Handout 3B: Nucleic Acid Synthesis

Protection of Nucleobases in DNA synthesis

• protection a A, G, C are often done via transient protection using TMS protection to protect OH

• Amines need to be protected, as they would interfere with the synthesis w/ their nucleophilicity

  • Thymine does not need a protecting group as it doesn’t contain an amine 

• Adenine and cytosine are protected at their amino group as a benzamide

• Guanine is protected at its amine as an isobutrylamide (benzamide is difficult to remove from a guanine)

TMS

• TMS = trimethylsilyl (Me₃Si)

• Reaction of an alcohol with TMS-Cl goes via an addition/elimination mechanism, via a pentavalent intermediate

• Si-O bonds are very strong and drive the reaction selectively over formation of Si-N bonds (Si-F bonds are even stronger than Si-F bonds)

• TBAF (tetrabutylammonium fluoride) is used to remove TMS from the R-O-TMS via an addition/elimination mechanism

Guanosine Protection

Adenosine Protection

Cytosine Protection

Thymine Protection

DMT Protection

• Once the nucleobases are protected, the 5’OH can be protected as RO-DMT (4,4’-dimethoxyltrityl)

• DMT is similar to trityl, but is easier to remove and is colored, which is used to monitor reaction progress

  • Selective to primary over secondary alcohols (due to steric bulk)

DMT Incorporation

Resin Incorporation

Next, the nucleoside w/ protection at its nucleobase and DMT at its 5’OH is attached to resin.

• Resin = CPG (Controlled pore glass)

• Silica gel, glass, CPG, etc. are (SiO₂)_n, a solid polymeric matrix of silicondioxide

• attaches nucleosides (protected sugar base units) covalently to a solid support to make purification easy and allows for stepwise synthesis

Resin Incorporation Mech

Functionalization of surface silanols (Si-OH)

• Triethoxysilane reacts with surface OH groups, creating covalent Si-O-Si bonds, so the silica surface now has amine (-NH₂) functional groups sticking out (to react later with other molecules)

DMT Deprotection

• Before coupling to the next nucleoside, the DMT group must be removed. 

  • Use trichloroacetic acid, which proceeds via SN1 mechanism

  • These conditions don’t affect the other PGs, the phosphate triesters or the linkage to the CPG resin

Phosphoarmidite

• The incoming nucleoside, protected at its nucleobase and at its 5’OH is then activated at its 3’OH as a phosphoramidite using cyanoethylchloro-N,N-diisopropylphosphoramidite 

  • on its own it’s not reactive enough to directly react with the 5’ OH on the growing DNA chain and has a poor leaving group

• The reaction at the phosphoryl chloride proceeds via an SN2-like mechanism on the P atom

• figure: activation of incoming nucleotides

Coupling

• Coupling then occurs using tetrazole as the nucleophilic catalyst 

  • Tetrazole has a pK_a of 14.5 in CH₃CN (solvent)

  • The charge is delocalized on all ring atoms

• The coupling reaction likely proceeds via nucleophilic catalysis, where the tetrazole anion is the nucleophilic catalyst

• Tetrazole protonates the amine on the phosphoramidite, turning the amine into a better leaving group 

• Then, tetrazole anion attacks P, and we have a reactive intermediate where tetrazole is a better leaving group than NR₂ and phosphorus becomes more electrophilic

• Coupling: now the 5’OH of the growing chain attacks, tetrazole leaves and the new P-O bond forms

Coupling Mechanism

Capping

• Following each coupling, capping is often done using acetic anhydride and 1-methylimidazole (nucleophilic catalyst)

• This makes purification easier, as most of the impurities are kept small and easy to separate from the large product

• Capping is done before oxidation

• Only free 5’OH, those that have not reacted with the previous nucleoside, will be capped; the rest are left unchanged

Phosphite Oxidation

• Following each coupling, the ensuing phosphite is oxidized to the phosphate, because the phosphite is not sufficiently stable to survive repeated cycles

• Phosphite ester → oxidation → phosphate ester

After all the couplings…

• Once all couplings are complete, the last DMT is removed (using trichloroacetic acid).

  • RO-DMT → acid → ROH

Concentrated ammonium hydroxide (NH₄OH) at 65ºC for one hour:

• cyanoethyl removal is via an E1 cb (elimination via conjugate base) reaction

  • All cyanoethyls are removed together at the end of the synthesis, not after each cycle

  • Base removes a proton → forms a carbanion → carbanion eliminates → kicks out the phosphate oxygen

• nucleobases are deprotected via transamidation (remember the amine groups were protected)

  • Ammonia replaces the protecting group

•  The cleavage from the resin is ester hydrolysis

Deprotection of Phosphotriester (Cyanoethyl Removal)

Deprotection of nucleobases

Cleavage

Summary of solid phase DNA synthesis:

1. Link nucleotide to CPG protected at the nucleobase (purine/pyrimidine) and the 5’OH 

2. Protect the incoming nucleotide at its nucleobase and its 5’OH

3. Incorporate the phosphoramidite into incoming nucleotide

4. Do the phosphoramidite coupling

5. Cap unreacted 5’OH’s

6. Oxidize the phosphite to the phosphate

7. Deprotect the new 5’OH

8. Repeat steps 2-7 for each nucleotide

9. Remove final DMT; then cleave oligo from the CPG and remove protecting groups from the nucleobases and the cyanoethyls from phosphotriesters