Sensitive Oligodeoxynucleotide Synthesis Using Dim and Dmoc as Protecting Groups.

In traditional oligodeoxynucleotide (ODN) synthesis, phosphate groups are protected with the 2-cyanoethyl group, and amino groups are protected with acyl groups. At the end of ODN synthesis, deprotection is achieved with strong bases and nucleophiles. Therefore, traditional technologies are not suitable for the synthesis of ODNs containing sensitive functionalities. To address the problem, we report the use of Dim and Dmoc groups, which are based on the 1,3-dithian-2-yl-methyl function, for phosphate and amine protection for the solid phase ODN synthesis. Using the new Dim-Dmoc protection, deprotection was achieved under mild oxidative conditions without using any strong bases and nucleophiles. As a result, the new technology is suitable for the synthesis of ODNs containing sensitive functions. To demonstrate feasibility, seven 20-mer ODNs including four that contain sensitive ester and alkyl chloride groups were synthesized, purified with RP HPLC, and characterized with MALDI-TOF MS and enzyme digestion essays. High purity ODNs were obtained.

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.

dM-Dim for Carboxylic Acid Protection.

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.

An amine protecting group deprotectable under nearly neutral oxidative conditions.

The 1,3-dithiane-based dM-Dmoc group was studied for the protection of amino groups. Protection was achieved under mild conditions for aliphatic amines, and under highly reactive conditions for the less reactive arylamines. Moderate to excellent yields were obtained. Deprotection was performed by oxidation followed by treating with a weak base. The yields were good to excellent. The new amino protecting group offers a different dimension of orthogonality in reference to the commonly used amino protecting groups in terms of deprotection conditions. It is expected to allow a collection of transformations to be carried out on the protected substrates that are unattainable using any known protecting groups.

Tritylation of Alcohols under Mild Conditions without Using Silver Salts.

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.

Oligodeoxynucleotide Synthesis Under Non-Nucleophilic Deprotection Conditions.

This protocol describes a method for the incorporation of sensitive functional groups into oligodeoxynucleotides (ODNs). The nucleophile-sensitive epigenetic N4-acetyldeoxycytosine (4acC) DNA modification is used as an example, but other sensitive groups can also be incorporated, e.g., alkyl halide, α-haloamide, alkyl ester, aryl ester, thioester, and chloropurine groups, all of which are unstable under the basic and nucleophilic deprotection and cleavage conditions used in standard ODN synthesis methods. The method uses a 1,3-dithian-2-yl-methoxycarbonyl (Dmoc) group that carries a methyl group at the carbon of the methoxy moiety (meDmoc) for the protection of exo-amines of nucleobases. The growing ODN is anchored to a solid support via a Dmoc linker. With these protecting and linking strategies, ODN deprotection and cleavage are achieved without using any strong bases and nucleophiles. Instead, they can be carried out under nearly neutral non-nucleophilic oxidative conditions. To increase the length of ODNs that can be synthesized using the meDmoc method, the protocol also describes the synthesis of a PEGylated Dmoc (pDmoc) phosphoramidite. With some of the nucleotides being incorporated with pDmoc-CE phosphoramidite, the growing ODN on the solid support carries PEG moieties and becomes more soluble, thus enabling longer ODN synthesis. The ODN synthesis method described in this protocol is expected to make many sensitive ODNs that are difficult to synthesize accessible to researchers in multiple areas, such as epigenetics, nanopore sequencing, nucleic acid-protein interactions, antisense drug development, DNA alkylation carcinogenesis, and DNA nanotechnology. © 2024 Wiley Periodicals LLC. Basic Protocol: Sensitive ODN synthesis Support Protocol 1: Synthesis of meDmoc-CE phosphoramidites Support Protocol 2: Synthesis of a pDmoc-CE phosphoramidite.

Oligonucleotide synthesis under mild deprotection conditions.

Over a hundred non-canonical nucleotides have been found in DNA and RNA. Many of them are sensitive toward nucleophiles. Because known oligonucleotide synthesis technologies require nucleophilic conditions for deprotection, currently there is no suitable technology for their synthesis. The recently disclosed method regarding the use of 1,3-dithian-2-yl-methyl (Dim) for phosphate protection and 1,3-dithian-2-yl-methoxycarbonyl (Dmoc) for amino protection can solve the problem. With Dim-Dmoc protection, oligodeoxynucleotide (ODN) deprotection can be achieved with NaIO4 followed by aniline. Some sensitive groups have been determined to be stable under these conditions. Besides serving as a base, aniline also serves as a nucleophilic scavenger, which prevents deprotection side products from reacting with ODN. For this reason, excess aniline is needed. Here, we report the use of alkyl Dim (aDim) and alkyl Dmoc (aDmoc) for ODN synthesis. With aDim-aDmoc protection, deprotection is achieved with NaIO4 followed by K2CO3. No nucleophilic scavenger such as aniline is needed. Over 10 ODNs including one containing the highly sensitive N4-acetylcytidine were synthesized. Work on extending the method for sensitive RNA synthesis is in progress.

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.

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 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.

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

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

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.

Synthesis of Oligodeoxynucleotides Containing Electrophilic Groups.

By use of 1,3-dithian-2-yl-methoxycarbonyl (Dmoc) as a protecting group and linker for oligodeoxynucleotide (ODN) synthesis, deprotection and cleavage are achieved under non-nucleophilic oxidative conditions. The nucleophile-sensitive thioester and α-chloroacetyl groups are conveniently incorporated into ODN sequences. The technology could be universally useful for electrophilic ODN synthesis.