3 Nucleic acids
3.1 Structure and synthesis of Nucleic acids
3.1.1 Structure and function of nucleic acids
The structure of oligonucleotides consists of individual nucleotides linked by a
DNA/RNA synthesis is now done routinely using established protocols, and can often be
obtained from commercial sources. The versatility of oligonucleotide synthesis is a central
technology for molecular biology, as it is used to generate specific primers and other reagents
necessary for these methods.
3.1.2 Chemical Synthesis of Nucleic acids
Several strategies have been developed for the synthesis of oligonucleotides. The central
problem to be solved in oligonucleotide synthesis is the efficient generation of the
phosphodiester. A brief summary in approximate chronological order is given below.
184.108.40.206 Phosphate coupling
Coupling using a phosphate was one of the earliest strategies applied. In the scheme below,
where X is a good leaving group.
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R Base O O
Nomenclature of O
phosphorous derivatives 1 OH
O P O X
O- O Base
O P O O
O P X Base
OH HO O
An example would be the use of TsCl to form the tosylated
phosphate ester, which is then reactive with the 3’-OH of
the other residue. Coupling reagents, such as carbodiimides
may also be used to accelerate this type of coupling.
Khorana received the Nobel prize for work on this problem
in 1968 (Chemistry).
220.127.116.11 Phosphite coupling
Phosphites have been used to generate phosphodiester
bonds. Letsinger, at Northwestern, identified that
phosphites under acidic conditions react very rapidly with
nucleophiles, and this principle was developed to generate
phosphites that could be oxidized to the phosphate diester
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The phosphite strategy has been expanded to phosphoramidites. This is the most commonly
applied system for oligonucleotide synthesis. The phosphonium species are much more reactive
with nucleophiles than the corresponding phosphite or phosphoramidite, which is desirable for
coupling reactions. One rationale for this is that the phosphite has fewer O lp, which would donate
electron density to the empty orbitals on P. However, the phosphonium which results from
protonation of a phosphite is much more reactive with water, making this pathway difficult to
implement. The phosphoramidites are less susceptible to reaction with water, and retain the
electrophilic character needed for coupling. The identity of the alkyl groups (R) can be selected
to allow easy conversion of the resulting pentavalent phosphorous species to the desired
18.104.22.168 Typical modern strategy for oligonucleotide synthesis
General reviews of the synthesis of oligonucleotides are available. Abriefsummaryofthe
key features used in modern oligonucleotide synthesis is given below.
22.214.171.124.1 Solid support
Analogous to the iterative strategy used in peptide synthesis (discussed in Sec 2), solid
support has advantages for the generation of oligonucleotides. Although polystyrene supports
have been used for oligonucleotide synthesis, it is more typical to use controlled pore glass
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OH OH OH
Si Si Si
O O OH
O O O OH
Si NH2 Si Si Si
O O O O
R O O
O cleavage synthesis O
HN conc. N3
Si OH OH
O O OH Si
O O OH
Si Si Si
O O Si Si Si
126.96.36.199.2 5’-OH Protection
The 5’-OH protecting group is an important feature of the strategy, as it will need to be
removed at every coupling step. Therefore, it should have relatively mild conditions for removal.
The Trityl group (Trt) is the most commonly employed protecting group for this purpose.
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The general coupling scheme relies on use of a phosphoramidite:
The specific implementation is shown below, where the 3’-OH is R1, and 5’-OH is R2. 1H
Tetrazole is used as an additive which generates a reactive P-tetrazolyl intermediate. The
tetrazole has a remarkably low pKa (4.9).
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Note that the resulting phosphite product after each coupling step is very reactive. To avoid
cleavage, the nucleotide chain must be capped and oxidized shortly after coupling.
Capping is used to prevent any unreacted 5’-OH groups from continuing to be used later in
the synthesis. Having a capping step should reduce the number of side products formed from
deletion of an individual residue. Typical capping conditions are acetic anhydride and N,N-
Oxidation of the phosphite to a phosphate is performed with iodine and water in the presence
of a strong base, lutidine.
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188.8.131.52.6 Cleavage and deprotection
The final cleavage step is performed using concentrated ammonia, to cleave the ester linkage
to the CPG, and simultaneously cleave the cyanoethylester. The elimination reaction can be
referred to as A N +E , oN an E cb 1E conj1gate base) mechanism.
184.108.40.206.7 Summary of iterative synthesis
A summary of the scheme for modern synthesis of a DNA sequence is shown below. This
scheme uses the convention that individual nucleotide residues are represented as a vertical line,
with the 5'-OH shown on an upward diagonal line, and the 3'-OH on a downward diagonal line.
ixE1cb mechanism is discussed on pg 1488-1489, March 6 Ed.
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3' 1. Solid support
HO OH O CPG
O HO DMT O
TCA 2. 5' deprotection
5. cleavage HO
2) NH (aq) N
O N 3. coupling
O O H P N
O P O R O
HO OH n
O P O
1) Ac O, DMAP
2 4. Capping and
2) 2 ,2 O, Oxidation
O O CPG
O P O
DMT O R O
3.2 Modification & reactivity of nucleic acids
3.2.1 Synthesis of modified oligonucleotides
Oligonucleotides have been dissected with a variety of strategies which allow for
modification of the nucleotide base, the ribose backbone, and the phosphodiester linkage.
Excellent reviews are available with additional examples.
220.127.116.11 Modified bases
Many modified bases have been generated, too many in fact to summarize in much detail
here., 5A few examples to illustrate the versatility available to nucleotide chemists are given
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18.104.22.168.1 5’-OH Modificiation
The most common strategy is to introduce modifications at the 5' end, this can most easily be
done using a modified phorphoramidate.
Alternatively, a thiol nucleophile can be installed in place of the5'OH,allowingselective
coupling at this position. Oligonucleotides with a 5’-SH or 5’-NH have b2en synthesized and
can be easily used for conjugation chemistry.
A number of useful reagents are available for introduction of other reactive handles.
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22.214.171.124.2 3’-OH Modification
To modify the 3' end of an oligonucleotide, the linkage to solid support can be exploited.
Commercially available reagents are available that allow simple incorporation of a thiol at the 3'
DMT O Base
S O O N CPG