4,5-BIS(ETHOXYCARBONYL)-[1,3]DIOXOLAN-2-YL AS A NEW ORTHOESTER-TYPE PROTECTING GROUP FOR THE 2′-HYDROXYL FUNCTION IN THE CHEMICAL SYNTHESIS OF RNA

We wish to report 4,5-bis(ethoxycarbonyl)-[1,3]dioxolan-2-yl as a new protecting for the 2′-hydroxyl function. Our cyclic orthoester-type group is compatible with the DMTr strategy for oligonucleotide synthesis. This group was introduced to the 2′-hydroxyl group of appropriately protected nucleoside derivatives in good yields under mild acidic conditions. Post-synthetic conversion of the moiety of this protecting group with an amine resulted in formation of a new amide moiety that is more stable to acid deprotection in aqueous solution, but it can still be easily removed by treatment with acids in organic solvents. In this article, we also describe the stability of not only the original and modified protecting groups but also internucleotidic phosphate linkages of protected RNA intermediates under deprotection conditions.     

Synthesis of RNA oligomer using 9-fluorenylmethoxycarbonyl (Fmoc) group for 5'-hydroxyl protection

An approach using a new combination of protecting groups in RNA oligomer synthesis is proposed, in which 5'-hydroxyl group of ribose moiety is temporarily protected with the alkaline labile 9-fluorenylmethoxycarbonyl (Fmoc) group and the 2'-hydroxyl group is protected with the acid labile 1-ethoxyethyl (EE) group. The adoption of this method presented great selectivity in removing the 5'-hydroxyl protecting group and facilitated the RNA oligomer synthesis. A RNA pentamer was synthesized by the phosphotriester method in solution.     

Selective 2''-benzoylation at the cis 2'',3''-diols of protected ribonucleosides. New solid phase synthesis of RNA and DNA-RNA mixtures.

5'-0-(Dimethoxytrityl)-2'-0-(benzoyl or 3,4,5-trimethoxybenzoyl)-base protected ribonucleosides have been prepared by selective benzoylation of the 2'-hydroxyl group. The isomerization of the 2'-benzoates to the 3'-benzoates was studied. The protected ribonucleosides have been converted to either methylphosphochloridites or methylphosphoamidites and used to synthesize oligoribonucleotides on silica gel solid support. The synthetic RNA were deprotected and isolated using conditions that minimize internucleotide cleavage. The use of 2'-benzoates as protecting groups for ribonucleosides has made it possible to easily prepare and isolate mixtures of DNA and RNA.     

Study on reactivity and protection of the alpha-hydroxyphosphonate moiety in 5'-nucleotide analogues: formation of the 3'-O-P-C(OH)-C4' internucleotide linkage

The recently described epimeric nucleosidyl-5'-C-phosphonates (alpha-hydroxyphosphonates) represent novel nucleotide analogues that can be incorporated into chimeric oligonucleotides by the phosphotriester condensation method. In order to prepare suitable protected monomer(s) we have studied condensation reaction between protected 2'-deoxythymidine and 2'-deoxythymidinyl-5'-C-phosphonate, both as model compounds, in dependence on the nature of the 5'-hydroxyl protecting group. We have found that the O-acetyl group is unstable in the presence of TPSCl or MSNT used as condensing agents for activation of the phosphorus moiety. This instability negatively influences the scope of the condensation process. On the other hand, introduction of the O-methoxycarbonyl group gave excellent results. The O-methoxycarbonyl group does not participate in the condensation process, and its quantitative introduction into the nucleotide analo gues is accomplished using a novel acylating agent, methoxycarbonyl tetrazole.     

2'(3')-O-glycyl oligoribonucleotides with sequences of the 3'-terminus of glycyl-tRNA: chemical synthesis and properties in partial reactions of protein biosynthesis

Specific syntheses of 2'(3')-O-aminoacyl oligoribonucleotides C-C-A-Gly (12), C-C-A(AcGly) (7), U-C-C-A-Gly (17), and C-U-C-C-A-Gly (19), which are the 3'-terminal sequences of Escherichia coli Gly-tRNA (or AcGly-tRNA, respectively) are described. Compounds 12, 17, and 19 were synthesized by employing the benzotriazolyl phosphotriester approach with the following protection groups on the components: benzoyl for the heterocyclic amino groups, 2-chlorophenyl group for internucleotide phosphate protection, dimethoxytrityl and levulinoyl groups for blocking of the 5'-hydroxyl, methoxytetrahydropyranyl group for protection of the 2'-hydroxyl functions, and N-(benzyloxycarbonyl)orthoglycinate as the masked aminoacyl group simultaneously protecting the 2',3'-cis diol group of the 3'-terminal adenosine moiety. The fully protected tri-, tetra-, and pentanucleotides were obtained via 5'-extension of di- and trinucleotide blocks after prior selective removal of the 5'-O-levulinoyl group with hydrazine. The blocked derivatives 11, 16, and 18 were totally deprotected by reactions with NH4OH, H+, and H2/Pd to yield the target compounds 12, 17, and 19 in good yields. C-C-A(AcGly) (7) was synthesized according to a stepwise procedure via activation of preformed diesters with (mesitylenesulfonyl)tetrazole. C-C-A-Gly (12), U-C-C-A-Gly (17), and C-U-C-C-A-Gly (19) were all acceptor substrates in the peptidyltransferase reaction with the Ac-Phe-tRNA-70S ribosome-poly(U) system. All three models also promoted EF-Tu-70S ribosome GTP hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

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 general and efficient approach for the construction of RNA oligonucleotides containing a 5'-phosphorothiolate linkage

Oligoribonucleotides containing a 5'-phosphorothiolate linkage have provided effective tools to study the mechanisms of RNA catalysis, allowing resolution of kinetic ambiguity associated with mechanistic dissection and providing a strategy to establish linkage between catalysis and specific functional groups. However, challenges associated with their synthesis have limited wider application of these modified nucleic acids. Here, we describe a general semisynthetic strategy to obtain these oligoribonucleotides reliably and relatively efficiently. The approach begins with the chemical synthesis of an RNA dinucleotide containing the 5'-phosphorothiolate linkage, with the adjacent 2'-hydroxyl group protected as the photolabile 2'-O-o-nitrobenzyl or 2'-O-α-methyl-o-nitrobenzyl derivative. Enzymatic ligation of the 2'-protected dinucleotide to transcribed or chemically synthesized 5' and 3' flanking RNAs yields the full-length oligoribonucleotide. The photolabile protecting group increases the chemical stability of these highly activated oligoribonucleotides during synthesis and long-term storage but is easily removed with UV irradiation under neutral conditions, allowing immediate use of the modified RNA in biochemical experiments.     

Diastereomeric process control in the synthesis of 2'-O-(2-methoxyethyl) oligoribonucleotide phosphorothioates as antisense drugs

Coupling of 2'-O-methoxyethylsubstituted nucleoside phosphoramidites to 5'-hydroxyl group of a nucleoside or nucleotide on solid support is under stereochemical process control and is independent of scale, concentration, synthesizer, ratio of amidite diastereomers, solid support etc. However, activators and phosphate protecting groups do play a role in influencing the ratio of phosphorothioate diesters obtained by sulfurization of phosphite triesters.     

Oligoribonucleotide synthesis by use of the 2-(methylthio)-phenylthiomethyl (MPTM) group

The 2-(methylthio)phenylthiomethyl (MPTM) group was developed as a new type of 2'-hydroxyl protecting group in oligoribonucleotide synthesis. The building monomer units of uridine and cytidine for the phosphotriester approach were synthesized from 2'-O-(1,3-benzodithiol-2-yl)-3',5'-O- (1,1,3,3-tetraisopropyldisiloxan-1,3-diyl)uridine and successfully utilized for the synthesis of CpUpG.     

Application of solid-phase chemistry for the synthesis of 3'-fluoro-3'-deoxythymidine

Reported solution-phase methods for the synthesis of 3'-fluoro-3'-deoxythymidine (FLT) are cumbersome, require purification of intermediates, and include several protecting/deprotecting steps. To circumvent these problems, a solid-phase strategy was designed for the synthesis of FLT. Thymidine was immobilized on trityl resin via the 5'-hydroxyl group. The subsequent mesylation of the free 3'-hydroxyl group in the presence of methanesulfonyl chloride afforded the polymer-bound 3'-O-mesylthymidine. Nucleophilic substitution of the mesyl moiety by hydroxyl group in the presence of sodium hydroxide produced the polymer-bound threothymidine. Fluorination with diethylaminosulfur trifluoride followed by acidic cleavage of the polymer-bound FLT in the presence of trifluoroacetic acid afforded FLT.     

Improved synthesis of trinucleotide phosphoramidites and generation of randomized oligonucleotide libraries

A new method to produce a set of 20 high quality trinucleotide phosphoramidites on a 5-10 g scale each was developed. The procedure starts with condensation reactions of P-components with N-acyl nucleosides, bearing the 3 '-hydroxyl function protected with 2-azidomethylbenzoyl, to give fully protected dinucleoside phosphates 13. Upon cleavage of dimethoxytrityl group from 13, dinucleoside phosphates 16 are initially transformed into trinucleoside diphosphates 19 and then the 2-azidomethylbenzoyl is selectively removed under neutral conditions to generate trinucleoside diphosphates 5 in excellent yield. Subsequent 3 '-phosphitylation affords target trinucleotide phosphoramidites 7. When mutagenic oligonucleotides are synthesized employing mixtures of building blocks 7 as well as following the new synthetic protocol, representative oligonucleotide libraries are generated in good yields.     

Solid-phase synthesis of H-Phe-Tyr-(pATAT)-NH2: a nucleopeptide fragment from the nucleoprotein of bacteriophage phi X174.

The preparation of the nucleopeptide H-Phe-Tyr-(pATAT)-NH2 could be realized via a solid phase phosphitetriester approach and by using the protected protecting group 2-(tert-butyldiphenylsilyoxymethyl)-benzoyl for the masking of the N6-amino function of deoxyadenosine. The latter protecting group can be removed under mild conditions with fluoride ion.     

Synthesis of L-3'-hydroxymethylribonucleosides

The target compounds were synthesized via the key intermediate carbohydrate 8, which was synthesized by first selectively protecting the 1'- and 2'-hydroxyl groups followed by selective tosylation of the 5'-hydroxyl group to obtain compound 3. The tosyl moiety was then replaced by a benzyl either to obtain 4. Compound 4 underwent Dess-Martin oxidation to afford the ketone 5. Compound 5 was subjected to Wittig olefination to afford the alkene 6 followed by regioselective hydroboration to obtain 7. Compound 7 was fully acetylated using acetic acid, acetic anhydride and sulfuric acid to obtain the key intermediate 8.     

A new linkage for solid phase synthesis of oligodeoxyribonucleotides.

An aryl diisocyanate has been used to attach an appropriately protected 2'-deoxyribonucleoside bearing a free 3'-hydroxyl group, to a long chain alkylamine controlled pore glass support via a urethane moiety, in a simple two step procedure. This obviates the need for the preparation and short column chromatographic purification of the 2'-deoxyribonucleoside-3'-O-succinates required for preparation of the widely used succinyl linked supports. The greater stability of the urethane bond compared to an ester bond led to substantially higher yields of oligodeoxyribonucleotides prepared by the solid phase phosphotriester method. More than twenty oligodeoxyribonucleotides have already been synthesized on the glass support bearing the new linkage.     

A solution to the problem of 3'----2'-O-phosphoryl migration in oligoribonucleotide synthesis

Oligoribonucleotides were obtained without contamination of 2'-5' linked regioisomers by means of polymer support when Thp group was employed as 2'-hydroxyl protecting group.     

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.     

Synthesis of branched ribonucleotides

A synthetic strategy for branched ribonucleotides, which have recently been discovered, was described. A fully protected adenosine unit (5) having the tris (4,5-dichlorophthalimido)trityl (CPTr), bis(anilino)phosphoryl (BAP), and bis(phenylthio)phosphoryl (BPTP) groups as the 5'-, 2'-, and 3'-hydroxyl protecting groups, respectively, was synthesized from adenosine by a five-step reaction involving a new method for the 2'-O-phosphorylation by the use of hexaethylphosphorous triamide. The selective deprotection of appropriate protecting groups from 5 followed by stepwise condensation with two different ribonucleoside derivatives (7 and 10) gave a protected branched ribonucleotide (11) via a 3'-phosphorylated 2'-5' dinucleotide (8). Deprotection of 11 and 8 gave a branched trinucleotide (12) and 3'-phosphorylated dinucleotide (13).     

Synthesis of oligonucleotides using the 2-(levulinyloxymethyl)-5-nitrobenzoyl group for the 5'-position of nucleoside 3'-H-phosphonate and -H-phosphonothioate derivatives.

Synthetic studies on phosphodiester, phosphorothioate, and phosphorodithioate-linked oligonucleotides in terms of 2-(levulinyloxymethyl)-5-nitrobenzoyl (LMNBz) group as the base-labile protecting group for the 5'-hydroxyl groups of nucleoside 3'-H-phosphonate and -H-phosphonothioate derivatives, are described.     

Synthesis of ‘head‐to‐tail’ cyclized peptides on solid support using a chelating amide as new orthogonal protecting group

The synthesis of ‘head‐to‐tail’ cyclized peptides requires orthogonal protecting groups. Herein, we report on the introduction of bis(2‐pyridylmethyl)amine (Bpa) as a new protecting group for carboxylic functions in SPPS. The synthesis of the Bpa‐protected aspartic acid was straightforward, and its utility was investigated under standard peptide synthesis conditions. The new protecting group was cleaved in a very mild way using Cu(OAc)₂ and 2‐(trimethylsilyl)ethanol as nucleophile in a microwave oven without affecting other groups. Hence, the new group is ideally suited for the synthesis of ‘head‐to‐tail’ cyclic peptides, as demonstrated for a cyclic pentapeptide and cyclic hexapeptides. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Rapid DNA synthesis by use of a new functionalized 5'-hydroxyl protecting group in the phosphorothio ester approach

This paper describes a new protecting group of the 5'-hydroxyl group, 3-(imidazol-l-ylmethyl)-4,4'-dimethoxytrityl (IDTr), which serves as potential catalytic site for internucleotide bond formation.