Synthesis and Properties of Mixed Anhydrides of AMP,ADP and ATP with Mesitoic Acid

Abstract

New affinity reagents for ATP-dependent enzymes are described. Optimal conditions are evolved for the synthesis of mixed anhydrides of AMP, ADP, ATP with mesitoic acid (MsCOp_nA, n = 1?3) and for their 1, N⁶-etheno, 2′, 3′-dialdehyde and photoactive analogues. UV, CD and fluorescence spectra of the compounds have been analyzed. Hydrolysis of MsCOp_nA (n = 1?3) and their etheno analogues over a wide pH range has been carried out.     

Oligonucleotides comprised of alternating 2'-deoxy-2'-fluoro-beta-D-arabinonucleosides and D-2'-deoxyribonucleosides (2'F-ANA/DNA 'altimers') induce efficient RNA cleavage mediated by RNase H

Chimeric oligonucleotides comprised of alternating residues of 2'-deoxy-2'-fluoro-D-arabinonucleic acid (2'F-ANA) and DNA were synthesized and evaluated for an important antisense property-the ability to elicit ribonuclease H (RNase H) degradation of complementary RNA. Experiments used both human RNase HII and Escherichia coli RNase HI. Mixed backbone oligomers comprising alternating three-nucleotide segments of 2'F-ANA and three-nucleotide segments of DNA were the most efficient at eliciting RNase H degradation of target RNA, and were significantly better than oligonucleotides entirely composed of DNA, suggesting that these mixed backbone oligonucleotides may be potent antisense agents.     

Studies in fluorescence histochemistry. VII. The mechanism of the complex reactions that may take place between protein carboxyl groups and hot mixtures of acetic anhydride and pyridine in the acetic anhydride-salicylhydrazide-zinc (or fluorescent ketone) method for localizing protein C-terminal carboxyl groups

Synopsis Theoretical arguments are marshalled with experimental evidence to support claims made previously (Stoward & Burns, 1967, 1968) that at 60°C acetic anhydride in the presence of pyridine transforms C-terminal carboxyl groups of proteins to methyl ketones and converts side-chain carboxyl groups to acid anhydrides. On balance the experimental evidence also supports another claim, namely that the methyl ketones thus formed from C-terminal carboxyl groups may be demonstrated specificallyin situ by the intense fluorescence they emit after treatment successively with aqueous solutions of salicylhydrazide and zinc acetate.The experiments carried out included ones favouring the exclusive formation of acid anhydrides, blocking of possible anhydrides with aromatic amines or alcohols, hydrolysis of anhydrides with alkalis, and prior methylation of carboxyl groups.     

Diblock-type supramacromolecule via biocomplementary hydrogen bonding

Control of nanostructure formation by a diblock-type supramacromolecule via biocomplementary hydrogen bonding has been achieved. Two different homopolymers, poly(4-trimethylsilylstyrene) and poly(styrene-d8), that are end-decorated with complementary oligonucleotides, i.e., thymidine phosphates and deoxyadenosine phosphates, were prepared by using the phosphoramidite method and blended successively. Association behavior in a blend solution was examined with NMR, and a cast bulk film obtained from the solution has been confirmed to show a nanophase-separated structure by transmission electron microscopy and X-ray scattering. Suppression of this nanostructure formation of a block-type supramacromolecule was also attained by adding a smaller agent as an inhibitor.     

A novel thymidine phosphoramidite synthon for incorporation of internucleoside phosphate linkers during automated oligodeoxynucleotide synthesis

A novel thymidine phosphoramidite synthon was synthesized and successfully used for incorporation of primary amino groups, attached through a triethylene glycol linker to the internucleoside phosphates, at desired locations during automated oligodeoxynucleotide synthesis. The synthesized amino-linker bearing oligonucleotides are stable under deprotection conditions and exhibit Watson-Crick base-pairing properties. Covalent labeling of oligonucleotides with carbocyanine near-infrared fluorochromes resulted in 2.5 times higher labeling yields when compared with oligonucleotides containing base-attached aminolinkers. We anticipate that the developed synthetic approach will be useful for nucleotide sequence-specific attachment of single or multiple ligands or reporter molecules.     

Chromosomal detection of simple sequence repeats (SSRs) using nondenaturing FISH (ND-FISH)

Simple Sequence Repeats (SSRs) are known to be scattered and present in high number in eukaryotic genomes. We demonstrate that dye-labeled oligodeoxyribonucleotides with repeated mono-, di-, tri, or tetranucleotide motifs (15-20 nucleotides in length) have an unexpected ability to recognize SSR target sequences in non-denatured chromosomes. The results show that all these probes are able to invade chromosomes, independent of the size of the repeat motif, their nucleotide sequence, or their ability to form alternative B-DNA structures such as triplex DNA. This novel and remarkable property of binding SSR oligonucleotides to duplex DNA targets permitted the development of a non-denaturing fluorescence in situ hybridization method that quickly and efficiently detects SSR-enriched chromosome regions in mitotic, meiotic, and polytene chromosome spreads of different model organisms. These results have implications for genome analysis and for investigating the roles of SSRs in chromosome structure and function.     

Construction of oligonucleotide arrays on a glass surface using a heterobifunctional reagent, N-(2-trifluoroethanesulfonatoethyl)-N-(methyl)-triethoxysilylpropyl-3-amine (NTMTA)

A rapid method for construction of oligonucleotide arrays on a glass surface, using a novel heterobifunctional reagent, N-(2-trifluoroethanesulfonatoethyl)-N-(methyl)-triethoxysilylpropyl-3-amine (NTMTA), has been described. The heterobifunctional reagent, NTMTA, carries two different thermoreactive groups. The triethoxysilyl group on one end is specific towards silanol functions on the virgin glass surface, while the trifluoroethanesulfonyl (tresyl) group on the other end of the reagent reacts specifically with aminoalkyl- or mercaptoalkyl- functionalized oligonucleotides. Immobilization of oligonucleotides on a glass surface has been realized via two routes. In the first one (A), 5′- aminoalkyl- or mercaptoalkyl-functionalized oligonucleotides were allowed to react with NTMTA to form a oligonucleotide-triethoxysilyl conjugate which, in a subsequent reaction with unmodified (virgin) glass microslide, results in surface-bound oligonucleotides. In the second route (B), the NTMTA reagent reacts first with a glass microslide whereby it generates trifluoroethanesulfonate ester functions on it, which in a subsequent step react with 5′-aminoalkyl or mercaptoalkyl oligonucleotides to generate support-bound oligonucleotides. Subsequently, the oligonucleotide arrays prepared by both routes were analyzed by hybridization experiments with complementary oligonucleotides. The constructed microarrays were successfully used in single and multiple nucleotide mismatch detection by hybridizing these with fluorescein-labeled complementary oligonucleotides. Further more, the proposed method was compared with the existing methods with respect to immobilization efficiency of oligonucleotides.     

The Synthesis of Moraprenyl and Dolichyl Hydrogen Succinates and Maleates

The reactions of moraprenol and dolichol with succinic and maleic anhydrides in the presence of pyridine or triethylamine were studied, and the conditions were found for the efficient synthesis of moraprenyl and dolichyl hydrogen succinates and maleates. These may be of interest as analogues of moraprenyl and dolichyl hydrogen phosphates with modified anionic groups.     

Synthesis of monomoraprenyl- and monodolichylsuccinates and maleates

The reactions of moraprenol and dolichol with succinic and maleic anhydrides in the presence of pyridine or triethylamine were studied, and the conditions were found for the efficient synthesis of moraprenyl and dolichyl hydrogen succinates and maleates. These may be of interest as analogues of moraprenyl and dolichyl hydrogen phosphates with modified anionic groups. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 2; see also http://www.maik.ru.     

Potentially prebiotic syntheses of condensed phosphates

In view of the importance of a prebiotic source of high energy phosphates, we have investigated a number of potentially prebiotic processes to produce condensed phosphates from orthophosphate and cyclic trimetaphosphate from tripolyphosphate. The reagents investigated include polymerizing nitriles, acid anhydrides, lactones, hexamethylene tetramine and carbon suboxide. A number of these processes give substantial yields of pyrophosphate from orthophosphate and trimetaphosphate from tripolyphosphate. Although these reactions may have been applicable in local areas, they are not sufficiently robust to have been of importance in the prebiotic open ocean.     

Preparation of DNA-duplexes, containing internucleotide thiophosphate groups in various positions of the recognition site for the EcoRII restriction endonuclease

Twenty-four 12-mer DNA duplexes, each containing a chiral phosphorothioate group successively replacing one of the internucleotide phosphate groups either in the EcoRII recognition site (5'CCA/TGG) or near to it, were obtained for studying the interaction of the restriction endonuclease EcoRII with internucleotide DNA phosphates. Twelve of the 12-mer oligonucleotides were synthesized as Rp and Sp diastereomeric mixtures. Six of them were separated by reversed-phase HPLC using various buffers. Homogeneous diastereomers of the other oligonucleotides were obtained by enzymatic ligation of the Rp and Sp diastereomers of 5- to 7-mer oligonucleotides preliminarily separated by HPLC with the corresponding short oligonucleotides on a complementary DNA template. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2003, vol. 29, no. 6; see also http://www.maik.ru.     

Inhibition of pancreatic ribonuclease by 2'-5' and 3'-5' oligonucleotides.

Forty different oligonucleotides were investigated as possible inhibitors of the depolymerizing activity of RNase A. The strongest inhibitors among the diribonucleoside 2'-5' mono- phosphates were: G2'-5'G, C2'-5'G and U2'-5'G, and among the diribonucleoside 3'-5' monophosphates: ApU, ApC and GpU. Of the eight trinucleotides investigated, ApApUp, ApApCp and ApGpUp were the strongest inhibitors. All four dinucleotides studied (ApUp, ApCp, GpUp and GpCp) were very strong inhibitors, ApUp being the strongest one. The results show that the nature of the various bases in the oligonucleotide has an effect on the degree of inhibition, and that the 3' phosphomonoester group increases the binding of the oligonucleotide to RNase A. These inhibitors can be used in physicochemical and biochemical studies of ribonuclease.     

Total synthesis of the structural gene for the precursor of a tyrosine suppressor transfer RNA from Escherichia coli. 3. Synthesis of deoxyribopolynucleotide segments corresponding to the nucleotide sequence 27-51.

Chemical syntheses of the four deoxyribodecanucleotides, d(T-C-G-A-A-G-T-C-G-A), d(C-G-T-C-A-T-C-G-A-C), d(T-G-A-C-G-G-C-A-G-A), and d(C-T-A-A-A-T-C-T-G-C) are described. These polynucleotides form, respectively, segments 7 to 10 in the plan adopted for the total synthesis of the DNA corresponding to the precursor for the Escherichia coli tyrosine tRNA. The syntheses used the principles of stepwise addition of protected mono- and oligonucleotides to the 3'-hydroxyl end of growing oligonucleotide chains. Detailed schemes used in the present syntheses are shown in Diagrams 1 to 4 in the text. The final products were subjected to extensive chromatography and were characterized as pure by chemical and enzymatic procedures.     

N-(3-Triethoxysilylpropyl)-4-(N'-maleimidylmethyl)cyclohexanamide (TPMC): a heterobifunctional reagent for immobilization of oligonucleotides on glass surface

A new heterobifunctional reagent, namely, N-(3-triethoxysilylpropyl)-4-(N'-maleimidylmethyl)cyclohexanamide (TPMC) was developed and its potentiality for fixing of thiol (-SH) modified oligonucleotides were tested. The covalent attachment of oligonucleotides with the reagent was achieved through its maleimide functionality at one end via stable thioether linkage while the other end bearing triethoxysilyl functionality has been utilized for coupling with the virgin glass surface with simplified methodologies. Immobilization of oligonucleotides was achieved by two alternating ways. The PATH-1 involves formation of conjugate of reagent and SH-modified oligonucleotides through thioether linkage and was subsequently immobilized on unmodified glass surface through triethoxysilyl group and alternatively, PATH-2 involves reaction of reagent first with unmodified glass surface to get maleimide functionality on the surface and then the SH-modified oligonucleotides were immobilized via thioether linkage. The specificity of immobilization was tested by hybridization study with complementary fluorescein labeled oligonucleotide strand.     

Yeast tRNAAsp tertiary structure in solution and areas of interaction of the tRNA with aspartyl-tRNA synthetase. A comparative study of the yeast phenylalanine system by phosphate alkylation experiments with ethylnitrosourea

Ethylnitrosourea is an alkylating reagent preferentially modifying phosphate groups in nucleic acids. It was used to monitor the tertiary structure, in solution, of yeast tRNAAsp and to determine those phosphate groups in contact with the cognate aspartyl-tRNA synthetase. Experiments involve 3' or 5'-end-labelled tRNA molecules, low yield modification of the free or complexed nucleic acid and specific splitting at the modified phosphate groups. The resulting end-labelled oligonucleotides are resolved on polyacrylamide sequencing gels and data analysed by autoradiography and densitometry. Experiments were conducted in parallel on yeast tRNAAsp and on tRNAPhe. In that way it was possible to compare the solution structure of two elongator tRNAs and to interpret the modification data using the known crystal structures of both tRNAs. Mapping of the phosphates in free tRNAAsp and tRNAPhe allowed the detection of differential reactivities for phosphates 8, 18, 19, 20, 22, 23, 24 and 49: phosphates 18, 19, 23, 24 and 49 are more reactive in tRNAAsp, while phosphates 8, 20 and 22 are more reactive in tRNAPhe. All other phosphates display similar reactivities in both tRNAs, in particular phosphate 60 in the T-loop, which is strongly protected. Most of these data are explained by the crystal structures of the tRNAs. Thermal transitions in tRNAAsp could be followed by chemical modifications of phosphates. Results indicate that the D-arm is more flexible than the T-loop. The phosphates in yeast tRNAAsp in contact with aspartyl-tRNA synthetase are essentially contained in three continuous stretches, including those at the corner of the amino acid accepting and D-arm, at the 5' side of the acceptor stem and in the variable loop. When represented in the three-dimensional structure of the tRNAAsp, it clearly appears that one side of the L-shaped tRNA molecule, that comprising the variable loop, is in contact with aspartyl-tRNA synthetase. In yeast tRNAPhe interacting with phenylalanyl-tRNA synthetase, the distribution of protected phosphates is different, although phosphates in the anticodon stem and variable loop are involved in both systems. With tRNAPhe, the data cannot be accommodated by the interaction model found for tRNAAsp, but they are consistent with the diagonal side model proposed by Rich & Schimmel (1977). The existence of different interaction schemes between tRNAs and aminoacyl-tRNA synthetases, correlated with the oligomeric structure of the enzyme, is proposed.     

DNA gold nanoparticle conjugates incorporating thiooxonucleosides: 7-deaza-6-thio-2'-deoxyguanosine as gold surface anchor

A new protocol for the covalent attachment of oligonucleotides to gold nanoparticles was developed. Base-modified nucleosides with thiooxo groups were acting as molecular surface anchor. Compared to already existing conjugation protocols, the new linker strategy simplifies the synthesis of DNA gold nanoparticle conjugates. The phosphoramidite of 7-deaza-6-thio-2'-deoxyguanosine (6) was used in solid-phase synthesis. Incorporation of the sulfur-containing nucleosides can be performed at any position of an oligonucleotide; even multiple incorporations are feasible, which will increase the binding stability of the corresponding oligonucleotides to the gold nanoparticles. Oligonucleotide strands immobilized at the end of a chain were easily accessible during hybridization leading to DNA gold nanoparticle network formation. On the contrary, oligonucleotides immobilized via a central position could not form a DNA-AuNP network. Melting studies of the DNA gold nanoparticle assemblies revealed sharp melting profiles with a very narrow melting transition.     

Chemical modification of the epsilon-amino groups of lysine residues in horseradish peroxidase and its effect on the catalytic properties and thermostability of the enzyme.

Chemical modification of horseradish peroxidase (donor:hydrogen-peroxide oxidoreductase, EC 1.11.1.7) (isoenzyme C) by anhydrides of mono- and dicarboxylic acids and picryl sulfonic acid has been performed. The effect of the modification on the catalytic activity, absorption and circular dichroism spectra of peroxidase has been studied. Rate constants of irreversible thermoinactivation (kin) for the native and modified peroxidase at 56--80 degrees C have been measured. The effective values of the thermodynamic activation parameters of thermoinactivation, delta H not equal to and delta S not equal to, have been also determined. A relationship between the number of modified epsilon-amino groups of lysine residues and the nature of the modifier on the one hand, and the conformation and thermostability of the enzyme on the other, is discussed. It has been shown that it is the degree of modification, rather than the nature of the modifier, that produces the major effect on the macromolecular conformation and the thermostability of the enzyme after modification. The conclusion is drawn that the thermostability of the modified enzyme increases due to the decrease of the conformational mobility in the protein moiety around the heme.     

Antimicrobial and biological effects of N-diphenylphosphoryl-P-triphenylmonophosphazene-II and di(o-tolyl) phosphoryl-P-tri(o-tolyl)monophosphazene-III on bacterial and yeast cells

The purpose of the study was to synthesize and evaluate the antimicrobial effects of two monophosphazenes, N-diphenylphosphoryl-P-triphenylmonophosphazene-II and N-di(o-tolyl)phosphoryl-P-tri(o-tolyl)monophosphazene-III on bacterial and yeast strains. The biological effects of these molecules were compared with a potential antioxidant vitamin E. According to results, the triphenyl monophosphazene-II has antimicrobial effect on all the bacterial and yeast cells, but tri(o-tolyl)monophosphazene-III has only antimicrobial effect on some bacterial cells. When the concentration of triphenyl monophosphazene-II was raised, it was observed that inhibition zone increased on the bacterial growth media. The biological effects of these molecules were compared to vitamin E in the Saccharomyces cerevisiae culture media. In 200 microg administered culture media, the cell density decreased in vitamin E, triphenyl monophosphazene-II and tri(o-tolyl)monophosphazene-III groups at the end of 24 and 48 h incubation times (p<0.001,p<0.05). While the cell densities in vitamin E and tri(o-tolyl)monophosphazene-II groups decreased partly at the end of 72 h incubation time (p<0.05), its level in triphenyl monophosphazene-II group increased (p<0.01) at the same incubation time. In 1,000 microg administered culture media, cell density was not found to differ between vitamin E and control groups at the end of 24h incubation time, but it was found that the cell densities in triphenyl monophosphazene and tri(o-tolyl)monophosphazene-III groups decreased at the same incubation time (p<0.001). The cell densities in tri(o-tolyl)monophosphazene-III group and triphenyl monophosphazene-II decreased at the end of 48 h incubation time (respectively, p<0.05,p<0.001). In 200 microg administered cell pellets, while the lipid level was not found to differ between control and vitamin E, the lipid level decreased in triphenyl monophosphazene-II and tri(o-tolyl)monophospazene-III groups (respectively, p<0.001,p<0.01). In 1,000 microg administered cell pellets, it was found that the lipid level decreased in vitamin E, triphenyl monophosphazene-II and tri(o-tolyl)monophosphazene-III groups (p<0.001,p<0.01).     

The linkage of sugar phosphate polymer to peptidoglycan in walls of Micrococcus sp. 2102.

1. Protein-free walls of Micrococcus sp. 2102 contain peptidoglycan, poly-(N-acetylglucosamine 1-phosphate) and small amounts of glycerol phosphate. 2. After destruction of the poly-(N-acetylglucosamine 1-phosphate) with periodate, the glycerol phosphate remains attached to the wall, but can be removed by controlled alkaline hydrolysis. The homogeneous product comprises a chain of three glycerol phosphates and an additional phosphate residue. 3. The poly-(N-acetylglucosamine 1-phosphate) is attached through its terminal phosphate to one end of the tri(glycerol phosphate). 4. The other end of the glycerol phosphate trimer is attached through its terminal phosphate to the 3-or 4-position of an N-acetylglucosamine. It is concluded that the sequence of residues in the sugar 1-phosphate polymer-peptidoglycan complex is: (N-acetylglucosamine 1-phosphate)24-(glycerol phosphate)3-N-acetylglucosamine 1-phosphate-muramic acid (in peptidoglycan). Thus in this organism the phosphorylated wall polymer is attached to the peptidoglycan of the wall through a linkage unit comprising a chain of three glycerol phosphate residues and an N-acetylglucosamine 1-phosphate, similar to or identical with the linkage unit in Staphylococcus aureus H.     

The hexa- and pentapeptide extension of proalbumin. I. Chemical synthesis of serum albumin propeptides

The proalbumin hexapeptide extension was synthesized beginning from the C-terminal end by stepwise N-terminal peptide chain elongation starting from N-tert-butyloxycarbonyl-(Ng-nitro)arginyl-(Ng-nitro)arginine 4-nitrobenzyl ester; [alpha](20)365-12 degrees C (c = 1; dimethylformamide). The other amino acids were incorporated by excess mixed anhydrides of Ddz-amino acids (Ddz; 3,5-dimethoxyphenylisopropyloxycarbonyl) yielding the fully protected hexapeptide in crystalline quality. After removal of the protective groups by acid treatment and hydrogenation the peptide was purified by Dowex ion-exchange and Sephadex chromatography. The purity was confirmed by thin-layer chromatography and amino acid analysis.