Biomedical subjects

Daniel C Capaldi

Publications and source records attributed to Daniel C Capaldi.

6 recordsLinked to original sources

PubMed2005

Characterization of high molecular weight impurities in synthetic phosphorothioate oligonucleotides.

Phosphorothioate oligonucleotides manufactured by standard phosphoramidite techniques using 2'-deoxyadenosine- or 2'-O-(2-methoxyethyl)-5-methylcytosine-loaded solid supports contain branched impurities consisting of two chains linked through the exocyclic amino group of the 3'-terminal nucleoside of one chain and the 3'-terminal hydroxyl group of another via a P(O)SH group. These impurities are not produced when a universal, non-nucleoside derivatized support is used.

5-Methylcytosine↗

PubMed2005

Formation of modified cytosine residues in the presence of depurinated DNA.

[Chemical reaction: See text] Depurination is an important degradation pathway for antisense phosphorothioate oligonucleotides under conditions of thermal stress. We present evidence showing that depurinated oligonucleotides react with cytosine-containing sequences giving products containing a 6-(2-deoxy-beta-D-erythro-pentofuranosyl)-3-(2-oxopropyl)imidazo[1,2-c]pyrimidin-5(6H)-one residue. Further, we demonstrate that the same product is formed upon treatment of 2'-deoxycytidine with 4-oxo-2-pentenal, the latter being an expected byproduct of serial elimination reactions at apurinic sites. In addition to being important for synthetic oligonucleotides, apurinic site formation in cellular DNA is a common occurrence. Because repair of these sites can result in the production of 4-oxo-2-pentenal, it is interesting to speculate whether 6-(2-deoxy-beta-D-erythro-pentofuranosyl)-3-(2-oxopropyl)imidazo[1,2-c]pyrimidin-5(6H)-one residues can form in vivo.

Base Sequence↗

PubMed2005

Trichloroacetaldehyde modified oligonucleotides.

Some commercial batches of dichloroacetic acid (DCA) contain traces of chloral (trichloroacetaldehyde). Using such DCA to effect detritylation during solid-phase oligonucleotide synthesis results in the formation of a family of process impurities in which the atoms of chloral (Cl3CCHO) are incorporated between the 5'-oxygen and phosphorus atoms of an internucleotide linkage. The structure was elucidated by HPLC with UV and MS detection, digestion of the oligonucleotide, synthesis of model compounds, and 1H and 31P NMR spectroscopy. By understanding the chemistry behind its formation, we are now able to limit levels of this impurity in synthetic oligonucleotides by limiting chloral in DCA.

Chloral Hydrate↗

PubMed2004

Formation of 4,4'-dimethoxytrityl-C-phosphonate oligonucleotides.

Incomplete sulfurization during solid-phase synthesis of phosphorothioate oligonucleotides using phosphoramidite chemistry was identified as the cause of formation of two new classes of process-related oligonucleotide impurities containing a DMTr-C-phosphonate (DMTr=4,4'-dimethoxytrityl) moiety. Phosphite triester intermediates that failed to oxidize (sulfurize) to the corresponding phosphorothioate triester react during the subsequent acid-induced (dichloroacetic acid) detritylation with the DMTr cation or its equivalent in an Arbuzov-type reaction. This leads to formation of DMTr-C-phosphonate mono- and diesters resulting in oligonucleotides modified with a DMTr-C-phosphonate moiety located internally or at the 5'terminal hydroxy group. DMTr-C-phosphonate derivatives are not detected when optimized sulfurization conditions are employed.

Chromatography, High Pressure Liquid↗

PubMed2003

Antisense phosphorothioate oligodeoxyribonucleotide targeted against ICAM-1: synthetic and biological characterization of a process-related impurity formed during oligonucleotide synthesis.

A phosphorothioate-linked oligonucleotide bearing a 3'-terminal phosphorothioate monoester has been synthesized and characterized. This oligonucleotide has been identified as a process-related impurity formed during synthesis of ISIS 2302. Biological properties of the compound have been determined. Based on these data, it can be concluded that this species (3'-TPT) has biological properties similar to parent drug.

Animals↗

PubMedDate not supplied

Synthesis of high quality phosphorothioate oligonucleotides as antisense drugs. Use of I-linker in the elimination of 3'-terminal phosphorothioate monoesters.

Detritylation of a 5'-O-DMT-2'-deoxyadenosine moiety attached to solid support under acidic condition leads to depurination during oligonucleotide synthesis. Deprotection followed by reversed phase HPLC purification leads to desired oligonucleotide contaminated with significant levels of 3'-terminal phosphorothiaote (3'-TPT) monoester (n-1)-mer. However, it is demonstrated that attachment of dA nucleoside through its exocyclic amino group to solid support leads to substantial reduction of 3'-TPT formation thereby improving the quality of oligonucleotide synthesized.

Base Sequence↗