Artificial DNA Directed toward Synthetic Metallofoldamers - Metallofoldamers: Supramolecular Architectures from Helicates to Biomimetics - page 312

interference with the automated DNA synthesis process, their full removal from the final
oligonucleotide, preferably in aqueous medium and without harming the DNA molecule,
must be tested in control experiments beforehand.
9.3.4 Automated Oligonucleotide Synthesis
Having the artificial phosphoramidite building block in hand, incorporation into oligonu-
cleotides is achieved by automated DNA synthesis on a commercially available machine
along with the natural nucleotides [7]. The machine, termed an oligonucleotide synthe-
sizer (or DNA synthesizer), is merely a computer-controled system of pumps, tubes and
valves working strictly anhydrously under a protecting gas in order to deliver all needed
reagents in the right amounts and temporal sequence. The underlying chemical principle
is solid-phase synthesis using porous glass or polystyrene beads as the solid support on
which the first nucleotide is usually already bound (Figure 9.7). The cyclic process lead-
ing to a stepwise growing of the single-stranded oligonucleotide sequence can be divided
into the following four steps:
1. In the coupling step, the free 5 0 -OH group of the last nucleotide of the bound nascent
strand reacts in a nucleophilic substitution reaction with the added phosphoramidite
(activated at the phosphorous center) under formation of a phosphorus(III) ester.
2. In the following capping step, unreacted oligonucleotide strands are inactivated for
further elongation (leading to false sequences) by blocking the free 5 0 -OH groups via
esterification with acetic anhydride from further reaction.
3. Subsequently, the unstable phosphorus(III) center is oxidized to a phosphorus(V) ester
using an iodine containing reagent.
4. In the following deprotection step, the 5 0 O -DMT group is cleaved from the newly
coupled nucleoside by the use of acid.
The remainder of the DMT protecting group, the 4,4 0 -dimethoxytrityl cation, gives an
intense red color to the solution leaving the solid support cartridge. This is collected in an
integrated photometer cell in a modern DNA synthesizer machine in order to estimate its
molar amount, which is proportional to the amount of deprotected oligonucleotide and
can be taken as a comparative measure for the coupling efficiency in each cycle. The
Figure 9.7 The cycle of automated DNA synthesis based on the phosphoramidite protocol.