A new approach to the synthesis of the 5'-deoxy-5'-methylphosphonate linked thymidine oligonucleotide analogues.

A new synthetic method for the preparation of the 5'-deoxy-5'-methylphosphonate linked thymidine oligonucleotides (5'-methylenephosphonate analogues) was developed. The method is based on the use of a phosphonate protecting group, 4-methoxy-1-oxido-2-picolyl, enabling intramolecular nucleophilic catalysis which together with the condensing agent, 2,4,6-triisopropylbenzenesulfonyl chloride, secures fast and efficient formation of the 5'-methylenephosphonate internucleosidic bonds. The produced protected oligomers were treated with thiophenol and triethylamine to remove the phosphonate protecting groups, cleaved from the solid support using concentrated aqueous ammonia, and purified by HPLC. Several thymidine oligonucleotide analogues with the chain length of up to 20 nucleotidic units, in which all internal 5'-oxygen atoms have been replaced by methylene groups directly bound to phosphorus, were synthesised using this methodology.     

Synthesis of oligonucleotides carrying 5'-5' linkages using copper-catalyzed cycloaddition reactions

There is considerable interest in coupling oligonucleotides to molecules and surfaces. Although amino- and thiol-containing oligonucleotides are being successfully used for this purpose, cycloaddition reactions may offer greater advantages due to their higher chemoselectivity and speed. In this study, copper-catalyzed 1,3-dipolar cycloaddition reactions between oligonucleotides carrying azido and alkyne groups are examined. For this purpose, several protocols for the preparation of oligonucleotides carrying these two groups are described. The non-templated chemical ligation of two oligonucleotides via copper-catalyzed [3+2] cycloaddition is described. By solid-phase methodology, oligonucleotides carrying 5'-5' linkages can be obtained in good yields.     

Oxalyl-CPG: a labile support for synthesis of sensitive oligonucleotide derivatives.

A procedure is described for linking nucleosides covalently to controlled pore glass or cross-linked polystyrene supports by means of an oxalyl anchor. Though stable to triethylamine and diisopropylamine, the nucleoside-oxalyl link can be cleaved within a few minutes at room temperature with ammonium hydroxide in methanol. This new anchor can be used in automated synthesis of conventional oligonucleotides. The primary value, however, is that it enables one to employ solid support methodology to synthesize a variety of base-sensitive oligonucleotide derivatives, as illustrated here by synthesis of oligomers with base protecting groups intact and with methyl phosphotriester groups at the internucleoside links.     

Synthesis of DNA fragments containing 5,6-dihydrothymine, a major product of thymine gamma radiolysis

5,6-Dihydrothymine is one of the most important products of base damage by gamma irradiation of DNA in anoxic conditions. This modified base is unstable in the deprotection conditions used for classical synthesis of oligonucleotides. For its incorporation in synthetic DNA fragments, a new set of amino protecting groups has been developed. The 5,6-dihydrothymidine phosphoramidite was successfully employed for the synthesis of two 14-mers and one 17-mer bearing this defect at positions corresponding to restriction enzymes sites. The presence of the modified base still intact in the oligonucleotides was evidenced by mass spectrometry in pyrolytic conditions.     

'Protected DNA Probes' capable of strong hybridization without removal of base protecting groups

We propose a new strategy called the ‘Protected DNA Probes (PDP) method’ in which appropriately protected bases selectively bind to the complementary bases without the removal of their base protecting groups. Previously, we reported that 4-N-acetylcytosine oligonucleotides (ac⁴C) exhibited a higher hybridization affinity for ssDNA than the unmodified oligonucleotides. For the PDP strategy, we created a modified adenine base and synthesized an N-acylated deoxyadenosine mimic having 6-N-acetyl-8-aza-7-deazaadenine (ac⁶az⁸c⁷A). It was found that PDP containing ac⁴C and ac⁶az⁸c⁷A exhibited higher affinity for the complementary ssDNA than the corresponding unmodified DNA probes and showed similar base recognition ability. Moreover, it should be noted that this PDP strategy could guarantee highly efficient synthesis of DNA probes on controlled pore glass (CPG) with high purity and thereby could eliminate the time-consuming procedures for isolating DNA probes. This strategy could also avoid undesired base-mediated elimination of DNA probes from CPG under basic conditions such as concentrated ammonia solution prescribed for removal of base protecting groups in the previous standard approach. Here, several successful applications of this strategy to single nucleotide polymorphism detection are also described in detail using PDPs immobilized on glass plates and those prepared on CPG plates, suggesting its potential usefulness.     

A Rapid Deprotection Procedure for Phosphotriester DNA Synthesis

Abstract

An equimolar solution of aldoxime and tetramethylguanidine at 70°C cleaves all base labile protecting groups from oligonucleotides.     

New Approach to the Synthesis of 2′(3′)-O-Aminoacyloligoribonucleotides Related to the 3′-Terminus of Aminoacyl Transfer Ribonucleic Acid

Abstract

A new methodology for the synthesis of 2′(3′)-0-aminoacyl oligonucleotides based on an unique combination of protecting groups is described. The blocking scheme allows a simple two step deblocking procedure, which provides easy access to the target compounds.     

On the rapid deprotection of synthetic oligonucleotides and analogs.

The efficiency of oligodeoxynucleotide deprotection is greatly enhanced using a combination of: (a) ethanolamine, and especially a mixture of hydrazine, ethanolamine and methanol, in place of the usual aqueous ammonia; (b) tert-butylphenoxyacetyl amino protecting groups, and (c) oxalyl link between the first nucleotide and the polymeric support. The extent of base modification, particularly of C, is shown to be extremely low, and the quality of deprotected oligonucleotides is as high as in the case of ammonia deprotection. This method is also shown to be applicable to the preparation of phosphorothioate and methylphosphonate oligodeoxynucleotides and oligoribonucleotides.     

Hybridization triggered cross-linking of deoxyoligonucleotides.

This paper reports details of the synthesis of oligodeoxynucleotides containing the modified base 5-methyl-N4,N4-ethanocytosine (Ce). The 9-fluorenylmethoxycarbonyl group is used as a protecting group for the exocyclic amines of dA and dC. This group can be removed rapidly under very mild conditions. Oligomers containing the Ce base form a cross-link when hybridized to their complementary deoxyoligonucleotides. Some of the scope and limitations of these cross-link forming oligonucleotides are reported.     

Application of new catalytic phosphate protecting groups for the highly efficient phosphotriester oligonucleotide synthesis.

An effective procedure for the synthesis of oligonucleotides by the phosphotriester method has been developed. The procedure is based on the use of phosphate protecting groups enabling O-nucleophilic intramolecular catalysis in the reaction of internucleotide bond formation under the action of arylsulfonyl chlorides and their derivatives. Using this new procedure, the time needed to perform one elongation step on polymer support is 7-8 min. The effectiveness of the methodology has been demonstrated in the synthesis of many oligodeoxyribonucleotides of different length with high yields.     

A New Protecting Group Strategy for Amino Groups in Oligonucleotide Chemistry: CEOC Group

Abstract

A new protecting group, 2-cyanoethyloxycarbonyl, or CEOC, has been developed for amino groups and utilized in synthesizing modified oligonucleotides. (CEOC)-oxy-succinimide reagent has been synthesized to introduce this protecting group. The protecting group is removed by standard oligonucleotide deprotection protocols. Using this approach, oligonucleotides have been synthesized with various types of alkylamine substituents.     

Preparation of Oligonucleotides Containing Non-natural Base Analogues

Abstract

The preparation of a protected derivative of 2-aza- 2′-deoxyinosine carrying a photolabile protecting group is described. The new derivative is useful to prepare oligonucleotides containing 2-azahypoxanthine. The synthesis of oligonucleotides containing 2-fluorohypoxanthine and O⁴-alkylthymine is also described.     

Engineered polymer-supported synthesis of 3'-carboxyalkyl-modified oligonucleotides and their applications in the construction of biochips for diagnosis of the diseases

An engineered polymer support 5 has been prepared for the solid-phase assembly of 3'-carboxyalkyl-modified oligonucleotides using commonly available reagents. A two-step deprotection procedure resulted in the quantitative cleavage of oligonucleotides from the support and removal of the protecting groups from phosphodiesters and exocyclic amino groups of the nucleic bases. The fully deprotected oligomers, obtained in high yield, were desalted and analyzed on RP-HPLC. After characterization by MALDI-TOF, these carboxyalkylated oligonucleotides were immobilized onto the epoxy-functionalized glass microslides to prepare biochips. The performance of these biochips was evaluated under different sets of conditions and then successfully validated by the detection of base mismatches and human infectious disease, bacterial meningitis, caused by N. meningitidis.     

Synthesis of Oligodeoxyribonucleotides Containing 2,6-Diaminopurine

Abstract

The preparation of a new protected derivative of 2,6-diaminopurine 2′-deoxyriboside carrying two phenoxyacetyl groups is described. The new derivative is useful to prepare oligonucleotides containing 2,6-diaminopurine and it is deprotected at the same time as the standard protecting groups of the natural bases.     

Synthesis Of RNA by the Rapid Phosphotriester Method Using Azido-Based 2′-O-Protecting Groups

The azidomethyl and 2-(azidomethyl)benzoyl as 2′-OH protecting groups are reported for preparation of oligoribonucleotides by the phosphotriester solid-phase method using O-nucleophilic intramolecular catalysis. The procedures for the synthesis of the corresponding monomer synthons were developed and the usefulness of the application of 2′-O-azidomethyl and 2′-O-2-(azidomethyl)benzoyl groups was examined in the synthesis of different RNA fragments with a chain length of 15–22 nucleotides. The azidomethyl group was found to be more preferable for effective synthesis of oligoribonucleotides. Hybridization properties of RNAs toward their complementary oligonucleotides were examined before and after the removal of 2′-O-azidomethyl groups.     

5-Fluoro-4-thiouridine phosphoramidite: new synthon for introducing photoaffinity label into oligodeoxynucleotides

The synthesis of phosphoramidite of 5-fluoro-4-thio-2'-O-methyluridine is described. An appropriate set of protecting groups was optimized including the 4-thio function introduced via 4-triazolyl as the 4-(2-cyanoethyl)thio derivative, and the t-butyldimethyl silyl for 2' and 3' hydroxyl protection, enabling efficient synthesis of the phosphoramidite. These protecting groups prevented unwanted side reactions during oligonucleotide synthesis. The utility of the proposed synthetic route was proven by the preparation of several oligonucleotides via automated synthesis. Photochemical experiments confirmed the utility of the synthon.     

Boranophosphate Oligonucleotides: New Synthetic Approaches

Abstract

Recently our laboratory reported a new backbone-modified class of oligonucleotides, with a borane (B₃3?) group replacing one of the non-bridging oxygen atoms. Here we present two new approaches to synthesize the boranophosphate oligonucleotides. All-stereoregular boranophosphate oligonucleotides can be prepared by enzymatic template extension reactions using nucleoside a-boranotriphosphates, which are good substrates for a number of polymerases. Larger scale synthesis of boranophosphate oligonucleotides can be carried out by effective chemical synthesis using the H-phosphonate approach, instead of previously used phosphoramidite methodology. The main advantage of H-phosphonate methodology is the ability to carry out one boronation reaction, after oligonucleotide chain elongation has been completed, using mild conditions without base damage and producing the desired boranophosphate oligonucleotides in high yield.     

Facile Synthesis of <Emphasis Type="Italic">N</Emphasis>-protected Amino Acid Esters Assisted by Microwave Irradiation

A highly efficient and safe methodology for synthesis of various N-protected amino acid ethyl esters have been established in this study. This methodology employs orthoesters as both esterification reagent and solvent for protected amino acids. The reactions were carried out under microwave irradiation in neutral conditions for only 2 min, resulting in highly pure crude products in most cases. This strategy works with a variety of N-protecting groups, such as acid labile protecting group: BOC and tBu; base labile protecting group: Fmoc; hydrogenation labile protecting group: Z and Na/NH₃ labile protecting group: Tos, thus providing facile access to numerous valuable building blocks for solid phase synthesis. Further reduction of the crude protected amino acid ethyl ester by sodium borohydride under mild conditions led to the corresponding protected β-amino alcohols with excellent yield, as demonstrated by three examples.     

Microwave-assisted rapid deprotection of oligodeoxyribonucleotides.

A novel method for the deprotection of oligodeoxyribonucleotides under microwave irradiation has been developed. The oligodeoxynucleotides having base labile, phenoxyacetyl (pac), protection for exocyclic amino functions were fully deprotected in 0. 2 M sodium hydroxide (methanol:water : : 1:1, v/v) = A and 1 M sodium hydroxide (methanol:water : : 1:1, v/v) = B using microwaves in 4 and 2 min, respectively. The deprotection of oligodeoxyribonucleotides carrying conventional protecting groups, dAbz, dCbzand dGpac, for exocyclic amino functions was achieved in 4 min in B without any side product formation. The deprotected oligonucleotides were compared with the oligomers deprotected using standard deprotection conditions (29% aq. ammonia, 16 h, 55 degrees C) with respect to their retention time on HPLC and biological activity.     

Use of an aminooxy linker for the functionalization of oligodeoxyribonucleotides

We describe the preparation of oligonucleotides containing a 5'-linker bearing an aminooxy group. Use of the trityl protecting group for the aminooxy moiety allows purification of the modified oligonucleotide by reverse phase HPLC and cleavage in mild acidic conditions. Derivatization with an aldehydic reporter group is efficient and rapid.