Protective groups in the chemical synthesis of oligoribonucleotides

The methods of chemical oligoribonucleotide synthesis and the protective groups used are reviewed. The latest data on the protection of 2′-OH functions of nucleotide derivatives used as monomers for the RNA synthesis are comprehensively discussed.     

The chemical synthesis of oligoribonucleotides with selectively placed 2'-O-phosphates

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.     

Prevention of chain cleavage in the chemical synthesis of 2''-silylated oligoribonucleotides.

Strong aqueous ammonium hydroxide used to remove N-acyl protecting groups from synthetic oligoribonucleotides causes removal of some alkylsilyl protecting groups from 2'-hydroxyls and leads to chain cleavage. This problem is most severe when 30% ammonium hydroxide is used and substantially reduced but still detectable when 3:1 ammonium hydroxide-ethanol is used. We have virtually eliminated this unwanted cleavage by incorporating the labile phenoxyacetyl amino protecting group on adenosine and guanosine. The N-benzoyl protecting group remains adequate for cytidine nucleosides. Synthetic oligoribonucleotides containing these N-acylated nucleosides and 2'-t-butyldimethylsilyl or 2'-triisopropylsilyl protecting groups can be deacylated by room temperature treatment in saturated anhydrous methanolic ammonia (8-12 h) without causing any detectable chain cleavage.     

The nonenzymatic hydrolysis of oligoribonucleotides. VII. Structural elements affecting hydrolysis

Several elements of oligoribonucleotide structure are important for efficient hydrolysis. We have found that the following factors influence oligoribonucleotide hydrolysis: (i) single-stranded structure of RNA flanking the scissile phosphodiester bond, (ii) the substituent on atom C-5 of the uridine adjacent to the cleaved internucleotide bond, (iii) the position of the scissile UA phosphodiester bond within a hairpin loop, (iv) the concentration of formamide, urea, ethanol and sodium chloride.     

The prebiotic synthesis and catalytic role of imidazoles and other condensing agents

In the past decade significant advances have been made in the synthesis of oligonucleotides and other polymers by means of imidazoles and other condensing agents. In spite of the current knowledge of the chemistry of imidazoles and their importance as prebiotic catalysts, their formation under primitive earth conditions has not been properly demonstrated. We have now been able to synthesize imidazole as well as its 2-methyl and 4-methyl derivatives under plausible prebiotic conditions. One method utilizes an aldehyde (formaldehyde or acetaldehyde), glyoxal and ammonia as the starting materials for the formation of imidazole and 2-methylimidazole. The other method uses a carbohydrate and ammonia as the key reagents for the synthesis of 4-methylimidazole. The importance of imidazole and related compounds (e.g., cyanamide) in the synthesis of oligonucleotides has been studied by us as well as others. Apparently the charge relay group (-N-C-N-) present in imidazoles, carbodiimides, cyanamide, or the histidine and arginine of enzyme active centers is essential for the synthesis of phosphodiester and pyrophosphate bonds.     

The prebiotic synthesis and catalytic role of imidazoles and other condensing agents

In the past decade significant advances have been made in the synthesis of oligonucleotides and other polymers by means of imidazoles and other condensing agents. In spite of the current knowledge of the chemistry of imidazoles and their importance as prebiotic catalysts, their formation under primitive earth conditions has not been properly demonstrated. We have now been able to synthesize imidazole as well as its 2-methyl and 4-methyl derivatives under plausible prebiotic conditions. One method utilizes an aldehyde (formaldehyde or acetaldehyde), glyoxal and ammonia as the starting materials for the formation of imidazole and 2-methylimidazole. The other method uses a carbohydrate and ammonia as the key reagents for the synthesis of 4-methylimidazole. The importance of imidazole and related compounds (e.g., cyanamide) in the synthesis of oligonucleotides has been studied by us as well as others. Apparently the charge relay group (–N–C–N–) present in imidazoles, carbodiimides, cyanamide, or the histidine and arginine of enzyme active centers is essential for the synthesis of phosphodiester and pyrophosphate bonds.     

A new 2'-hydroxyl protecting group for the automated synthesis of oligoribonucleotides.

We have developed a new type of 2'-hydroxyl protecting group for the automated machine synthesis of RNA oligomers: a 2-hydroxyisophthalate formaldehyde acetal (HIFA). The unique feature of this protecting group is that, as the bis ester, it is relatively stable to the acidic conditions that are used for repeated removal of dimethoxytrityl groups during chain elongation, but the final deprotection step in alkali, which cleaves the chain from the support and removes the base and phosphate protecting groups, converts it to the bis carboxylate and this can be removed relatively rapidly by treatment with mild acid. Conversion of the bis ester to the bis carboxylic acid increases the rate of acid-catalyzed hydrolysis of the acetal by 42-fold at pH 1, and, possibly, by 1320-fold at pH 3. The bis ester is 112 times more stable than the 1-(2-fluorophenyl)-4-methoxypiperidin-4-yl group (Fpmp) towards hydrolysis at pH 1, while the bis acid is only 2.35 times more stable than Fpmp at pH 3. In synthesis of the dimers UpU and UpG, with a coupling time of 5 min, the dimethoxytrityl cation assay indicated coupling yields of > 98%.     

A new approach to the synthesis of branched and branched cyclic oligoribonucleotides.

The six-step synthesis of the di-triethylammonium salt of 5[prime]-O -trityl-6-N-pivaloyladenosine-2[prime]-(H -phosphonate)-3'-[(2-chlorophenyl) phosphate]9 from 3', 5'- O -(1,1,3,3-tetraisopropyldisiloxan-1,3-diyl)-6-N-pivaloyla denosine1in 68% overall yield is described. Compound9is converted into a branched pentaribonucleoside tetraphosphate 24 and a branched cyclic pentaribonucleotide ('lariat') 25 by solution phase triester chemistry involving both H-phosphonate and conventional phosphotriester coupling reactions. The monomeric building block 9 is proposed as a universal synthon for the preparation of branched and branched cyclic oligoribonucleotides derived from adenosine.     

Conformational analysis of arabinonucleosides and nucleotides. A comparison with the ribonucleosides and nucleotides.

Conformations of arabino nucleosides and nucleotides have been analyzed by semiempirical energy calculations. It is found that the change in the configuration of the O(2')-hydroxyl group in arabinoses compared to riboses exerts significant influence on the conformational priorities of the glycosyl and the exocyclic C(4')-C(5') bond torsions. While the anti conformations for the bases are preferred, the anti in equilibrium or formed from syn interconversion is considerably hampered compared to ribosides due to large energy barrier. Further the preferred anti glycosyl torsions are shifted to higher values for C(3')-endo puckers and in ribosides. While the gauche+ conformation around the C(4')-C(5') bond is favored for C(3')-endo arabinosides, it is strongly stabilized for C(2')-endo arabinosides only in the presence of the intrasugar hydrogen bond O(2')-H ... O(5'). The net attractive electrostatic interactions between the phosphate and the base stabilizes the preferred conformations of 5'-arabinonucleotides also.     

Some aspects of oligoribonucleotides synthesis via the H-phosphonate approach.

This review gives a short account of selected aspects of oligoribonucleotide synthesis via the H-phosphonate method. It includes: (i) recent methods for the preparation of suitably protected ribonucleoside 3'-H-phosphonates (the phosphonylation step), (ii) some chemical and stereochemical features of the formation of H-phosphonate internucleosidic linkages, and (iii) stereoselective synthesis of oligoribonucleoside phosphorothioates using chemo-enzymatic approach.     

The H-phosphonate approach to the solution phase synthesis of linear and cyclic oligoribonucleotides.

The solution phase synthesis of the tetraribonucleoside triphosphate r(ApCpGpU) 18 and the corresponding cyclic tetraribonucleotide 19 is described. The synthetic methodology is based on 5'- O -(DMTr)-2'- O -(Fpmp)-ribonucleoside-3'- H -phosphonate building blocks 10. Coupling, which is rapid and quantitative, is effected with di-(2-chlorophenyl) phosphorochloridate 5 at -40 degreesC; it is followed by in situ treatment with 2-(4-methyl-phenyl)sulphanyl-1 H -isoindole-1,3(2 H )-dione 6b. The resulting sulphur transfer reaction also proceeds rapidly and quantitatively at -40 degreesC. The same coupling and sulphur transfer steps are used in the cyclization reaction, but a 5'- H -phosphonate intermediate 24 is involved. The final three-step unblocking process involves treatment with (i) E -2-nitrobenzaldoxime 7 and N 1, N 1, N 3, N 3-tetramethylguanidine (TMG) 8 in aceto-nitrile, (ii) concentrated aqueous ammonia at 50 degreesC and (iii) 0.5 mol/dm3sodium acetate buffer (pH 4.0) at 40 degreesC. The fully unblocked products 18 and 19 were characterized by NMR spectroscopy and by enzymatic digestion.     

Simplified methods for large scale enzymatic synthesis of oligoribonucleotides.

We report simplified methods for large scale enzymatic synthesis of oligoribonucleotides using polynucleotide phosphorylase. The main features of the method are use of RPC-5 chromatography, including chromatography at two pH values to deal with the problem of primer phosphorolysis, rapid dialysis for large scale desalting, simplified methods for enzyme removal, and high resolution 1H and 31P NMR for product identification and demonstration of purity. The capacity of the method is adequate to allow beginning with grams of material in the first polymerization step, so that product yields of several milligrams, sufficient for many physical studies, are possible after as many as three separate polymerization reactions.     

Design and synthesis of novel carbocyclic versions of 2'-spirocyclopropyl ribonucleosides as potent anti-HCV agents

The discovery of 2'-spirocyclopropyl-ribocytidine as a potent inhibitor of RNA synthesis by NS5B (IC(50) = 7.3 μM), the RNA polymerase encoded by hepatitis C virus (HCV), has led to the synthesis and biological evaluation of carbocyclic versions of 2'-spiropropyl-nucleosides from cyclopentenol 6. Spirocyclopropylation of enone 7 was completed by using (2-chloroethyl)-dimethylsulfonium iodide and potassium t-butoxide to form the desired intermediate 9a. The synthesized nucleoside analogues, 18, 19, 26, and 27, were assayed for their ability to inhibit HCV RNA replication in a subgenomic replicon Huh7 cell line. The synthesized cytosine nucleoside 19 showed moderate anti-HCV activity (IC(50) = 14.4 μM).     

A study of the efficiency and the problem of sulfonation of several condensing reagents and their mechanisms for the chemical synthesis of deoxyoligoribonucleotides.

The efficiencies and problems of sulfonation of several condensing reagents for deoxyoligoribonucleotide synthesis have been studied. While 2,4,6-triisopropylbenzenesulfonyl chloride (TPSC1) gave very low yield and slow rate of coupling, the new 1:3 mixture of TPSC1 and tetrazole afforded excellent yield and extremely fast rate of reaction with the lowest rate of sulfonation. The difference and advantages of this mixture over triisopropylbenzenesulfonyl tetrazole (TPSTe) and mesitylenesulfonyl tetrazole (MSTe) are discussed. Mechanisms for the coupling reactions with these condensing reagents to account for the difference in their rates and yields of coupling are discussed.     

Studies in prebiotic synthesis. VII

Summary The purine nucleosides (adenosine, guanosine, inosine, xanthosine) are formed when the corresponding purine bases and D-ribose are heated together in the presence of certain salts and minerals. The salts remaining after the evaporation of seawater are particularly effective in these syntheses. The relevance of these reactions for prebiological evolution is discussed.     

Use of new phosphonylating and coupling agents in the synthesis of oligodeoxyribonucleotides via the H-phosphonate approach.

New phosphonylating and coupling agents for the synthesis of oligodeoxyribonucleotides via H-phosphonate approach have been developed. Tris(1,1,1,3,3,3-hexafluoro-2-propyl) phosphite, prepared by the reaction of lithium salt of 1,1,1,3,3,3-hexafluoro-2-propoxide with PCl3, reacts with deoxyribonucleosides in the presence of a catalytic amount of triethylamine to produce in the high yield the corresponding deoxyribonucleoside 3'-H-phosphonate units. The use of a new coupling reagent, 1,3-dimethyl-2-chloro-imidazolinium chloride (DMCI) for the internucleotidic H-phosphonate bond formation via the H-phosphonate approach is also discussed in detail.     

The S,X-acetals in nucleoside chemistry. I. The synthesis of 2'- and 5'-O-methylthiomethyl ribonucleosides

Ribonucleoside 2'- and 5'-methylthiomethyl derivatives were synthesized from selectively protected nucleosides by the action of dimethyl sulfoxide-acetic anhydride-acetic acid mixture.