Organic Chemistry

(Dana P.) #1

064 CHAPTER 27 Nucleosides, Nucleotides, and Nucleic Acids


The groups of cytosine, adenine, and guanine are nucleophiles and therefore
must be protected to prevent them from reacting with a newly added monomer. The
groups of cytosine and adenine are protected as benzamides and the group
of guanine is protected as a butanamide. Thymine does not contain any nucleophilic
groups, so it does not have to be protected.

After the oligonucleotide is synthesized, the protecting groups on the phosphates
and the protecting groups on the bases must be removed, and the oligonucleotide has
to be detached from the solid support. This can all be done in a single step using aque-
ous ammonia. Because a hydrogen bonded to a carbon adjacent to a cyano group is
relatively acidic (Section 19.1), ammonia can be used as the base in the elimination re-
action that removes the phosphate protecting group.

Currently, automated DNA synthesizers can synthesize nucleic acids containing as
many as 130 nucleotides in acceptable yields, adding one nucleotide every 2 to 3 min-
utes. Longer nucleic acids can be prepared by splicing together two or more individu-
ally prepared strands. To ensure a good overall yield of oligonucleotide, the addition
of each monomer must occur in high yield This can be accomplished by
using a large excess of monomer. This, however, makes oligonucleotide synthesis very
expensive because the unreacted monomers are wasted.

H-Phosphonate Monomers
A second method for synthesizing oligonucleotides with specific base sequences,
using H-phosphonate monomers, has the advantage over the phosphoramidite
method in that the monomers are easier to handle and phosphate protecting groups

( 7 98%).

N C OR

HH

NH 3 +NH 4

OR

O PO +

OR

OR

−O PO

N C

NH 2

NH 2

N

N N

N

N

N

O

OO

H 2 N N

HN N

HN

N

O

O

NH N

HN N

N

N

N

N

N

N

unprotected bases protected bases

CH 3 CH 2 CH 2 CCl

O

CCl

O

CCl

O

CH 3 CH 2 CH 2 C

+ HCl

+ HCl

O

N

C

HN

+ HCl

O

C

cytosine

adenine

guanine

NH 2 NH 2

NH 2
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