Synthesis of mixed oligodeoxyribonucleotides following the solid phase method.

A method for the synthesis of mixed dimers, trimers and oligonucleotides on a solid support using monomeric protected nucleoside phosphochloridites (1a-d) has been developed and the different nucleoside reagents, and the results show that yields of different oligomers in a mixture could be directly correlated to the concentration of the four reagents. Separation of mixed oligomers on a reversed phase C18 column has also been studied.     

alpha-DNA. VII. Solid phase synthesis of alpha-anomeric oligodeoxyribonucleotides.

An efficient procedure for the synthesis of unnatural alpha-anomeric oligodeoxyribonucleotides is described. This solid-phase procedure is based on the use of alpha-nucleoside phosphoramidites and alpha-nucleoside derivatized solid supports corresponding to the four natural bases and allow rapid synthesis of oligonucleotides up to 20 alpha-deoxynucleotide units in length. After HPLC purification, a 15-mer: alpha-d(CCTCTCGTTCTTTAC) and a 20-mer: alpha-d(ATACTTGAGGAAGAGGTGTT) were obtained respectively in 27 and 29% overall yields. Their purity, nucleoside composition and primary structure were ascertained by HPLC and Maxam-Gilbert sequence analyses.     

Solid phase synthesis of polynucleotides. VI. Further studies on polystyrene copolymers for the solid support.

A simple solid phase method for the synthesis of oligodeoxyribonucleotides has been developed using the phosphotriester approach. Mononucleotide coupling units are sequentially added to the polystyrene copolymer with 1% divinylbenzene and two kinds of oligonucleotides, d(CACGACCCCTCCACGT) and d(AACTGGTATTACTGGGCG), are synthesized in a relatively high yield. One cycle of the mononucleotide addition is about 70 minutes, and this method is particularly suitable for the automation of the synthesis upon availability of an automatic synthesizer.     

A new general approach for the simultaneous chemical synthesis of large numbers of oligonucleotides: segmental solid supports.

A new approach is described which will allow the simultaneous synthesis of large numbers of pre-defined oligonucleotide chains. No machine aid is needed. The simultaneous syntheses can be performed by one person and do not require much more time than is currently needed for the synthesis of just one oligonucleotide in existing strategies. The general idea is the following: One uses noninterchangeable polymeric entities from each of which enough OD units can be isolated after completion of the syntheses. Whenever growing chains on different entities have to be elongated with the same building block these entities are gathered in the same reaction vessel. After such a common reaction cycle the entities are separated and now combined according to the next common building blocks etc. The practicability of this approach is demonstrated by the synthesis of d(T-A-A-T-A-T-T-A) and d(T-A-G-T-A-C-T-A) on cellulose filter disks following the phosphotriester approach.     

Eutectic mixed monolayers in equilibrium with phospholipid-bilayers and triolein-liquid phase.

Triolein (TO) and phospholipids (egg yolk phosphatidylcholine, egg yolk phosphatidylethanolamine, and bovine brain phosphatidylserine) had low mutual solubilities and separated into the TO-liquid phase and phospholipid-bilayers. Spreading pressures of the TO-phospholipid mixture (i.e., surface pressures of the mixed monolayer in equilibrium with the phase-separating lipid mixture) at the air/saline interface were independent of the lipid composition. On the other hand, collapse pressures of the mixed monolayer of TO and phospholipid (i.e., surface pressures of the mixed monolayer in equilibrium with the TO-liquid phase) at the interface changed with the monolayer composition and were lower than the spreading pressure. The experimental data indicated the spreading and collapse pressures as offering a phase diagram for the presence of equilibrium between the mixed monolayer, the phospholipid-bilayers and the TO-liquid phase. The diagram showed that TO and the phospholipids were miscible in the mixed monolayer, forming an eutectic mixed monolayer. When the mixed monolayer initially had the eutectic composition, no collapse of the monolayer was detected until the surface pressure reached the value of the spreading pressure. No specific complex between TO and the phospholipid is required to explain the stability and collapse of the mixed monolayers. The bulk immiscibility of the lipids elucidated by the spreading pressure-measurements, immediately leads to the phase behaviors observed.     

Improvements to solid phase phosphotriester synthesis of deoxyoligonucleotides.

A solution of benzenesulphonic acid (3%, w/v) in a dimethylformamide and dichloromethane mixture (9:1, v/v) is shown to be a very effective reagent for the detritylation of deoxyoligonucleotides attached to a solid support. The levels of depurination with this reagent were lower than those observed with other reagents such as trichloroacetic acid. Coupling reactions are described using above ambient temperatures with no detectable increase in side products. Both procedures have been successfully incorporated into an automated system, which can compete with the rate of synthesis by the phosphite approach.     

Cleavage kinetics and anchor linked intermediates in solid phase peptide amide synthesis.

Kinetics and cleavage conditions of peptide amide synthesis were studied using the anchor molecules 5-(4'-aminomethyl-3',5'-dimethoxyphenoxy)valeric acid (4-ADPV-OH) and 5-(2'-aminomethyl-3'-5'-dimethoxyphenoxy) valeric acid (2-ADPV-OH). Unexpectedly the anchor amide alanyl-4-ADPV-NH2 was isolated and characterized as an intermediate during the cleavage with trifluoroacetic acid (TFA) of alanyl-4-ADPV-alanyl-aminomethyl-polystyrene to yield the alanine amide. As a matter of fact the NH--CH alpha bond of the alanyl spacer has to be cleaved to form this intermediate. Using TFA-dichloromethane (1:9) alanyl-4-ADPV-NH2 was obtained as a cleavage product in 50% yield within 60 min, whereas the isomeric alanyl-2-ADPV-NH2 was formed more slowly under these mild conditions. At high TFA concentration no difference between the 2- and 4-ADPV anchor was observed in the rate of formation of the free alanine amide. The presence of tryptophan amide in the cleavage mixture resulted in an anchor alkylated tryptophan amide, which remains stable in acidic solution but disappears rapidly in the presence of the resin. A low TFA/high TFA cleavage procedure is recommended for peptide amid synthesis applying the ADPV anchor.     

Replication of mammalian DNA in vitro. Evidence for initiation from fiber autoradiography

We have used fiber autoradiography to examine the DNA product made in vitro in a lysed cell system. CHO cells were treated with 0.01% Brij-58 and the lysates were incubated at 30 degrees C in a complete reaction mixture for in vitro DNA synthesis with [3H]thymidine triphosphate ([3H]TTP) as the radioactive tracer. Fiber autoradiograms prepared from the DNA showed that it was synthesized on tandemly arranged replication units that were of average size of 20 micrometers, very similar to the size of units found in vivo. The rate of replication fork movement was 25--50% of the in vivo rate. More than 80% of forks stopped functioning by 15 min, and 95% stopped by 60 min. This suggests that synthesis is halted by premature terminations. Evidence for new initiations was provided by replication units with labeled origins in DNA synthesized in an in vitro reaction in which radioactivity was omitted for the first 10 min of incubation. This, plus the observations that the distance between initiation points (replication unit size) is not increased and that premature termination accounts largely for the cessation of synthesis, suggest that significant initiation takes place in this in vitro replication system.     

Selective isolation of in vitro phase II conjugates using a lipophilic anionic exchange solid phase extraction method

Identification, characterization and structure elucidation of human metabolites of drug candidates is crucial for the pharmaceutical industry to assess their activity against the therapeutic target of interest and potential toxicological effects. It often requires in vitro synthesis of microgram quantities of metabolites of interest with enzymatic preparations, pre-concentration of the reaction mixture by solid phase extraction (SPE), metabolite isolation using HPLC systems coupled to fraction collectors prior to nuclear magnetic resonance characterization. The method reported herein is a rapid and simple technique using solely off-line mixed phase anionic exchange lipophilic SPE cartridges to selectively isolate glucuronide and sulfate metabolites from their parent compound. This approach capitalizes on the pKa differences between the parent compound, devoided of acidic moieties, and the negatively charged glucuronide and/or sulfate metabolites. Once loaded on the SPE cartridge, the incubation mixture is washed successively with a basic aqueous solution, methanol to elute the non-anionic parent compounds, and then with an acidic methanolic solution to protonate and recover the phase II conjugates. Over 100 microg (>95% purity) of 17 alpha-ethynylestradiol-3-glucuronide and 6-gingerol-4'-glucuronide were successfully isolated using this technique, as well as glucuronide and a sulfate conjugates of 1-{4'-[(1R)-2,2-difluoro-1-hydroxyethyl]biphenyl-4-yl}cyclopropanecarboxamide (DHBC) synthesized in-house. Their structures were confirmed by Ultra Performance Liquid Chromatography coupled to Quadrupole-Time of flight (UPLC-QTof) and nuclear magnetic resonance analysis.     

Rapid synthesis of oligodeoxyribonucleotides. IV. Improved solid phase synthesis of oligodeoxyribonucleotides through phosphotriester intermediates.

A phosphotriester solid phase method on a polyamide support has been used to prepare oligodeoxyribonucleotides up to 12 units long. Compared to solid phase phosphodiester synthesis the new methodology is quicker, more flexible and gives 10-60-fold better overall yields.     

Polymerization of glucans by enzymatically active membranes

Conventional enzyme membrane reactors are not appropriate for a continuous synthesis of macromolecules and simultaneous product release. By immobilizing the enzyme in sufficiently large pores of a membrane an ensemble of miniaturized bioreactors is created. Product molecules are continuously removed from the enzyme by the flow of the reaction mixture across the membrane. Additionally, by varying the flow rate, it ought to be possible to influence the substrate as well as the enzyme-product residence times and thereby the product macromolecule's size. In this paper we present the first results of experiments involving enzymatic 1,4-alpha-glucan synthesis, using sucrose as substrate, maltooligosaccharides (DP 3-6) as primers, and membrane-immobilized amylosucrase. Epoxy groups for a covalent enzyme immobilization were generated on polypropylene microfiltration membranes by heterogeneous photoinitiated graft polymerization of glycidyl methacrylate. The influence of primer concentration and flow rate through the enzyme-membrane on amylosucrase activity, molecule growth, and coupling efficiency for glucose (% of coupled glucose versus free glucose) were investigated. The enzymatically mediated chain elongation of maltooligosaccharides by the successive addition of glucose units was achieved for the first time in a transmembrane process utilizing amylosucrase membranes.     

Design, construction and application of a fully automated equimolar peptide mixture synthesizer.

A fully automated peptide synthesizer has been constructed that is capable of the synthesis of equimolar peptide mixtures and the simultaneous synthesis of 36 individual peptides. The synthesizer was constructed from a workstation of our own design utilizing a Zymark robot arm. A Macintosh II computer coordinates the movements of the robotic arm, the switching of over 40 solenoid valves and the monitoring of sensors in the workstation. The robot hands are used to deliver solvents from pressurized spigot lines and to pipet amino acid solutions from reservoirs to an array of reaction vessels. Liquid dispensing, reagent mixing and solvent removal are controlled from a multifunction I/O board in the computer. The design features of the synthesizer are presented, as well as the characterization of multiple individual peptides, a simple mixture of 19 components, and a complex mixture of 15,625 components.     

Studies on the inhibition of synthesis of the tyrosine-rich proteins of wool.

Three treatments known to produce weak wool were imposed on sheep, and the effects on the synthesis of high-tyrosine wool proteins were noted. The treatments were: intravenous infusion of the amino acid mimosine (a potential chemical defleecing agent), intravenous injection of the synthetic steroid Opticortenol (dexamethasone-21-trimethylacetate), and the abomasal infusion of methionine into sheep consuming a diet of wheat. All three treatments caused a partial suppression of high-tyrosine protein synthesis. The inhibition caused by mimosine could not be prevented by the simultaneous infusion of tyrosine or phenylalanine, suggesting that in this system mimosine is not acting as a tyrosine antagonist. The role of phenylalanine in controlling the synthesis of the high-tyrosine proteins in wool was also investigated. Although the infusion of an amino acid mixture minus phenylalanine reduces the level of these proteins, supplements of phenylalanine or tyrosine do not stimulate their synthesis, irrespective of the initial level in the fibre. The improtance of aromatic amino acids in the regulation of the high-tyrosine proteins is therefore uncertain. Suppression of the high-tyrosine proteins is usually accompanied by a stimulation in the synthesis of the ultra-high-sulphur proteins, although there does not seem to be a simple stoichiometric relationship between the two protein types.     

Combining the enantioselectivity of a cyclodextrin and a diamide selector in a mixed binary gas‐chromatographic chiral stationary phase

The present work reports on the investigation of a mixed binary chiral stationary phase (CSP) prepared by simultaneous attachment of permethylated‐β‐cyclodextrin ( D selector) and resorcinarene with pendant l ‐ or d ‐valine diamide groups ( L′ and D′ selectors, respectively) to a polysiloxane matrix via platinum‐catalyzed hydrosilylation. The gas‐chromatographic investigation of a number of racemates on the four different CSPs ( D, D′, DD′, and DL′ ) showed that the enantioselectivity of the individual chiral selectors was retained in the mixed binary CSPs. As a consequence, hydrocarbons, underivatized alcohols, ketones, and almost all proteinogenic amino acid derivatives could be separated simultaneously on each of the mixed CSPs. Matched and mismatched cases of enantioseparation on the mixed binary CSPs were observed but turned out to be of minor importance for enantiomeric separation. In general, more racemates were separated with α ≥ 1.02 on the mixed phases as compared to the single phases. In order to analyze the influence of the presence of the diamide selector on the enantioselectivity of the cyclodextrin selector, a mixed ternary CSP containing the selector D and a racemic mixture of the selectors D′ and L′ [ D ( D′L′ )] was prepared and investigated. Merits and limitations of the approach of mixed binary CSPs are discussed. © 2005 Wiley‐Liss, Inc. Chirality     

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.     

Acute phase protein stimulation by alpha 1-antichymotrypsin-cathepsin G complexes. Evidence for the involvement of interleukin-6.

Incubation of alpha 1-antichymotrypsin-cathepsin G complexes with human lung fibroblasts caused a nearly 5-fold increase in synthesis of the cytokine interleukin-6. In turn, the fibroblast-conditioned medium induced significant synthesis of the acute phase proteins haptoglobin, fibrinogen, and alpha 1-antichymotrypsin in human Hep G2 cells, whereas a mixture of interleukin-1 and conditioned medium was considerably less stimulatory. These data indicate that proteinase-proteinase inhibitor complexes formed between plasma serpins and their target enzymes could play major roles in signaling for acute phase protein synthesis in response to injury.     

On enhancing productivity of bioethanol with multiple species

The present work is initiated to investigate whether a defined culture comprising a mixture of three yeast species, Kluyveromyces marxianus, Saccharomyces cerevisiae, and Pichia stipitis can ferment a mixture of sugars to produce bioethanol at rates higher than those achieved by pure cultures of the same. For this purpose, we develop models of single species based on the hybrid cybernetic model framework, and simulate fermentations in the mixed culture by combining individual models. An underlying assumption is that the behavior of each species is determined only by the common environment independently of the presence and metabolism of other species. Model performance is thoroughly assessed using the experimental data available in the literature. The dynamic behavior of mixed cultures in mixed culture experiments are accurately predicted by the model reflecting faithfully the simultaneous/sequential uptake patterns of mixed substrates. This model is then used to investigate performance of various possible reactor configurations. With the foregoing species of organisms, mixed culture itself does not lead to a significant increase of bioethanol productivity. Rather, the model shows that substantial improvement is acquired by sequential use of different, properly chosen organisms during fermentation. Thus, the successive use of K. marxianus and P. stipitis is shown to increase bioethanol productivity up to about 58% in comparison to fermentation by single species alone.     

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.     

A direct evidence for the involvement of poly(A) in protein synthesis

A radioactive polyadenylated globin mRNA was translated in either rabbit reticulocyte lysate or wheat germ extract under various conditions. When globin mRNA was translated, globin synthesis was directly proportional to the rate of loss in A units from the poly(A) tail. On the other hand, when globin poly(A) mRNA was incubated under non-translated conditions, no loss of A units was detected. The presence of ribonuclease inhibitor in the reaction mixture did not alter either the rate of globin synthesis or the loss in A units from the poly(A) tail. The present data suggests a correlation between protein synthesis and loss in A units from the poly(A) tail.     

A kinetic method for the simultaneous determination of isoenzymes activities in mixture. Application to A2 and A3 horseradish peroxidases

A graphical method for the simultaneous determination of the activity of two isoenzymes in a mixture, is presented. The method is based on the different kinetic behaviour of the isoenzymes to the changes in the substrate concentrations. Having determined the reaction rates for the enzyme mixture at different substrate concentrations, the activity of both isoenzymes can be derived graphically. An algebraic method for two or more isoenzymes is mentioned, as well. The applications of the graphical and the algebraic method to A2 and A3 horseradish isoperoxidases demonstrated that the difference between the actual activities of the two isoperoxidases and those determined by the proposed method was around 5% of the actual activities. The scope of application of this method could be extended to isoenzymes of clinical importance.