Nucleic Acids in Chemistry and Biology

Synthesis of Oligonucleotides 153

4.1.4.4 Purification of the Oligonucleotides. The average yield for each step during an oligonu-

cleotide synthesis is usually in excess of 98%, but for a long oligonucleotide this will correspond to a sig-

nificant quantity of impurities and truncated oligonucleotides. The efficient removal of these impurities is

an important process in the synthesis of oligonucleotides, and powerful separation methods have been

developed for purification of microgram to milligram quantities of oligonucleotides.

4.1.4.4.1 Polyacrylamide Gel Electrophoresis (PAGE). PAGE separates oligonucleotides

according to their unit charge difference (see Section 11.4.3). Oligonucleotides are applied to thick gels

(1–2 mm) and after electrophoresis, the presence of the oligonucleotides may be detected with short wavelength

(254 nm) UV light and the appropriate band cut out. The oligonucleotide may then be removed from the gel

either by a soaking buffer or by electro-elution, followed by a desalting step using either a desalting column

or dialysis. This method of purification is suitable for oligonucleotides of any length: short oligonucleotides

being separated in a high percentage polyacrylamide gel (e.g.20%) and longer oligonucleotides separated

using lower polyacrylamide gel concentrations.

4.1.4.4.2 High Performance Liquid Chromatography (HPLC). HPLC is particularly suit-

able for purification of oligonucleotides. Ion exchange chromatography resolves predominantly by charge

difference, and can be used both analytically and preparatively for oligonucleotides up to about 100

residues long. Reversed phase HPLC separates according to hydrophobicity, but the elution profile is less

predictable than ion exchange chromatography. A common and more reliable method is to purify oligonu-

cleotides before removal of the 5
-terminal DMT group, where the oligonucleotide will be resolved from

the shorter non-DMT containing impurities. The 5
-DMT group is then cleaved after purification, and may

be removed by a reversed-phase desalting cartridge or on a small gel filtration column.

There have been a number of recent protecting group strategies and improved reagents for the synthe-

sis cycle devised to improve the yields of oligonucleotides even further. For example, in a recent method

developed by Caruthers, aryloxycarbonyl protection is used for the 5
-hydroxyl group and DMT protec-

tion for the nucleobase exocyclic amino groups (Figure 4.12). After coupling in the usual manner, treatment

of the extended chain with peroxy anions at pH 9.6 simultaneously cleaves the 5
-carbonate protection and

oxidises the internucleoside phosphite linkage. In this way the number of steps in the synthesis cycle is

reduced to two, resulting in a shorter nucleotide addition cycle.

4.2 Synthesis of Oligoribonucleotides

The development of effective chemical methods for the synthesis of oligoribonucleotides has been slower

than for oligodeoxyribonucleotides, largely because of the need to find a suitable protecting group for the

O B

O

O B

O

O

O

RO

P

O

NC

O

CPG

O B

O

O B

O

HO

P

O

NC

O

CPG

O

R = 4-chlorophenyl or 3-(trifluoromethyl)phenyl

MCPBA, pH 9.6

Figure 4.12 Simultaneous oxidation of the internucleotide linkage and removal of the 5
-aryloxycarbonyl protecting
group carried out with m-chloroperbenzoic acid (MCPBA) in the presence of lithium hydroxide and
2-amino-2-methyl-1-propanol at pH 9.6 by the phosphoramidite method