Combinatorial solid phase peptide synthesis and bioassays

Solid phase peptide synthesis method, which was introduced by Merrifield in 1963, has spawned the concept of combinatorial chemistry. In this review, we summarize the present technologies of solid phase peptide synthesis (SPPS) that are related to combinatorial chemistry. The conventional methods of peptide library synthesis on polymer support are parallel synthesis, split and mix synthesis and reagent mixture synthesis. Combining surface chemistry with the recent technology of microelectronic semiconductor fabrication system, the peptide microarray synthesis methods on a planar solid support are developed, which leads to spatially addressable peptide library. There are two kinds of peptide microarray synthesis methodologies: pre-synthesized peptide immobilization onto a glass or membrane substrate and in situ peptide synthesis by a photolithography or the SPOT method. This review also discusses the application of peptide libraries for high-throughput bioassays, for example, peptide ligand screening for antibody or cell signaling, enzyme substrate and inhibitor screening as well as other applications.     

Construction of synthetic genes using PCR after automated DNA synthesis of their entire top and bottom strands.

A new method is described for the direct construction of synthetic genes by applying a modified version of the polymerase chain reaction (PCR) to crude oligonucleotide mixtures made by automated solid phase DNA synthesis. Construction of the HIV-1 393 bp rev gene and the 655 bp nef gene by this method is illustrated. The sequences for the entire top and bottom strands of rev were each programmed into an automated DNA synthesizer. Following DNA synthesis, the two crude oligonucleotide solutions were mixed together, specific primers were added, and the target gene was amplified by a modified PCR technique. Although the longer (greater than 200 bases) strands comprise a very small percentage of the total DNA after solid phase synthesis, this method uses PCR to 'find' and amplify such strands to create the target gene. The rev gene constructed by this method was found to contain 4 sequence errors, which were subsequently corrected by site-directed mutagenesis. In order to evaluate the source of sequence errors, several nef genes were made from the top and bottom strand DNA synthesis solutions using independent PCR's. Results suggest that sequence errors arose from both DNA synthesis and PCR. The utility of this method in producing a functional gene is demonstrated by expression of rev in E.coli.     

Chemical Synthesis of 80-mer Thymidylic Acid

Chemical synthesis of long-chain oligodeoxyribonucleotides was studied by the method of block condensation in a liquid phase. A method for elongating the chain and the synthesis of oligothymidylic acids up to 80-mer are described.     

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.     

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.     

Isolation and characterization of D-serine deaminase constitutive mutants by utilization of D-serine as sole carbon or nitrogen source.

Mutants constitutive for D-serine deaminase (Dsdase) synthesis were isolated by utilizing D-serine as sole nitrogen or carbon source in the chemostat. This method generated only regulatory constitutive (dsdC) mutants. The altered dsdC gene product in these strains is apparently able to bind D-serine more efficiently than the wild-type dsdC+ gene product--a selective advantage. Constitutive synthesis of Dsdase in all of these dsdC mutants is extremely sensitive to catabolite repression, and catabolite repression is reversed by the addition of D-serine. Of the 15 mutants generated by this method, none are suppressible by supD, supE, or supF. Mutations to a low level of constitutivity (maximal specific activity of 9) occur much more frequently than mutations to a high level (maximal specific activity of 79). High level constitutive synthesis of Dsdase results from the synthesis of an altered dsdC gene product--not from loss of ability to form the dsdC product. Dsdase synthesis is not regulated by the nitrogen supply in the medium, as nitrogen starvation does not result in the derepression of Dsdase synthesis.     

人脑利钠肽的Fmoc固相合成

探讨生物活性肽人脑利钠肽(BNP)的固相合成工艺,并为工业化合成提供理论依据.本文以二氯三甲基树脂(以下简称为二氯树脂)为载体,采用9-芴甲氧羰基(Fmoc)保护的氨基酸,以1-氧-3-双二甲胺羰基苯骈三氮唑四氟化硼盐(TBTU)/1-羟基苯并三氮唑(HOBT)/二异丙基乙胺(DIEA)缩合,以碘作为环化试剂,用切割试剂将BNP粗品从树脂上切割下来.通过MALDI-MS质谱仪检测,所合成环肽的分子量与理论分子量一致,使用RP-HPLC液相色谱仪对合成的环肽进行纯化,得到的BNP纯度达到97%以上.本合成工艺具有快捷、简便、高效的特点,适合于大批量的生产目的肽.     

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.     

Rapid determination of DNA synthesis in adherent cells grown in microtiter plates

A specific, rapid, and economical method for measuring the extent of DNA synthesis in adherent rat hepatoma H4-II-E cells grown in 96-well microtiter plates is described. The adherent cells were pulsed for 1 h with [methyl-3H]thymidine, released from the substratum by trypsinization, and collected on fiberglass filters with a MASH II cell harvester. The amount of radioactivity incorporated was directly proportional to the number of cells per well. Growth curves generated by measuring [methyl-3H]thymidine incorporation and counting the number of cells per well were identical. Experiments with inhibitors of DNA, protein, and RNA synthesis demonstrated that this method selectively measured DNA synthesis. In addition, [3H]thymidine uptake showed excellent correlation with autoradiographic assessment of DNA synthesis. This specific and sensitive method for determining DNA synthesis in microtiter cultures should facilitate studies of effects of various growth-controlling agents on epithelial, fibroblastic, and other cells which grow as adherent cells in culture.     

General method for rapid synthesis of multicomponent peptide mixtures

A method is suggested for the synthesis of multicomponent peptide mixtures. The method is a solid phase synthesis modified in order to give a closely equimolar mixture of peptides with predetermined sequences. The main point of modification is that before every coupling cycle the resin is divided into equal parts and each portion is coupled with a different amino acid. Then the portion are mixed and before the next coupling cycle the resin is again distributed into equal portions. The method is illustrated by the synthesis of a mixture of 27 tetrapeptides and that of 180 pentapeptides.     

Ecdysteroids affect in vivo protein metabolism of the flight muscle of the tobacco hornworm (Manduca sexta)

Ecdysteroid growth promotion of the dorsolongitudinal flight muscle of Manduca sexta was studied by measuring in vivo protein metabolism using both "flooding-dose" and "non-carrier" techniques. These procedures differ in that the former method includes injection of non-labelled phenylalanine (30 micromoles/insect) together with the [3H]amino acid. Injected radioactivity plateaued in the haemolymph within 7 min. With the flooding-dose method, haemolymph and intramuscular specific radioactivities were similar between 15 min and 2 h. Incorporation of [3H]phenylalanine into muscle protein was linear with either method between 30 and 120 min. Fractional rates (%/12 h) of synthesis with the flooding-dose technique were best measured after 1 h because of the initial delay in radioactivity equilibration. Estimation of body phenylalanine turnover with the non-carrier method showed 24-53%/h which was negligible with the flooding-dose method. Since the two methods yielded similar rates of protein synthesis, the large injection of non-labelled amino acid did not alter the rate of synthesis. Because the flooding-dose technique requires only a single time point measurement, it is the preferred method. The decline and eventual cessation of flight-muscle growth was mostly a consequence of declining protein synthesis though degradation increased between 76-86 h before eclosion and was relatively rapid. This decline in muscle growth could be prevented by treating pupae with 20-hydroxyecdysone (10 micrograms/insect). Protein accretion was promoted by a decline of up to 80% in protein breakdown, which was offset in part by a concurrent though much smaller decrease in protein synthesis. Therefore, ecdysteroids may increase flight-muscle growth by inhibiting proteolysis.     

The polyamide method of solid phase peptide and oligonucleotide synthesis

A versatile system of solid-phase peptide synthesis based on polar polyamide resins, a range of reversible peptide-resin linkage agents, and Nα-t-butoxycarbonyl or fluorenylmethoxycarbonyl-amino acids has been developed. Principles used in the design of the method are discussed and illustrated by synthesis of a number of natural peptides. Application of these principles to oligodeoxyribonucleotide synthesis has provided for the first time a practical solid phase method in the nucleotide field.     

Nucleophilic catalysis in the oligonucleotide synthesis

Rapid procedure for the synthesis of oligonucleotides by the phosphotriester method has been developed. It is based on the use of O-nucleophilic intramolecular catalysis. The application of this method to automated solid-phase oligonucleotide synthesis allows to perform one elongation cycle for 6-7 min.     

Effect of growth hormone on fibrinogen synthesis

A method described by McFarlane (1963a) for the measurement of the absolute rate of synthesis of liver-made plasma proteins was used to show that, within a few hours of giving bovine growth hormone to rats, fibrinogen synthesis increased significantly without a change in albumin synthesis.     

A theory for the estimation of DNA synthesis rates by flow cytometry

A method is presented for estimating the rate of DNA synthesis of a cell population by examining the DNA histogram generated by flow cytometry (FCM). The model is based on the use renewal equations to estimate the steady-state fraction of cells in each DNA compartment. The fraction of cells in each compartment is shown to be related to the Laplace transform of the transit time through that compartment. Two methods are introduced for estimating the rate of DNA synthesis utilizing different transit time distributions. One method is shown to be a simplification of the method of Dean and Anderson. The other method allows for variability in the DNA synthesis rate. The effects of quiescent cells are considered and attention is paid to the various assumptions underlying the estimation.     

一种多肽固相合成方法与纯化策略研究

目的：多肽与小分子化学药物相比,具有生物活性高、特异性强、不容易产生耐药性等特点,是目前新型药物研发的重点领域。多肽的合成直接影响到多肽药物的作用机制以及药物效果,因此需要建立一种更加便捷、高效的多肽合成方法。方法：采用Fmoc固相合成法合成多肽HF01,通过比较氨基酸连接的反应体系以及氨基酸脱保护的反应体系,从中确定最优体系。利用乙酰化基团进行肽链末端保护,经肽链剪切制备干燥的粗肽,最后采用高效液相色谱仪与高分辨质谱仪联用对粗肽进行纯化。结果：确定多肽合成的连接和脱保护反应体系,并获得纯度高达98.3%的线性多肽。结论：建立了一种高效、便捷的多肽合成及纯化方法,提高了实验室合成多肽的效率,为多肽类药物的研发提供技术支撑。     

Selective metabolic labelling of intracellular parasite proteins using ricin

The characterization of protein synthesis by eukaryotic intracellular parasites is inherently difficult because it may represent only a small fraction of host cell protein synthesis. Here, Mark Carrington, Yael Shochat and Anne Gurnett describe a method for overcoming this problem through the use of the toxin ricin to inhibit host cell protein synthesis specifically.     

Microwave-assisted Ribosylation of modified heterocyclic bases by Vorbrüggen method

During the last decades the nucleoside synthesis has proven to be important. The modified silyl-Hilbert-Johnson nucleoside synthesis modified by Vorbrüggen is one of the most often used methods. We have studied N-glycosilation of modifieded heterocyclic bases by Vorbrüggen method with microwave irradiation and we were able to shorten the reaction time and obtain higher yields. The method was demonstrated by fluoroquinolone and purine.