Paosphorothioate Analogues of Inositol Phosphates

Abstract

Novel analogues of the intracellular second messenger D-myo-inositol 1,4,5-trisphosphate, which possess phosphorothioate groups in place of phosphate groups have been synthesized. They exhibit unusual biological properties which will be of considerable application in understanding the phosphoinositide cycle.     

Efficient Solid Phase Synthesis of Cleavable Oligodeoxynucleotides Based on a Novel Strategy for the Synthesis of 5′‐S‐(4,4′‐Dimethoxytrityl)‐2′‐deoxy‐5′‐thionucleoside Phosphoramidites

The incorporation of a specific cleavage site into an oligodeoxynucleotide can be achieved by utilizing the four 5′‐S‐(4,4′‐dimethoxytrityl)‐2′‐deoxy‐5′‐thionucleoside 3′‐(2‐cyanoethyl diisopropylphosphoramidites) 5 and 15a – c (Fig. 1). Based on the silver ion assisted cleavage of P? S and C? S bonds, we synthesized oligodeoxynucleotides with an achiral 5′‐phosphorothioate linkage 3′–O–P–S–5′ by the solid‐phase phosphoramidite procedure. The efficient cleavage of these modified oligodeoxynucleotides can be detected by HPLC, PAGE, and surface plasmon resonance (SPR) spectrometry. The liberated 5′‐thiol moiety can be used directly for post‐reaction labeling with appropriately functionalized reporter groups.     

Phosphorothioate Analogues : Probes for Studying DNA Conformation

Abstract

Oligonucleotides containing stereospecifically incorporated phosphorothioate residues have been synthesised and their conformation studied using CD spectroscopy.     

Collision-induced reporter fragmentations for identification of covalently modified peptides

Collision-induced reporter fragmentations of the currently most important covalent peptide modifications as detected by tandem mass spectrometry are summarized. These fragmentations comprise the formation of reporter ions, which are preferentially immonium ions, immonium ion-derived fragments or side chain fragments. In addition, the reporter neutral loss reactions for covalently modified amino acid residues are summarized. For each individual covalent modification which can be recognized by a reporter fragmentation, the accurate mass shift and the gross formula shift of the modified amino acid residue are given. The same set of data is provided for the reporter fragmentations. Finally, an extensive accurate mass and gross formula list is presented as supplementary material, describing mostly regular and modified y₁ and dipeptide a and b ions, which are helpful for identification of the peptide ends of covalently modified peptides. Figure When modified peptides are fragmented by collision-induced dissociation in a tandem mass spectrometer, the modification is either lost as part of a charged fragment, so that a reporter ion for the modification is generated or it is lost as part of a neutral fragment, so that a modification-specific reporter neutral loss is observed in the fragment ion spectrum. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Chien-Wen Hung and Andreas Schlosser contributed equally to this work.     

Chromo- and Fluoroionophores Based on Diaza-Crown Ethers for Alkaline Earth Metal Ions

Abstract

Two new metal-selective dyes were synthesized and used to develop methods for determining Ca²⁺ and Mg²⁺. A chromogenic diaza-18-crown-6 with an alizarin group at each crown nitrogen is a calcium-selective ionophore used for extraction-spectrophotometry. The detection limit for the technique is 1 × 10^?5 M Ca²⁺. Fluorometric determination of Mg²⁺ is performed using a diaza-15-crown-5 derivatized with two methyiumbelliferone groups. This method allows for the one-phase determination of Mg²⁺ down to 7 × 10^?6 M.     

Two organophosphorus pesticides: methyl parathion and dicapthon

Structural studies performed in this laboratory of organophosphorus pesticides continue with these related compounds. The –NO₂ groups of methyl parathion (systematic name: dimethyl 4‐nitrophenyl phosphorothioate, C₈H₁₀NO₅PS) and dicapthon (systematic name: 2‐chloro‐4‐nitrophenyl dimethyl phosphorothioate, C₈H₉ClNO₅PS) make dihedral angles of 10.67 (8) and 5.8 (1)°, respectively, with the planes of their attached rings, which accompanies angular distortion at the ring C atoms to which the –NO₂ groups are attached. Similar distortions are observed at the C atom to which the thiophosphate groups are attached. Significant differences in distances and angles around the phenolic O, versus the –OMe groups, explain why it is the site of hydrolysis for these compounds. A comparison of a torsion angle involving the thiophosphate group and phenolic O atom with similar pesticide structures is given and indicates steric influences on that angle.     

Dinucleoside (5′ → 3′)-O,S-Phosphorodithioates - New Class of Dinucleotide Analogues

Abstract

Nucleoside 3′-0- and 5′-O-phosphorodithioates have been recently described by Caruthers et al.¹ as a new type of nucleotide analogues. These compounds have also been obtained in our Laboratory by one-pot dithiaphospholane approach.² We now report on the transformation of some of these derivatives into new class of dinucleotide analogues. We have found that nucleoside 3′-O-phosphorodithioates (1) react in DMF solution with 5′-bromo-5′-deoxythymidine to give in high yield corresponding dinucleoside (5′→3′)-O,S-phosphorodithioates (2) - first examples of a new class of dinucleotide analogues possessing the internucleotide phosphorothioate linkage with one of the sulfur atoms in a 3′-bridging position.     

Sequence-specific cleavage of RNA by designed ribozymes

Abstract

Ribozymes that distinguish a single base change in RNA were synthesized and used to specifically cleave c-Ha-ras messenger RNA. Using phosphorothioate containing oligonucleotide substrates, we have shown that Mg²⁺ binds to the pro-R oxygen of the phosphate and that the RNA cleavage reaction occurs via an in-line mechanism. Oligoribonucleotides containing a modified nucleoside are described.     

Measurement Of Ethiofos (Wr 2721) In Plasma: Preliminary Pharmaco-Kinetics in the Beagle

Abstract

A specific, sensitive high performance liquid Chromatographie method for ethiofos [S-2-(3-aminopropylamino)ethyl phosphorothioate, WR 2721, I] in plasma is described. The detection limit is 0.05 (μg/mL (0.23 (μM). Application of the method to the development of pharma-cokinetic parameters following IV administration of the drug to beagle dogs is demonstrated. Presented are pharmacokinetics of unchanged drug in plasma on 10-min constant-rate infusion of 150 mg/kg to two dogs, two studies in each dog. Following the cessation of drug infusion the plasma concentration versus time profile was best described by a two-compartment pharmacokinetic model. Mean pharmacokinetic parameters were: terminal elimination half-life=16.0 min, volume of central compartment=120 mL/kg and clearance=11.0 mL/min/kg.     

Palladium-catalysed cross-coupling reactions in the synthesis of some high polarizability materials

Abstract

Liquid crystal materials of high optical anisotropy consist of moieties of high electron density in conjugation with each other along the molecular length. Such structures are conducive to convergent synthesis methods. Here we report the synthesis of a range of novel materials by the strategic use of palladium-catalysed cross-coupling methods. In addition to the traditional use of bromide and iodide leaving groups, invaluable use of the triflate leaving group and the importance of selective cross-coupling methods using both arylboronic acids and alkynylzinc chloride derivatives is illustrated. This systematic methodology allows the separate synthesis of the appropriate sub-units that can be efficiently coupled together to provide high overall product yields.     

Rapid Covalent Method for Fabrication of Optical pH Sensitive Membranes

ABSTRACT

A new method for covalent immobilization of pH indicators was developed.

Indicators, containing amino groups, are immobilized covalently on transparent hydrolyzed acet[ydot]lcellulose membranes. All the steps were performed at ambient temperature. The method was rapid and allows for preparation of the final membranes within one day.     

A two-step sulfurization for efficient solution-phase synthesis of phosphorothioate oligonucleotides

A solution-phase approach for the synthesis of phosphorothioate oligonucleotides that circumvents the use of chromatographic purifications of protected phosphorothioate intermediates was developed. Implementation of a two-step sulfurization protocol in the phosphoramidite method allows efficient isolation of the intermediate phosphorothioates by extractions. The viability of this approach is demonstrated by the synthesis of a hexameric phosphorothioate oligonucleotide fragment.     

Hexamethyldisilathiane in Novel Chemical Transformations: Concept of “Counterattack Reagent”

Abstract

Deliberate design on utilization of each moiety in the “counterattack reagent,” Me₃SiSSiMe₃, allows it to accomplish multistep chemical transformations in one flask.     

New approach to the determination phosphorothioate oligonucleotides by ultra high performance liquid chromatography coupled with inductively coupled plasma mass spectrometry

This text presents a novel method for the separation and detection of phosphorothioate oligonucleotides with the use of ion pair ultra high performance liquid chromatography coupled with inductively coupled plasma mass spectrometry The research showed that hexafluoroisopropanol/triethylamine based mobile phases may be successfully used when liquid chromatography is coupled with such elemental detection. However, the concentration of both HFIP and TEA influences the final result. The lower concentration of HFIP, the lower the background in ICP-MS and the greater the sensitivity. The method applied for the analysis of serum samples was based on high resolution inductively coupled plasma mass spectrometry. Utilization of this method allows determination of fifty times lower quantity of phosphorothioate oligonucleotides than in the case of quadrupole mass analyzer. Monitoring of ³¹P may be used to quantify these compounds at the level of 80 μg L⁻¹, while simultaneous determination of sulfur is very useful for qualitative analysis. Moreover, the results presented in this paper demonstrate the practical applicability of coupling LC with ICP-MS in determining phosphorothioate oligonucleotides and their metabolites in serum within 7 min with a very good sensitivity. The method was linear in the concentration range between 0.2 and 3 mg L⁻¹. The limit of detection was in the range of 0.07 and 0.13 mg L⁻¹. Accuracy varied with concentration, but was in the range of 3%.     

KINMODEL (AGDC): a multipurpose computational method for kinetic treatment

KINMODEL (AGDC) is a kinetic computational methodology that is valid for the treatment of any reaction mechanism and that allows the determination of different kinetic and non-kinetic parameters from the experimental data acquired by monitoring absorbance at one or several different wavelengths. It is a numerical computational model that can be applied to any reaction mechanism, with the advantage that on changing the treatment from one mechanism to another it is not necessary to modify even a single line of the program code since it automatically establishes and solves the set of differential rate equations. It is able to treat a broad set of reaction mechanisms in the individual and joint determination of the following groups of parameters (a) the individual rate constants of the different reaction mechanisms; (b) the values of the molar absorption coefficients (which are very valuable in the case of intermediate species and their identification) of all the species involved in the mechanism, and (c) the concentrations of the species participating in the mechanism. The program can be used by non-experts in the field and it is able to treat mechanisms involving ambiguities in the solutions and in the identification of parameters when kinetic constants and molar absorption coefficients are optimized together, and it allows a discrimination to be made between the possible mechanisms responsible for the course of the reaction after the residuals have been analyzed statistically, automatically choosing the one that best fits the kinetic data.     

A New Approach to Dendrimers made from p-tert-Butyl Calix[4]arenes

A new family of hyperbranched molecules and dendrimers has been constructed from a diamide–dicalix derivative prepared from monocarbomethoxymethyl p-tert-butyl calix[4]arene and tris(2-aminoethyl)amine (‘tren’) via amide-formation reactions. The selective 1,3-di-O-functionalization of p-tert-butyl calix[4]arene moieties allows the synthesis of first- (G1) and second-generation (G2) calix-dendrimers. Replacement of the quadridentate amine by a trithia-ether-triamine-mono-ol, ’hyten’, again results in acylation of the amino groups, but with the generation of a central cavity with different complexing properties.

A new family of hyperbranched molecules and dendrimers has been constructed from a diamide–dicalix derivative prepared from monocarbomethoxymethyl p-tert-butyl calix[4]arene and tris(2-aminoethyl)amine (‘tren’) via amide-formation reactions. The selective 1,3-di-O-functionalization of p-tert-butyl calix[4]arene moieties allows the synthesis of first- (G1) and second-generation (G2) calix-dendrimers     

Adenosine Reagentless Electrochemical Aptasensor Using a Phosphorothioate Immobilization Strategy

This work deals with the characterization of a phosphorothioate anchoring strategy for aptamer molecules linked to gold, in the context of electrochemical sensors, using adenosine aptamer as model system. Surface density of immobilized phosphorothioate oligonucleotide sequences has been explored for a range of oligonucleotide concentrations (0.055–55 μM), finding a consequent variation of molecular surface density (3.5×10¹¹–2.8×10¹³ molecules/cm²). Most suitable aptamer concentration for adenosine recognition was also explored and found to be around 5.5 μM. As proof of concept of phosphorothioate strategy, electrochemical response to adenosine concentration was measured using a ferrocene‐labeled oligonucleotide sequence, and phosphorothioate anchoring thermal stability was compared to thiol immobilization.     

Effect of position of the lateral fluoro substituent on the mesophase behaviour of aryl 4-alkoxyphenylazo benzoates in pure and binary mixtures

Five groups of 4-substituted phenyl 4?-(2?- (or 3″-) substituted-4?-alkoxyphenylazo) benzoates (In_a-c to Vn_a-c) were investigated in which, within each group, the length of the terminal alkoxy group varies between 8 and 16 carbons, while the other terminal substituent, X, is a polar group that alternatively changed between the electron-donating CH₃O and the electron-withdrawing Br group, in addition to the un-substituted analogue (X = H). The lateral substituent (Y) in the five groups I–V varies, respectively, between H, 3-CH₃, 2-CH₃, 3-F and 2-F. Their mesophase stabilities were determined by differential scanning calorimetry and phases identified by polarised light microscopy. The two newly prepared groups of compounds (IVn_a-c and Vn_a-c) are structurally characterised by infrared, ¹H-NMR, mass spectroscopy, thermogravimetric and elemental analyses. Binary phase diagrams were constructed for each pair of isomers from groups IV and V bearing the same wing substituents but the lateral F is attached to different positions (2? or 3″).     

Rapid Chromatographic Analysis of Nucleosides and N-Bases in Physiologic Fluids

Abstract

A high-resolution anion-exchange system has been developed to analyze specifically for the nucleosides and bases present in physiologic fluids. This sytem employs a unique coupled-column operation that allows rapid column stripping and regeneration on completion of each run. Results obtained in the analysis of urine for nucleosides and bases are presented.     

Chiral recognition in NMR spectroscopy using crown ethers and their ytterbium(III) complexes

Chiral crown ethers 1 and 5 are useful enantiomeric discriminating agents in ¹H NMR spectroscopy for neutral and protonated primary amines, amino acids, and amino alcohols. The presence of the carboxylic acid groups in 1 and 5 provide sites at which ytterbium(III) can bind. Adding ytterbium(III) nitrate to crown–substrate mixtures in methanol-d₄ causes shifts in the spectra of substrates and often enhances the chiral discrimination in the ¹H NMR spectrum. The enhancement in enantiomeric discrimination that occurs in the presence of ytterbium(III) allows lower concentrations of the crown ether to be used in chiral recognition studies. Several amide derivatives of 1 were prepared and evaluated as chiral NMR discriminating agents, although except for 1e, these were less effective than 1.