Synthesis and Hybridization Properties of Modified Oligonucleotides with PNA-DNA Dimer Blocks

Abstract

Different modified PNA-DNA dimer-analogous synthons (I and II) were synthesized as phosphoramidites. These dimer units were assembled by a 5′-modified deoxythymidine and a modified PNA monomer. These synthons were used in the routine coupling procedure for oligonucleotides. Therefore no PNA coupling chemistry is necessary to synthesize PNA-DNA chimeric oligonucleotides. Various deoxyoligonucleotides were synthesized introducing the dimer blocks I and II at different positions in the sequences. Melting temperatures of the modified oligonucleotides with their complementary DNA analogues were determined.

Backbone modifications of oligonucleotides are required in the antisense strategy for protection against endonucleolytic cleavage in biological environment. Peptide nucleic acids (PNA fragments) are known to be nuclease resistant analogues, which show stable and discriminating hybridization. For this reason we prepared chimeric PNA-DNA oligomers by incorporation of two different modified PNA-DNA dimer blocks (Scheme A) into oligonucleotides. Melting temperatures of the modified oligonucleotides with their complementary DNA were determined.     

Chemical synthesis of diastereomeric diadenosine boranophosphates (ApbA) from 2'-O-(2-cyanoethoxymethyl)adenosine by the boranophosphotriester method

We have synthesized diastereomerically pure diadenosine 3',5'-boranophosphates (Ap(b)A) by using the boranophosphotriester method from ribonucleosides protected with the 2'-hydroxy protecting group 2-cyanoethoxymethyl (CEM). Melting curves of the triple-helical complex of the dimer Ap(b)A and 2poly(U) at high ionic strength revealed that presumptive (Sp)-Ap(b)A had a much higher affinity and presumptive (Rp)-Ap(b)A a much lower affinity for poly(U) than the natural dimer ApA did. In contrast, the affinities of these dimers for poly(dT) were similar. Both the (Rp)- and the (Sp)-boranophosphate diastereomers showed much higher resistance to digestion by snake venom phosphodiesterase and nuclease P1 than ApA did. They have potential for use as synthons to be incorporated into boranophosphate oligonucleotides. In particular, because oligonucleotides containing Sp boranophosphate nucleotides are expected to bind more strongly and specifically to RNA than natural oligoribonucleotides do, they may find application in the isolation and detection of functional RNA in basic research and diagnostics.     

Morpholino phosphorodiamidate oligonucleotides in zebrafish: a recipe for functional genomics?

Morpholino phosphorodiamidate anti-sense oligonucleotides (MPOs) have recently emerged as a tool for gene-specific knockdown. MPOs have a great potential for both therapeutic applications and functional genomics. In particular, zebrafish are well suited for gene function studies using MPOs owing to their rapid external development, transparent embryos and the ease of delivery of the intervening MPOs. This paper describes principles of MPO action and the application of MPOs for gene function studies and therapeutics. In the field of functional genomics, the MPO strategy has been most successfully used to study gene function in zebrafish. Over 35 mutations have been successfully phenocopied and over 30 novel gene functions have been analysed in zebrafish using MPOs during the last two years. The essential controls that are required to avoid misinterpretation of experimental data when using MPOs for gene function analysis will also be described.     

The photochemical and electrochemical properties of chiral porphyrin dimer and self-aggregate nanorods of cobalt(II) porphyrin dimer

In this paper, the novel chiral porphyrin dimer ligand and its cobalt(II) porphyrin dimer were synthesized by using a glutamate bridging group. The FT-IR and Raman spectra of the chiral porphyrin dimer were investigated. Furthermore, the photochemical and electrochemical properties of dimer were studied. In addition, we prepared the nanorods of the cobalt(II) porphyrin dimer using liquid-solid-solution (LSS) technologies. The shape and dimension of the spontaneous aggregates of cobalt(II) porphyrin dimer were characterized by the transmission electron microscopy (TEM). The results show the diameter and shape of the aggregates can be controlled by refining the stocked solution temperature.     

Methoxymethyl and (p-nitrobenzyloxy)methyl groups in the synthesis of oligoribonucleotides by the phosphotriester method

An efficient synthetic method for monomer ribonucleotide synthons containing 2'-O-methoxymethyl and 2'O-(p-nitrobenzyloxy)methyl groups used for oligonucleotide phosphotriester method with O-nucleophilic intramolecular catalysis at the stage of formation of internucleotide bond is developed. It is shown that synthons containing protecting 2'-O-(p-nitrobenzyloxy)methyl group may be used for automatic synthesis of phosphotriester oligoribonucleotides with high yields and synthons containing methoxymethyl group--to get 2'-O-modified oligonucleotides.     

Facile and Rapid Deprotection Conditions for the Cleavage of Synthetic Oligonucleotides from 1,4-Anhydroerythritol-Based Universal Polymer Support

In our previous report [Kumar, P.; Dhawan, G.; Chandra, R.; Gupta, K.C. Polyamine-assisted rapid and clean cleavage of oligonucleotides from cis-diol bearing universal support. Nucl. Acids Res. 2002, 30, e130 (1-8)], we demonstrated polyamine-mediated deprotection of oligonucleotides from cis-diol group bearing universal polymer support (I). However, vulnerability of the conventional dC^bz to modifications under these conditions compelled us to employ dC^ac during synthesis of oligonucleotide using conventional synthons. Here, a new set of simple and rapid deprotection conditions has been developed for the complete cleavage of oligonucleotides from the 1,4-anhydroerythritol-based universal polymer support employing conventional dC^bz synthon. Using manganese-imidazole complex in aqueous ammonium hydroxide (～30%), fully deprotected oligonucleotide sequences were obtained in 40 min, which were analyzed on reverse phase-HPLC and compared with the standard oligomers in terms of their retention time. Finally, their biological compatibility was established by analyzing PCR amplified products of npsA gene of N. meningitidis.     

Polyamine-assisted rapid and clean cleavage of oligonucleotides from cis-diol bearing universal support

Polyamine-assisted deprotection conditions have been developed for the rapid and clean cleavage of oligonucleotide chains from a cis-diol group bearing universal polymer support, making it compatible with modern oligonucleotide synthesis via all types of phosphoramidite synthons, including base labile protecting group bearing synthons as well. The synthesized oligonucleotides were found to be comparable with the corresponding standard oligomers with respect to their retention time on HPLC, mass on MALDI-TOF and biological activity in PCR amplification.     

Microbial conversion of indene to indandiol: a key intermediate in the synthesis of CRIXIVAN

Indene is oxidized to mixtures of cis- and trans-indandiols and related metabolites by Pseudomonas putida and Rhodococcus sp. isolates. Indene metabolism is consistent with monooxygenase and dioxygenase activity. P. putida resolves enantiomeric mixtures of cis-1,2-indandiol by further selective oxidation of the 1R, 2S-enantiomer yielding high enantiomeric purity of cis-(1S, 2R)-indandiol, a potential intermediate in the synthesis of indinavir sulfate (CRIXIVAN), a protease inhibitor used in the treatment of AIDS. Molecular cloning of P. putida toluene dioxygenase in Escherichia coli confirmed the requirement for the dihydrodiol dehydrogenase in resolving racemic mixtures of cis-indandiol. Rhodococcus sp. isolates convert indene to cis-(1S, 2R)-indandiol at high initial enantiomeric excess and one isolate also produces trans-(1R, 2R)-indandiol, suggesting the presence of monooxygenase activity. Scale up and optimization of the bioconversions to these key synthons for chiral synthesis of potential intermediates for commercial manufacture of indinavir sulfate are described.     

A convenient method for the synthesis of phosphoramidite non-nucleotide inserts for preparation of functionalized oligonucleotides

A simple approach to the synthesis of amidophosphite synthons of achiral non-nucleotide inserts using 4-(2-(4,4??-dimethoxytrityloxy)ethyl)morpholine-2,3-dione as a backbone of key precursor was suggested. Non-nucleotide synthons were synthesized using this approach that were suitable for synthesis of acridine-containing oligonucleotide derivatives, as well as oligonucleotides with branched carbohydrate-phosphate backbone.     

Circular permutation of the granulocyte colony-stimulating factor receptor agonist domain of myelopoietin

Myelopoietins (MPOs) are a family of engineered dual interleukin-3 (IL-3) and granulocyte colony-stimulating factor (G-CSF) receptor agonists that are superior in comparison to the single agonists in their ability to promote the growth and maturation of hematopoietic cells of the myeloid lineage. A series of MPO molecules were created which incorporated circularly permuted G-CSF (cpG-CSF) sequences with an IL-3 receptor (IL-3R) agonist moiety attached at locations that correspond to the loops that connect the helices of the G-CSF four-helix bundle structure. The cpG-CSF linkage sites (using the original sequence numbering) were residue 39, which is at the beginning of the first loop connecting helices 1 and 2; residue 97, which is in the turn connecting helices 2 and 3; and residues 126, 133, and 142, which are at the beginning, middle, and end, respectively, of the loop connecting helices 3 and 4. The N- and C-terminal helices of each cpG-CSF domain were constrained, either by direct linkage of the termini (L0) or by replacement of the amino-terminal 10-residue segment with a seven-residue linker composed of SGGSGGS (L1). All of the MPO molecules stimulated the proliferation of both IL-3-dependent (EC50 = 13-95 pM) and G-CSF-dependent (EC50 = 35-710 pM) cell lines. MPOs with the IL-3R agonist domain linked to cpG-CSFs in the first (residue 39) or second (residue 133) long overhand loops were found by CD spectroscopy to have helical contents similar to that expected for a protein comprised of two linked four-helix bundles. The MPOs retained the ability to bind to the IL-3R with affinities similar to that of the parental MPO. Using both a cell surface competitive binding assay and surface plasmon resonance detection of binding kinetics, the MPOs were found to bind to the G-CSF receptor with low nanomolar affinities, similar to that of G-CSF(S17). In a study of isolated cpG-CSF domains [Feng, Y., et al. (1999) Biochemistry 38, 4553-4563], domains with the L1 linker had lower G-CSF receptor-mediated proliferative activities and conformational stabilities than those which had the L0 linker. A similar trend was found for the MPOs in which the G-CSFR agonist activity is mostly a property of the cpG-CSF domain. Important exceptions were found in which the linkage to the IL-3R agonist domain either restored (e.g., attachment at residue 142) or further decreased (linkage at residue 39) the G-CSFR-mediated proliferative activity. MPO in which the IL-3R agonist domain is attached to the cpG-CSF(L1)[133/132] domain was shown to be more potent than the coaddition of the IL-3R agonist and G-CSF in stimulating the production of CFU-GM colonies in a human bone marrow-derived CD34+ colony-forming unit assay. Several MPOs also had decreased proinflammatory activity in a leukotriene C4 release assay using N-formyl-Met-Leu-Phe-primed human monocytes. It was found that circular permutation of the G-CSF domain can alter the ratio of G-CSFR:IL-3R agonist activities, demonstrating that it is a useful tool in engineering chimeric proteins with therapeutic potential.     

Immobilization of (1S,2R)-(+)-2-amino-1,2-diphenylethanol derivates on aminated silica gel with different linkages as chiral stationary phases and their enantioseparation evaluation by HPLC

A chiral selector was prepared through the reaction between (1S,2R)-(+)-2-amino-1,2-diphenylethanol and phenyl isocyanate. This selector was immobilized on aminated silica gel, respectively, with bifunctional group linkers of 1,4-phenylene diisocyanate, methylene-di-p-phenyl diisocyanate, and terephthaloyl chloride to produce corresponding three chiral stationary phases. The prepared compounds and chiral stationary phases were characterized by FT-IR, elemental analysis, (1)H NMR, and solid-state (1)H NMR. The enantioseparation ability of these chiral stationary phases was evaluated with structurally various chiral compounds. The chiral stationary phase prepared with 1,4-phenylene diisocyanate as linker showed excellent enantioseparation ability. The influence of different linkages on the enantioseparation was discussed.     

Superinfection exclusion reveals heteroimmunity between Pseudomonas aeruginosa temperate phages

Temperate siphophages (MP29, MP42, and MP48) were isolated from the culture supernatant of clinical Pseudomonas aeruginosa isolates. The complete nucleotide sequences and annotation of the phage genomes revealed the overall synteny to the known temperate P. aeruginosa phages such as MP22, D3112, and DMS3. Genome-level sequence analysis showed the conservation of both ends of the linear genome and the divergence at the previously identified dissimilarity regions (R1 to R9). Protein sequence alignment of the c repressor (ORF1) of each phage enabled us to divide the six phages into two groups: D3112 group (D3112, MP29, MP42, and MP48) and MP22 group (MP22 and DMS3). Superinfection exclusion was observed between the phages belonging to the same group, which was mediated by the specific interaction between the c repressor and the cognate operator. Based on these, we suggest that the temperate siphophages prevalent in the clinical strains of P. aeruginosa represent at least two distinct heteroimmunity groups.     

Characterization of medium chain length (R)-3-hydroxycarboxylic acids produced by Streptomyces sp. JM3 and the evaluation of their antimicrobial properties

(R)-3-Hydroxycarboxylic acids, chiral enantiomers of bacterial polyhydroxyalkanoates (PHA), may be valuable synthons for the production of numerous industrial materials such as β-lactams, fungicides, flavors, pheromones and vitamins. In this study, (R)-3-hydroxycarboxylic acid [(R)-3HAs)] synthons were produced by Streptomyces sp. JM3 (JN166713) under batch fermentation. Initial confirmation of PHA production was achieved by matrix assisted laser desorption ionization-time of flight mass spectroscopy and gas chromatography/mass spectroscopy (GC/MS). Subsequently, (R)-3HAs were produced by in vivo depolymerization and the monomers were separated using acid precipitation and anion exchange chromatography. The (R)-3HAs were identified by GC/MS as 3-trimethylsiloxy esters of decanoic, octanoic and butanoic acids. This was further supported by (13)C nuclear magnetic resonance spectrometry. The (R)-3HAs exhibited antimicrobial activity against Escherichia coli O157:H7, Listeria monocytogenes (ATCC 7644) and Salmonella typhimurium (ATCC 14028) with minimum inhibitory concentration ranging from 12.5 to 25?mg?ml(-1). However, the minimum bactericidal concentration data suggest that the (R)-3HAs may be bactericidal for E. coli O157:H7 and bacteriostatic for S. typhimurium and L. monocytogenes. Furthermore, the major purified synthon was shown to minimize the invasion of fibroblasts by S. typhimurium (ATCC 14028) [p?相似文献   

The use of molecular modelling in the understanding of configurational specificity (R or S) in asymmetric reactions catalyzed by Saccharomyces cerevisiae or isolated dehydrogenases

This method gives a general ideal how to use crystallographic information of enzymes to understand reactions catalyzed by these biocatalysts, commonly used by biochemists to produce chiral products. The interactions of three acetoacetic esters with the enzymes L-lactate dehydrogenase and alcohol dehydrogenase were studied through molecular modelling computer program. These artificial substrates have been widely used to produce chiral synthons. Through this methodology it was possible to understand the conformational specificity of these enzymes with respect to the products and how these enzymes can be inhibited by modifying the structures of the artificial substrates. Also, it was possible to predict whether some type of artificial substrate will suffer reduction by cells that contain these dehydrogenases and what kind of configuration (R or S) the final product will have.     

PNA-DNA chimeras containing 5-alkynyl-pyrimidine PNA units. Synthesis, binding properties, and enzymatic stability

Three chimeric dimer synthons (oeg_t(NH)T, oeg_up(NH)T and oeg_uh(NH)T) containing thymine (t), 5-(1-propynyl)-uracil (up) and 5-(1-hexyn-1-yl)-uracil (uh) PNA units with N-(2-hydroxyethyl)glycine (oeg) backbone were synthesized in solution and incorporated into T20 oligonucleotide analogues, using standard P-amidite chemistry. Insertion of dimer blocks led to destabilization of duplexes with dA20 target. The smallest Tm drops were found for chimeras containing oeg_up(NH)T dimers. Incorporation of the chimeric synthons into the 3'-end of T20 brought about growing resistance to 3'-exonucleolytic (SV PDE) cleavage in the order of oeg_t(NH)T < oeg_up(NH)T < oeg_uh(NH)T. Due to different endonuclease activities of 3'- and 5'-exonucleases applied, placing of five consecutive dimers at the 5'-terminus resulted in a relatively smaller, but also side-chain dependent, stabilization towards the hydrolysis by 5'-exonuclease (BS PDE). Neither exonucleases (SV and BS PDE) nor an endonuclease (Nuclease P1) could hydrolyse the unnatural phosphodiester bond linking the 3'-OH of thymidine to the terminal OH of N-(2-hydroxyethyl)glycine PNA backbone.     

Methoxymethyl and (p-nitrobenzyloxy)methyl groups in synthesis of oligoribunucleotides by the phosphotriester method

An efficient method to synthesize monomer ribonucleotide synthons containing 2′-O-methoxymethyl and 2′-O-(p-nitrobenzyloxy)methyl groups is developed. These synthons are applied to the oligonucleotide phosphotriester method using O-nucleophilic intramolecular catalysis at the stage of the internucleotide bond formation. The former synthons may be used for the automatic synthesis of 2′-modified oligonucleotides; the latter synthons made be used for the synthesis of phosphotriester oligoribonucleotides in high yields.     

Chiral cruciferous phytoalexins: preparation, absolute configuration, and biological activity

Synthesized by an efficient one-pot spirocyclization method, two chiral cruciferous phytoalexins, 1-methoxyspirobrassinin (2) and 1-methoxyspirobrassinol methyl ether (4a), were prepared through optical resolution using the chiral HPLC method of corresponding racemates. The absolute configuration of natural (+)-2 was elucidated as R by using the direct comparison of ECD and VCD spectra with those of known (S)-(-)-spirobrassinin (1). Another chiral phytoalexin, (-)-4a, had its absolute configuration 2R,3R elucidated through the comparison of observed and calculated VCD. Interestingly, the absolute configurations of natural (S)-(-)-spirobrassinin (1) and (R)-(+)-1-methoxyspirobrassinin (2) were opposite of each other, even though their structures are almost similar, with the exception of an N-methoxy group. A significant difference in the antiproliferative activity between (2R,3R)-(-) and (2S,3S)-(+)-4a was observed.     

A New Approach to the Synthesis of Trinucleotide Phosphoramidites - Synthons for the Generation of Oligonucleotide/Peptide Libraries

Abstract

Trinucleotide phosphoramidites that correspond to the codons of all 20 amino acids were synthesized in high yield in 5g scale. Precursors of those amidites - trinucleotide phosphotriesters - have been prepared using the phosphotriester approach without protection of the 3′-hydroxyl function. More than 10 oligonucleotides up to 90 bases long have been synthesized by a phosphite-triester approach using new synthons. The 67-mer (12 random codons) has been used to generate a display library of 2 × 10⁸ complexity.     

Genetic and phenotypic diversity of (R/S)-mecoprop [2-(2-methyl-4-chlorophenoxy)propionic acid]-degrading bacteria isolated from soils

Twelve mecoprop-degrading bacteria were isolated from soil samples, and their genetic and phenotypic characteristics were investigated. Analysis of 16S rDNA sequences indicated that the isolates were related to members of the genus Sphingomonas. Ten different chromosomal DNA patterns were obtained by polymerase-chain-reaction (PCR) amplification of repetitive extragenic palindromic (REP) sequences from the 12 isolates. The isolates were found to be able to utilize the chiral herbicide mecoprop as a sole source of carbon and energy. While seven of the isolates were able to degrade both (R)- and (S)-mecoprop, four isolates exhibited enantioselective degradation of the (S)-type and one isolate could degrade only the (R)-enantiomer. All of the isolates were observed to possess plasmid DNAs. When certain plasmids were removed from isolates MP11, MP15, and MP23, those strains could no longer degrade mecoprop. This compelling result suggests that plasmid DNAs, in this case, conferred the ability to degrade the herbicide. The isolates MP13, MP15, and MP24 were identified as the same strain; however, they exhibited different plasmid profiles. This indicates that these isolates acquired different mecoprop-degradative plasmids in different soils through natural gene transfer.     

Hybridase cleavage of RNA. IV. Oligonucleotide probes containing 2'-deoxy-2'-fluoronucleoside and arabinofuranosylcytosine]

A synthesis of synthons which allow one to introduce 2'-deoxy-2'-fluoropyrimidine derivatives into the oligodeoxynucleotide chain by means of the standard solid phase phosphoramidite method has been developed. Oligonucleotides with 1-beta-D-arabinofuranosylcytosine were synthesized using either aC derivative with the unprotected 2'-OH group or O2,2'-anhydro-4-thiouridine. The synthesis of seven modified oligonucleotides (7 to 11 nucleotide residues) is described and their ability to form duplexes with complementary DNA have investigated as well as RNase H hydrolysis of hybrids formed by the E. coli 5S RNA and the obtained oligonucleotide probes.