Synthesis of DNA oligonucleotides containing 5-(mercaptomethyl)-2'-deoxyuridine moieties

Recently thiolated oligonucleotides have attracted significant interest due to their ability to efficiently undergo stable bond formation with gold nanoparticles and surfaces to form DNA conjugates. In this respect we became interested in the synthesis of oligonucleotides that bear short thioalkyl functions located at the nucleobase. Here we present a strategy for the synthesis of DNA oligonucleotides that bear 5-(mercaptomethyl)-2'-deoxyuridine moieties. The building blocks were synthesized in a straightforward manner from thymidine. Only moderate changes of standard protocols for automated DNA synthesis are required for the generation of modified oligonucleotides containing the thiolated building blocks.     

Hydrazide oligonucleotides: new chemical modification for chip array attachment and conjugation

We report the synthesis of new phosphoramidite building blocks and their use for the modification of oligonucleotides with hydrazides. The reaction of these hydrazide oligonucleotides with active esters and aldehydes is demonstrated for solution conjugation and immobilization. Compared with the established amino modified oligonucleotides, hydrazides show enhanced reactivity at neutral and acidic buffer conditions. One method to introduce hydrazides is using amidites with preformed, protected hydrazides. A completely novel approach is the generation of the hydrazide functionality during the oligonucleotide cleavage and deprotection with hydrazine. Therefore, building blocks for the introduction of esters as hydrazide precursors are described. For the enhanced attachment on surfaces branched modifier amidites, which introduce up to four reactive groups to the oligonucleotide, are applied. The efficiency of branched hydrazide oligonucleotides compared with standard amino modified oligonucleotides for the immobilization of DNA on active electronic Nanogen chips is demonstrated.     

Nucleosides and oligonucleotides with diynyl side chains: the huisgen-sharpless cycloaddition "click reaction" performed on DNA and their constituents

Phosphoramidite building blocks related to the four canonical DNA constituents and oligonucleotides with octadiynyl side chains (5b-8b) or dipropargyl ether residues (5c) were synthesized. Nucleosides and oligonucleotides were functionalized at the terminal triple bonds employing the Huisgen-Sharpless cycloaddition "click reaction."     

Alpha-hydroxybenzylphosphonate modified oligonucleotides: synthesis, properties, and a novel route via monomer building blocks

In general, alpha-hydroxybenzylphosphonate modified 2'-deoxyadenosine-thymidine dimer building blocks 1, 2 are utilized for the incorporation into alpha-hydroxybenzylphosphonate pro-oligonucleotides. For a universal application of our pro-oligonucleotide concept on biologically relevant oligonucleotides a route for the synthesis of modified monomer building blocks 3 was developed and is presented herein.     

A General Approach for the Hydroxy Group Functionalization of Synthetic Oligonucleotides

Abstract

Generally applicable non-nucleosidic solid supports and phosphoramidite building blocks that enable attachment of various tethers to the hydroxy groups of oligonucleotides were prepared. The key feature of the structure of these reagents is an ester bond of moderate reactivity which allows postsynthetic introduction of tethers by reaction with substituted alkylamines.     

Construction of Double-helical DNA Structures Based on Dinucleotide Building Blocks

Abstract

We present a new method for building full 3-D structures of DNA sequences. A database of the conformational properties of dinucleotide steps has been compiled using X-ray crystal structures of oligonucleotides. The protocol uses these dinucleotides as building blocks to generate three dimensional structures of any required sequence in any required conformation.     

Synthesis of building blocks for solid-phase introduction of diethylenetriaminepentaacetic acid (DTPA) to oligonucleotides and oligopeptides

Synthesis of building blocks that allow site-specific incorporation of diethylenetriaminepentaacetic acid (DTPA) to oligonucleotides and oligopeptides using phosphoramidite and Fmoc chemistries, respectively, is described.     

Synthesis of oligonucleotide building blocks of 2'-deoxyguanosine bearing a C8-arylamine modification

C8-Arylamine-dG adducts were synthesized by palladium-catalyzed cross-coupling reactions. The corresponding 5'-O-DMTr-3'-O-phosphoramidite-C8-arylamine-dG adducts were synthesized as potential building blocks for the automated synthesis of site-specifically modified oligonucleotides.     

2'-O,4'-C-ethylene-bridged nucleic acids (ENA): highly nuclease-resistant and thermodynamically stable oligonucleotides for antisense drug.

To develop antisense oligonucleotides, novel nucleosides, 2'-O,4'-C-ethylene nucleosides and their corresponding phosphoramidites, were synthesized as building blocks. The 1H NMR analysis showed that the 2'-O,4'-C-ethylene linkage of these nucleosides restricts the sugar puckering to the N-conformation as well as the linkage of 2'-O,4'-C-methylene nucleosides which are known as bridged nucleic acids (BNA) or locked nucleic acids (LNA). The ethylene-bridged nucleic acids (ENA) showed a high binding affinity for the complementary RNA strand (DeltaT(m)=+5.2 degrees C/modification) and were more nuclease-resistant than natural DNA and BNA/LNA. These results indicate that ENA have better properties as antisense oligonucleotides than BNA/LNA.     

Synthesis and evaluation of RNA transesterification efficiency using stereospecific serinol-terpyridine conjugates

Six novel artificial ribonucleases were synthesized employing a stereochemically pure abasic serinol backbone residue for attachment of the RNA transesterification agent copper(II) terpyridine. These stereochemically pure abasic residues were synthesized as phosphoramidite building blocks from the parent L-serine and D-serine starting building blocks and incorporated into oligonucleotides via solid-phase DNA synthesis. These artificial ribonucleases were constructed to determine if the stereochemistry of the alpha carbon of an abasic serinol residue has influence over RNA transesterification through selective placement of a pendant transesterification agent in either the major or minor groove. The novel artificial ribonucleases and previously synthesized artificial ribonucleases were challenged with a 28-mer and 159-mer RNA substrate. It was determined that the stereochemistry of the carbon atom derived from the alpha-carbon of serine did not influence the extent of cleavage in these studies using copper(II) terpyridine conjugated artificial ribonucleases.     

Triple-helix mediated excimer and exciplex formation

The synthesis and properties of triple-helical hybrids containing non-nucleosidic polyaromatic building blocks are described. Clamp-type oligonucleotides containing a non-nucleosidic pyrene linker form stable triple helices with a polypurine target strand containing a terminal pyrene or phenanthrene moiety. Stacking interactions between the unnatural building blocks enhance triplex stability and lead to strong excimer or exciplex formation, which is monitored by fluorescence spectroscopy.     

Porphyrin substituted phosphoramidites: new building blocks for porphyrin-oligonucleotide syntheses

Thymidine phosphoramidites containing trispyridylphenyl and tetraphenylporphyrin chromophores attached via a short amide linker in the 3'-position have been synthesized and used as building blocks in solid-phase synthesis of self-complementary 8-mer oligonucleotides 3'-T-5'-GCGCGCA-3' and 5'-ACGCGCGT-3'. To our knowledge, these are the first porphyrin-oligonucleotide conjugates carrying the porphyrin chromophores in the 3'-position. Chain assembly was achieved by automated solid-phase synthesis and by inexpensive straightforward 'in flask' modification of commercially available solid supported oligonucleotides. This approach allows the synthesis of modified oligonucleotides without using costly instrumentation for automated DNA synthesis. Porphyrin-containing self-complementary oligonucleotides are expected to be a valuable model for drug binding studies and determination of conformational changes in DNA sequences using circular dichroism.     

1,5-Anhydro-2-Deoxy-D-Altritol Oligonucleotides as Conformationally Restricted Analogues of Rna

Abstract

As part of a project concerning the investigation of new hexitol nucleic acids (HNA), the 1,5-anhydro-2-deoxy-D-altritol nucleoside building blocks with a uracil, cytosine, adenine and guanine base moiety were synthesized. The uracil analogue was used for the automated synthesis of corresponding oligonucleotides. Hybridization capabilities of these altritol nucleic acids (ANA) are illustrated by the Tm values obtained for the (a ^hU)₁₃/(dA)₁₃ duplex.     

Synthesis of modified nucleotide building blocks containing electrophilic groups in the 2'-position

Chemical syntheses of 2'-O-(allyloxycarbonyl)methyladenosine, 2'-O-(methoxycarbonyl)methyladenosine and 2'-O-(2,3-dibenzoyloxy)propyluridine 3'-2-cyanoethyl-N,N-diisopropyl phosphoramidite building blocks are described. These monomers were used successfully to incorporate carboxylic acid, 1,2-diol and aldehyde functionalities into synthetic oligonucleotides.     

Second Generation Antisense Oligonucleotides—Inhibition of PKC-α and c-raf Kinase Expression by Chimeric Oligonucleotides Incorporating 6″-Substituted Carbocyclic Nucleosides and 2″-O-Ethylene Glycol Substituted Ribonucleosides

Abstract

6′-substituted carbocyclic deoxyribonucleosides and 2′-O-ethylene glycol substituted ribonucleosides have been evaluated as building blocks for antisense oligonucleotides. Within the former class 6′-hydroxy substituted building blocks in combination with internucleoside phosphorothioate linkages have the potential to enhance antisense activity. 2′-O-ethylene glycol substituted ribonucleosides generally allow for the construction of potent antisense oligonucleotides with reduced phosphorothioate content, but differences exist in their effects on biological activity in cell culture in spite of virtually identical effects on RNA-binding affinity. Activity enhancement was most pronounced for a 2′-O-methoxyethyl substituent.     

Evaluation of oligonucleotides containing two novel 2'-O-methyl modified nucleotide monomers: a 3'-C-allyl and a 2'-O,3'-C-linked bicyclic derivative.

The two ribo-configured nucleosides 1-(3-C-allyl-2-O-methyl-beta-D-ribo-pentofuranosyl)thymine 3 and (1S,5R,6R,8R)-5-hydroxy-6-(hydroxymethyl)-1-methoxy-8-(thymin-1-yl )- 2,7-dioxabicyclo[3.3.0]octane 6 have been transformed into their corresponding phosphoramidites, 5 and 8 respectively, and used as building blocks for the synthesis of modified oligonucleotides. The oligonucleotides were shown to hybridize with decreased binding affinity towards complementary single stranded DNA and RNA.     

Oligonucleotide N-alkylphosphoramidates: synthesis and binding to polynucleotides

A few different methods for the preparation of oligonucleotide N-alkylphosphoramidates were compared directly. One of these, involving the use of protected nucleoside phosphites as building blocks, provided the requisite N-alkylphosphoramidates via oxidation of the intermediate dinucleoside methyl phosphites with iodine in the presence of the appropriate alkylamine. This method was found to have several attractive features, including the use of building blocks identical with those employed for the synthesis of DNA and compatibility with procedures and instruments employed for the stepwise synthesis of oligonucleotides by solution and solid-phase methods. This procedure was used to make several di-, tri-, and tetranucleotide N-alkylphosphoramidates derived from deoxyadenosine and thymidine; alkyl substituents included N,N-dimethyl, N-butyl, N-octyl, N-dodecyl, and N-(5-aminopentyl). The aminoalkyl derivative of d(TpT) (24) was used to demonstrate the feasibility of introducing an intercalative agent to the alkylphosphoramidate moiety of such derivatives. The oligonucleotide N-alkylphosphoramidates were separated into their component diastereomers and characterized structurally by a number of techniques including circular dichroism, high-field 1H NMR spectroscopy, FAB mass spectrometry, and enzymatic digestion to authentic nucleosides and nucleotides. Physicochemical characterization of several di- and trinucleotide alkyl-phosphoramidates revealed that the adenine nucleotide analogues formed stable complexes with poly-(thymidylic acid). The stabilities of these complexes were found to increase with increasing chain length of the N-alkylphosphoramidate substituents. The finding that N-alkylphosphoramidate substituents can enhance the binding of certain oligonucleotides to their complementary polynucleotides suggests the existence of a novel source of polynucleotide affinity.     

New Building Blocks for the Preparation of Branched Oligonucleotides

Abstract

New building blocks 2 and 3 were prepared and successfully employed for the synthesis of branched oligonucleotides 5 and 6. The structure of oligomers obtained was confirmed by electrospray ionisation mass spectrometry.     

Synthesis and hybridization properties of inverse oligonucleotides.

The synthesis of adenine and thymine cyclopentylethyl nucleosides is presented. This novel constrained monomeric building block is very difficult to incorporate into oligonucleotides. It was introduced in 13mer oligodeoxynucleotide sequences at a single position using H-phosphonate chemistry. Phosphoramidite chemistry completely failed in this particular case. The H-phosphonate building blocks were obtained starting from the corresponding phosphoramidites. Stability of duplexes with RNA and DNA is significantly reduced.     

Methoxyoxalamido chemistry in the synthesis of novel amino linker and spacer phosphoramidites: a robust means for stability, structural versatility, and optimal tether length

We have developed a general route to the synthesis of novel amino linker and spacer phosphoramidites utilizing methoxyoxalamido (MOX) chemistry. The synthesis makes use of readily available and inexpensive primary aliphatic amino alcohols and diamines to produce a rich and diverse variety of phosphoramidites. Among these are monomers with exceptionally long (up to 56 atoms in length) amphipathic tethering arms. The chemistry bestows exceptional control over the physical characteristics within the tethers through the selection of appropriate building blocks. Furthermore, MOX chemistry enables fairly rapid assembly of these discrete-length tethers in a modular fashion. All novel phosphoramidites were successfully used in automated syntheses of 5'-modified oligonucleotides.