Achim H. Krotz

On the formation of longmers in phosphorothioate oligodeoxyribonucleotide synthesis

Abstract The extent of longmer formation in phosphorothioate oligodeoxyribonucleotide synthesis through amidete chemistry on solid support depends on base composition, contact time and acidity of the promotor used for activation of the phosphoramidite. A longmer formation mechanism that involves dedimethoxytritylation of the phosphite triester intermediate is proposed.

Phosphorothioate oligonucleotides: Largely reduced (N-1)-mer and phosphodiester content through the use of dimeric phosphoramidite synthons

Abstract Phosphorothioate oligonucleotides synthesized through an assembly of dimeric phosphoramidite synthons on controlled pore glass solid support show a significantly improved impurity profile compared to oligomers synthesized through a coupling of standard monomer phosphoramidites. A greater than 70% reduction of the (n-1)-mer population and a ca 50% reduction of phosphodiester linkages has been achieved.

Efficient synthesis of deoxyribonucleotide phosphorothioates by the use of DMT cation scavenger1

Abstract Triethylsilane in the presence of dichloroacetic acid in dichloromethane is an efficient DMT cation scavenger during the synthesis of deoxyribonucleotide phosphorothioates and leads to increased overall yields.

Synthesis of Antisense Oligonucleotides with Minimum Depurination

Abstract The removal of 4,4′-dimethoxytrityl (DMTr) groups from oligonucleotides at low pH and the acid lability of the glycosidic linkage of purine nucleotides constitute an inherent conflict in preparative oligonucleotide chemistry. The use of a mildly acidic NaOAc buffer (10 mM, pH 3.0–3.2) allows adjustment of the pH in a range where the progress of the DMTr removal reaction can be monitored conveniently by HPLC and the optimum reaction time can be calculated. As a result, oligonucleotides with minimum depurination are obtained.

Polysulfide Reagent in Solid-Phase Synthesis of Phosphorothioate Oligonucleotides: Greater than 99.8% Sulfurization Efficiency

A solution of sulfur (0.1 M) and sodium sulfide (0.01 M) in 3-picoline, referred to as polysulfide reagent, rapidly converts trialkyl and triaryl phosphite triesters to the corresponding phosphorothioate derivatives. Greater than 99.8% average stepwise sulfurization efficiency is obtained in the solid-phase synthesis of DNA and RNA phosphorothioate oligonucleotides via the phosphoramidite approach.

Improved Impurity Profile of Phosphorothioate Oligonucleotides Through the Use of Dimeric Phosphoramidite Synthons

Abstract Phosphorothioate oligonucleotides synthesized through assembly of dimeric phosphoramidite synthons show a significantly improved impurity profile compared to oligomers synthesized through coupling of standard monomer phosphoramidites. A greater than 70% reduction of the (n-1)-mer population and a ca. 50% reduction of phosphodiester linkages has been achieved.

Dimethoxytrityl Removal in Organic Medium: Efficient Oligonucleotide Synthesis Without Chlorinated Solvents

Abstract Oligonucleotides are finding widespread utility in various applications in diagnostics and molecular biology and as therapeutic agents. In standard synthesis of such oligonucleotides through phosphoramidite coupling, removal of the typical acid-labile 4,4′-dimethoxytrityl 5′-protecting group (DMTr), from the support-bound oligonucleotide plays a crucial role in each synthesis cycle in achieving high product yield and oligonucleotide quality. Although several reagents have been developed for this purpose, many have limited applicability to automated oligonucleotide synthesis on solid supports. The most commonly used reagents today are dilute solutions (2–15%) of an organic acid, typically trichloroacetic acid (TCA, pKa 0.8) or dichloroacetic acid (DCA, pKa 1.5) in dichloromethane. The high volatility (boiling point 40 °C) of dichloromethane and its high toxicity and carcinogenicity pose a hazard for personnel and the environment. In addition, as oligonucleotide synthesizers are now available to al...

Synthesis and deprotection of β-silylethyl protected O,O,O- and O,O,S-trialkylphosphorothioates

Abstract Functionalized 2-(diphenylmethylsilyl)ethyl protected thymidyl-thymidine phosphorothioate dimers are easily accessible and stable under conditions used in oligophosphorothioate synthesis. Deprotection with ammonium hydroxide occurs through β-fragmentation. Methylamine and tetrabutyl-ammonium fluoride rapidly and selectively remove the DPSE protecting group of O , O , O - and O , O , S -trialkylphosphorothioates.

Synthesis of antisense oligonucleotides. using environmentally friendly and safe deprotection procedures

It is demonstrated that mixed-sequence phosphorothioate oligodeoxyribonucleotides can be synthesized on scales up to 80 mmol without using chlorinated solvents like dichloromethane, while preserving both high yield and purity of the product. A solution of dichloroacetic acid in toluene cleanly and efficiently removes 4,4′-dimethoxytrityl groups from the 5′-terminus of the growing oligonucleotide chain during synthesis on solid support. Ammonium hydroxide treatment at room temperature at atmospheric pressure furnishes deprotected oligonucleotides reducing the risk that pressurized reaction glass vessels pose. To ensure facile separation of polymer beads (Primer HL 30) and oligonucleotide solution, minimum agitation of the reaction mixture is applied.

Preparation of oligonucleotides without aldehyde abasic sites

High-quality oligonucleotides are obtained by selective modification of sequences containing aldehyde apurinic sites with a new chromatographic tag followed by RP-HPLC separation. Hydroxylamine derivative 1 of a water soluble nonionic surfactant modifies oligonucleotides selectively at abasic sites leading to significantly increased retention.