Coupling reagents, nucleotide

DNA synthesizers operate on a principle similar to that of the Merrifield solid-phase peptide synthesizer (Section 26.8). In essence, a protected nucleotide is covalently bonded to a solid support, and one nucleotide at a time is added to the growing chain by the use of a coupling reagent. After the final nucleotide has been added, all the protecting groups are removed and the synthetic DNA is cleaved from the solid support. Five steps are needed ... 

In the early solution phase syntheses of oligonucleotides, coupling of phosphate diesters was used. A mixed 3 -ester with one aryl substituent, usually o-chlorophenyl, was coupled with a deprotected 5 -OH nucleotide. The coupling reagents were sulfonyl halides, particularly 2,4,6-tri-i-propylbenzenesulfonyl chloride,53 and the reactions proceeded by formation of reactive sulfonate esters. Coupling conditions... 

More recently, automated gene synthesizers have been developed that operate on principles similar to the Merrifield solid-phase technique for peptides. A protected nucleotide is covalently bonded to a polymer. Other protected nucleotides are then added sequentially to the chain, using a coupling reagent. Eventually, the protecting groups are removed, and the synthetic oligonucleotide is then detached from the solid support. 

Reagents A Convenient Route to sec- and tert- 6-Alkylpurines. Tetrahedron Lett. 1996, 37, 1285-1288. (c) Dvorakova, H. Dvorak, D. Holy, A. Synthesis of Acyclic Nucleotide Analogues Derived from 6-(sec- or tert-Alkyl) purines via Coupling of 6-Chloropurine Derivatives with Organocuprates. Collect. Czech. Chem. Commun. 1998, 63, 2065-2074. (d) Hocek, M. Holy, A. Perfluoroalkylation of 6-Iodopurines by Trimethyl(perfluoro-alkyl)silanes. Synthesis of 6-(Perfluoroalkyl)purine Bases, Nucleosides and Acyclic Nucleotide Analogues. Collect. Czech. Chem. Commun. 1999, 64, 229-241. 

The reagent is used for the synthesis of phosphomonoesters of alcohols, particularly of nucleotides. It is coupled to the alcoholic component in pyridine using dicyclo hexylcarbodiimide (DCC) as condensing agent, as illustrated for the case of 5 -0-tritylthymidine. Acid hydrolysis eliminates the protective trityl group, and very mild... 

The authors further found that y-subunit rotation was blocked when the Fq complex was modified by dicyclohexylcarbodiimide (DCCD), the lipid-soluble carboxyl reagent that is known to inhibit proton transport by reacting with a carboxylate residue on one of the c-subunits in Fq (Glu in MFo Asp in EcFq). These results demonstrate that the reconstituted EcFi can rebind to Fq to form a functional, membrane-bound EcFo F complex and that y-subunit rotation in F, is functionally coupled to Fq. This is also consistent with the long-held notion of a long-range conformational interaction between Fq within the membrane and the catalytic nucleotide-binding site on the extrinsic F complex. 

---