Synthesis of High-Quality Antisense Drugs. Addition of Acrylonitrile to Phosphorothioate Oligonucleotides:  Adduct Characterization and Avoidance

Capaldi, Daniel C.; Gaus, Hans; Krotz, Achim H.; Arnold, J. O.; Carty, Ricaldo L.; Moore, Max N.; Scozzari, Anthony N.; Lowery, Kirsten; Cole, Douglas L.; Ravikumar, Vasulinga T.

doi:10.1021/op020090n

Cited by 72 publications

(56 citation statements)

References 35 publications

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“…Characterization and determination of the source origin of the impurities is the first step in the development of mitigation measures for prevention and elimination of their formation. Phosphorothioate oligonucleotide impurities can be classified in several categories depending on the type of reaction leading to their formation: (1) failure sequences due to coupling inefficiency, incomplete capping, or detritylation (shortmers) [20,21]; (2) molecules larger that the full length product (longmers) [22,23]; and (3) other impurity products formed by depurination, deamination, sulfur loss, thermal stress, adduct formation, and other reactions [24][25][26][27][28]. It is possible for some of these impurities to be formed by mixed consecutive or parallel secondary reactions initiated by solvent and raw materials initial impurities, process variables, or the chemical nature of the target oligonucleotide structure.…”

Section: Introductionmentioning

confidence: 99%

A Novel and Intuitive Method of Displaying and Interacting with Mass Difference Information: Application to Oligonucleotide Drug Impurities

Roussis

2015

J. Am. Soc. Mass Spectrom.

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Abstract.A new method is presented for determining relationships between components in complex analytical systems. The method uses the mass differences between peaks in high resolution electrospray ionization (ESI) mass spectra. It relates peaks that share common mass differences. The method is based on the fundamental assumption that peaks in the spectra having the same exact mass difference are related by the same chemical moiety/substructure. Moreover, the presence (or absence/loss) of the same chemical moiety from a series of molecules may reflect similarities in the mechanisms of formation of each molecule. The determined mass differences in the spectra are used to automatically differentiate the types of components in the samples. Contour plots and summary plots of the summed total ion signal as a function of the mass difference are generated, which form powerful tools for the rapid and automated determination of the components in the samples and for comparisons with other samples. For the first time, in this work a unique profile contour plot has been developed that permits the interactive interrogation of the mass range by mass difference data matrix to obtain valuable information about components that share a common mechanism of formation, and all possible mechanisms of formation linked to a selected precursor molecule. The method can be used as an additional and complementary method to the existing analytical methods to determine relationships between components in complex chemical systems.

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Section: Introductionmentioning

confidence: 99%

A Novel and Intuitive Method of Displaying and Interacting with Mass Difference Information: Application to Oligonucleotide Drug Impurities

Roussis

2015

J. Am. Soc. Mass Spectrom.

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“…The twostep deprotection procedure made it possible to avoid the reaction of heterobase residues with acrylonitrile generated during the deprotection of phosphate residue (Capaldi et al 2003). For the simultaneous removal of the TBDMS and neoO-dPS protecting groups in τm 5 U-modified RNA, Et 3 N•3HF was effectively employed.…”

Section: Resultsmentioning

confidence: 99%

Chemical synthesis of the 5-taurinomethyl(-2-thio)uridine modified anticodon arm of the human mitochondrial tRNA^Leu(UUR) and tRNA^Lys

Leszczyńska

Leonczak

Woźniak

et al. 2014

RNA

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5-Taurinomethyluridine (τm 5 U) and 5-taurinomethyl-2-thiouridine (τm 5 s 2 U) are located at the wobble position of human mitochondrial (hmt) tRNA Leu(UUR) and tRNA Lys , respectively. Both hypermodified units restrict decoding of the third codon letter to A and G. Pathogenic mutations in the genes encoding hmt-tRNA Leu(UUR) and hmt-tRNA Lys are responsible for the loss of the discussed modifications and, as a consequence, for the occurrence of severe mitochondrial dysfunctions (MELAS, MERRF). Synthetic oligoribonucleotides bearing modified nucleosides are a versatile tool for studying mechanisms of genetic message translation and accompanying pathologies at nucleoside resolution. In this paper, we present site-specific chemical incorporation of τm 5 U and τm 5 s 2 U into 17-mers related to the sequence of the anticodon arms hmt-tRNA Leu(UUR) and hmttRNA Lys , respectively employing phosphoramidite chemistry on CPG support. Selected protecting groups for the sulfonic acid (4-(tert-butyldiphenylsilanyloxy)-2,2-dimethylbutyl) and the exoamine function (-C(O)CF 3 ) are compatible with the blockage of the canonical monomeric units. The synthesis of τm 5 s 2 U-modified RNA fragment was performed under conditions eliminating the formation of side products of 2-thiocarbonyl group oxidation and/or oxidative desulphurization. The structure of the final oligomers was confirmed by mass spectroscopy and enzymatic cleavage data.

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“…[24][25][26] Table 1. At end of each synthesis, the support was thoroughly dried to determine the crude weight yield [27] and then treated with a solution of triethylamine:CH 3 CN (1:1, v/v) at room temperature for 2 hours to remove the β-cyanoethyl protecting groups, [28,29] then treated with 30% aqueous ammonium hydroxide solution for 12 hours at 55 • C to effect release from support and base deprotection. Yield (expressed in mg of oligonucleotide/µmole of support) [27] and analytical RP-HPLC (full length determination) data were collected for each synthesis.…”

Section: Synthesis Of Universal Linker and Loading To Solid Supportmentioning

confidence: 99%

A Novel Universal Linker for Efficient Synthesis of Phosphorothioate Oligonucleotides

Wang

Olsen

Ravikumar

2007

Nucleosides, Nucleotides and Nucleic Acids

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A versatile and conformationally preorganized universal linker molecule is reported here for efficient synthesis of phosphorothioate oligonucleotides. With respect to nucleoside loaded support, comparable yield and quality based on ion-pair LC-MS are obtained for both deoxy and 2'-O-methoxyethyl modified phosphorothioate oligonucleotides. No 3'-phosphate or phosphorothioate monoester or any modification of universal molecule still attached to oligonucleotide was observed. [structure: see text]

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Synthesis of High-Quality Antisense Drugs. Addition of Acrylonitrile to Phosphorothioate Oligonucleotides: Adduct Characterization and Avoidance

Cited by 72 publications

References 35 publications

A Novel and Intuitive Method of Displaying and Interacting with Mass Difference Information: Application to Oligonucleotide Drug Impurities

A Novel and Intuitive Method of Displaying and Interacting with Mass Difference Information: Application to Oligonucleotide Drug Impurities

Chemical synthesis of the 5-taurinomethyl(-2-thio)uridine modified anticodon arm of the human mitochondrial tRNA^Leu(UUR) and tRNA^Lys

A Novel Universal Linker for Efficient Synthesis of Phosphorothioate Oligonucleotides

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Synthesis of High-Quality Antisense Drugs. Addition of Acrylonitrile to Phosphorothioate Oligonucleotides: Adduct Characterization and Avoidance

Cited by 72 publications

References 35 publications

A Novel and Intuitive Method of Displaying and Interacting with Mass Difference Information: Application to Oligonucleotide Drug Impurities

A Novel and Intuitive Method of Displaying and Interacting with Mass Difference Information: Application to Oligonucleotide Drug Impurities

Chemical synthesis of the 5-taurinomethyl(-2-thio)uridine modified anticodon arm of the human mitochondrial tRNALeu(UUR) and tRNALys

A Novel Universal Linker for Efficient Synthesis of Phosphorothioate Oligonucleotides

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Chemical synthesis of the 5-taurinomethyl(-2-thio)uridine modified anticodon arm of the human mitochondrial tRNA^Leu(UUR) and tRNA^Lys