The Chemical Synthesis of Biochemically Active Oligoribonucleotides Using Dimethylaminomethylene Protected Purine H-Phosphonates

Abstract

Dimethylaminomethylene was applied as the protecting group for the exocyclic amino groups of adenosine and guanosine in the automated chemical synthesis of oligoribonucleotides on a polymer bound support. The dimethyl-aminomethylene protecting group can be removed at room temperature under conditions where the concomitant loss of the 2′-protection group can be excluded. The transformation of 2′-O-(t-butyldimethylsilyl)-5′-O-(4,4′-dimethoxytrityl) protected nucleosides to 3′-H-phosphonates yields synthons, well suited for the automated chemical synthesis of oligoribonucleotides. Using these H-phosphonate monomers, a coupling time of two minute: is sufficient to obtain average coupling yields of more than 98 %. Synthesized RNA is recognized as a substrate in an enzymatic reaction, forms the expected secondary structures and is suitable for NMR structural investigations.     

Some Aspects on Acyclonucleoside Synthesis

Abstract

An acyclonucleoside synthesis was investigated on the regioselective introduction of an acyclochain. We found that iodotrimethylsilane catalyzed the reaction of acyclochain introduction as well as its migration from S² to N¹ of 2-thiothymine and from N⁷ to N⁹ position of guanine. By taking the findings into account, several acyclo-nucleosides were synthesized in a simple one-pot procedure.     

19F NMR of RNA. The Structural and Chemical Aspects of 5-Fluoro-cytidine and-uridine Labelling of Oligoribonucleotides

Abstract

Results of PM3 semiempirical calculation revealed that energy and hydrogen bonds geometry of 1-methyl-5-fluoro-uracil and -cytosine base-pairs with 9-methyl-adenine and -guanine respectively are virtually the same as for the natural bases. Analysis of proton coupling constants proved that the sugar puckering of 5-fluorouridine and 5-fluorocytidine is analogous to non-modified ribonucleosides. 5-Fluorocytidine was regioselectively introduced to oligoribonucleotides, prepared using 2′-O-tert-dimethylsilyl protection, via post-synthetic quantitative ammonolysis of 4-O-methyl-5-fluorouridine derived precursor.

     

Progress in the Synthesis of Cyclic Deoxyribo- and Oligoribonucleotides

Abstract

The preparation of purine-rich sequences of cyclic DNA, up to a 28-mer, has been achieved. The products were purified by HPLC and PAGE (larger circles) and fully characterized. Cyclic RNA synthesis can be carried out using the same methodology as for cyclic DNA, provided that a single deoxynucleoside or a 2′-O-methylribonucleoside is placed at the 3′-end of the linear precursor.     

Parameters for the Calculation of Proton NMR Chemical Shifts of Oligoribonucleotides

Abstract

A set of empirical parameters which allows the prediction of the proton NMR chemical shifts at 70 C of non-exchangeable heterobase and anomeric protons in oligoribonucleotides has been constructed. The set is based on the highly flexible nature of oligoribonucleotide single strands and the wide range of conformational states which can be populated at relatively high temperatures (70 C or greater). A pairwise subtractive procedure, using 129 ribonucleotide oligomers (all 16 dimers, all 64 trimers, 37 tetramers, and 12 pentamers), shows that significant contributions to the observed chemical shift of protons in a given nucleoside residue are made by first, second, and third neighbors on the 3′ and the 5′ sides. The majority of the neighbors cause shielding effects with the exception of some first neighbors on the 5′ side of a given residue. The magnitude of the shielding effects is greatest for the purine heterobases and follows the order A>G>C>U, with first neighbors on the 3′ side showing more pronounced effects than second neighbors and these in turn showing larger effects than third neighbors. Second neighbors on the 5′ side showed consistently greater shieldings than first neighbors, a result attributed to the deshielding effects of the first 5′ neighbor phosphate group. The parameter Tables are applied to the prediction of proton chemical shifts in one heptamer, four hexamers, and two pentamers and give average absolute differences between predicted and observed shifts less than 0.030 ppm. The parameter approach represents an excellent method of generating initial assignments of proton chemical shifts for any single strand oligoribonucleotide.     

Improvements in the Synthesis of L-Ribonucleosides for the Preparation of Mirror-Image Oligoribonucleotides

Abstract

Different improvements are described for the chemical synthesis of L-ribonucleosides corresponding to the four natural bases. These nucleosides properly protected were used to synthesize successfully a 27-base long L-oligoribonucleotide.     

Synthesis of a Novel,Optically Active Uridine Analog Containing a 1,4-Dioxane Sugar Moiety. Synthesis of the Corresponding Dinucleotide

A new optically active uridine nucleoside analogue in which a substituted 1,4-dioxane ring functioned as the sugar analogue was prepared from L-tartaric acid. The nucleoside analogue was further converted into the corresponding protected dinucleotide.     

A New Approach to the Synthesis of Linear and Cyclic Oligoribonucleotides

Abstract

The tetraribonucleoside triphosphate 15 and the cyclic tetraribonucleotide 16 have been prepared by a recently reported triester approach in solution, involving H-phosphonate coupling.     

Some Mechanistic Aspects on Fmoc Solid Phase Peptide Synthesis

Peptides are biomolecules that may have several biological activities which makes them important to the environment in which they operate. Sometimes it is necessary for larger amounts of peptides to carry out some studies, like biological tests, NMR structural research or even interaction studies between peptides with other molecules. Expression can be an alternative for that. However, synthesis is specially useful when unnatural modifications or introduction of site specific tags are required. Synthetic peptides have been used for different studies such as cell signaling, development of epitope-specific antibodies, in cell-biology, biomarkers for diseases etc. Many different methodologies for peptide synthesis can be found in the literature. Solid phase peptide synthesis (SPPS) has been largely used and can be an excellent alternative to achieve larger quantities of these biomolecules. In this mini review, we aim to describe the SPPS and explain some of the mechanistic aspects and reagents involved in all phases of the synthesis: the use of resin, the ninhydrin test, some of the protecting groups, coupling reagents for peptide bond formation and the cleavage process.     

Large Scale Synthesis of Oligoribonucleotides on High-Loaded Polystyrene (HLP) Support

Abstract

Large quantities of oligoribonucleotides (up to 200 μmole) were synthesized on the high-loaded polystyrene (HLP) support with phosphoramidite nucleosides and 5-ethylthio-1H-tetrazole as activator. The HLP support significantly reduces solvent and reagent consumption. RNA synthesized on HLP support at large scale was shown to have full biological activity by a comparative ribozyme-substrate assay.     

Synthesis of the Oligoribonucleotides Incorporating 8-Oxo-Guanosine and Evaluation of their Base Pairing Properties

6-O-7-N-Bis(diphenylcarbamoyl)-2-N-phenoxyacetyl-5′-O-dimethoxytrityl-2′-O-{[(triisopropyl- silyl)oxy]methyl}-8-oxoguanosine-3′-yl-β-cyanoethyl-N,N-diisopropylphosphoramidite (5) was synt- hesized as a new phosphoramidite precursor unit for the synthesis of RNA. Compound 5 was successfully incorporated into the middle of the RNA sequences, and the synthesized RNAs were identified by MALDI-TOF mass measurements. Their properties were evaluated for formation of the RNA duplex and RNA/DNA heteroduplex. ORNs 1 and 4 containing 8-oxo-G can form base pairs with rC or dC in an anti conformation, while it can also interact with rA or dA in a syn conformation in the RNA duplex or RNA/DNA heteroduplex. The described synthetic method is therefore a useful procedure for the synthesis of ORN containing 8-oxo-G and contributes to the study of 8-oxo-G in RNA.     

Synthesis and Properties of DNA Containing Cyclonucleosides

Here, we present efficient syntheses of the R and S diastereomers of 8,5′-cyclo-2′-deoxyadenosine and 6,5′-cyclo-2′-deoxyuridine. We incorporated these interesting nucleosides into DNA to study how the cyclo linkage affects the stability of duplex formation.     

Medical Aspects of Chemical Warfare

The first-aid treatment of mass casualties from nerve gas relies mainly upon the use of drugs, and provision for their self-injection is recommended. Means for giving artificial respiration must also be provided, even though its large-scale use is regarded as impracticable. Prophylactic oxime (2 g. PAM chloride orally) is recommended if the situation permits. Some nerve gases are extremely rapid in action, and following exposure (or suspicion of exposure) 4 mg. of atropine and 2 g. of PAM chloride should be injected intramuscularly without delay. Preferably, atropine should be given intravenously. At the same time any clothing contaminated with liquid nerve gas should be removed and the skin cleansed thoroughly with a suitable fluid. Following this, the casualty should be watched closely for one hour. If poisoning develops despite these measures, or is already established, injection of atropine should be continued at short intervals until improvement occurs.