The Chemical Synthesis of Oligoribonucleotides with Selectively Placed 2′-O-Phosphates

Abstract

A phosphoramidite, solid support method for the chemical synthesis of oligoribonucleotides containing 2′-O-phosphate at a selected position is presented. Synthesis of these oligoribonucleotides is based on uridine- and adenosine-(2′-O-phosphate)-3′-phosphoramidites, and a new condition for removal of 2′-O-phosphate protecting groups, which does not cleave internucleotide bonds. The structure of oligoribonucleotides with 2′-O-phosphate has been proven by enzymatic digestions and dephosphorylation by yeast 2′-phosphotransferase.     

Synthesis of Adenosine,Guanosine and Cytidine Monomer Building Units Bearing the [[2-(Methylthio)-phenyl]thio]methyl (MPTM) Group as The 2′-Hydroxyl Protecting Group

Abstract

A new 2′-hydroxyl protecting group, [[2-(methylthio)phenyl]thio]methyl (MPTM), was introduced into the 2′-position of adenosine, guanosine and cytidine building units required for the synthesis of oligoribonucleotides.     

The Protection of The 2′-Hydroxyl Function in Oligoribonucleotide Synthesis

Abstract

A new, easily accessible and achiral 2′-ketal protective group has been designed for the use in the chemical synthesis of oligoribonucleotides; the proposed 2′-ketal group⁽¹⁾ has the additional advantage that it could be easily functionalized to the diamide (6) with aq. ammonia at the penultimate step of deblocking of oligoribonucleotides which makes it more acid-labile than the parent 2′-ketal group during the final acid-promoted deprotection step.     

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.     

Synthesis and Analytical Characterization of RNA-Polyethylene Glycol Conjugates

Abstract

Polyethylene glycols with degrees of polymerization from 5 to more than 100 were incorporated into synthetic oligoribonucleotides by automated solid phase synthesis at 3′-terminal, 5′-terminal and internal positions. The conjugates were characterized by chromatographic, electrophoretic and mass-spectrometric methods. The influence of coupling site, polymer size and number of coupled polymers per oligonucleotide on the molecular properties of the conjugates is investigated.     

An Improved Method for The Preparation Of The Phosphoramidites Of Modified 2′-Deoxynucleotides: Incorporation Of 8-Oxo-2′-Deoxy-7H-Guanosine Into Synthetic Oligomers

Abstract

3′ -O-(Diisopropylamino-2-cyanoethoxyphosphinyl)-5′ -0-(4, 4′ -dimethoxytrityl)-N²-isobutyryl-8-oxo-2′-deoxy-7H-guanosine was synthesized and used for the introduction of an 8?0×0?2′-deoxy-7H-guanosine residue into a DNA oligomer by means of automated synthesis. A modification of the preparation of the phosphoramidite markedly improves the coupling efficiency in the oligomer synthesis in this and several other cases that were tested.     

Synthesis of Oligoribonucleotides by the H-Phosphonate Approach Using Base Labile 2′-O-Protecting Groups. V. Recent Progress in Development of the Method

Abstract

A simple and inexpensive method for the preparation of oligoribonucleotides using N-isopropoxyacety(phenoxyacetyl)-2′-O-(2-chlorobenzoyl)-5′-O-dimethoxytritylnucleoside H-phosphonate building blocks has been developed.     

Some Aspects of the Chemical Synthesis of Oligoribonucleotides

Abstract

The present position regarding the protection of the 2′- and 5′-hydroxy functions in the chemical synthesis of oligoribonucleotides is discussed.     

How to Synthesize A tRNA?

Abstract

New blocking group combinations have been investigated to achieve an automated synthesis of a tRNA and structural analogs on solid-support. The use of the 4-methoxytetrahydropyranyl group for 2′-OH-protection and the dansylethoxycarbonyl group for the 5′-OH position shows in the phosphoramidite approach good results. In the arabino series the 2-(4-nitrophenyl)ethoxycarbonyl group is a perfect 2′-OH blocking group which can be combined with the dimethoxytrityl residue in the usual manner to give high yields and pure materials.     

Synthesis of 8-Bromo- and 8-Azido-2′-deoxyadenosine-5′-O-(1-thiotriphosphate)

Abstract

Treatment of 3′-O-methoxyacetylated 8-bromo-2′-deoxyadenosine (5), with a twofold excess of salicyl phosphorochloridite (6), and subsequent reaction with bis(tri-n-butylammonium) pyrophosphate and oxidation with sulfur followed by removal of the protecting group gives predominantly 8-bromo-2′-deoxyadenosine-5′-O-(1-thiotriphosphate) (7), and minor amounts of the corresponding brominated monothiophosphate. Alternatively, the photoreactive dATP analog 8-azido-2′-deoxyadenosine-5′-O-(1-thiotriphosphate) (11), is obtained by phosphorylation of unprotected 8-azido-2′-deoxyadenosine (9) with a 1.8 molar equivalent excess of thiophosphoryl chloride and bis(tri-n-butylammonium) pyrophosphate. A protection of the nucleobase 6-amino group is not required. The photoaffinity labeling reagent 11, was characterized by ³¹P-NMR and ion-spray mass spectroscopy and its photolysis upon long wavelength UV irradiation was studied. Both α-thioderivatives of 2′-deoxyadenosine triphosphates can be incorporated into plasmid DNA by T7 DNA polymerase. Thus, they can be used for interference studies of protein binding and for cross-linking with amino acids in protein-nucleic acid-complexes.     

Synthesis and Evaluation of Oligonucleotides of High Purity

Abstract

We have developed and evaluated methods for the production of highly pure oligonucleotides.

Presently the solid phase synthesis in an automated DNA synthesiser applying the phosphoramidite chemistry can be regarded as a standard. During the synthesis several undesirable by-products arise:

- incomplete coupling (1%) leads to 5′-truncated sequences. These sequences are acetylated at their 5′-hydroxyl group to prevent further elongation in subsequent coupling steps, but this “capping step” is incomplete, the capping-yield is 90%, leading to accumulation of sequences of the length n-1 with internal deletions.

- the glycosidic bond to N-protected purines, especially adenine, is susceptible to acid leading to depurination and subsequently to strand scission during alkaline deprotection of the oligonucleotide. This gives rise to 3′- and to 5′-truncated sequences. The 3′-truncated sequences will not be removed by standard Rp HPLC as they are tritylated.

- the reactions involved in synthesis and deprotection may cause base modifications (full length product with damaged bases).

- insufficient deprotection procedures may result in incomplete removal of protecting groups, especially from the bases (full length products with altered bases).

We have set up two different schemes (Fig. 1 and Fig. 2) for synthesis and purification, which should provide highly pure oligonucleotides with the potential of adapting to large scale production:

- accumulation of n-1 sequences (failure of capping) will be avoided by a double capping procedure using phosphite in the first capping step and an acetic anhydride capping reagent in the second capping step, as described in the literature¹.

- 3′-truncated sequences are removed by different methqds in the two schemes. In scheme I (Fig. 1) the 3′-truncated sequences can be washed off, as the 3′-full length product still is anchored to the solid support after deprotection. In scheme II (Fig. 2) the 3′truncated sequences are digested by snake venom phosphodiesterase. The 3′-full length product is protected against digestion by a 3′ - 3′-inverted end. An oligo with a correct 3′-end is, in both schemes, eventually obtained by cleaving with RNase between the ribo unit and the requested DNA-sequence.

- 5′-truncated sequences are removed by Rp HPLC using the DMTr group of the last coupling step (trityl-on synthesis) as a hydrophobic tag.

Very labile protecting groups will be used to avoid problems with deprotection.     

The Zn2+ Complex of 1,4,7,10-Tetraazacyclododecane as an Artificial Nucleobase

{2-Deoxy-3-O-[2-cyanoethoxy(diisopropylamino)phosphino]-5-O-(4,4′-dimethoxytrityl)-α-D- erythro-pentofuranosyl}-N-{2-[4,7,10-tris(2,2,2-trifluoroacetyl)-1,4,7,10-tetraazacyclododecan-1- yl]ethyl}acetamide (1) was prepared and incorporated into a 2′-O-methyl oligoribonucleotide. The hybridization of this oligonucleotide with complementary 2′-O-methyl oligoribonucleotides incorporating one to five uracil bases opposite to the azacrown structure was studied in the absence and presence of Zn²⁺. Introduction of Zn²⁺ moderately stabilized the duplex with U-bulged targets.     

Synthesis of 2′-O-Photocaged Ribonucleoside Phosphoramidites

The chemical synthesis and incorporation of the phosphoramidite derivatives of 2?′-O-photocaged ribonucleosides (A, C, G and U) with o-nitrobenzyl, α-methyl-o-nitrobenzyl or 4,5-dimethoxy-2-nitrobenzyl group into oligoribonucleotides are described. The efficiency of UV irradiated uncaging of these 2′-O-photocaged oligoribonucleotides was found in the order of α-methyl-o-nitrobenzyl < 4,5-dimethoxy-2-nitrobenzyl < 2′-O-o-nitrobenzyl.     

Partial Protection of Carbohydrate Derivatives. Part 24.1 Synthesis of Adenylyl-(2′–5′)-Adenylyl-(2′–5′)-Adenosine Using a 3′-O-(Tetrahydropyran-2-YL) Adenosine Derivative

Abstract

The synthesis of the title compound was performed using a 3′-O-(tetrahydropyran-2-yl) adenosine derivative as the starting material, i.e., a coupling reaction of triethylammonium N ⁶-benzoyl-5′-O-dimethoxytrityl-3′-O-(tetrahydropyran-2-yl) adenosine 2′-(4-chlorophenyl)phosphate with N ⁶-benzoyl-2′,3′-di-O-benzoyladenosine, followed by a sequence of reactions, O-dedimethoxytritylation, a coupling reaction with the former triethylammonium salt, and complete deblocking of the resultant 2′, 5′-triadenylic acid derivative.     

Extending the applicability of carboxyfluorescein in solid-phase synthesis

Optimized coupling protocols are presented for the efficient and automated generation of carboxyfluorescein-labeled peptides. Side products, generated when applying earlier protocols for the in situ activation of carboxyfluorescein, were eliminated by a simple procedure, yielding highly pure fluorescent peptides and minimizing postsynthesis workup. For the cost-efficient labeling of large compound collections, coupling protocols were developed reducing the amount of coupling reagent and fluorophore. To enable further chemical derivatization of carboxyfluorescein-labeled peptides in solid-phase synthesis, the on-resin introduction of the trityl group was devised as a protecting group strategy for carboxyfluorescein. This protecting group strategy was exploited for the synthesis of peptides labeled with two different fluorescent dyes, essential tools for bioanalytical applications based on fluorescence resonance energy transfer (FRET). Tritylation and optimized labeling conditions led to the development of a fluorescein-preloaded resin for the automated synthesis of fluorescein-labeled compound collections with uniform labeling yields.     

Methoxymethyl and (p-nitrobenzyloxy)methyl groups in synthesis of oligoribunucleotides by the phosphotriester method

An efficient method to synthesize monomer ribonucleotide synthons containing 2′-O-methoxymethyl and 2′-O-(p-nitrobenzyloxy)methyl groups is developed. These synthons are applied to the oligonucleotide phosphotriester method using O-nucleophilic intramolecular catalysis at the stage of the internucleotide bond formation. The former synthons may be used for the automatic synthesis of 2′-modified oligonucleotides; the latter synthons made be used for the synthesis of phosphotriester oligoribonucleotides in high yields.     

Purification of 5′-O-Trityl-on Oligoribonucleotides. Investigation of Phosphate Migration During Purification and Detritylation.

Abstract

Synthetic oligoribonucleotides (RNA) are efficiently prepared with 2′-O-tert-butyldimethylsilyl nucleoside 3′-O-phosphoramidites with labile base-protection; A_dmf or A_Pac, G_dmf, C_ibu, U. After cleavage from the polystyrene support, the exocyclic amine protecting groups are removed with conc. NH₄OH: ethanol/3:1 by heating at 55°C for 3–5 h. The 2′-O- silyl protecting groups are removed with tetra-n-butylammonium fluoride in THF or more conveniently with neat triethylamine trihydrofluoride. To gain the advantages of increased capacity on reverse phase HPLC and the convenience of cartridge based purification (OPC, Oligonucleotide Purification Cartridge), the 5′ trityl was left on the RNA as the final protecting group to be removed. The mild conditions which are effective for trityl removal are shown to preserve 3′-5′ phosphate linkage integrity in RNA. The absence of phosphate migration is demonstrated by model studies, utilizing N⁴ -isobutyryl-5′-O-DMT-3′-O-TBDMS-2′-O-(2-cyanoethyl-N,N-diisopropylphosphoramidite) as a control monomer and digestion by 3′-5′ selective P1 nuclease and alkaline phosphatase and HPLC analysis. Oligoribonucleotides were analyzed by Microgel capillary electrophoresis, anion-exchange HPLC, and the enzymatic digest/HPLC method.

     

Dimethoxytrityl Removal in Organic Medium: Efficient Oligonucleotide Synthesis Without Chlorinated Solvents

Abstract

Oligonucleotides are finding widespread utility in various applications in diagnostics and molecular biology and as therapeutic agents. In standard synthesis of such oligonucleotides through phosphoramidite coupling, removal of the typical acid-labile 4,4′-dimethoxytrityl 5′-protecting group (DMTr), from the support-bound oligonucleotide plays a crucial role in each synthesis cycle in achieving high product yield and oligonucleotide quality. Although several reagents have been developed for this purpose, many have limited applicability to automated oligonucleotide synthesis on solid supports. The most commonly used reagents today are dilute solutions (2–15%) of an organic acid, typically trichloroacetic acid (TCA, pKa 0.8) or dichloroacetic acid (DCA, pKa 1.5) in dichloromethane. The high volatility (boiling point 40 °C) of dichloromethane and its high toxicity and carcinogenicity pose a hazard for personnel and the environment. In addition, as oligonucleotide synthesizers are now available to allow syntheses of up to 0.5 mole scale, the quantities of chlorinated waste generated have become quite large. In this context we became interested in replacing dichloromethane as deblocking reagent solvent with a less harmful solvent while preserving product yield and quality. We now report that it is not necessary to use halogenated solvents such as dichloromethane in the deblocking step of automated oligonucleotide synthesis in order to obtain high yields of high quality oligonucleotide product.     

Synthesis of RNA Fragments Using the H-Phosphonate Method and 2′-(2′-Chlorobenzoyl) Protection

Abstract

The performance of 2′-(2-chlorobenzoyl) protected ribonucleoside H-phosphonates in the synthesis of oligoribonucleotides has been studied.     

Chemical synthesis of biologically active oligoribonucleotides using beta-cyanoethyl protected ribonucleoside phosphoramidites.

The preparation of fully protected diisopropylamino-beta-cyanoethyl ribonucleoside phosphoramidites with regioisomeric purity greater than 99.95% is described. It is demonstrated that the combination of standard DNA protecting groups, 5'-O-DMT, N-Bz (Ade and Cyt), N-iBu (Gua), beta-cyanoethyl for phosphate, in conjunction with TBDMS for 2'-hydroxyl protection, constitutes a reliable method for the preparation of fully active RNA. Average stepwise coupling yields in excess of 99% were achieved with these synthons on standard DNA synthesizers. Two steps completely deprotect the oligoribonucleotide and workup is reduced to a fifteen minute procedure. Further, it is shown that the deprotected oligoribonucleotides are free from 5'-2' linkages. This methodology was applied to the chemical synthesis of a 24-mer microhelix, a 35-mer minihelix and two halves of a catalytic 'Hammerhead Ribozyme'. These oligoribonucleotides were directly compared in two distinct biochemical assays with enzymatically (T7 RNA polymerase) prepared oligoribonucleotides and shown to possess equal or better activity.