Many hydroxyl and amine moieties make nucleic acids very animated molecules with rich chemistries of their own that can interfere with the phosphite triester reactions used to couple the nucleotide monomers; therefore, protection strategies are necessary in chemical synthesis to mask the functional groups on the monomers so that the only significant reaction is the desired 3' to 5' sequential condensation of monomers to the growing oligonucleotide. (Note that enzymatic polymerizations occur with the opposite directionality and require no masking of monomer functionality.) These synthetic protecting groups must be chosenso that they can be removed easily to reveal the natural nucleotides.
The fully protected monomers for nucleic acid synthesis are generally called phosphoramidites. In traditional protection schemes the nucleophilic amino functions on the bases are protected with either isobutyryl (N-2 of guanine) or benzoyl (N-6 of adenine and N-4 of cytidine) groups, both of which can be removed at the completion of synthesis by ammoniolysis. However, recent advances have lead to the wide spread use of phenoxyacetyl (PAC) protection of adenosine, dimethyl formadine (Dmf) protection of gaunosine, and acyl protection of cytosine to yield oligonucleotides which can be deprotected rapidly under very mild conditions.The 5' primary hydroxyl of the ribose sugar is protected with a dimethoxytrityl (DMT) ether moiety which is removed by acids at the beginning of each coupling cycle. The efficiency of synthesis at each coupling cycle can be monitored by detecting the release of the chromophore trityl cation. For the synthesis of nucleic acids with the natural phosphodiester backbone, the 3' secondary hydroxyl function of the ribose sugar is derivatized with a highly reactive phosphitylating agent. The phosphate oxygen on this moiety is usually masked by ß-cyanoethoxy and diisopropylamine protecting groups. By insulating the phosphate oxygen with alternative groups, modified phosphate backbones may be accessed. Finally, for ribonucleic acids the secondary 2'-hydroxyl of the ribose is shielded throughout the chemical synthesis by the tert-butyl dimethyl silyl (TBDMS) group.
The genius of the protecting groups for automated nucleic acid synthesis is that they yield nearly lesion free natural nucleic acids with high efficiency through simple hydrolysis, nucleophilic displacement, and redox chemistries. In a standard synthesis cycle, the nucleotide chain grows from an initial protected nucleoside deriviatized via its terminal 3' hydroxyl to a solid support. Reagents and solvents are pumped through the support to induce the consecutive removal and addition of sugar protecting groups in order to isolate the reactivity of a specific chemical moiety on the monomer and affect its stepwise addition to the growing oligonucleotide chain. This design eliminates the need to purify synthetic intermediates or unreacted reagents because they are simply rinsed off the column at the end of each chemical step. Assembly of the protected oligonucleotide chain is carried out in four chemical steps: deblocking, activation-coupling, oxidation, and capping.
1. DEBLOCKING. The synthesis cycle begins with the removal of the acid labile DMT ether from the 5'-hydroxyl of the 3'-terminal nucleoside. This is usually accomplished by using trichloroacetic acid (TCA) or dichloroacetic acid (DCA) in dichloromethane. The resulting tritylcation chromophore can be quantitiated to determine coupling efficiency. After deblocking,the 5'-hydroxyl is the only reactive nucleophile capable of participating in the subsequent coupling step. Since the nitrogenous bases of the growing DNA chain are susceptible to acid catalyzed depurination, the deblocking step is short, and an acetonitrile rinse thoroughly removes the deblocking agent from the support. Also, coupling efficiency and accuracy are increased by this wash since premature detritylation of the incoming phosphoramidite monomer is prevented.
2. ACTIVATION-COUPLING. Following deblocking of the 5'-hydroxyl group, the next protected phosphoramiditeis delivered to the reaction column along with the weakly acidic activator tetrazole (pKa = 4.8). Nucleophilic attack of the previously freed 5'-hydroxyl upon the incoming monomer's diisopropylamine-protected phosphorus, which was activated via protonation by tetrazole, elongates the nucleic acid chain. Because this protonated phosphoramidite is so reactive, the coupling reaction is usually complete within 30 seconds. A molar excess of tetrazole over the phosphoramidite ensures complete activation, and a molar excess of phosphoramidite to free 5' hydroxyls of the growing chain promotes efficient coupling. To optimize the coupling efficiency, the amounts of reagents injected and the coupling time can be varied.
3. CAPPING. In spite of these efficiency measures, a small percentage of the support-bound nucleoside's 5'-hydroxyls do not couple to the incoming activated monomer. They must be rendered inactive to minimize deletion products and simplify the purification process. Usually, acetic anhydride and N-methyl-imidazole dissolved in pyridine and tetrahydrofuran (THF) act to create an acylating agent that "caps" the unextended 5'-hydroxyls. The 5' acetyl ester cap is unreactive in all subsequent cycles and is removed during the final ammonia deprotection step. Additional acetonitrile washing subsequent to capping can increase synthetic yield. After coupling and capping the internucleotide linkageis a trivalent phosphite triester that is extremely unstable and must be oxidized to a phosphotriester which will ultimately yield natural DNA.
4. OXIDATION. In the last step of the cycle, the unstable phosphite triester linkages are oxidized to a more stable phosphotriester by 0.02 M iodine dissolved in water:pyridine:THF. An iodine-pyridine adduct forms to the phosphite triester which is subsequently displaced by water to yield phosphorus oxidized to the pentavalent state.Pyridine also neutralizes the hydrogen iodide byproduct. Because the oxidizer contains water, the support is rinsed several times with acetonitrile following this reaction. One cycle of monomer addition is then complete, and another cycle begins with the removal of the 5' DMT from the previously added monomer.
At the end of the synthesis, the final trityl can either be removed with a final acid wash ("trityl-off"), or can be left on for purification purposes ("trityl-on"). The oligonucleotide itself is removed from the support with concentrated ammonium hydroxide. Additionally, this treatment deprotects the phosphorus by ß-elimination of the cyanoethyl group and removes the protecting groups on the heterocyclic bases to yield a single stranded nucleic acid.
RNA chemical synthesisis identical to that used for DNA except for the presence of an additional protecting group at the 2' hydroxyl of ribose. This position is usually protected with tertbutyldimethyl silyl (TBDMS) groups which are stable throughout the synthesis. They are removed at the final deprotection step by the basic fluoride ion. The remaining positions on both the sugar and the bases are protected in the same fashion as for DNA. By adjusting several parameters in the DNA synthesis protocol includingthe coupling times, monomer delivery rate, frequency of washing steps, and types of capping reagents, stepwise coupling efficiencies of up to 99% can be obtained.
For longer oligonucleotide synthesis (>100 bases), 1) Use dichloroaceticacid (DCA) for deblocking as opposed to trichloroacetic acid (TCA) if the synthesizer is compatible with this reagent. Depurination (cleavage of the glycosidic bond) under acidic conditions is a prominent side reaction that ultimately limits DNA synthesis; 2) Modify the synthesis protocol to increase the coupling time of the phosphoramidite. Also, additional methylene chloride wash steps included prior and subsequent to deblocking along with increased acetonitrile washing subsequent to capping leads to increased yields; 3) Increase the phosphoramidite concentration to enhance the coupling efficiency.
