Organic Chemistry

062 CHAPTER 27 Nucleosides, Nucleotides, and Nucleic Acids

Phosphoramidite Monomers

One current method synthesizes oligonucleotides using phosphoramidite monomers.

The group of each phosphoramidite monomer is attached to a para-

dimethoxytrityl (DMTr) protecting group. The particular group (Pr) used to protect the

base depends on the base.

The of the oligonucleotide to be synthesized is attached to a controlled-

pore glass solid support and the oligonucleotide is synthesized from the When a

monomer is added to the nucleoside attached to the solid support, the only nucleophile

in the reaction mixture is the group of the sugar bonded to the solid support.

This nucleophile attacks the phosphorus of the phosphoramidite, displacing the amine

and forming a phosphite. The amine is too strong a base to be expelled without being

protonated. Protonated tetrazole is the acid used for protonation because it is strong

enough to protonate the diisopropylamine leaving group, but not strong enough to re-

move the DMTr protecting group. The phosphite is oxidized to a phosphate using

ortert-butylhydroperoxide. The DMTr protecting group on the of the dinu-

cleotide is removed with mild acid. The cycle of (1) monomer addition, (2) oxidation,

and (3) deprotection with acid is repeated over and over until a polymer of the desired

length and sequence is obtained. Notice that the DNA polymer is synthesized in the

direction, which is opposite to the direction DNA is synthe-

sized in nature.

3 ¿ ¡ 5 ¿ (5¿¡ 3 ¿)

5 ¿-end

I 2

5 ¿-OH

3 ¿-end.

3 ¿-nucleoside

5 ¿-OH

3-D Molecule:

Phosphoramidite

O

OR

C

C

N

RO NR 2

P

O N DMTr CH 3 O

OCH 3

=

O

P

DMTr O base Pr

a phosphoramidite

a phosphoramidite monomer

used for

oligonucleotide synthesis

O

O

O N

N

base

O

O

HO base

Pr

Pr

C P

monomer

solid support

H+ tetrazole

O

O

O

O

N

DMTr O DMTr

base

O

O

O base

Pr

Pr

C P

a phosphite

solid support

I 2