Synthesis and Binding of Stable Bisubstrate Ligands for Phosphoglycerate Kinase

Stable bisubstrate ligands of phosphoglycerate kinase (PGK) have been synthesised with AMP or ADP conjugated to hydrolytically-stable, symmetrical analogues of 1,3-bisphosphoglycerate and their binding to yeast PGK evaluated. Their Kds decrease with net negative charge, with a penta-anionic analogue 7 showing highest affinity - in accordance with its approximation to the transition state for the reaction catalysed by PGK.

Reversible Photocontrol of Deoxyribozyme-Catalyzed RNA Cleavage under Multiple-Turnover Conditions

Lights, camera, action! Photoswitchable nucleoside analogues containing o-, m-, or p-azobenzenes can be inserted in the catalytic core of RNA-cleaving 10-23 deoxyribozymes by replacing a nonconserved residue (see picture). Irradiation of the modified deoxyribozymes at 366 nm enhances RNA cleavage rates up to ninefold, thus achieving the rates observed for the unmodified deoxyribozyme.

Solid-phase synthesis of terminal oligonucleotide-phosphoramidate conjugates

A novel phosphoramidite, N,N-diisopropylamino-2-cyanoethyl-9-anthracenemethyl phosphoramidite 1, was prepared and coupled with the terminal 5'-hydroxyl of support-bound T10 and the putative phosphite triester intermediate was subsequently reacted with iodine in the presence of either water or a series of primary and secondary amines. The reactivity of 1 compared to a previously reported benzyl phosphoramidite 2 was also investigated: oxidation of the product of coupling 2 with CPG-T10-5'OH under aqueous conditions resulted in greater than 30% of the benzyl moiety being retained. In contrast, essentially complete loss of the 9-anthracenemethyl group was observed using 1 under the same conditions. Oligonucleotides modified with a terminal phosphate monoester, lipophilic, fluorescent or cationic groups were thus prepared.

Novel methodology for the preparation and purification of oligonucleotides incorporating phosphorothiolate termini

A novel phosphoramidite; N,N-diisopropylamino-2-cyanoethyl-ortho-methylbenzylphosphoramidite 1, was prepared. The reaction of 1 with DMTrT and subsequent derivatisation of the phosphite triester product under solution-phase, Michaelis–Arbuzov conditions was investigated. Coupling of 1 with the terminal hydroxyl groups of support-bound oligodeoxyribonucleotides and subsequent reaction with an activated disulfide yielded oligonucleotides bearing a terminal, phosphorothiolate-linked, lipophilic moiety. The oligomers were readily purified using RP-HPLC. Silver(I)-mediated cleavage of the phosphorothiolate linkage and desalting of the oligonucleotides were performed readily in one step to yield cleanly the corresponding phosphate monester-terminated oligomers.