Use of Phthaloyl Protecting Group for the Automated Synthesis of 3'-[(Hydroxypropyl)amino] and 3'-[(Hydroxypropyltriglycyl] Oligonucleotide Conjugates

Abstract

The chemical stability of oligonucleotides (ODNs) containing 3'-propanolamine was investigated. Invariably, all the ODNs synthesized from Fmoc-protected 3-aminopropane-1,2-diol-CPG support gave a mixture of three compounds at the end of automated synthesis as analyzed by denaturing PAGE and HPLC. On the basis of analytical procedures, these compounds were identified to be 3'-[N-acetyl-N-(hydroxypropyl)amino],3'-[(hydroxypropyl)amino], and 3'-hydroxyl ODNs. The instability of the amino protecting group under the synthesis conditions was responsible for this observed heterogeneity. In order to evaluate the stability, a comparative study on the chemical stability of the ODN containing amino-protecting groups such as [(9-fluorenylmethyl)oxy]carbonyl (Fmoc), trifluoroacetyl (TFA), and phthaloyl was undertaken. The results indicate that the phthaloyl group provided the best stability for the synthesis of 3' amine-modified ODNs, and the protecting group is cleaved and deprotected in concentrated ammonium hydroxide:40% aqueous methylamine, 1:1, for 5-10 min, at 56 degrees C. The 3'-hydroxypropyl)triglycyl] ODN conjugates were also synthesized from Fmoc- and phthaloyl-protected (hydroxypropyl)triglycine-CPG supports.

A 15-Base Acridine-Conjugated Oligodeoxynucleotide Forms Triplex DNA with Its IL-2Rα Promoter Target with Greatly Improved Avidity

“…The full formula is shown in Figure 1B. In the course of the NaOH deprotection of AG15C-3′-NH 2 , some of the 3′amino groups are eliminated (Vu et al, 1995), leading to the appearance of two electrophoretically distinct products (Figure 1C, lane 4). As determined by mass spectrometry, the upper band (referred to as AG15C-3′-NH 2 , Figure 1A) is the expected 3′-amino derivative, whereas the lower band lacks the 3-amino-2-hydroxypropyl phosphate group.…”

“…The 3′-amino group was included in the DAMAconjugated oligonucleotides to protect them from exonucleolytic degradation in future in vivo experiments (McShan et al, 1992;Orson et al, 1991). However, preparation of the oligo resulted in a mixture of the 3′amino protected and unprotected forms (Vu et al, 1995). The two species were separated on a gel (Figure 1C) and their identities confirmed by mass spectral analysis.…”

Solid‐Phase Supports for Oligonucleotide Synthesis

“…The amino group in the aminoglycerol linker was also protected with trifluoroacetyl (TFA) and with a phthaloyl group to give, respectively, solid supports 111b and 111c (Vu et al, ). The phthaloyl group was stable under the conditions of extended capping and detritylation, and was successfully removed by treatment with AMA for 5 to 10 min at 56°C.…”

Solid-phase synthesis of base-sensitive oligonucleotides

“…32 Protected amino group (28) is further acylated upon the repetitive capping steps (29). In the deprotection conditions, the orthogonal protection is cleaved but the acetyl cap remains intact (30). To overcome this shortcoming, the acetic anhydride capping step may be omitted but also alternative capping reagents may be considered.…”