Derivatization of unprotected polynucleotides

Abstract

A simple and efficient method for attaching amines to the terminal 5'-phosphate of unprotected oligonucleotides or nucleic acids in aqueous solution is described. The method is applicable to low molecular-weight amines, polypeptides, or proteins. The terminal 5'-phosphate of an oligonucleotide or nucleic acid reacts with a water-soluble carbodiimide in imidazole buffer at pH 6 to give good yields of the 5'-phosphorimidazolide. Exposure of the phosphorimidazolide to amine-containing molecules in aqueous solution results in the production of a wide range of stable phosphoramidates in high yield. The exposure of polynucleotides to carbodiimide does not result in significant breakage of phosphodiester bonds or damage to nucleoside bases. The biological activity of a drug resistant plasmid is not affected. The direct condensation of polynucleotides with amines in 1-methylimidazole buffer is also possible. However, it is not a satisfactory preparative method if the ligand is sensitive to carbodiimide.

An approach to gene-specific transcription inhibition using oligonucleotides complementary to the template strand of the open complex

“…All ORNs were subjected to matrix-assisted laser desorption ionization-time of flight MS and visualized on polyacrylamide gel (20%) to confirm their sequence and purity. Treatment of phosphor ylated ORNs (30 nmol in water) with 5-[␤-alanylamido]-OP (32), in the presence of carbodiimide (33) and methylimidizole (pH 6.7) overnight at room temperature followed by polyacrylamide gel purification afforded OP-ORNs whose structures were confirmed by MS. Phenanthroline attachment to the 2Ј position of 5Ј-GU(OP)GGA-3Ј required the synthesis of 2Ј-aminouridine from cyclouridine as described by McGee et al (34,35). Carbodiimide coupling of the amine to 5-(propanoic acid)-OP afforded the 2Ј amidophenanthroline uridine derivative.…”

Surface‐enhanced Raman scattering for medical diagnostics and biological imaging

“…38 Briefly, ¾10 µg of the oligonucleotides were dried by vacuum evaporation in a microcentrifuge tube, then 50 µl of 0.2 M imidazole (1,3-diaza-2,4-cyclopentadiene) at pH 8.0 and 50 µl of 50 mM 1-ethyl-3-(dimethylaminopropyl)carbodiimide (EDC) in 2-(N-morpholino)ethanesulfonic acid (MES) buffer (pH 8.0) were added and incubated at 50°C for 3 h. This treatment resulted in the production of 5 0 -phosphorimidazolides intermediates. The 5 0 -phosphorimidazolides were then reacted with equal volumes of CFV (0.20 M aqueous solution) for 18 h at 50°C.…”

Synthesis of adenosine derivatives as transcription initiators and preparation of 5′ fluorescein- and biotin-labeled RNA through one-step in vitro transcription

“…The linkers in these derivatives differ in both size and hydrophobicity. Synthesis of 5Ј-amino derivatives of adenosine phosphoramidate followed the standard direct phosphate-amine coupling procedure by the water-soluble carbodiimide, N-(3-dimethylaminopropyl)-NЈ-ethylcarbodiimide (EDAC; Chu et al 1983). Coupling efficiencies as determined by HPLC are excellent, ranging from 70% to 95% after a 2-h reaction at room temperature.…”

“…Direct coupling (Chu et al 1983) of diamines with AMP by the water-soluble carbodiimide, EDAC, was used to synthesize a series of six 5Ј-amino derivatives of adenosine phosphoramidate. The reactions and structures are shown in Figure 2A.…”