Synthesis of Oligodeoxynucleotides Containing Sensitive Electrophiles

Oligodeoxynucleotides (ODNs) containing electrophilic groups are useful in many studies including antisense drug development and DNA/protein interaction. Due to the use of strong nucleophiles for cleavage and deprotection, traditional ODN synthesis methods are not suitable for their preparation. To solve this problem, a new ODN synthesis technology using the 1,3- dithiane-2-yl-methoxycarbonyl (Dmoc) function as protecting groups and linker has been developed. Furthermore, Dmoc-derivatives were developed to demonstrate the feasibility of the technology. The Dmoc and Dmoc derivative functions are stable under all ODN synthesis conditions using the phosphoramidite chemistry. Upon oxidation of the sulfides in them, because of the drastically increased acidity of H-2, the groups and linker are readily cleaved under nearly non-nucleophilic conditions. Many sensitive electrophiles were able to be incorporated on DNA strands successfully using these technologies. These include but are not limited to sensitive thioester, ethyl ester, alkyl chloride, and α-chloroacetamide moieties

Electrophilic oligodeoxynucleotide synthesis using dM-Dmoc for amino protection

Solid-phase synthesis of electrophilic oligodeoxynucleotides (ODNs) was achieved using dimethyl-Dmoc (dM-Dmoc) as amino protecting group. Due to the high steric hindrance of the 2-(propan-2-ylidene)-1,3-dithiane side product from deprotection, the use of excess nucleophilic scavengers such as aniline to prevent Michael addition of the side product to the deprotected ODN during ODN cleavage and deprotection was no longer needed. The improved technology was demonstrated by the synthesis and characterization of five ODNs including three modified ones. The modified ODNs contained the electrophilic groups ethyl ester, α-chloroamide, and thioester. Using the technology, the sensitive groups can be installed at any location within the ODN sequences without using any sequence- or functionality-specific conditions and procedures

Formation of Hindered Arylcarbamates using Alkyl Aryl Carbonates under Highly Reactive Conditions

Hindered O-tert-alkyl N-arylcarbamates were conveniently prepared by treating arylamines with aryl tert-alkyl carbonates in the presence of a strong base. The new method avoids the use of sensitive and difficult-to-access dialkyl dicarbonates and isocyanates, which are most commonly used in known methods. Instead, the stable and readily accessible alkyl aryl carbonates are used. Therefore, the new method is particularly suitable for the synthesis of N-arylcarbamates that contain a complex O-alkyl moiety. Using the method, electron-rich and electron-poor, and primary and secondary arylamines can all be conveniently converted to their carbamates with acceptable yields. The method was also found equally effective for the synthesis of the less hindered O-secondary and O-primary alkyl N-arylcarbamates

dM-Dim for carboxylic acid protection

© 2018 Elsevier Ltd The 1,3-dithian-2-yl-methyl (Dim) and its analogous groups including dimethyl-Dim (dM-Dim) can provide a new dimension of orthogonality for carboxylic acid protection. They can be deprotected under nearly neutral oxidative conditions. In this paper, the protection of carboxylic acid with dM-Dim, deprotection of dM-Dim ester with sodium periodate, stability of dM-Dim protected carboxylic acid under acidic and basic conditions, and selective deprotection of dM-Dim protected carboxylic acids in the presence of tertiary butyl and methyl esters are presented

Tritylation of alcohols under mild conditions without using silver salts

© 2016 Elsevier Ltd Secondary alcohols were conveniently tritylated under mild conditions within a short running time with tritylium trifluoroacetate generated in situ from trityl alcohols and trifluoroacetic anhydride. No expensive silver salts were needed for the reactions. Four secondary alcohols were tritylated with both mono- and dimethoxy trityl alcohols giving good to excellent isolated yields. The reaction was also tested on four nucleoside derivatives that have primary alcohols. Satisfactory results were also obtained

Incorporation of Sensitive Ester and Chloropurine Groups into Oligodeoxynucleotides through Solid-Phase Synthesis

Nucleosides containing ester groups that are sensitive to nucleophiles were incorporated into oligodeoxynucleotides (ODNs) through solid phase chemical synthesis. The sensitive esters are located on a purine nucleobase. They are the esters of ethyl, 2-methoxyethyl, 4-methoxyphenyl and phenyl groups, and a thioester. These esters cannot survive the deprotection and cleavage conditions used in known ODN synthesis technologies, which involve strong nucleophiles such as ammonium hydroxide and potassium methoxide (potassium carbonate in anhydrous methanol). To incorporate these sensitive groups into ODNs, the Dmoc (i. e. dimethyl-1,3-dithian-2-ylmethoxycarbonyl) phosphoramidites and linker were used for solid phase synthesis, which allowed ODN deprotection and cleavage to be carried out under non-nucleophilic oxidative conditions. Sixteen ODN sequences containing these groups were synthesized and characterized with MALDI MS. In addition, the synthesis and characterization of three ODNs containing a nucleophile sensitive 6-chloropurine using the same strategy are described

aDim-aDmoc Protection for ODN Synthesis Allows Deprotection under Non-nucleophilic and Nearly Neutral Conditions

Over a hundred non-canonical nucleotides have been found in DNA and RNA. Many of these modified nucleotides are sensitive toward nucleophiles and bases. Because known solid phase DNA and RNA synthesis technologies require strongly nucleophilic and basic conditions for deprotection and cleavage, there is no technology for the synthesis of DNAs and RNAs containing these sensitive nucleotides. The Dim-Dmoc technology has been developed to overcome the challenge. With Dim-Dmoc protection, oligodeoxynucleotide (ODN) deprotection has be achieved with sodium periodate oxidation followed by β-elimination induced by the weak base aniline. Some sensitive groups have been shown to be stable under the deprotection conditions. Besides serving as a base, aniline also serves as a nucleophilic scavenger for the side products of Dim-Dmoc deprotection, which prevents the side products from reacting with the deprotected ODN. For this reason, excess aniline is needed. In this article, we report the use of alkyl Dim (aDim) and alkyl Dmoc (aDmoc) protecting groups for ODN synthesis. With aDim-aDmoc protection, deprotection is demonstrated to be achievable with sodium periodate oxidation followed by the non-nucleophilic base potassium carbonate at pH 8. No scavenger for the side products of deprotection is needed. Over 10 ODNs with length ranging from 10-mer to 23-mer were synthesized, and importantly, the ODNs could be easily purified with RP HPLC. It was further demonstrated that the highly sensitive N4-acetylcytidine nucleoside could survive the oxidative deprotection conditions, and ODNs containing this sensitive nucleotide could be readily synthesized and purified without the need of any special cautions. Work on extending the method for the synthesis of sensitive RNAs such as those containing the biologically important ac4C is in progress

Parallel, Large-Scale, and Long Synthetic Oligodeoxynucleotide Purification Using the Catching Full-Length Sequence by Polymerization Technique

© 2018 American Chemical Society. The catching by polymerization synthetic oligodeoxynucleotide (ODN) purification technique was shown to be potentially suitable for high-throughput purification by the purification of 12 ODNs simultaneously, to be convenient for large-scale purification by purification at the 60 μmol synthesis scale, and to be highly powerful for purification of long ODNs by purification of ODNs as long as 303-mers. LC-MS analysis indicated that the ODNs purified with the technique have excellent purity

Dim and Dmoc Protecting Groups for Oligodeoxynucleotide Synthesis

This protocol provides details for the preparation of nucleoside phosphoramidites with 1,3-dithian-2-yl-methyl (Dim) and 1,3-dithian-2-yl-methoxycarbonyl (Dmoc) as protecting groups, and a linker with Dmoc as the cleavable function, then using them for solid phase synthesis of sensitive oligodeoxynucleotides (ODNs). Using these Dim-Dmoc phosphoramidites and Dmoc linker, ODN synthesis can be achieved under typical conditions using phosphoramidite chemistry with slight modifications, and ODN deprotection and cleavage can be achieved under mild conditions involving oxidation with sodium periodate at pH 4 followed by aniline at pH 8. Under the mild deprotection and cleavage conditions, many sensitive functional groups including but not limited to esters, thioesters, alkyl halides, N-aryl amides, and α-chloroamides-which cannot survive the basic and nucleophilic deprotection and cleavage conditions such as concentrated ammonium hydroxide and dilute potassium methoxide used in typical ODN synthesis technologies-can survive. Thus, it is expected that the Dim-Dmoc ODN synthesis technology will find applications in the synthesis of ODNs that contain a wide range of sensitive functional groups. © 2020 Wiley Periodicals LLC. Basic Protocol: Synthesis, deprotection, cleavage, and purification of sensitive oligodeoxynucleotides Support Protocol 1: Synthesis of Dim-Dmoc nucleoside phosphoramidites Support Protocol 2: Preparation of CPG with a Dmoc linker Support Protocol 3: Synthesis of a phosphoramidite containing a sensitive alkyl ester group