Monomers containing 2′-<Emphasis Type="Italic">O</Emphasis>-alkoxymethyl groups as synthons for the oligonucleotide synthesis by the phosphotriester method

A general scheme for the synthesis of ribonucleotides containing an alkoxymethyl group at the 2′-O-position of ribose and an O-nucleophilic catalytic 4-methoxy-1-oxido-2-picolyl phosphate-protecting group has been developed for the introduction into oligonucleotides during their solid-phase synthesis by the phosphotriester method. The scheme has been tested in the synthesis of monomers with 2′-O-modifying groups as examples: 2-azidoethoxymethyl, propargyloxymethyl, and 3,4-cyclocarbonatebutoxymethyl groups.     

Synthesis of oligoribonucleotides containing 2'-O-methoxymethyl group by the phosphotriester method

An effective procedure for the synthesis of ribonucleotide monomers containing a 2 '-О-methoxymethyl-modifying group was developed. These monomers were used for the synthesis of RNA fragments by the solid-phase phosphotriester method under O-nucleophilic intramolecular catalysis. The properties of 2 '-О-methoxymethyl-containing oligoribonucleotides were examined.     

Synthesis of Oligoribonucleotides Containing 2′-O-Methoxymethyl Group by the Phosphotriester Method

An effective procedure for the synthesis of ribonucleotide monomers containing a 2 ′-О-methoxymethyl-modifying group was developed. These monomers were used for the synthesis of RNA fragments by the solid-phase phosphotriester method under O-nucleophilic intramolecular catalysis. The properties of 2 ′-О-methoxymethyl-containing oligoribonucleotides were examined.     

Synthesis And Binding Study Of Phosphonate Analogues Of Pnas And Their Hybrids With Pnas

Abstract

The preparation of monomers for the synthesis of phosphonate analogues of peptide nucleic acids containing the four natural nucleobases: thymine, cytosine, adenine and guanine, has been ccomplished. The monomers obtained were used for the automated online solid phase synthesis of pure phosphono-PNA oligomers as well as chimeras consisting of phosphono-PNA and PNA resudies. The hybridization properties of these oligonucleotide mimics to complementary DNA and RNA fragments were studied.     

Synthesis of the 3'-thio-nucleosides and subsequent automated synthesis of oligodeoxynucleotides containing a 3'-S-phosphorothiolate linkage

Oligodeoxynucleotides containing 3'-S-phosphorothiolate (3'-PS) linkages have become useful tools for probing enzyme-catalyzed cleavage processes in DNA. This protocol describes the synthesis of the phosphorothioamidite monomers derived from thymidine and 2'-deoxycytidine, and their application to a fully automated procedure for synthesising oligodeoxynucleotides containing 3'-PS linkages. The synthesis of the 5'-protected-3'-amidites is achievable in 2 weeks with the DNA synthesis and purification taking another 1 week.     

The synthesis of, and studies on, 2'-C-modified nucleotides

The synthesis of uridine monomers containing either a 2'-deoxy-2'-C-methylcyano or ethylcyano group is described. These monomers are intended for incorporation into oligonucleotides to investigate a proposed duplex-stabilising effect exerted by 2'-tethered amide groups.     

A New Approach for the Efficient Synthesis of Oligodeoxyribonucleotides Containing the Mutagenic DNA Modification 7,8-Dihydro-8-oxo-2′-deoxyguanosine at Predefined Positions

Abstract

A combination of H-phoshonate and phosphoramidite chemistry has been applied for the automated solid-phase synthesis of oligodeoxyribonucleotides containing 7, 8-dihydro-8-oxo-2′-deoxyguanosine (8-oxodG) residues at predefined positions. The unmodified part of the oligomers has been synthesized by using protected standard phosphoramidites, for the incorporation of 8-oxodG the synthon 2-N-acetyl-5′-0-(4,4′-dimethoxytrityl)-7,8-dihydro-2′-deoxyguanosin-8-one-3′-H-phosphonate, prepared in a five step synthesis via 8-bromo-2′-deoxyguanosine, has been used. This approach combines the advantages of both DNA synthesis strategies in that a high yield of full length oligomers is obtained and unreacted, protected 8-oxodG monomers can be recycled, respectively.     

β-PNA: peptide nucleic acid (PNA) with a chiral center at the β-position of the PNA backbone

Peptide nucleic acid (PNA) monomers with a methyl group at the β-position have been synthesized. The modified monomers were incorporated into PNA oligomers using Fmoc chemistry for solid-phase synthesis. Thermal denaturation and circular dichroism (CD) studies have shown that PNA containing the S-form monomers was well suited to form a hybrid duplex with DNA, whose stability was comparable to that of unmodified PNA–DNA duplex, whereas PNA containing the R-form monomers was not.     

Synthesis and Evaluation of Novel Oligodeoxynucleotides Containing 3′-C-(3-Benzoyloxypropyl)thymidine and Bicyclo Nucleoside Derivatives

Abstract

The stereoselective synthesis of 3′-C-Allyluridine derivative 2 has been accomplished. This nucleoside was used as a key synthon for the synthesis of oligodeoxynucleotides containing 3′-C-(3-benzoyloxypropyl)thymidine (X) or bicyclo nucleoside (Y+Z) monomers. Preliminary thermal experiments are reported.     

THE SYNTHESIS OF,AND STUDIES ON, 2′-C-MODIFIED NUCLEOTIDES

The synthesis of uridine monomers containing either a 2′-deoxy-2′-C-methy- lcyano or ethylcyano group is described. These monomers are intended for incorporation into oligonucleotides to investigate a proposed duplex-stabilising effect exerted by 2′-tethered amide groups.     

Rapid automated synthesis of polydeoxynucleotides

A rapid automatic method of synthesis of deoxypolynucleotides from 5'-O-dimethoxytritylnucleoside-3'-H-phosphonates is described. An improved construction of synthesizer "Gene-2" adapted for this method has been developed. The modified scheme of synthesis included detritylation with trifluoroacetic acids in dichloromethane, washing with acetonitrile instead of pyridine--acetonitrile mixture and one-step oxidation with iodine solution in acetic acid and pyridine instead of two-step oxidation in the presence of amines. By means of this method, more then 160 polynucleotides containing 8 to 83 monomers were prepared for various biochemical goals including synthesis of promotor 9(260 bp) of the mouse metallothionein-I gene and of promotor and leader sequence (120 bp) of gene of the E. coli alkaline phosphatase.     

Synthesis and evaluation of some properties of chimeric oligomers containing PNA and phosphono-PNA residues.

In an attempt to improve physico-chemical and biological properties of peptide nucleic acids (PNAs), particularly water solubility and cellular uptake, the synthesis of chimeric oligomers consisted of PNA and phosphono-PNA analogues (pPNAs) bearing the four natural nucleobases has been accomplished. To produce these chimeras, pPNA monomers of two types containing N-(2-hydroxyethyl)phosphonoglycine, or N-(2-aminoethyl)phosphonoglycine backbone, were used in conjunction with PNA monomers representing derivatives of N-(2-aminoethyl)glycine, or N-(2-hydroxyethyl)glycine. The oligomers obtained were composed of either PNA and pPNA stretches or alternating PNA and pPNA monomers. The examination of hybridization properties of PNA-pPNA chimeras to DNA and RNA complementary strands in comparison with pure PNAs, and pPNAs as well as DNA-pPNA hybrids and DNA fragments confirmed that these chimeras form stable complexes with complementary DNA and RNA fragments. They were found to be resistant to degradation by nucleases. All these properties together with good solubility in water make PNA-pPNA hybrids promising for further evaluation as potential therapeutic agents.     

Liquid Phase Synthesis of Peptide Nucleic Acid (or Polyamide Nucleic Acid) Dimers

Abstract

The liquid phase synthesis of “polyamide nucleic acid” (PNA) dimers containing the purine nucleic acid bases adenine and guanine has been achieved in good yields. This strategy was elaborated in order to circumvent difficult direct coupling of protected PNA monomers. This method can be applied to the liquid phase synthesis of short protected polyPNAs fragments, which can then selectively be deprotected.     

Large-scale synthesis of "Cpep" RNA monomers and their application in automated RNA synthesis

Small interfering RNAs (siRNA) are the latest candidates for oligonucleotide-based therapeutics. Should siRNA be successful in clinical trials, a huge demand for synthetic RNA is anticipated. We believe that 1-(4-chlorophenyl)-4-ethoxypiperidin-4-yl (Cpep) is an ideal 2'-protecting group for large-scale syntheses. Unlike 2'-silyl groups, mild acid hydrolysis instead of fluoride ion is used for the 2'-deprotection. The syntheses of 2'-Cpep protected nucleosides (A, C, G, and U) has been accomplished on a 0.5 Kg scale. The 2'-Cpep monomers were transformed into 3'-O-phosphoramidites for conventional automated solid-phase synthesis. Cost-effective processes for large-scale synthesis of Cpep monomers and initial automated solid-phase synthesis are demonstrated.     

Advanced bacterial polyhydroxyalkanoates: Towards a versatile and sustainable platform for unnatural tailor-made polyesters

Polyhydroxyalkanoates (PHAs) are biopolyesters that generally consist of 3-, 4-, 5-, and 6-hydroxycarboxylic acids, which are accumulated as carbon and energy storage materials in many bacteria in limited growth conditions with excess carbon sources. Due to the diverse substrate specificities of PHA synthases, the key enzymes for PHA biosynthesis, PHAs with different material properties have been synthesized by incorporating different monomer components with differing compositions. Also, engineering PHA synthases using in vitro-directed evolution and site-directed mutagenesis facilitates the synthesis of PHA copolymers with novel material properties by broadening the spectrum of monomers available for PHA biosynthesis. Based on the understanding of metabolism of PHA biosynthesis, recombinant bacteria have been engineered to produce different types of PHAs by expressing heterologous PHA biosynthesis genes, and by creating and enhancing the metabolic pathways to efficiently generate precursors for PHA monomers. Recently, the PHA biosynthesis system has been expanded to produce unnatural biopolyesters containing 2-hydroxyacid monomers such as glycolate, lactate, and 2-hydroxybutyrate by employing natural and engineered PHA synthases. Using this system, polylactic acid (PLA), one of the major commercially-available bioplastics, can be synthesized from renewable resources by direct fermentation of recombinant bacteria. In this review, we discuss recent advances in the development of the PHA biosynthesis system as a platform for tailor-made polyesters with novel material properties.     

LARGE-SCALE SYNTHESIS OF “Cpep” RNA MONOMERS AND THEIR APPLICATION IN AUTOMATED RNA SYNTHESIS

Small interfering RNAs (siRNA) are the latest candidates for oligonucleotide-based therapeutics. Should siRNA be successful in clinical trials, a huge demand for synthetic RNA is anticipated. We believe that 1-(4-chlorophenyl)-4-ethoxypiperidin-4-yl (Cpep) is an ideal 2′-protecting group for large-scale syntheses. Unlike 2′-silyl groups, mild acid hydrolysis instead of fluoride ion is used for the 2′-deprotection. The syntheses of 2′-Cpep protected nucleosides (A, C, G, and U) has been accomplished on a 0.5 Kg scale. The 2′-Cpep monomers were transformed into 3′-O-phosphoramidites for conventional automated solid-phase synthesis. Cost-effective processes for large-scale synthesis of Cpep monomers and initial automated solid-phase synthesis are demonstrated.     

New reagents and solid support for automated oligonucleotide synthesis

The optimal system for the rapid, efficient, convenient, and economical synthesis and purification of synthetic oligonucleotides has been advancing. By recognizing the very rapid reaction kinetics and taking advantage of an efficient, low volume delivery system, cycle times have decreased to about 5.5 minutes, without compromising synthesis performance. A new set of base protecting groups for cyanoethylphosphoramidite nucleoside monomers have been developed, which decreases the post-synthesis time requirements. A particular form of polystyrene has also been developed as a solid support for automated oligonucleotide synthesis. Typical sequencing or PCR primers (20mers) now require less than 2 hours for synthesis and 2 hours for cleavage and deprotection.     

Multiple-copy genes: production and modification of monomeric peptides from large multimeric fusion proteins

A vector system has been designed for obtaining high yields of polypeptides synthesized in Escherichia coli. Multiple copies of a synthetic gene encoding the neuropeptide substance P (SP) (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Met-NH2) have been linked and fused to the lacZ gene. Each copy of the SP gene was flanked by codons for methionine to create sites for cleavage by cyanogen bromide (CNBr). The isolated multimeric SP fusion protein was converted to monomers of SP analog, each containing a carboxyl-terminal homoserine lactone (Hse-lactone) residue (Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Hse-lactone), upon treatment with CNBr in formic acid. The Hse-lactone moiety was subjected to chemical modifications to produce an SP Hse amide. This method permits synthesis of peptide amide analogs and other peptide derivatives by combining recombinant DNA techniques and chemical methods.     

Gene silencing activity of siRNA molecules containing phosphorodithioate substitutions

Chemically synthesized small interfering RNAs (siRNAs) have been widely used to identify gene function and hold great potential in providing a new class of therapeutics. Chemical modifications are desired for therapeutic applications to improve siRNA efficacy. Appropriately protected ribonucleoside-3'-yl S-[β-(benzoylmercapto)ethyl]pyrrolidino-thiophosphoramidite monomers were prepared for the synthesis of siRNA containing phosphorodithioate (PS2) substitutions in which the two non-bridging oxygen atoms are replaced by sulfur atoms. A series of siRNAs containing PS2 substitutions have been strategically designed, synthesized, and evaluated for their gene silencing activities. These PS2-siRNA duplexes exhibit an A-form helical structure similar to unmodified siRNA. The effect of PS2 substitutions on gene silencing activity is position-dependent, with certain PS2-siRNAs showing activity significantly higher than that of unmodified siRNA. The relative gene silencing activities of siRNAs containing either PS2 or phosphoromonothioate (PS) linkages at identical positions are variable and depend on the sites of modification. 5'-Phosphorylation of PS2-siRNAs has little or no effect on gene silencing activity. Incorporation of PS2 substitutions into siRNA duplexes increases their serum stability. These results offer preliminary evidence of the potential value of PS2-modified siRNAs.     

A-hydroxyphosphonate oligonucleotides: a promising DNA type?

The synthesis of monomers (S)-1, (R)-1 and 2 derived from (5'S)-, (5'R)-2'-deoxythymidine-5'-C-phosphonic acids and 2',5'-dideoxythymidine-5'-C-phosphonic acids was elaborated. The protection of the 5'-hydroxyl by the methoxycarbonyl group was a key step of the synthesis. Prepared monomers were used for the solid-phase assembly of several types oligothymidylate 15-mers (S)-3, (S)-4, (S)-5, (R)-4 and (R)-5 containing the chiral 3'-O-P-CH(OH)-5' internucleotide linkage. Their hybridization properties with dA15 and rA15 were studied as well as their resistance against nuclease cleavage.