Synthesis of Isonitrile Derivatives of Diglycerides and Carbohydrates as Intermediates for Multicomponent Ugi Reaction

Russian Journal of Bioorganic Chemistry - Recently, there has been an increase in interest in multicomponent reactions, mainly due to the possibility of assembling of complex organic molecules in...     

Scalable 96-well Plate Based iPSC Culture and Production Using a Robotic Liquid Handling System

Continued advancement in pluripotent stem cell culture is closing the gap between bench and bedside for using these cells in regenerative medicine, drug discovery and safety testing. In order to produce stem cell derived biopharmaceutics and cells for tissue engineering and transplantation, a cost-effective cell-manufacturing technology is essential. Maintenance of pluripotency and stable performance of cells in downstream applications (e.g., cell differentiation) over time is paramount to large scale cell production. Yet that can be difficult to achieve especially if cells are cultured manually where the operator can introduce significant variability as well as be prohibitively expensive to scale-up. To enable high-throughput, large-scale stem cell production and remove operator influence novel stem cell culture protocols using a bench-top multi-channel liquid handling robot were developed that require minimal technician involvement or experience. With these protocols human induced pluripotent stem cells (iPSCs) were cultured in feeder-free conditions directly from a frozen stock and maintained in 96-well plates. Depending on cell line and desired scale-up rate, the operator can easily determine when to passage based on a series of images showing the optimal colony densities for splitting. Then the necessary reagents are prepared to perform a colony split to new plates without a centrifugation step. After 20 passages (~3 months), two iPSC lines maintained stable karyotypes, expressed stem cell markers, and differentiated into cardiomyocytes with high efficiency. The system can perform subsequent high-throughput screening of new differentiation protocols or genetic manipulation designed for 96-well plates. This technology will reduce the labor and technical burden to produce large numbers of identical stem cells for a myriad of applications.     

Synthesis of 4-azasteroids by an intramolecular Ugi reaction

In this paper we report the use of an intramolecular Ugi reaction to synthesize new 4-azacholestanes diversely substituted both at N-4 and C-5. Both the scope and the stereochemical outcome of this approach were studied by varying the nature of the components necessary for this multicomponent reaction. In sight of our results we concluded that this methodology can be applied to obtain 4-azasteroids targeted to find new biologically active compounds.     

云南松SSR-PCR反应体系的建立与优化

为了建立适宜云南松SSR-PCR的反应体系和扩增程序,利用近缘种火炬松的引物,采用正交设计L16(45)对云南松SSR-PCR反应体系的5因素(Taq酶、Mg2+、模板DNA、dNTP、引物)在4个水平上进行优化,筛选出各反应因素的最佳水平,建立了适于云南松的SSR反应体系.在10μL的反应体系中,模板DNA的用量为30.0 ng,Taq DNA聚合酶的用量为1.0 U,Mg2+的浓度为2.0 mmol/L,dNTPs浓度为0.4 mmol/L,引物的浓度为0.2 μmol/L.扩增程序为:94℃预变性4 min;94℃变性45 s,48℃退火30 s,72℃延伸30 s,30个循环;72℃延长10 min,4℃保存.最后利用1个居群对该体系进行稳定性验证,结果可用于云南松SSR标记的研究.     

利用正交设计建立与优化北美驼绒藜ISSR-PCR反应体系

利用正交试验设计的方法,对北美驼绒藜ISSR-PCR反应的5因素(Taq酶、dNTP、引物、Mg²⁺和模板DNA)4水平进行试验,试验结果运用MINITAB软件进行分析,建立了适合北美驼绒藜的既稳定又谱带多的ISSR-PCR最佳反应体系,即20 μL的反应体系中含有1×buffer,1.5 U Taq酶,0.2 mmol·L^-1 dNTP,0.5 μmol·L^-1引物,2.5 mmol·L^-1 Mg²⁺和10 ng模板DNA。这一优化的ISSR-PCR反应体系的建立,为今后利用ISSR技术进行驼绒藜属植物种质资源分类、遗传图谱构建和遗传变异奠定了技术基础。     

A New Method for the Synthesis of 2′-O-Benzyladenosine Using Mitsunobu Reaction

Abstract

A new method to introduce a benzyl group onto the 2′-OH of purine ribonucleoside is described. Thus, 6-chloropurine 3′-O-benzoylriboside and its 5′-O-trityl congener were condensed with benzyl alcohol using the Mitsunobu reaction to give the 2′-O-benzyl derivative. The yields were varied from 4.6 to 62.9% depending on the solvent used. The product was converted to adenosine, indicating that the stereochemistry at C-2′ is retained.     

四数獐牙菜ISSR-PCR反应体系的正交优化

采用正交试验与单因素设计相结合的方法,对四数獐牙菜ISSR-PCR反应体系中的4种主要因素(Mg2+、TaqDNA聚合酶、dNTP及引物)进行优化筛选,PCR结果用统计软件SPSS16.0分析。结果显示,Mg2+、TaqDNA聚合酶、dNTP这3因素的不同水平对PCR反应结果都有显著影响,其中Mg2+的浓度影响最大。筛选出各反应因素的最佳水平,建立四数獐牙菜ISSR-PCR反应的最佳体系(25μL)为3.0 mmol/L Mg2+,250μmol/L dNTP,0.6μmol/L引物,1UTaqDNA聚合酶,40 ngDNA,2.5μL10×buffer。这一体系的建立为今后利用ISSR技术进行四数獐牙菜遗传多样性分析以及物种保护奠定了技术基础。     

Synthesis of Programmable Main-chain Liquid-crystalline Elastomers Using a Two-stage Thiol-acrylate Reaction

This study presents a novel two-stage thiol-acrylate Michael addition-photopolymerization (TAMAP) reaction to prepare main-chain liquid-crystalline elastomers (LCEs) with facile control over network structure and programming of an aligned monodomain. Tailored LCE networks were synthesized using routine mixing of commercially available starting materials and pouring monomer solutions into molds to cure. An initial polydomain LCE network is formed via a self-limiting thiol-acrylate Michael-addition reaction. Strain-to-failure and glass transition behavior were investigated as a function of crosslinking monomer, pentaerythritol tetrakis(3-mercaptopropionate) (PETMP). An example non-stoichiometric system of 15 mol% PETMP thiol groups and an excess of 15 mol% acrylate groups was used to demonstrate the robust nature of the material. The LCE formed an aligned and transparent monodomain when stretched, with a maximum failure strain over 600%. Stretched LCE samples were able to demonstrate both stress-driven thermal actuation when held under a constant bias stress or the shape-memory effect when stretched and unloaded. A permanently programmed monodomain was achieved via a second-stage photopolymerization reaction of the excess acrylate groups when the sample was in the stretched state. LCE samples were photo-cured and programmed at 100%, 200%, 300%, and 400% strain, with all samples demonstrating over 90% shape fixity when unloaded. The magnitude of total stress-free actuation increased from 35% to 115% with increased programming strain. Overall, the two-stage TAMAP methodology is presented as a powerful tool to prepare main-chain LCE systems and explore structure-property-performance relationships in these fascinating stimuli-sensitive materials.     

phenoSeeder - A Robot System for Automated Handling and Phenotyping of Individual Seeds

The enormous diversity of seed traits is an intriguing feature and critical for the overwhelming success of higher plants. In particular, seed mass is generally regarded to be key for seedling development but is mostly approximated by using scanning methods delivering only two-dimensional data, often termed seed size. However, three-dimensional traits, such as the volume or mass of single seeds, are very rarely determined in routine measurements. Here, we introduce a device named phenoSeeder, which enables the handling and phenotyping of individual seeds of very different sizes. The system consists of a pick-and-place robot and a modular setup of sensors that can be versatilely extended. Basic biometric traits detected for individual seeds are two-dimensional data from projections, three-dimensional data from volumetric measures, and mass, from which seed density is also calculated. Each seed is tracked by an identifier and, after phenotyping, can be planted, sorted, or individually stored for further evaluation or processing (e.g. in routine seed-to-plant tracking pipelines). By investigating seeds of Arabidopsis (Arabidopsis thaliana), rapeseed (Brassica napus), and barley (Hordeum vulgare), we observed that, even for apparently round-shaped seeds of rapeseed, correlations between the projected area and the mass of seeds were much weaker than between volume and mass. This indicates that simple projections may not deliver good proxies for seed mass. Although throughput is limited, we expect that automated seed phenotyping on a single-seed basis can contribute valuable information for applications in a wide range of wild or crop species, including seed classification, seed sorting, and assessment of seed quality.Seeds play a major role in keeping continuity between successive generations (Esau, 1977) and are key for the distribution and evolution (Moles et al., 2005) of higher plants. Fertile seeds carry an embryo and may contain nutrient storage tissues in cotyledons, endosperm, and/or perisperm, supporting germination and seedling development at early developmental stages. Although this is true for all seed plants, various traits of seeds, such as size, shape, weight, and chemical composition, can be very different between plant species or accessions. For example, the Arabidopsis (Arabidopsis thaliana) accession Cape Verde Islands was reported to yield on average 40% fewer seeds than Landsberg erecta, but they are almost twice as heavy (Alonso-Blanco et al., 1999). Considering today’s plant species, single-seed mass may vary over a range of 11.5 orders of magnitude (Moles et al., 2005). Seed mass is under strong genetic control, whereas the total number of seeds of a plant is largely affected by the environment (Paul-Victor and Turnbull, 2009). It has been demonstrated that the size, mass, and shape of Arabidopsis seeds may be regulated by brassinosteroid (Jiang et al., 2013), and it was shown recently that seed size in rice (Oryza sativa) can be influenced by the epiallele Epi-rav6 (Zhang et al., 2015). The ability of plants to switch between small and larger seeds may be understood as an adaptation to novel environments (Igea et al., 2016). However, it is still not fully understood whether, or to what extent, the variability of seed traits within plant species or genotypes has an impact on the development and further performance of a plant.When comparing biometric seed data of different dimensions such as length (one-dimensional), projected area (two-dimensional [2D]), or volume and mass (both three-dimensional [3D]), one can argue that mass is the most relevant parameter as a proxy for the amount of reserves a seed provides for the offspring. This might be true even when considering that the type of reserves, such as proteins, carbohydrates, or lipids (Rolletschek et al., 2015), and also different seed tissues, such as seed coat, embryo, or endosperm, may contribute differently to seed mass (Alonso-Blanco et al., 1999). While seed mass and time to germination (radicle protrusion) do not necessarily correlate (Norden et al., 2009), in particular under greenhouse conditions, higher seed mass may be advantageous for seedling establishment under adverse environmental conditions (Moles et al., 2005). For example, shade-tolerant species showed largely higher seed masses than cogeneric species growing in open habitats, indicating that seedlings under low-light conditions need more reserves than under good light (Salisbury, 1974). Seedlings of wild radish (Raphanus raphanistrum) emerged more likely from heavier seeds than from small seeds under field conditions but not in the greenhouse (Stanton, 1984), and for Arabidopsis, seed mass was reported to be higher in populations growing naturally at higher altitudes taken as a proxy for harsher conditions (Montesinos-Navarro et al., 2011).Seed mass can be measured individually (Stanton, 1984), but it is generally collected as an average value of batches of 50 to 1,000 seeds (Jako et al., 2001; Jofuku et al., 2005; Montesinos-Navarro et al., 2011; Tanabata et al., 2012). Alternatively, 2D scans are analyzed to determine parameters such as seed length, width, area, and perimeter length as a measure for seed size (Tanabata et al., 2012). This approach can be implemented in high-throughput facilities to obtain projected areas of seed grains combined with genome-wide association studies (Yang et al., 2014). Although projected seed area can easily be measured with a common office scanner (Herridge et al., 2011; Tanabata et al., 2012; Moore et al., 2013), it is not necessarily a precise or reliable measure of the true seed size because it may depend on the shape (Alonso-Blanco et al., 1999) and the orientation of a seed at scan (see “Results”). These issues also apply when using 2D projections to calculate length-to-width ratios as a simple shape factor (Tanabata et al., 2012). Projected seed area also has been used to calculate seed mass, assuming a fixed relationship between these parameters (de Jong et al., 2011; Herridge et al., 2011). This may hold with sufficient accuracy when averaging a large number of seeds but might be misleading when considering individual seeds.From a physical point of view, volume should be a much better proxy for mass than 2D traits. Although it has been stated that for 65 species analyzed seed masses can be compared easily with seed volumes (Moles et al., 2005), it is not clear how these seed volumes were determined. Volumes can be assessed using advanced methods such as x-ray computed tomography (CT) on fruits (Stuppy et al., 2003) or synchrotron radiation x-ray tomographic microscopy applied in paleobiological studies (e.g. on fruits and seed; Friis et al., 2014). Nuclear magnetic resonance (NMR) methods are used to measure water uptake in kidney beans (Phaseolus vulgaris) and adzuki beans (Vigna angularis; Kikuchi et al., 2006) or to estimate seed weight and content (Borisjuk et al., 2011; Rolletschek et al., 2015) rather than volumes. To our best knowledge, affordable methods to measure seed volumes directly are not achievable so far. For that reason, we have set up a volume-carving method for 3D seed shape reconstruction that is described briefly here and in more detail in a recent publication (Roussel et al., 2016).While traits derived from scanning procedures can easily be assigned to individual seeds (Herridge et al., 2011), further handling and processing of phenotyped single seeds is not as simple, in particular for tiny ones like those of Arabidopsis. The aim of this work was to develop an automated seed-handling system that can analyze single seeds of very different sizes or shapes, from Arabidopsis seeds up to barley (Hordeum vulgare) seeds or even bigger. The phenoSeeder system is designed to pick and place seeds, to achieve basic morphometric traits (one-dimensional and 2D data from projections, 3D reconstruction data, and mass) of each individual seed, and to store all analyzed seed traits in a database. Another goal is to use phenoSeeder for seed-to-plant tracking approaches and to analyze whether, or which, particular seed traits have an impact on plant development and performance under various environmental conditions. We describe the main features of the phenoSeeder technology and present results obtained with seeds of three accessions of Arabidopsis, rapeseed (Brassica napus), and barley, respectively. When analyzing the data, we focused particularly on correlations between projected seed area, seed volume, and seed mass, with the hypothesis that the respective seed volume may better correlate with mass than the projected area.     

基因工程酶法生产N-氨基甲酰-D-对羟基苯甘氨酸反应条件的优化(英文)

为了实现利用生物酶转化法生产D 对羟基苯甘氨酸 ,以工程菌E .coliBL2 1 pMD T7 dht细胞作酶源 ,对底物对羟基苯海因到中间体N 氨基甲酰 D 对羟基苯甘氨酸的酶转化条件进行了优化 .酶转化的最适温度为 37℃ ,最适pH为 9 0 .在Tris HCl、磷酸盐、碳酸盐和硼酸盐 4种缓冲体系中 ,底物对羟基苯海因的转化率相近 .菌体细胞经适当冻融后 ,底物对羟基苯海因的转化率被提高 .水溶性有机溶剂DMSF、DMF和Tween 80使对羟基苯海因的转化率降低 .转化时底物和菌体的合适比例为 30g L对羟基苯海因和 10g L湿菌体 .经工程菌E .coliBL2 1 pMD T7 dht细胞催化 ,底物的转化率在 13h内可达到 96 % .所制备的产物熔点、旋光性和红外光谱等与标准品一致     

Novel Synthesis of Optically Active Bishomotyrosine Derivatives Using the Friedel-Crafts Reaction in Triflic Acid

We report here a novel synthesis of optically active bishomotyrosine. The bishomotyrosine skeleton was constructed by using a Friedel-Crafts reaction between phenol and optically active N-Tfa-Glu(Cl)-OMe in triflic acid under the mild condition. Reduction and subsequent deprotection then afforded bishomotyrosine derivatives without any loss of optical purity.     

Optimization of Synthesis of Ethyl Isovalerate Using Rhizomucor miehei Lipase

Immobilized lipase from Rhizomucor miehei (Lipozyme IM-20) was used to catalyze the esterification reaction between isovaleric acid and ethanol to synthesize ethyl isovalerate in n-hexane. Response surface methodology based on a four-variable, five-level, central composite rotatable design was employed to optimize four important reaction variables—enzyme/substrate (E/S) ratio, substrate concentration, incubation time, and temperature—affecting the synthesis of ethyl isovalerate. The optimum conditions predicted for achieving maximum ester yield (500 mM) are as follows: E/S ratio, 48.41 g/mol; substrate concentration, 1 M; reaction time, 60 h; and temperature, 60°C. The predicted value matched well with the experimentally obtained value of 487 mM.     

Parameter Estimation of Fractional-Order Chaotic Systems by Using Quantum Parallel Particle Swarm Optimization Algorithm

Parameter estimation for fractional-order chaotic systems is an important issue in fractional-order chaotic control and synchronization and could be essentially formulated as a multidimensional optimization problem. A novel algorithm called quantum parallel particle swarm optimization (QPPSO) is proposed to solve the parameter estimation for fractional-order chaotic systems. The parallel characteristic of quantum computing is used in QPPSO. This characteristic increases the calculation of each generation exponentially. The behavior of particles in quantum space is restrained by the quantum evolution equation, which consists of the current rotation angle, individual optimal quantum rotation angle, and global optimal quantum rotation angle. Numerical simulation based on several typical fractional-order systems and comparisons with some typical existing algorithms show the effectiveness and efficiency of the proposed algorithm.     

盐藻RAPD反应条件的优化

从盐藻中提取基因组DNA作为模板进行RAPD反应的优化试验 ,获得PCR扩增反应的最佳条件 :随机引物为CPS32 0 (5′ GGCTCATGTG 3′) ;2 0 μl体系中Mg2 2 .0mmol/L ,退火温度 35℃ ;Taq酶活性值 0 .8U。     

响应面法优化脯氨酸羟化酶转化反应工艺条件

通过优化脯氨酸羟化酶表达条件和转化反应条件,提高其转化反应效率。采用单因素法筛选脯氨酸羟化酶的最佳诱导温度、诱导剂浓度和转化反应条件,并采用响应面法预测影响转化反应各因素的最佳条件。结果显示,经过筛选和验证,蛋白表达最适诱导温度为28℃,IPTG浓度为0.2 mmol/L;转化反应最佳条件为:120 mmol/L 2-(N-吗啡啉)乙磺酸(pH 6.6)、1.5% Nonidet P-40、200 mmol/L L-脯氨酸、200 mmol/L α-酮戊二酸,6 mmol/L L-抗坏血酸、6.0 mmol/L 硫酸亚铁,最适反应温度为27℃,振荡速率为152 r/min。在最佳条件下,转化反应进行48 h后产物反式-4-羟基-L-脯氨酸的转化率可达100%,为合成反式-4-羟基-L-脯氨酸奠定了坚实的实验基础。     

Efficiency of Parallel Direct Optimization

Tremendous progress has been made at the level of sequential computation in phylogenetics. However, little attention has been paid to parallel computation. Parallel computing is particularly suited to phylogenetics because of the many ways large computational problems can be broken into parts that can be analyzed concurrently. In this paper, we investigate the scaling factors and efficiency of random addition and tree refinement strategies using the direct optimization software, POY, on a small (10 slave processors) and a large (256 slave processors) cluster of networked PCs running LINUX. These algorithms were tested on several data sets composed of DNA and morphology ranging from 40 to 500 taxa. Various algorithms in POY show fundamentally different properties within and between clusters. All algorithms are efficient on the small cluster for the 40-taxon data set. On the large cluster, multibuilding exhibits excellent parallel efficiency, whereas parallel building is inefficient. These results are independent of data set size. Branch swapping in parallel shows excellent speed-up for 16 slave processors on the large cluster. However, there is no appreciable speed-up for branch swapping with the further addition of slave processors (>16). This result is independent of data set size. Ratcheting in parallel is efficient with the addition of up to 32 processors in the large cluster. This result is independent of data set size.     

Synthesis of Aromatic Carbonyl Compounds by Friedel-Crafts Reaction Using BF3 Catalyst

Friedel-Crafts reaction using BF₃ catalyst was investigated in order to synthesize various carbonyl compounds.     

Synthesis of artemisinin dimers using the Ugi reaction and their in vitro efficacy on breast cancer cells

The Ugi four-component reaction was used to prepare a series of artemisinin monomers and dimers. We found that the endoperoxide group in artemisinin remains intact during the reaction. The new artemisinin dimers showed potent anti-cancer activity against two human breast cancer cell lines, MDA-MB-231 and BT-474. One of the Ugi artemisinin dimers showed an IC₅₀ value of 12 nM when tested on BT474 cells, more than 600 times more potent than artesunate. Furthermore, the same Ugi artemisinin dimer showed a low toxicity when tested on MCF10A, a nontumorigenic cell line, resulting in a selectivity index of more than 8000.