Nucleic Acids in Chemistry and Biology

152 Chapter 4

This cycle is repeated the requisite number of times for the length of the oligonucleotide required, with
each deoxynucleoside phosphoramidite added in the desired sequence. Synthesis by this method is carried
out in the 3
→ 5
direction.
The traditional method for the synthesis of oligonucleotides outlined above is in the 3
→ 5
direction.
However, there are applications where it is desirable to reverse this direction of synthesis, for example,
when oligonucleotides are required with their 5
-end attached to a support such as on a microarray chip
or to a bead. In such cases, synthesis in the 5
→ 3
direction has been made possible by the use of
5
-phosphoramidite building blocks. The overall chemical strategy for synthesis remains unchanged, but
the functional groups on the 3
- and 5
-hydroxyl groups are exchanged.

4.1.4.3 Deprotection and Removal of Oligonucleotides from the Support. Unless there is a need to

purify the oligonucleotides by reversed phase chromatography (see Section 4.1.4.4), the 5
-DMT group must
first be removed using the same conditions as those used during oligonucleotide synthesis. If phosphoramidite
chemistry has been used, then deprotection and removal of the oligonucleotide from the solid support is carried
out in a single step. The solid support-bound oligonucleotide is treated with concentrated ammonia for 30 min
and the column is then washed with a further portion of ammonia solution. This serves to cleave the oligonu-
cleotide from the solid support. Nucleobase and phosphate protecting groups (2-cyanoethyl is removed from
the phosphate by a -elimination reaction, Figure 3.47) are then removed by heating an ammoniacal solu-
tion at 50°C overnight. Shorter deprotection times and lower temperatures are used when the mild-depro-
tection groups (PAC, etc.) are used. If methyl phosphoramidites are used, then the methyl group may be
removed by treatment with thiophenolate ion (generated with thiophenol and triethylamine) prior to treatment
with ammonia (Figure 3.46b). Lyophilisation of the ammonia solution gives the crude oligonucleotide.
In phosphotriester chemistry, the phosphate aryl group is selectively displaced by use of syn-2-nitrobenz-
aldoximate ion or by 2-pyridine-carbaldoximate ion (Figure 3.48). The product of this reaction undergoes
elimination in the presence of water. Removal of the base protecting groups and cleavage of the oligonu-
cleotide from the solid support is then carried out with ammonia as described above.

B 1

O

O

DMTO

B^1

O

O

HO

B^2

O

O

DMTO

NC OP

NiPr 2

B^1

O

O

O

B^2

O

O

DMTO

NC OP

B^1

O

O

O

B^2

O

O

DMTO

NC O P

B^1

O

O

O

B^2

O

O

DMTO

NC OOP

1. Deprotection
   TCA/CH 2 Cl 2
   2. Condensation
   tetrazole or
   4,5-dicyanoimidazole/CH 3 CN


2. Capping
   Ac 2 O/N-methylimidazole/
   THF/pyridine


3. Oxidation
   I 2 /H 2 O/THF/pyridine

Figure 4.11 Basic steps in a cycle of nucleotide addition by the phosphoramidite method

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