New insights into the de novo gene synthesis using the automatic kinetics switch approach

Here we present a simple, highly efficient, universal automatic kinetics switch (AKS) gene synthesis method that enables synthesis of DNA up to 1.6 kbp from 1 nM oligonucleotide with just one polymerase chain reaction (PCR) process. This method eliminates the interference between the PCR assembly and amplification in one-step gene synthesis and simultaneously maximizes the amplification of emerged desired DNA by using a pair of flanked primers. In addition, we describe an analytical model of PCR gene synthesis based on the thermodynamics and kinetics of DNA hybridization. The kinetics difference between standard PCR amplification and one-step PCR gene synthesis is analyzed using this model and is validated using real-time gene synthesis with eight gene segments (318-1656 bp). The effects of oligonucleotide concentration, stringency of annealing temperature, annealing time, extension time, and PCR buffer conditions are examined systematically. Analysis of the experimental results leads to new insights into the gene synthesis process and aids in optimizing gene synthesis conditions. We further extend this method for multiplexing gene assembly with a total DNA length up to 5.74 kbp from 1 nM oligonucleotide.     

A concise synthesis of benzimidazoles via the microwave-assisted one-pot batch reaction of amino acids up to a 10-g scale

An efficient method for the synthesis of aminomethyl benzimidazoles is developed by using a one-pot batch reaction between amino acids and o-phenylenediamines. This reaction proceeds smoothly in an unmodified household microwave oven, even though scale-up is to 10 g. A desirable method for the quick synthesis of benzimidazoles, which are used as a kind of important intermediates in drug synthesis, is provided by the scale-up utilization of amino acid resource.     

Radioautographic demonstration of the intracellular and extracellular bactericidal action of the neutrophils

The authors described a method of quantitative radioautographic analysis of RNA synthesis level in the bacteria. The method allows to define by inhibition of RNA synthesis a bactericidal action of neutrophils during phagocytosis. The essence of a method consists in the introduction for a short period of a label and development of radioautographs at semithin section by paraphenylenediamine which form a small and numerous silver grains above bacteria which retained an ability to RNA synthesis. Preparations obtained in this way is studied in a light optical microscope using a double illumination: from below through a condenser and from above through an objective.     

Simple and an efficient method for the synthesis of 1-[2-dimethylamino-1-(4-methoxy-phenyl)-ethyl]-cyclohexanol hydrochloride: (+/-) venlafaxine racemic mixtures

A novel synthetic method was developed for the synthesis of venlafaxine using inexpensive reagents. An improvement in the method, in the yield was achieved for the conversion of the venlafaxine. This is an improved version, simple and efficient method for the large-scale synthesis of venlafaxine.     

Programmable reactivity-based one-pot oligosaccharide synthesis

A detailed protocol is described for the application of a programmable one-pot oligosaccharide synthesis methodology to the synthesis of fucosyl GM1. This serves as a general example of the application of this method to the synthesis of any desired oligosaccharide. The method relies on a large database of relative reactivities for differentially protected tolyl thioglycoside donor molecules and a computer program to suggest the best order of addition for assembly of the oligosaccharide in optimal yield and with the fewest operations. The product is a protected form of the desired oligosaccharide isolated in 47% yield, which is then deprotected using standard procedures to provide fucosyl GM1 oligosaccharide (1) in 44% yield. The total time for synthesis of 1 from building blocks 3, 4 and 5 is approximately 4 d, whereas synthesis of the same compound by traditional stepwise procedures would take significantly longer. Protocols for the synthesis of thioglycoside building blocks 3 and 4 are also described.     

A review on the biosynthesis of metallic nanoparticles (gold and silver) using bio-components of microalgae: Formation mechanism and applications

The synthesis of nanoparticles (NP) using algae has been underexploited and even unexplored. In recent times, there are few reports on the synthesis of NP using algae, which are being used as a bio-factory for the synthesis. Moreover, the algae are a renewable source, so that it could be effectively explored in the green synthesis of NP. Hence, this review reports on the biosynthesis of NP especially gold and silver NP using algae. The most widely reported NP from algae are silver and gold than any other metallic NP, which might be due to their enormous biomedical field applications. The NP synthesized by this method is mainly in spherical shape; the reports are revealing the fact that the cell free extracts are highly exploited for the synthesis than the biomass, which is associated with the problem of recovering the particles. Besides, mechanism involving in the reduction and stabilization is well demonstrated to deepen the knowledge towards enhancement possibilities for the synthesis and applications.     

A new, efficient, and simple method for the one-pot synthesis of alpha-acetoxyphosphonates from aldehydes under solvent-free conditions

A simple, efficient, and new method has been developed for the synthesis of alpha-acetoxyphosphonates from aldehydes through a one-pot reaction of aldehydes with diethylphosphite in the presence of acetic anhydride under solvent-free conditions using magnesium oxide. This method is easy, rapid, and high yielding for the one-pot synthesis of alpha-acetoxyphosphonates from aldehydes.     

DNA合成技术及应用

DNA从头合成技术是指以寡核苷酸链为起始的合成DNA片段的技术,其不断进步是合成生物学快速发展的基石之一。常规使用的连接介导的DNA合成技术和PCR介导的DNA合成技术日益成熟,精确合成长度已经达到0．5—1kb。微阵列介导的DNA合成技术不断发展,其低成本、高通量的特点吸引了人们的注意;而酵母体内DNA合成技术的成功探索也为体外DNA合成提供了一种补偿方法。DNA合成在优化密码子用于异源表达、构建异源代谢途径、合成人工基因组以及合成减毒病毒用于疫苗研制等方面有广泛应用。综述了DNA从头合成技术的研究进展,并介绍了DNA合成的前沿应用。     

Microwave assisted high yielding preparation of N-protected 2'-deoxyribonucleosides useful for oligonucleotide synthesis

A rapid and high yielding method for the synthesis of precursors of synthons for DNA synthesis, N-protected 2'-deoxyribonucleosides is described, which occur under mild conditions using microwave irradiation. The desired material, N-protected nucleosides, was obtained in 93-96% yield in few minutes. The final products were then characterized by 1H-NMR and MALDI-TOF and compared with the standard samples. The method is amenable to small to moderate scale of synthesis.     

Psoralen plus near-ultraviolet light: a possible new method for measuring DNA repair synthesis

A new method is proposed to inhibit semiconservative DNA synthesis in cultured cells while DNA repair synthesis is being measured. The cells are treated with the DNA-crosslinking agent Trioxalen (4,5,8-trimethylpsoralen) plus near-ultraviolet light, and consequently 99.5% inhibition of replicative DNA synthesis is achieved. Additional DNA-damaging agents induce thymidine incorporation into the double-stranded regions of the DNA. The new method gave results very similar to those obtained with the benzoylated naphthoylated DEAE (BND) cellulose method using three human fibroblast strains, of which one had deficient capacity for DNA repair synthesis following treatment with gamma rays and methyl methanesulfonate. The advantages of the new method are simplicity and rapidity, as well as the high extent to which replicative DNA synthesis is inhibited.     

脂肪酶催化的末端官能化聚酯合成研究进展

脂肪酶催化的末端官能化聚酯合成是高分子合成领域中极具吸引力且发展非常迅速的重要技术：重点介绍引发剂法、终止剂法以及酶促化学偶联合成末端官能化聚酯研究所取得的主要进展。     

Determination of protein synthesis in vivo using labeling from deuterated water and analysis of MALDI-TOF spectrum.

This paper describes a method of determining protein synthesis and turnover using in vivo labeling of protein with deuterated water and analysis of matrix-assisted laser desorption time-of-flight mass spectrometer (MALDI-TOF) spectrum. Protein synthesis is calculated using mass isotopomer distribution analysis instead of precursor to product amino acid enrichment ratio. During protein synthesis, the incorporation of deuterium from water changes the mass isotopomer distribution (isotope envelop) according to the number of deuterium atoms (0, 1, 2, 3, etc.) incorporated, and the distribution of the protein with 0, 1, 2, 3,... atoms of deuterium follows a binomial distribution. A mathematical algorithm by which the distribution of deuterium isotopomers can be extracted from the observed MALDI-TOF spectrum is presented. Since deuterium isotopomers are unique to newly synthesized proteins, the quantitation of their distribution provides a method for the quantitation of newly synthesized proteins. The combined use of postsource decay sequence identification and mass isotopomer distribution analysis makes the use of in vivo labeling with deuterated water a precise method to determine specific protein synthesis.     

Silica gel: an improved support for the solid-phase phosphotriester synthesis of oligonucleotides.

The phosphotriester method for the stepwise synthesis of deoxyoligonucleotides has been employed using HPLC-grade silica gel (Porasil B) as the solid support. The procedure results in a convenient flow-through system for the synthesis of oligomers where all the reaction steps including the zinc bromide method of detritylation are compatible with the selected support. Deoxyoligonucleotides of 25-30 nucleotides in length can be synthesized in high yields utilising stable phosphotriester intermediates. Ease of handling of the solid support allows convenient synthesis of mixed oligonucleotide sequences.     

A convenient approach to synthesizing peptide C-terminal N-alkyl amides

Peptide C-terminal N-alkyl amides have gained more attention over the past decade due to their biological properties, including improved pharmacokinetic and pharmacodynamic profiles. However, the synthesis of this type of peptide on solid phase by current available methods can be challenging. Here we report a convenient method to synthesize peptide C-terminal N-alkyl amides using the well-known Fukuyama N-alkylation reaction on a standard resin commonly used for the synthesis of peptide C-terminal primary amides, the peptide amide linker-polyethylene glycol-polystyrene (PAL-PEG-PS) resin. The alkylation and oNBS deprotection were conducted under basic conditions and were therefore compatible with this acid labile resin. The alkylation reaction was very efficient on this resin with a number of different alkyl iodides or bromides, and the synthesis of model enkephalin N-alkyl amide analogs using this method gave consistently high yields and purities, demonstrating the applicability of this methodology. The synthesis of N-alkyl amides was more difficult on a Rink amide resin, especially the coupling of the first amino acid to the N-alkyl amine, resulting in lower yields for loading the first amino acid onto the resin. This method can be widely applied in the synthesis of peptide N-alkyl amides.     

Microwave-assisted Boc-solid phase peptide synthesis of cyclic cysteine-rich peptides.

In this study we describe the first protocols for the synthesis of cystine-rich peptides in the presence of microwave radiation with Boc-solid phase peptide synthesis (SPPS). This method is exemplified for macrocyclic peptides known as cyclotides, which comprise approximately 30 amino acids and incorporate a cystine knot arrangement of their three disulfide bonds. However, the method is broadly applicable for a wide range of peptides using Boc-SPPS, especially for SPPS of large peptides via native chemical ligation. Microwave radiation produces peptides in high yield and with high purity, and we were able to reduce the time for the assembly of approximately 30 mer peptide chains to an overnight reaction in the automated microwave-assisted synthesis.     

Preparation of template plasmids for cell-free protein synthesis using a regenerated anion-exchange column

In this report, we describe how reactions of cell-free protein synthesis can be successfully conducted using plasmids prepared with regenerated anion-exchange columns. When washed, stripped, and equilibrated with appropriated buffers, regenerated columns were able to be used repeatedly to prepare plasmids with consistent yield and purity. The regenerated columns exhibited comparable performance to a fresh column with respect to the efficiency of protein synthesis using the plasmids prepared from them. Overall, we expect that the presented results will contribute significantly to economizing the technology of cell-free protein synthesis as a practical method for protein production in preparative scales.     

Effects of sodium azide on replicative and repair DNA synthesis in barley embryos

Sodium azide inhibited protein synthesis and replicative DNA synthesis at doses used for mutation induction (0.1–1 mM) in barley embryos. During subsequent treatment for 24 h using lower doses of azide, partial recovery from this inhibition was observed, especially in the case of replicative DNA synthesis. Contrary to expectation, cysteine acted as a weak enhancer of the inhibitory effect of azide on DNA replication. Under conditions of minimal replicative DNA synthesis, DNA-repair synthesis was detected after the azide action as measured by the BND-cellulose method. Benazzmide — an inhibitor of poly(ADP-ribose)-polymerase — stimulated the azide-dependent repair synthesis approximately 2-fold.     

Asymmetrically Substituted myo-Inositols. XXXIX. The Synthesis of Conjugate of 2',3'-Didehydro-3'-deoxythymidine with Phosphatidylinositol: A New Nucleoside Phospholipid with Potential Anti-HIV Activity

A partially protected phosphatidylinositol with a free hydroxyl group in the cyclitol moiety was synthesized by phosphorylation of a tetrasubstituted myo-inositol using the H-phosphonate and phosphoamidite methods. The H-phosphonate method was advantageous for the synthesis of selectively protected monophosphoinositide due to a lesser number of stages. Two schemes for the conjugation of 2',3'-didehydro-3'-de oxythymidine with phosphatidylinositol using succinic acid as a linker were tested in the synthesis of the target nucleoside phospholipid.