定点突变提高IL-4免疫毒素对淋巴瘤的选择性和杀伤活性

采用软件分析选择与IL-4分子结合与活性相关的重要位点13T,121R,通过定点突变得到IL-4突变基因cpIL4(13D121E),将其与绿脓杆菌外毒素突变基因PE38KDEL融合,成功地构建了编码免疫毒素cpIL4(13D121E)-PE38KDEL的融合基因.该基因在原核表达系统中得到了高效表达,表达量占细胞全蛋白的30%以上.表达产物经亲和色谱和阴离子交换色谱纯化后,进行细胞毒性实验,证明其对表达型IL-4受体的淋巴瘤细胞Daudi具有良好的细胞毒作用,活性是同类型IL-4免疫毒素的2倍,而对表达型IL-4受体的内皮细胞活性较低.     

Dendroclefts: Optically Active Dendritic Receptors for the Selective Recognition and Chiroptical Sensing of Monosaccharide Guests

The enantiomerically pure dendritic receptors with cleft-type recognition sites (dendroclefts) of generation zero ((−)- G0 ), one ((−)- G1 ), and two ((−)- G2 ) (Fig. 1) were prepared for the complexation of monosaccharides via H-bonding. They incorporate a rigid, optically active 9,9′-spirobi[9H-fluorene] core bearing 2,6-bis(carbonylamino)pyridine moieties as H-bonding sites in the 2,2′-positions. The dendritic shells in (−)- G1 and (−)- G2 are made out of a novel type of dendritic wedges of the first ( 8 ; Scheme 2) and second ( 13 ; Scheme 3) generations, which contain only donor O-atoms and are attached to the H-bonding edges of the core via glycine spacers (Scheme 4). The formation of stable 1 : 1 complexes (association constants K_a between 100 and 600 M ⁻¹, T=298 K; Table 2) between the three receptors and pyranosides in CHCl₃ was confirmed by ¹H-NMR and CD binding titrations as well as by Job plot analyses. The degree of dendritic branching was found to exert a profound effect on the stereoselectivity of the recognition processes. The binding enantioselectivity decreases with increasing degree of branching, whereas the diastereoselectivity increases. The ¹H-NMR analysis showed that the N−H⋅⋅⋅O H-bonds between the amide NH groups around the core and the sugar O-atoms become weakened with increasing dendritic generation, presumably due to steric factors and competition from intramolecular H-bonding between these amide groups and the O-atoms of the dendritic shell. The chiroptical properties of the dendroclefts respond to guest binding in a stereoselective manner. Whereas large differential changes are seen in the circular dichroism (CD) spectra of (−)- G0 and (−)- G1 upon complexation of the enantiomeric monosaccharides (Figs. 3 and 4), the CD spectra of the higher-generation derivative (−)- G2 respond to a lesser extent to guest complexation (Fig. 5). This is indicative of a different binding geometry, more remote from the core chromophore. With their higher masses, the dendroclefts (−)- G1 and (−)- G2 are readily recycled from host-guest solutions by gel-permeation chromatography. The strong CD sensory response and the easy recyclability suggest applications of chiral dendroclefts as sensors for biologically important molecules.     

新的4’-去甲表鬼臼衍生物的合成及其抗癌活性

根据鬼臼毒衍生物和5-氟尿嘧啶的抗癌机理和构效关系,合成了7个新的4β- 5-氟尿嘧啶取代-4’-去甲表鬼鬼臼毒衍生物。在抑制金属基质蛋白酶I和胶原酶I 活性测试中,化合物2.4和2.6的抑制活性比鬼臼乙叉甙（VP-16）和5-氟尿嘧啶（ 5-Fu）强的3倍和5倍,在治疗癌细胞转移方面值得进一步探讨。     

新的4＇-去甲表鬼臼衍生物的合成及其抗癌活性

根据鬼臼毒衍生物和5-氟尿嘧啶的抗癌机理和构效关系,合成了7个新的4β-5-氟尿嘧啶取代4＇-去甲表鬼臼毒衍生物.在抑制金属基质蛋白酶I和胶原酶I活性测试中,化合物2.4和2.6的抑制活性比鬼臼乙叉甙(VP-16)和5-氟尿嘧啶(5-Fu)强3倍和5倍,在治疗癌细胞转移方面值得进一步探讨.     

Enhanced Activity and Substrate Specificity by Site-Directed Mutagenesis for the P450 119 Peroxygenase Catalyzed Sulfoxidation of Thioanisole

P450 119 peroxygenase was found to catalyze the sulfoxidation of thioanisole and the sulfonation of sulfoxide in the presence of tert-butyl hydroperoxide (TBHP) for the first time with turnover rates of 1549 min⁻¹ and 196 min⁻¹ respectively. Several mutants were designed to improve the peroxygenation activity and thioanisole specificity by site-directed mutagenesis. The F153G/T213G mutant gave an increase of sulfoxide yield and a decrease of sulfone yield. Moreover the S148P/I161T/K199E/T214V mutant and the K199E mutant with acidic Glu residue contributed to improving the product ratio of sulfoxide to sulfone. Addition of short-alkyl-chain organic acids to the P450 119 peroxygenase-catalyzed sulfur oxidation of thioanisole was investigated. Octanoic acid was found to induce a preferred sulfoxidation of thioanisole catalyzed by the F153G/T213G mutant to give approximately 2.4-fold increase in turnover rate with a k_cat value of 3687 min⁻¹ relative to that of the wild-type, and by the F153G mutant to give the R-sulfoxide up to 30 % ee. The experimental control and the proposed mechanism for the P450 119 peroxygenase-catalyzed sulfoxidation of thioanisole in the presence of octanoic acid suggested that octanoic acid could partially occupy the substrate pocket; meanwhile the F153G mutation could enhance the substrate specificity, which could lead to efficiently regulate the spatial orientation of thioanisole and facilitate the formation of Compound I. This is the most effective catalytic system for the P450 119 peroxygenase-catalyzed sulfoxidation of thioanisole.     

Synthesis and Cytotoxic Activity of Novel Water-soluble Prodrugs of Combretastatin A-4

Combretastatin A-4 (CA4) 1 is a natural product isolated from the bark of a South African tree Combretum caffrum1-3. This substance is a potent antimitotic agent and can strongly inhibit the polymerization of tubulin by binding to the colchicine-binding s…     

A Novel Purification Procedure for Active Recombinant Human DPP4 and the Inability of DPP4 to Bind SARS-CoV-2

Proteases catalyse irreversible posttranslational modifications that often alter a biological function of the substrate. The protease dipeptidyl peptidase 4 (DPP4) is a pharmacological target in type 2 diabetes therapy primarily because it inactivates glucagon-like protein-1. DPP4 also has roles in steatosis, insulin resistance, cancers and inflammatory and fibrotic diseases. In addition, DPP4 binds to the spike protein of the MERS virus, causing it to be the human cell surface receptor for that virus. DPP4 has been identified as a potential binding target of SARS-CoV-2 spike protein, so this question requires experimental investigation. Understanding protein structure and function requires reliable protocols for production and purification. We developed such strategies for baculovirus generated soluble recombinant human DPP4 (residues 29–766) produced in insect cells. Purification used differential ammonium sulphate precipitation, hydrophobic interaction chromatography, dye affinity chromatography in series with immobilised metal affinity chromatography, and ion-exchange chromatography. The binding affinities of DPP4 to the SARS-CoV-2 full-length spike protein and its receptor-binding domain (RBD) were measured using surface plasmon resonance and ELISA. This optimised DPP4 purification procedure yielded 1 to 1.8 mg of pure fully active soluble DPP4 protein per litre of insect cell culture with specific activity >30 U/mg, indicative of high purity. No specific binding between DPP4 and CoV-2 spike protein was detected by surface plasmon resonance or ELISA. In summary, a procedure for high purity high yield soluble human DPP4 was achieved and used to show that, unlike MERS, SARS-CoV-2 does not bind human DPP4.     

Synthesis and Antimicrobial Activity of Some Novel 7-Chloro-4-aminoquinoline Derivatives

Russian Journal of General Chemistry - A number of novel 7-chloro-4-aminoquinoline derivatives have been efficiently synthesized by nucleophilic aromatic substitution reaction of...     

Identification of Residue Involved in Nucleotidyltransferase Activity of LinA from Staphylococci by Site-directed Mutagenesis

The lincosamide class of antibacterials is widely used for the treatment of a broad spectrum of infections, and one prevalent route of resistance to lincosamides in pathogenic gram-positive cocco is antibiotic modification. Enzymes encoded by lin genes, belonging to nucleotidyltransferase superfamily, catalyze adenylylation to inactivate lincosamides. LinA can adenylylate lincosamides at either 3?-or 4?-OH of the methylthiolincosamide sugar. The crystal structure of LinA/lincomycin has confirmed its active site. However, the residue interacting with nucleotidyl donors remains elusive. Here, we modeled the complex structure of LinA/lincomycin/Mg~(2+)/AMPCPP to reveal a putative pocket for nucleotidyl donors and suggested the residue R45 in this pocket involved in the recognition of donor substrates NTP and catalysis. ITC and enzyme activity assays show that the mutation of residue R45 impairs LinA nucleotidyltransferase activity in vitro. This work provides insights into the molecular mechanism of the nucleotide binding and transferring activity of antibiotic NTases.     

Synthesis and Anti-HIV Activity of a Novel Series of Isoquinoline-Based CXCR4 Antagonists

An expansion of the structure–activity relationship study of CXCR4 antagonists led to the synthesis of a series of isoquinolines, bearing a tetrahydroquinoline or a 3-methylpyridinyl moiety as head group. All compounds were investigated for CXCR4 affinity and antagonism in competition binding and calcium mobilization assays, respectively. In addition, the anti-HIV activity of all analogues was determined. All compounds showed excellent activity, with compound 24c being the most promising one, since it displayed consistently low nanomolar activity in the various assays.     

新型1,1-二氧代-4H-苯并[1,2,4]-噻二嗪类衍生物的合成及其抗肿瘤活性

以2-硝基苯磺酰氯为起始原料,经多步反应合成了12种新型1,1-二氧代-4H-苯并[1,2,4]-噻二嗪类衍生物,产物收率高,水溶性好,并经¹H NMR、¹³C NMR、MS-ESI和元素分析法确证结构。 用MTT法测试了这类衍生物对肝癌细胞HepG-2生长的抑制作用,结果显示目标化合物能不同程度地抑制肿瘤细胞生长,其中7-氨甲基-3-环丙基-1,1-二氧代-4H-苯并[1,2,4]-噻二嗪 (7c)对肝癌细胞HepG-2的抗性显著,最高抑制率达到79.3%。     

新型喹唑啉类化合物的合成与抗癌活性的初步研究

以香兰素为起始原料,经过氰基化、醚化、硝化、还原和环化反应,合成了一系列新型的含二苯乙烯结构单元的4-芳胺基喹唑啉类衍生物。化合物的结构经IR、1H NMR和13C NMR确证,并采用MTT法对SMMC-7721(人肝癌细胞)、SK-OV-3(人卵巢癌细胞)等几种常见肿瘤细胞进行了初步体外抗肿瘤活性实验。结果表明合成的化合物均具有良好的抑制肿瘤细胞生长的作用。     

W544F定点突变提高苏云金杆菌Cry1Ac蛋白的稳定性

W544是Cry1Ac蛋白上独特于其它Cry类蛋白的一个氨基酸, 它与F578和F604一起组成一个“螺旋桨状”的疏水簇, 通过疏水相互作用维持蛋白的三维结构稳定. 本研究通过定点突变将W544保守地替换为苯丙氨酸, SDS-PAGE分析结果表明其纯化的原毒素对紫外照射、胰蛋白酶处理和室温存贮的稳定性相对于野生Cry1Ac都有一定程度的提高; 经原子力显微镜观察, 发现W544F产生的晶体两个顶点间的垂直距离比野生型Cry1Ac约长0.6 μm, 且晶体表面不及野生型光滑; 此外, W544F与野生Cry1Ac的杀虫活性相似, 但经过紫外光照射9 h后, 其保留的杀虫活性比野生型高4倍以上. W544F突变较好地解决了Cry1Ac毒素蛋白田间应用不持久的问题, 具有重要的应用价值.     

Synthesis and Antifungal Activity of Novel Quinazolin-4(3H)-one Derivatives

A novel group of 6-iodoquinazolin-4(3H)-one derivatives was prepared starting from 6-iodo-2-ethoxy-4H-3,1-benzoxazin-4-one (3) via action of various nitrogen nucleophiles such as primary and secondary amines, hydrazine hydrate, and its derivatives. The 3-amino-2-hydrazinyl-6-iodoquinazolin-4(3H)-one (15) was used as a key starting material to prepare new heterocyclic compounds. The structures of all synthesized compounds were inferred from the infrared, mass spectral, and ¹H NMR spectral data as well as elemental analysis. The fungicidal activities of the target compounds were preliminarily evaluated.     

1-(取代吡唑-4-甲酰基)吡唑类化合物的合成及生物活性研究

设计合成了25个新型1-取代吡唑甲酰基吡唑系列衍生物,化合物结构经¹HNMR、元素分析、IR和MS确证.生物活性测试结果表明,该类化合物具有一定除草活性,讨论了其结构与活性的关系.     

含有2,4-二卤代联苯基的新型唑类衍生物的合成及其抗真菌活性

以2,4-二卤代联苯为原料,经傅-克酰基化、还原、取代等反应步骤,合成了含有2,4-二卤代联苯基的新型唑类目标化合物3a~3k,其结构用红外光谱(IR)、高分辨质谱(HMRS)、核磁共振氢谱(¹H NMR)、核磁共振碳谱(¹³C NMR)等技术手段进行了表征。 测试了目标化合物的体外抗真菌活性,结果表明,所有目标化合物对所测试的致病真菌均有一定程度的抗真菌活性。 其中化合物3a~3k对红色发癣菌和石膏样毛癣菌的抗真菌活性和两性霉素B相当,化合物3b、3c、3e、3f、3h、3i、3k对白色念珠菌的抗真菌活性优于或等于酮康唑。     

Gallium Oxide Nanorods: Novel,Template‐Free Synthesis and High Catalytic Activity in Epoxidation Reactions

Gallium oxide nanorods with unprecedented small dimensions (20–80 nm length and 3–5 nm width) were prepared using a novel, template‐free synthesis method. This nanomaterial is an excellent heterogeneous catalyst for the sustainable epoxidation of alkenes with H₂O₂, rivaling the industrial benchmark microporous titanosilicate TS‐1 with linear alkenes and being much superior with bulkier substrates. A thorough characterization study elucidated the correlation between the physicochemical properties of the gallium oxide nanorods and their catalytic performance, and underlined the importance of the nanorod morphology for generating a material with high specific surface area and a high number of accessible acid sites.     

Preparation and Catalytic Activity of a Novel Nanocrystalline ZrO2@C Composite for Hydrogen Storage in NaAlH4

Sodium alanate (NaAlH₄) has attracted intense interest as a prototypical high‐density hydrogen‐storage material. However, poor reversibility and slow kinetics limit its practical applications. Herein, a nanocrystalline ZrO₂@C catalyst was synthesized by using Uio‐66(Zr) as a precursor and furfuryl alcohol (FA) as a carbon source. The as‐synthesized ZrO₂@C exhibits good catalytic activity for the dehydrogenation and hydrogenation of NaAlH₄. The NaAlH₄‐7 wt % ZrO₂@C sample released hydrogen starting from 126 °C and reabsorbed it starting from 54 °C, and these temperatures are lower by 71 and 36 °C, respectively, relative to pristine NaAlH₄. At 160 °C, approximately 5.0 wt % of hydrogen was released from the NaAlH₄‐7 wt % ZrO₂@C sample within 250 min, and the dehydrogenation product reabsorbed approximately 4.9 wt % within 35 min at 140 °C and 100 bar of hydrogen. The catalytic function of the Zr‐based active species is believed to contribute to the significantly reduced operating temperatures and enhanced kinetics.     

Gallium Oxide Nanorods: Novel,Template‐Free Synthesis and High Catalytic Activity in Epoxidation Reactions

Gallium oxide nanorods with unprecedented small dimensions (20–80 nm length and 3–5 nm width) were prepared using a novel, template‐free synthesis method. This nanomaterial is an excellent heterogeneous catalyst for the sustainable epoxidation of alkenes with H₂O₂, rivaling the industrial benchmark microporous titanosilicate TS‐1 with linear alkenes and being much superior with bulkier substrates. A thorough characterization study elucidated the correlation between the physicochemical properties of the gallium oxide nanorods and their catalytic performance, and underlined the importance of the nanorod morphology for generating a material with high specific surface area and a high number of accessible acid sites.     

Synthesis of Novel Functionalized 4-Aza-2,3-Didehydropodophyllotoxin Derivatives with Potential Antitumor Activity

Novel arylamino alcohols were synthesized and these alcohols were used to prepare 12 novel N-(2-hydroxy-ethyl)-2,3-didehydroazapodophyllotoxins, in one step, by simple reflux in ethanol. Isolated yields in the range of 50-70% were obtained.