The “Speedy” Synthesis of Atom‐Specific 15N Imino/Amido‐Labeled RNA

Although numerous reports on the synthesis of atom‐specific ¹⁵N‐labeled nucleosides exist, fast and facile access to the corresponding phosphoramidites for RNA solid‐phase synthesis is still lacking. This situation represents a severe bottleneck for NMR spectroscopic investigations on functional RNAs. Here, we present optimized procedures to speed up the synthesis of ¹⁵N(1) adenosine and ¹⁵N(1) guanosine amidites, which are the much needed counterparts of the more straightforward‐to‐achieve ¹⁵N(3) uridine and ¹⁵N(3) cytidine amidites in order to tap full potential of ¹H/¹⁵N/¹⁵N‐COSY experiments for directly monitoring individual Watson–Crick base pairs in RNA. Demonstrated for two preQ₁ riboswitch systems, we exemplify a versatile concept for individual base‐pair labeling in the analysis of conformationally flexible RNAs when competing structures and conformational dynamics are encountered.     

Efficient Automated Solid‐Phase Synthesis of DNA and RNA 5′‐Triphosphates

A fast, high‐yielding and reliable method for the synthesis of DNA‐ and RNA 5′‐triphosphates is reported. After synthesizing DNA or RNA oligonucleotides by automated oligonucleotide synthesis, 5‐chloro‐saligenyl‐N,N‐diisopropylphosphoramidite was coupled to the 5′‐end. Oxidation of the formed 5′‐phosphite using the same oxidizing reagent used in standard oligonucleotide synthesis led to 5′‐cycloSal‐oligonucleotides. Reaction of the support‐bonded 5′‐cycloSal‐oligonucleotide with pyrophosphate yielded the corresponding 5′‐triphosphates. The 5′‐triphosphorylated DNA and RNA oligonucleotides were obtained after cleavage from the support in high purity and excellent yields. The whole reaction sequence was adapted to be used on a standard oligonucleotide synthesizer.     

Non‐Hydrolyzable RNA–Peptide Conjugates: A Powerful Advance in the Synthesis of Mimics for 3′‐Peptidyl tRNA Termini

Translation of specific small peptides on the ribosome can confer resistance to macrolide antibiotics. To reveal the molecular details of this and related phenomena, stable RNA–peptide conjugates that mimic peptidyl‐tRNA would be desirable, especially for ribosome structural biology. A flexible solid‐phase synthesis strategy now allows efficient access to these highly requested derivatives without restriction on the RNA and peptide sequences.

     

Synthesis of a DNA Promoter Segment Containing All Four Epigenetic Nucleosides: 5‐Methyl‐, 5‐Hydroxymethyl‐, 5‐Formyl‐, and 5‐Carboxy‐2′‐Deoxycytidine

A 5‐formyl‐2′‐deoxycytidine (fdC) phosphoramidite building block that enables the synthesis of fdC‐containing DNA with excellent purity and yield has been developed. In combination with phosphoramidites for 5‐methyl‐dC, 5‐hydroxymethyl‐dC, and carboxy‐dC, it was possible to prepare a segment of the OCT‐4 promoter that contains all four epigenetic bases. Because of the enormous interest in these new epigenetic bases, the ability to insert all four of them into DNA should be of great value for the scientific community.     

Liquid‐Phase Synthesis of 2′‐Methyl‐RNA on a Homostar Support through Organic‐Solvent Nanofiltration

Due to the discovery of RNAi, oligonucleotides (oligos) have re‐emerged as a major pharmaceutical target that may soon be required in ton quantities. However, it is questionable whether solid‐phase oligo synthesis (SPOS) methods can provide a scalable synthesis. Liquid‐phase oligo synthesis (LPOS) is intrinsically scalable and amenable to standard industrial batch synthesis techniques. However, most reported LPOS strategies rely upon at least one precipitation per chain extension cycle to separate the growing oligonucleotide from reaction debris. Precipitation can be difficult to develop and control on an industrial scale and, because many precipitations would be required to prepare a therapeutic oligonucleotide, we contend that this approach is not viable for large‐scale industrial preparation. We are developing an LPOS synthetic strategy for 2′‐methyl RNA phosphorothioate that is more amenable to standard batch production techniques, using organic solvent nanofiltration (OSN) as the critical scalable separation technology. We report the first LPOS‐OSN preparation of a 2′‐Me RNA phosphorothioate 9‐mer, using commercial phosphoramidite monomers, and monitoring all reactions by HPLC, ³¹P NMR spectroscopy and MS.     

The 2‐Cyano‐2,2‐dimethylethanimine‐N‐oxymethyl Group for the 2′‐Hydroxyl Protection of Ribonucleosides in the Solid‐Phase Synthesis of RNA Sequences

The reaction of 2‐cyano‐2‐methyl propanal with 2′‐O‐aminooxymethylribonucleosides leads to stable and yet reversible 2′‐O‐(2‐cyano‐2,2‐dimethylethanimine‐N‐oxymethyl)ribonucleosides. Following N‐protection of the nucleobases, 5′‐dimethoxytritylation and 3′‐phosphitylation, the resulting 2′‐protected ribonucleoside phosphoramidite monomers are employed in the solid‐phase synthesis of three chimeric RNA sequences, each differing in their ratios of purine/pyrimidine. When the activation of phosphoramidite monomers is performed in the presence of 5‐benzylthio‐1H‐tetrazole, coupling efficiencies averaging 99 % are obtained within 180 s. Upon completion of the RNA‐chain assemblies, removal of the nucleobase and phosphate protecting groups and release of the sequences from the solid support are carried out under standard basic conditions, whereas the cleavage of 2′‐O‐(2‐cyano‐2,2‐dimethylethanimine‐N‐oxymethyl) protective groups is effected (without releasing RNA alkylating side‐products) by treatment with tetra‐n‐butylammonium fluoride (0.5 m) in dry DMSO over a period of 24–48 h at 55 °C. Characterization of the fully deprotected RNA sequences by polyacrylamide gel electrophoresis (PAGE), enzymatic hydrolysis, and matrix‐assisted laser desorption/ionization (MALDI) mass spectrometry confirmed the identity and quality of these sequences. Thus, the use of 2′‐O‐aminooxymethylribonucleosides in the design of new 2′‐hydroxyl protecting groups is a powerful approach to the development of a straightforward, efficient, and cost‐effective method for the chemical synthesis of high‐quality RNA sequences in the framework of RNA interference applications.     

Synthesis,Dynamic Combinatorial Chemistry,and PCR Amplification of 3′–5′ and 3′–6′ Disulfide‐linked Oligonucleotides

Disulfide dithymidines linked 3′–5′ or 3′–6′ were synthesized and incorporated into oligonucleotides through a combined phosphotriester and phosphoramidite solid‐phase oligonucleotide synthesis approach. The disulfide links are cleaved and formed reversibly in the presence of thiols and oligonucleotides. This link was shown to be sequence‐adaptive in response to given templates in the presence of mercaptoethanol. The artificial 3′–5′ and 3′–6′ disulfide link was tolerated by polymerases in the polymerase chain reaction (PCR). By using sequencing analysis, we show that single mutations frequently occurred randomly in the amplification products of the PCR.     

Synthesis and Solid‐state Reaction of 1‐[3′‐(Benzotriazol‐2″‐yl)‐4′ ‐hydroxybenzoyl]‐3‐methyl‐5‐pyrazolones

A new kind of UV stabilizers, 1‐(3′‐(benzotriazol‐2″‐yl)‐4′‐hydroxy‐benzoyl)‐3‐methyl‐5‐pyrazolones (1a‐d), was synthesized with the aim to bind them chemically to certain polymers. The reaction of 1d with substituted benzaldehydes 4 in the molten state at 150°C and in the solid state at room temperature produced the condensation products l‐(3′‐(5″‐chlorobenzotriazol‐2″‐yl)‐4′‐hydroxyl‐5′‐chlorobenzoyl)‐3‐methyl‐4‐arylmethylene‐5‐pyrazolones (2) and 4,4′‐arylmethylene‐bis [1‐(3′‐(5″‐chloro‐benzotriazol‐2″‐yl)‐4′‐hydroxy‐5′‐chloro‐benzoyl)‐3‐methyl‐5‐pyrazolone] s (3), respectively, as the major product. On the other hand, the reaction of 1d with 4 at 50°C in chloroform solution proceeded non‐selectively to give a mixture of 2 and 3.     

A Continuous Flow Process Using a Sequence of Microreactors with In‐line IR Analysis for the Preparation of N,N‐Diethyl‐4‐(3‐fluorophenylpiperidin‐4‐ylidenemethyl)benzamide as a Potent and Highly Selective δ‐Opioid Receptor Agonist

This article describes the design, optimisation and development of a continuous flow synthesis of N,N‐diethyl‐4‐(3‐fluorophenylpiperidin‐4‐ylidenemethyl)benzamide, a potent δ‐opioid receptor agonist developed by AstraZeneca. The process employs a sequence of flow‐based microreactors, with integrated purification employing solid‐supported reagents and in‐line IR analytical protocols using a newly developed ReactIR flow cell. With this monitoring device, initiation of the fourth input flow stream can be precisely controlled during the synthesis.     

Decomposition of 3′‐azido‐2′,3′‐dideoxythymidine 5′‐monophosphate (AZTMP) prodrugs in biological media studied by on‐line solid‐phase extraction coupled to liquid chromatography mass spectrometry

Using a column‐switching HPLC method previously described, we studied the behavior of some mononucleotide prodrugs (pronucleotides) of 3′‐azido‐2′,3′‐dideoxythymidine in various biological media. From UV data, this method allowed quantification of transient metabolites resulting from prodrug bioconversion. The kinetic data related to the successive steps were calculated according to pseudo‐first‐order kinetic models and optimized using mono‐ or poly‐exponential regressions. Various metabolites were identified by co‐injection with authentic samples and/or ESI‐MS coupling. The results led us to propose, for each considered pronucleotide, a global decomposition pathway ending in the selective delivery of the corresponding mononucleotide. Associated to the determination of other parameters (lipophilicity, aqueous solubility), the present study contributes to the search of suitable pharmacological properties for further in vivo evaluations. Copyright © 2009 John Wiley & Sons, Ltd.     

Increased Affinity of 2′‐O‐(2‐Methoxyethyl)‐Modified Oligonucleotides to RNA through Conjugation of Spermine at Cytidines

Structural modification at the 2′‐O‐position of riboses in oligonucleotide therapeutics is of critical importance for their use as drugs. To date, the methoxyethyl (MOE) substituent is the most important and features in dozens of antisense oligonucleotides that have been tested in clinical trials. Yet, the search for new improved modifications continues in a quest for increased oligonucleotide potency, improved transport in vivo and favorable metabolism. Recently, we described how the conjugation of spermine groups to pyrimidines in oligonucleotides vastly increases their affinity for complementary RNAs through accelerated binding kinetics. Here we describe how spermines can be linked to the exocyclic amino groups of cytidines in MOE‐oligonucleotides employing a straightforward ‘convertible nucleoside approach’ during solid phase synthesis. Singly‐ or doubly‐modified oligonucleotides show greatly enhanced affinity for complementary RNA, with potential for a new generation of MOE‐based oligonucleotide drugs.     

The Synthesis of Methylated,Phosphorylated, and Phosphonated 3′‐Aminoacyl‐tRNASec Mimics

The twenty first amino acid, selenocysteine (Sec), is the only amino acid that is synthesized on its cognate transfer RNA (tRNA^Sec) in all domains of life. The multistep pathway involves O‐phosphoseryl‐tRNA:selenocysteinyl‐tRNA synthase (SepSecS), an enzyme that catalyzes the terminal chemical reaction during which the phosphoseryl–tRNA^Sec intermediate is converted into selenocysteinyl‐tRNA^Sec. The SepSecS architecture and the mode of tRNA^Sec recognition have been recently determined at atomic resolution. The crystal structure provided valuable insights that gave rise to mechanistic proposals that could not be validated because of the lack of appropriate molecular probes. To further improve our understanding of the mechanism of the biosynthesis of selenocysteine in general and the mechanism of SepSecS in particular, stable tRNA^Sec substrates carrying aminoacyl moieties that mimic particular reaction intermediates are needed. Here, we report on the accurate synthesis of methylated, phosphorylated, and phosphonated serinyl‐derived tRNA^Sec mimics that contain a hydrolysis‐resistant ribose 3′‐amide linkage instead of the natural ester bond. The procedures introduced allow for efficient site‐specific methylation and/or phosphorylation directly on the solid support utilized in the automated RNA synthesis. For the preparation of (S)‐2‐amino‐4‐phosphonobutyric acid–oligoribonucleotide conjugates, a separate solid support was generated. Furthermore, we developed a three‐strand enzymatic ligation protocol to obtain the corresponding full‐length tRNA^Sec derivatives. Finally, we developed an electrophoretic mobility shift assay (EMSA) for rapid, qualitative characterization of the SepSecS‐tRNA interactions. The novel tRNA^Sec mimics are promising candidates for further elucidation of the biosynthesis of selenocysteine by X‐ray crystallography and other biochemical approaches, and could be attractive for similar studies on other tRNA‐dependent enzymes.     

Chemical Synthesis, 3D Structure,and ASIC Binding Site of the Toxin Mambalgin‐2

Mambalgins are a novel class of snake venom components that exert potent analgesic effects mediated through the inhibition of acid‐sensing ion channels (ASICs). The 57‐residue polypeptide mambalgin‐2 (Ma‐2) was synthesized by using a combination of solid‐phase peptide synthesis and native chemical ligation. The structure of the synthetic toxin, determined using homonuclear NMR, revealed an unusual three‐finger toxin fold reminiscent of functionally unrelated snake toxins. Electrophysiological analysis of Ma‐2 on wild‐type and mutant ASIC1a receptors allowed us to identify α‐helix 5, which borders on the functionally critical acidic pocket of the channel, as a major part of the Ma‐2 binding site. This region is also crucial for the interaction of ASIC1a with the spider toxin PcTx1, thus suggesting that the binding sites for these toxins substantially overlap. This work lays the foundation for structure–activity relationship (SAR) studies and further development of this promising analgesic peptide.