Ultrafast Cleavage and Deprotection of Oligonucleotides Synthesis and Use of CAc Derivatives

Abstract

We have investigated the use of alkylamines as fast cleavage and deprotection reagents for the solid phase synthesis of oligonucleotides and found methylamine/ammonium hydroxide (or methylamine) as an efficient reagent. The transamination side product formed with the commonly used dC^bz has been eliminated by the use of dC^Ac phosphoramidite. This system has successfully been used in the synthesis of oligonucleotides and oligonucleoside phosphorothioates. DMT dC^Ac hydrogen phosphonate and DMT ribo C^Ac-2′-O Me phosphoramidite also have been prepared and used in the synthesis of oligonucleotides.     

An Improved Post-Synthetic Substitution Approach for Synthesis of Oligodeoxynucleotides Containing Labile 4-Substituted Thymines

Abstract

A simple procedure is described for the preparation of a versatile oligodeoxynucleotide which contains 4-phenylthiothymidine. This versatile oligomer has been successfully used for synthesis of oligonucleotides containing labile 5-methyl-N⁴, N⁴-ethanocytosine (7) or 4-azido-5-methyl-2-pyrimidinone-1-β-(2′-deoxyriboside) (8).     

A Novel and Convenient Method for the Synthesis of Free 5′-Thiol Modified Oligonucleotides

Abstract

The synthesis of free 5′-thiol-modified oligonucleotides using a 4,4′,4″-trimethoxytrityl (TMTr)-protected linker and standard Poly-Pak^TM purification has been described.     

Carbohydrate Modifications in Antisense Oligonucleotide Therapy: New Kids on the Block

Abstract

Chemical modifications to improve the efficacy of an antisense oligonucleotide are designed to increase the binding affinity to target RNA, to enhance the nuclease resistance, and to improve cellular delivery. Among the different sites available for chemical modification in a nucleoside building block, the 2′-position of the carbohydrate moiety¹ has proven to be the most valuable for various reasons: (1) 2′-modification can confer an RNA-like 3′-endo conformation to the antisense oligonucleotide. Such a preorganization for an RNA like conformation^2,3,4,5 greatly improves the binding affinity to the target RNA; (2) 2′-modification provides nuclease resistance to oligonucleotides; (3) 2′-modification provides chemical stability against potential depurination conditions pharmacology evaluations and correlation with pharmacokinetic changes are emerging from these novel chemical modifications. Analytical chemistry of modified oligonucleotides before and after biological administration of antisense oligonucleotides with techniques such as capillary gel electrophoresis (CGE) and mass spectrometry help to determine the purity as well as the in vivo fate of these complex molecules. Large-scale synthesis is becoming a tangible reality for antisense oligonucleotides. Nucleic acid chemists and biologists alike are beginning to understand the structure-biological activity in terms of basic physical-organic parameters such as the gauche effect, the charge effect and conformational constraints. Synthesis of chimeric designer oligonucleotides bringing the attractive features of different modifications to a given antisense oligonucleotide sequence to generate synergistic interactions is forthcoming³⁰. These advances along with the potential availability of complete human genome sequence information promise a bright future for the widespread use of nucleic acid based therapeutics.     

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.     

SYNTHESIS OF 5-(2,2′-BIPYRIDINYL AND 2,2′-BIPYRIDINEDIIUMYL)-2′-DEOXYURIDINE NUCLEOSIDES: PRECURSORS TO METALLO-DNA CONJUGATES

ABSTRACT

The synthesis of 2,2′-bipyridinyl-2′-deoxyuridine metal-chelator nucleosides (Bipy-dU) with either ethynyl or ethylenyl linkers was now been accomplished. These new nucleosides will permit the construction of a number of corresponding metallo-DNA conjugates where many types of metals can be complexed to the 2,2′-bipyridinyl chelator group and the resulting metallo-dU conjugates incorporated into DNA oligonucleotides. Additionally this paper also reports the synthesis of a di-N-alkylated bipyridinediiumyl-2′-deoxyuridine nucleoside (Bipy²⁺-dU) with an ethylenyl linker. The Bipy²⁺-dU nucleoside was found to decompose under basic conditions precluding its use in standard automated DNA-synthesis by the phosphoramidite method. No such restrictions apply to the two Bipy-dU nucleosides reported here for use as metal chelators.     

Base Modified Oligodeoxynucleotides. II. Increase of Stability to Nucleases by 5-Alkyl-, 5-(1-Alkenyl)-, and 5-(1-Alkynyl)-pyrimidines

Abstract

The effect of pyrimidine base substitution on the sensitivity of oligonucleotides to nucleases has been studied with two series of self complementary deoxyoligonucleotides containing n-alkyl, n-(1-alkenyl) or n-(1-alkynyl) groups at C5 of pyrimidines, (dA-r⁵dU)₁₀ and (dG-r^sdC)₆. The rate of hydrolysis by snake venom phosphodiesterase and in human serum decreased with increasing length and unsaturation of the substituent.     

Synthesis and Enzymatic Studies of Modified Oligonucleotides with Abiological Monomer Units

Abstract

The synthesis and the enzymatic studies of modified oligonucleotides containing a PNA modified PNA-DNA dimer block and a new acyclic racemic serinol nucleoside is described. We show that both, the PNA-DNA dimer block¹ and the modified PNA-spacer (acyclic serinol nucleoside)² can be used as modified templates for the enzymatic generation of single stranded DNA. Degradation studies of the oligonucleotides containing the PNA-DNA dimer block with snake venom phosphodiesterase show that the modified oligonucleotides are stable towards exonucleolytic degradation.     

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.     

A Convenient High Yield Synthesis of N4-Isobutyryl-2′-O-Methylcytidine and Its Monomer Units For Incorporation into Oligonucleotides

Abstract

We designed an efficient three step procedure for the synthesis of N⁴-isobutyryl-2′-O-methylcytidine. This protected nucleoside was then used to prepare a methylphosphonamidite monomer for incorporation into oligonucleotides. Transamination at the C⁴ position of cytidine using ethylenediamine, which has been reported for the N⁴-benzoyl cytidine, was not observed with N⁴-isobutyryl protected 2′-O-methylcytidine.     

Phosphotriester Synthesis of Oligonucleotides with the Use of N- and O-Nucleophilic Intramolecular Catalysis

Abstract

An effective method for the synthesis of oligonucleotides by the phosphotriester approach has been developed. It is based on the use of phosphate protecting groups enabling O-nucleophilic intramolecular catalysis.     

An Improved Method for the Preparation of Methyl dichlorophosphite. A Key Reagent In the Phosphite Method of Oligonucleotide Synthesis

Abstract

The reaction between phosphorus trichloride (PCl₃) and trimethyl phosphite (CH₃O)3^p, has been examined by ³¹Pnmr, in order to achieve a simple and efficient procedure for the formation of methyl dichlorophosphite (CH₃OPCl₂), which is a key intermediate in the synthesis of oligonucleotides. The yield of the reaction was also studied on a preparative scale and it was found that the optimal condition is obtained when the reactants molar ratio is 1:1.     

Synthesis of (Dicarbonyl)(η5-cyclopentadieny1)iron -Derived Nucleoside Phosphonate Esters

Abstract

New methods for generating phosphonate linkages in oligonucleotides are under investigation. Among reagents that react selectively with phosphite triesters and H-phosphonates are (dicarbonyl)(η⁵-cyclopentadienyl)(η²-olefin)iron complexes. We have found that (dicarbonyl)(η⁵-cyclopentadienyl)(η²-ethylene)iron reacts with 5′-O-dimethoxytritylnucleoside 3′-phosphite esters to yield (dicarbonyl)(η⁵-cyclopentadienyl)iron(ethyl)phosphonates. The (dicarbonyl)(η⁵-cyclopentadienyl)iron-derived nucleoside phosphonates are stable to the conditions commonly used to construct oligonucleotides by phosphoramidite methodology.     

The Application of H-Phosphonate Chemistry in the HELP Synthesis of Oligonucleotides

Abstract

The use of the H-phosphonate chemistry for its possible utilization in the liquid phase synthesis of oligonucleotides has been investigated.     

Digoxigenated oligonucleotide probes specific for simple repeats in DNA fingerprinting and hybridization in situ

Summary A fast, reproducible and non-hazardous technique for non-isotopic DNA fingerprinting is presented. The method is based on digoxigenated oligonucleotides, which are specific for simple repetitive DNA sequences. The use of digoxigenin/ anti-digoxigenin detection avoids many drawbacks inherent in e.g. the biotin/streptavidin system which often causes a poor signal-to-background ratio. Synthesis and purification of digoxigenated oligonucleotides and their use in filter hybridization are described in detail. Hybridization patterns obtained with four different radioactively labeled oligonucleotides have been compared with those of the respective digoxigenated probes. When slightly less stringent hybridization conditions are applied for digoxigenated oligonucleotides than for those labeled with ³²P, the signal intensities are satisfying but additional minor bands occur as a result of the reduced strigency. With one explainable exception, these bands increase the information content of the fingerprint. In addition, hybridization of the digoxigenated (CAC)₅ probe has been performed in situ with human metaphase chromosomes. The hybridization patterns in many mitoses resemble R-bands.     

2′- and 3′- Biotin Conjugated Nucleoside Building Blocks: Synthesis of Biotinylated Oligonucleotides

Abstract

The vitamin biotin plays a significant role in biological assays based on its unusually high affinity [K_D=10^?15M] to streptavidin and avidin. This assay can be used for monitoring cellular trafficking of antisense oligonucleotides using hiotin conjugation. In addition to the above diagnostic application, biotin conjugation to macromolecules could be used as a vitamin-mediated delivery system for macromolecules into cells. Complexation of avidin to hiotin-oligonucleotides (phosphodiesters or PNA) have been used to enhance the uptake of oligonucleotides¹. Appropiiate placement of biotin in oligonucleotides could also provide increased nuclease resistance.     

Synthesis and Mass Spectrometry Analysis of Oligonucleotides Bearing 5-Formyl-2′-Deoxyurldlne in Their Structure

Abstract

Two oligonucleotides containing FdU (1) have been synthesized. The use of the “Pac-amidites” for the natural nucleosides has allowed the incorporation of the oxidized thymine residue without protection of the aldehydic function. The oligonucleotide composition was confirmed by enzymatic digestion and electrospray mass spectrometry.     

Biological Activities of Oligonucleotides Linked to Poly(L-Lysine)

Abstract

We have developped a method to couple oligonucleotides to poly(L-lysine). This tool has been tested with anti m-RNA synthetic oligodeoxyribonucleotides and 2′-5′(A)_n and allows oligonucleotides to enter intact cells.     

B-Z Transition of Synthetic Oligonucleotides Containing Non-Z Forming Elements

Abstract

Ten oligonucleotides of the length 8–12 base pairs have been synthesized, which contain, in addition to the obligatory sequences CG/CG, sequences not favorable for the transition to the Z conformation (AT pairs, GG/CC or AA/TT sequences). Conformational transitions of these oligonucleotides in high concentrations of NaClO₄ in the absence and in the presence of Ni²⁺ were investigated using CD spectroscopy.

The B-Z transition is affected by the length and sequence of the oligonucleotide. Increasing the NaC1O₄ concentration alone the transition of only one of the oligonucleotides studied, (CGCGCGTGC ACGCGCG)₂, can be induced. Other oligonucleotides remain in the B conformation or only partial transition to the Z conformation can be observed.

Most other oligonucleotides can be converted into the Z conformation at intermediate concentrations of NaC1O₄ (2.0?3.2 M) by an addition of Ni²⁺ ions. In some cases, however, Ni²⁺ can destabilize the double stranded structure of the sample. We have studied the effect of the presence of A.T pairs in the G.C containing oligonucleotides and the effect of the presence of pu-pu/pyr-pyr sequences. The presence of the latter sequences in the Z form implicates the formation of a Z-Z′ junction which makes the transition quite difficult. Despite the fact that some oligonucleotides contained several structural elements not favorable for the transition, we did not find any sequence which would completely block the ability of the oligonucleotide to adopt the Z conformation.     

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.