Chapter 27, Problem 32DSP

Oligonucleotide Synthesis

In Section $\text{27}\text{.6}$ we noted that synthetic oligonucleotides of defined sequence were commercially

available for use as primers for PCR and as probes for cloning DNA. Here we will examine how these oligonucleotides are prepared.

The method bears many similarities to the Merrifield solid-phase synthesis of peptides. A starter unit is attached to a solid support, nucleosides are attached one-by-one until the sequence is complete, whereupon the target oligonucleotide is removed from the support and purified. Like solid-phase peptide synthesis, the preparation of oligonucleotides relies heavily on protecting groups and bond-forming methods.

The starter units are nucleosides in which amine groups on the DNA bases have been protected by acylation.

Thymidine lacks an $-{\text{NH}}_{\text{2}}$ group, so needs no protecting group on its pyrimidine base.

These $\text{N-protecting groups}$ remain in place throughout the synthesis. They are the first ones added and the last ones removed. None of the further “chemistry” that takes place involves the purine or pyrimidine rings.

The $\text{5'-OH}$ group of the $\text{2'-deoxyribose}$ portion of the nucleosides is primary and more reactive toward ether formation than the $\text{3'-OH}$ group, which is secondary. This difference allows selective protection of the $\text{5'-OH}$ as its $\text{4,4'-dimethoxytriphenylmethyl}$

(DMT) ether.

The nucleoside that is to serve as the $\text{3'}$ end of the final oligonucleotide is attached to a

controlled-pore glass (CPG) bead by ester formation between its unprotected $\text{3'-OH}$ and a linker unit already attached to the CPG. In order for chain elongation to proceed in the $\text{3'}\to \text{5'}$ direction, the DMT group that protects the $\text{5'-OH}$ of the starter unit is removed by treatment with dichloroacetic acid.

The stage is now set for adding the second nucleoside. The four blocked nucleosides prepared

earlier are converted to their corresponding $\text{3'-phosphoramidite}$

derivatives. An appropriate A, C, T, or G phosphoramidite is used in each successive stage of the elongation cycle.

Each phosphoramidite is coupled to the anchored nucleoside by a reaction in which the free $\text{5'-OH}$ of the anchored nucleoside displaces the diisopropylamino group from phosphorus (Figure $\text{27}\text{.15}$).The coupling is catalyzed by tetrazole, which acts as a weak acid to protonate the diisopropylamino group.

The product of the coupling is a phosphite; it has the general formula $\text{P}{\left(\text{OR}\right)}_{\text{3}}$. It is oxidized to phosphate $\left[\text{P}\left(\text{O}\right){\left(\text{OR}\right)}_{\text{3}}\right]$

in the last step of Figure $\text{27}\text{.15}$.

The $\text{5'-OH}$ of the newly added nucleoside is then deprotected to prepare the bound dinucleotide for the next elongation cycle.

Once all the nucleosides are in place and the last DMT is removed, treatment with aqueous

ammonia removes the acyl and cyanoethyl groups and cleaves the oligonucleotide from the CPG

support.

Structure 1 is the one given for tetrazole in Figure $\text{27}\text{.15}$. Structures $\text{2}$ and $\text{3}$ have the

same molecular formula $\left({\text{CH}}_{\text{2}}{\text{N}}_{\text{4}}\right)$ and the same number of electrons as $1$. How are these

structures related?

A. $\text{1}$, $\text{2}$, and $\text{3}$ are constitutional isomers.

B. $\text{1}$, $\text{2}$, and $\text{3}$ are resonance contributors of the same compound.

C. $\text{1}$ and $\text{2}$ are resonance contributors of the same compound; $\text{3}$ is an isomer of $\text{1}$ and $\text{2}$.

D. $\text{1}$ and $\text{3}$ are resonance contributors of the same compound; $\text{2}$ is an isomer of $\text{1}$ and $\text{3}$.

Section 27.6 Many important compounds contain two or more nucleotides joined together by

a phosphodiester linkage. The best known are those in which the phosphodiester joins the $5\text{'}\text{-oxygen}$ of one nucleotide to the $3\text{'}\text{-oxygen}$ of the other.

Oligonucleotides contain about $50$ or fewer nucleotides held together by

phosphodiester links; polynucleotides can contain thousands of nucleotides.