5倍的聚丙烯酸钠制备的铂晶体, 形貌更加规整, 以球形颗粒为主, 粒子直径在6～8 nm之间.     

Synthesis,characterization and antimicrobial activity of maltol functionalized chitosan derivatives

Chitin was isolated from prawn shell powder through demineralization and deproteinization process. Chitosan was synthesized from isolated chitin by deacetylation process and characterized by Fourier Transform Infrared (FTIR) spectra which showed close agreement with commercial chitosan. Physicochemical features such as moisture content, ash content, degree of deacetylation and molecular weight has been measured. The prepared chitosan was found to have comparatively higher molecular weight than the commercial chitosan. Functionalization of NH₂ group of chitosan with C?=?O group of maltol and ethyl maltol by refluxing equimolar quantities of respective ketones was performed. These synthesized derivatives of chitosan were characterized by their FTIR, ¹³C-Nuclear magnetic resonance (NMR) spectroscopy, elemental analysis, X-Ray Diffraction (XRD), Thermogravimetric analysis (TGA) and Differential thermal analysis (DTA) instrumental techniques. Antibacterial screening results of the synthesized chitosan and its derivatives indicate that these compounds are active against Escherichia coli bacteria.     

An amine-derivatized, DOTA-loaded polymeric support for Fmoc solid phase peptide synthesis

An amine-derivatized DOTA has been used to modify the surface of a polymeric support for conventional solid phase peptide synthesis (SPPS) following standard Fmoc chemistry methods. This methodology was used to synthesize a peptide-DOTA conjugate that was demonstrated to be a PARACEST MRI contrast agent. Therefore, this synthesis methodology can facilitate Fmoc SPPS of molecular imaging contrast agents.     

Facile Regioselective Synthesis of a Novel Chitosan–Pexiganan Conjugate with Potential Interest for the Treatment of Infected Skin Lesions

A pexiganan–chitosan conjugate was designed to combine the exceptional bioadhesion and tissue-regenerating abilities of chitosan with the excellent antibiotic properties of pexiganan. We herein report our first results on the successful synthesis, including Fourier transform infrared (FT-IR) and amino acid analysis of such conjugate, which was prepared by regioselective covalent attachment of a Cys-containing pexiganan analog to the chitosan's amino groups. Further results from ongoing research will be reported.     

Protective,Biostimulating, and Eliciting Effects of Chitosan and Its Derivatives on Crop Plants

Chitosan is a biodegradable and biocompatible polysaccharide obtained by partial deacetylation of chitin. This polymer has been gaining increasing popularity due to its natural origin, favorable physicochemical properties, and multidirectional bioactivity. In agriculture, the greatest hopes are raised by the possibility of using chitosan as a biostimulant, a plant protection product, an elicitor, or an agent to increase the storage stability of plant raw materials. The most important properties of chitosan include induction of plant defense mechanisms and regulation of metabolic processes. Additionally, it has antifungal, antibacterial, antiviral, and antioxidant activity. The effectiveness of chitosan interactions is determined by its origin, deacetylation degree and acetylation pattern, molecular weight, type of chemical modifications, pH, concentration, and solubility. There is a need to conduct research on alternative sources of chitosan, extraction methods, optimization of physicochemical properties, and commercial implementation of scientific progress outcomes in this field. Moreover, studies are necessary to assess the bioactivity and toxicity of chitosan nanoparticles and chitosan conjugates with other substances and to evaluate the consequences of the large-scale use thereof. This review presents the unique properties of chitosan and its derivatives that have the greatest importance for plant production and yield quality as well as the benefits and limitations of their application.     

Integrin‐Targeting Knottin Peptide–Drug Conjugates Are Potent Inhibitors of Tumor Cell Proliferation

Antibody–drug conjugates (ADCs) offer increased efficacy and reduced toxicity compared to systemic chemotherapy. Less attention has been paid to peptide–drug delivery, which has the potential for increased tumor penetration and facile synthesis. We report a knottin peptide–drug conjugate (KDC) and demonstrate that it can selectively deliver gemcitabine to malignant cells expressing tumor‐associated integrins. This KDC binds to tumor cells with low‐nanomolar affinity, is internalized by an integrin‐mediated process, releases its payload intracellularly, and is a highly potent inhibitor of brain, breast, ovarian, and pancreatic cancer cell lines. Notably, these features enable this KDC to bypass a gemcitabine‐resistance mechanism found in pancreatic cancer cells. This work expands the therapeutic relevance of knottin peptides to include targeted drug delivery, and further motivates efforts to expand the drug‐conjugate toolkit to include non‐antibody protein scaffolds.     

Preparation of stable,camphor-derived,optically active allylic sulfoxides

(+)-Camphor has been stereospecifically converted in three steps into a single isoborneol allyl sulfoxide derivative, which upon heating to 145° is quantitatively converted into its sulfoxide epimer; the anions of these compounds undergo stereospecific conjugate addition to cyclopent-2-en-1-one.