Nucleic Acids in Chemistry and Biology

Synthesis of Oligonucleotides 151

sites) are beyond the scope of this Chapter. It is sufficient to note that a long spacer is used to extend the sites
away from the surface and ensure accessibility to all reagents. One type of spacer is illustrated (Figure 4.10).
The loading of amino groups on the glass is best kept within a narrow band of 30–80mol g^1 , below which
the reactions become irreproducible and above which they are subject to steric crowding between chains.
Highly cross-linked polystyrene beads have the advantage of good moisture exclusion properties, and allow
efficient oligonucleotide synthesis on an extremely small scale (10 nmole).
The 3
-terminal deoxyribonucleoside of the oligonucleotide to be synthesised is attached to the solid
support viaan ester linkage by conversion of the protected 5
-O-DMT derivative into its corresponding active
succinate ester, which is subsequently reacted with amino groups on the support (Figure 4.10). An assem-
bled oligonucleotide is released from the support by treatment with ammonia. Several other types of
derivatised solid supports are now available, which are obtainable through reagent suppliers.

4.1.4.2 Assembly of Oligonucleotide Chains. Assembly of the protected oligonucleotide chain is

carried out by packing a small column of deoxynucleoside-loaded support and flowing solvents and
reagents through in predetermined sequence. Columns containing only a few milligrams (10 nmole) up to
tens of grams (1 mmole or more) can be used. Small-scale assembly is usually accomplished by use of
a commercial DNA Synthesiser. Machine specifications vary, but the basic steps for oligonucleotide syn-
thesis are as shown in Figure 4.11.
Step 1. Detritylation (removal of the 5
-DMT group) is carried out with dichloroacetic or trichloroacetic
acid in dichloromethane. The orange colour from the dimethoxytrityl cation liberated from this step is
compared by intensity in a UV–Visible spectrometer to obtain the coupling efficiencyof the previous step.
Step 2. Activation of the phosphoramidite occurs when it is mixed with coupling agent (4,5-dicyanoim-
idazole, tetrazole or a derivative such as S-ethyl thiotetrazole) in acetonitrile solution (see Figure 3.56).
Step 3. Addition of the activated phosphoramidite to the growing chain.
Step 4. Capping is a safety step introduced to block chains that have not reacted during the coupling
reaction and also limits the number of failure sequences. A fortuitous benefit of this step is that phosphity-
lation of the O-6-position of guanosine is reversed. This is carried out using a mixture of two solutions:
acetic anhydride/2,6-lutidine and N-methylimidazole each in tetrahydrofuran (THF).
Step 5. Oxidation of the intermediate phosphite to the phosphate triester is achieved with iodine and
water in THF. Pyridine or 2,6-lutidine is added to neutralise the hydrogen iodide liberated.

B

O

HO

DMTO

B

O

O

DMTO

O

O

OH

B

O

O

DMTO

O

O

NO 2

B

O

O

DMTO

O

O

HN N

H

O O

O

OAc

H 2 N N

H

O O

O

OAc

O O O

NN

CH 3

CH 3

HO

NO 2

CPG

CPG

Figure 4.10 Attachment of a 5
-protected nucleoside to a solid support of controlled pore glass (CPG) functionalised

by a long chain alkylamine