Biochemistry II, BCH 3034L
DNA Synthesis Chemistry
Synthesis Cycles
• Each cycle of base addition consist of four steps.
Detritylation
Coupling
Capping
Oxidation
• These reaction steps are repeated in the above order
until all bases are added. Following synthesis, the
DNA chain must be cleaved from the solid support
and deprotected.
1
Synthesis Cycle
The Solid Support - CPG
• The support used in the DNA synthesis is controlledpore glass (CPG) and polystyrene.
• The polystyrene has an aminomethyl linker attached
to its surface.
• The support coupling efficiency is about 98%.
• The supports are covalently derivatized with one of
the four nucleosides.
• The reactive group on these nucleosides are blocked
or protected to prevent unwanted side reactions.
• They are all blocked at the 5′-hydroxyl with a
dimethoxytrityl (DMT) group.
2
DMT-protected nucleoside
Phosphoramidites nucleosides
• Phosphoramidites are chemically modified nucleosides used
as the building blocks for synthesis chemistry.
• Standard amidites use a benzoyl group to protect adenosine
and cytidine.
• A isobutyryl group is used to protect guanosine.
• Thymidine is unreactive and does not need a protecting group
because there are no exocyclic amines.
• These protecting groups prevent side reactions and are
removed with ammonia after completing of synthesis.
3
Protected exocyclic base amines for standard
phosphoramidites
Detritylation
• Just prior to detritylation the support is washed with
acetonitrile to eliminate traces of the preceding
reagents.
• Several flushes of argon eliminate the acetonitrile.
• The first step in oligonucleotide synthesis is removal
of the acid labile, dimethoxytrityl (DMT) protecting
group in the 5′ -hydroxyl of the support-bound
nucleoside.
• Treatment with a protic acid, TCA, will deprotect or
detritylate the 5′ end.
• This reaction will yield a reactive 5′ hydroxyl, which
can couple with a phosphoramidite.
4
Detritylation
Coupling
• Phosphoramidites are added to the support-bound
nucleotide chain one base at a time, until all bases in
the sequence are coupled.
5
Coupling
• The phosphoramities
nucleosides have the
following functional groups:
A diisopropylamino on a 3′
trivalent phosphorus moiety.
A β-cyanethyl protecting group
on the 3′ phosphorus moiety,
which prevents side reactions.
Coupling
• The phosphoramidites and tetrazole are
simultaneously delivered to the column. Several
deliveries of tetrazole are delivered to the column.
• When these reagents mix, the mild acid, tetrazole
transfers a proton to the nitrogen of the diisopropyl
group on the 3′ phosphorus.
• This protonated amine makes a very good leaving
group.
6
Coupling
Capping
A small percentage (about 2%) of support-bound
nucleotide will fail to undergo addition.
These truncated, or failure sequences, will remain
attached to the support.
They can propagate in subsequent coupling steps
producing a sequence with one less base.
Capping the remaining free hydroxyls by acetylation
eliminates this problem.
Capped failure sequences are prevented from
participating in the rest of the synthesis reactions.
7
Capping
Two reagents, acetic anhydride and 1-methylimidazole
are simultaneously delivered to the column.
The reagents react at the 5′ -hydroxyls, rendering
them unreactive for the remainder of the synthesis
reaction.
The capping time to acetylene about 2% unreacted 5′
hydroxyls is very brief.
The excess is removed by argon reverse flush.
Capping of unreacted chains
8
Oxidation
• The newly formed nucleotide is a phosphite (trivalent
phosphorus).
• The phosphite linkage is unstable and susceptible to
acid and base cleavage.
• Therefore, immediately after capping, the trivalent
phosphite is oxidized to a stable pentavalent
phosphate triester.
• Iodine and pyridine are used as a mild oxidant in a
basic tetrahydrofuran (THF) solution.
Oxidation of the trivalent phosphorous
9
Synthesis
Cycle
10