Chemical Synthesis Of Oligonucleotides-hemant.ppt 4q1hp
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Chemical Synthesis of Oligonucleotides
Hemant A ID MS 09/05 IIRBS
Historical OutLine
Prof Todd{1955} (Cambridge) pioneered the work in the area of Nucleic acid synthesis Indian Nobel Laureate Prof H.G. Khorana also worked there and went on to win Worldwide fame. The structural chemistry of nucleotides had been progressing steadily till the landmark year of 1953 (W&C) It was the discovery of internucleotidic linkages in Dr Todd’s lab coupled with Rosamund Franklin’s XRD that helped usher in the “Double Helix era”
A HISTORY OF DNA
Discovery of the DNA double helix A. Frederick Griffith – Discovers that a factor in diseased bacteria can transform harmless bacteria into deadly bacteria (1928)
B.
Rosalind Franklin - X-ray photo of DNA. (1952)
C.
Watson and Crick - described the DNA molecule from Franklin’s X-ray. (1953)
Kornberg et al followed this up soon with his discovery and characterization of the enzyme DNA polymerase in 1958. This discovery also incited a new drive to achieve Organic synthesis of Peptides. This was in the hope that the availability of synthetic polynucleotides of known size and structure would provide opportunities for chemical, physico-chemical, and enzymatic studies of nucleic acids. It was not in vain
Riboses
OH
OH HO
HO
O
O H
H
H
H
H OH
H
H
H
H OH
OH
Bases NH2 N
N H
O
N
N
NH
N
N H
Adenine NH2
N
Guanine O
N
N H
Cytosine
NH2
O
NH
O
N H
Thymine
NH
O
N H
Uracil
O
Nucleosides
NH2
O
N N
N
NH
N
N
HO HO O H
O
H
H
H
H
OH
OH
H OH
H
H
H
O
Nucleotides Nucleic acids consist of nucleotides that have a sugar, nitrogen base, and phosphate NH2 O N
N NH
N
O
O N
O -O
P
O
O H
O-
-O
O
O
H
H
H OH
P
H
H
OH
H OH
9
H
OH
N
NH 2
The First Synthesis 1.
Initial approaches were restricted to simple combination of nucleotides and nucleosides to form polynucleotide chains Later on two significant approaches were formulated :Step by step addition of mononucleotides to a growing polypeptide chain O
O
N
N
NH2 N
N
N
HO
H
H
O
O
H
N
H
H O
H
O
HO O
H H
N H
NH2
O
O H
NH2
N
NH2 N
OH
NH
NH
P
O
O-
O
H O P
O
O
H
H
O
N
O-
O
H O-
P
NH
O
N
H
O
H
H
H O-
H
O
O
O
H
P
NH
O-
O
N
O
O H
H
H
O
P O-
H O-
H
O
H
H
H O
H
O
H
P O-
O-
O
Such an addition is done by a polymerase enzyme in the presence of an Mg2+
The deoxy ribonucleotide adds to the monophosphate polymer and the result is an (n+1) polymer and a pyrophosphate.
Khorana et al achieved this first in amino acids by a single amino acid bound to a solid a protected amino acid, and a condensing agent, dicyclohexylcarbodiimide (DCC) which catalyzed the reaction. O H2N
CH
C
O
CH2 O
CH2
H N
S O CH3
CH
C
CH
CH3
O CH2 CH3
N C
N
DCC
dicyclohexylcarbodiimide
O
O H N O
CH
C
CH
CH3
H N
CH CH2
O CH2
CH2
CH3
S CH3
C
O
OH
The following scheme is adopted to protect the Amino group
The reaction would link the protected amino acid to the solid in only one way and the resulting di-peptide was then deprotected for the addition of the next amino acid. In this way, the synthetic polypeptide grew at the direction of the synthesis through the required cycle of steps
This process was not efficient and couldn’t synthesize very long nucleotides.
2. By creating individual oligonucleotide blocks and then bringing them together to form the subsequent chains This theory advocates the protection of several groups at the same time while only the necessary ones were left unprotected as a nucleotide in it’s own has many reactive sites NH2 N
NH2
N
N
HO N
HO
O
O
H O P O-
O
H
H
H
H
O
H
H
O H
H
-O
P
H O-
O-
O
N
N
Strategies…..
1. Protection is done on the following groups :-
•
Amine
•
Hydroxyl
•
Phosphate
The oligotide monomers involved here are called phoshoramidites. They are actually phosphorylated nucleotides with protecting groups attached to the 3 main groups.
Another notable variation is that the synthesis proceeds from 3’ to 5’ end as the link to the solid is made on the 3’ carbon. The may be a micron sized glass bead. NH 2
N N
N
N
HO O H
H
O
H
H
O
Si
R H H N
O N H
O
O
S i
Si
Si Si
This allows the researcher to customize the above method according to his needs.
Phophoramidite synthesis begins with the 3’-most nucleotide and proceeds through a series of cycles composed of fours steps that are repeated until the 5’-most nucleotide is attached.
These steps are deprotection, coupling, capping, and stabilization.
Deprotection
The trityl group is removed using trichloroacetic acid. O
O
HN
N
O
N O O H
H
O
H
H
H
P
O
O
N
C N
O
HN
-O
N
O
Cl Cl
O
N
Cl HO
TrichloroAcetate
O H
H
O
H
H
H
P O
N
C N
Coupling Reaction
Berner et al. showed that tetrazole, a weak acid, attacks the coupling phosphoramidite nucleoside forming a tetrazolyl phosphoramidite intermediate . This structure then reacts with the hydroxyl group of the nucleoside attached to the G bead and the 5’ to 3’ linkage is formed . The unbound base and by-products are washed out, the tetrazole is reconstituted, and the process continues………… The use of tetrazole increased coupling efficiency to greater than 99% which allowed longer and longer oligonucleotides to be synthesized. Berner S, Muhlegger K, and Seliger H. (1989) Studies on the role of tetrazole in the activation of phosphoramidites. Nucleic Acids Res, 17(3): 853 864.
O O HN HN N N N
O
O
N
THPO
THPO
O O H
H
H N
H
O
H
H
H N
N
H
H
H
H
N
H
O
N
N
N
O
P
N
O
P N
O
N NH
C
N
N
C
N
N
N
N H 2
HO O
O
H
H
O
OH
H
H
HN
N
O
N
O
THPO
O
O H
H
O
H
N NH H
H
HN N
N
NH2
O
N
P
O H
H
O
OH
O
N O C
H
O THPO
N
N
O
NH
H
H
O
H
H N
H
N
N
HO
N N
O
P O
H
H
O
OH
H C N
N
NH 2
Step 1 : Tetrazole attack O O HN HN N N N N
O
O THPO
THPO
O O H
H
H N H
H
N
N
H N
H
H
O
H
H
H
H
O
N N
N
P
N
O
P N
O
C
N
N
C N
Step 2 coupling the nucleotide bound to a
N
N
N
N
THPO N
H H
N THPO
P
N
O O
H H
H O
HN
P O
O
O H HN O
N
O
N
N
H H
N C
H
O
H
HO
N
N C
H
O N
O
O H N
O
N
N HN NH2
OH H
NH2 NH2
HN O
HN
N O
N
N
N
H H
OH
N
N
H
O H
H
O
N
HO
N
O
C
O
N HN
OH
O
O
O O
H
C H
N H
N
O H O
HN
H
N H
O
O
P H
O
O
P
H THP O
H
H
O H
N
N
THPO HN
N
N
N N
N
Synthesis Scale and Yield
Scale refers to the amount of starting material which is composed solely of the 3’-most nucleotide of a sequence attached to a solid used to make the oligonucleotide. Yield refers to the amount of final product recovered after all the synthesis, processing, and purification steps associated with the oligonucleotide have been completed.
Hemant A ID MS 09/05 IIRBS
Historical OutLine
Prof Todd{1955} (Cambridge) pioneered the work in the area of Nucleic acid synthesis Indian Nobel Laureate Prof H.G. Khorana also worked there and went on to win Worldwide fame. The structural chemistry of nucleotides had been progressing steadily till the landmark year of 1953 (W&C) It was the discovery of internucleotidic linkages in Dr Todd’s lab coupled with Rosamund Franklin’s XRD that helped usher in the “Double Helix era”
A HISTORY OF DNA
Discovery of the DNA double helix A. Frederick Griffith – Discovers that a factor in diseased bacteria can transform harmless bacteria into deadly bacteria (1928)
B.
Rosalind Franklin - X-ray photo of DNA. (1952)
C.
Watson and Crick - described the DNA molecule from Franklin’s X-ray. (1953)
Kornberg et al followed this up soon with his discovery and characterization of the enzyme DNA polymerase in 1958. This discovery also incited a new drive to achieve Organic synthesis of Peptides. This was in the hope that the availability of synthetic polynucleotides of known size and structure would provide opportunities for chemical, physico-chemical, and enzymatic studies of nucleic acids. It was not in vain
Riboses
OH
OH HO
HO
O
O H
H
H
H
H OH
H
H
H
H OH
OH
Bases NH2 N
N H
O
N
N
NH
N
N H
Adenine NH2
N
Guanine O
N
N H
Cytosine
NH2
O
NH
O
N H
Thymine
NH
O
N H
Uracil
O
Nucleosides
NH2
O
N N
N
NH
N
N
HO HO O H
O
H
H
H
H
OH
OH
H OH
H
H
H
O
Nucleotides Nucleic acids consist of nucleotides that have a sugar, nitrogen base, and phosphate NH2 O N
N NH
N
O
O N
O -O
P
O
O H
O-
-O
O
O
H
H
H OH
P
H
H
OH
H OH
9
H
OH
N
NH 2
The First Synthesis 1.
Initial approaches were restricted to simple combination of nucleotides and nucleosides to form polynucleotide chains Later on two significant approaches were formulated :Step by step addition of mononucleotides to a growing polypeptide chain O
O
N
N
NH2 N
N
N
HO
H
H
O
O
H
N
H
H O
H
O
HO O
H H
N H
NH2
O
O H
NH2
N
NH2 N
OH
NH
NH
P
O
O-
O
H O P
O
O
H
H
O
N
O-
O
H O-
P
NH
O
N
H
O
H
H
H O-
H
O
O
O
H
P
NH
O-
O
N
O
O H
H
H
O
P O-
H O-
H
O
H
H
H O
H
O
H
P O-
O-
O
Such an addition is done by a polymerase enzyme in the presence of an Mg2+
The deoxy ribonucleotide adds to the monophosphate polymer and the result is an (n+1) polymer and a pyrophosphate.
Khorana et al achieved this first in amino acids by a single amino acid bound to a solid a protected amino acid, and a condensing agent, dicyclohexylcarbodiimide (DCC) which catalyzed the reaction. O H2N
CH
C
O
CH2 O
CH2
H N
S O CH3
CH
C
CH
CH3
O CH2 CH3
N C
N
DCC
dicyclohexylcarbodiimide
O
O H N O
CH
C
CH
CH3
H N
CH CH2
O CH2
CH2
CH3
S CH3
C
O
OH
The following scheme is adopted to protect the Amino group
The reaction would link the protected amino acid to the solid in only one way and the resulting di-peptide was then deprotected for the addition of the next amino acid. In this way, the synthetic polypeptide grew at the direction of the synthesis through the required cycle of steps
This process was not efficient and couldn’t synthesize very long nucleotides.
2. By creating individual oligonucleotide blocks and then bringing them together to form the subsequent chains This theory advocates the protection of several groups at the same time while only the necessary ones were left unprotected as a nucleotide in it’s own has many reactive sites NH2 N
NH2
N
N
HO N
HO
O
O
H O P O-
O
H
H
H
H
O
H
H
O H
H
-O
P
H O-
O-
O
N
N
Strategies…..
1. Protection is done on the following groups :-
•
Amine
•
Hydroxyl
•
Phosphate
The oligotide monomers involved here are called phoshoramidites. They are actually phosphorylated nucleotides with protecting groups attached to the 3 main groups.
Another notable variation is that the synthesis proceeds from 3’ to 5’ end as the link to the solid is made on the 3’ carbon. The may be a micron sized glass bead. NH 2
N N
N
N
HO O H
H
O
H
H
O
Si
R H H N
O N H
O
O
S i
Si
Si Si
This allows the researcher to customize the above method according to his needs.
Phophoramidite synthesis begins with the 3’-most nucleotide and proceeds through a series of cycles composed of fours steps that are repeated until the 5’-most nucleotide is attached.
These steps are deprotection, coupling, capping, and stabilization.
Deprotection
The trityl group is removed using trichloroacetic acid. O
O
HN
N
O
N O O H
H
O
H
H
H
P
O
O
N
C N
O
HN
-O
N
O
Cl Cl
O
N
Cl HO
TrichloroAcetate
O H
H
O
H
H
H
P O
N
C N
Coupling Reaction
Berner et al. showed that tetrazole, a weak acid, attacks the coupling phosphoramidite nucleoside forming a tetrazolyl phosphoramidite intermediate . This structure then reacts with the hydroxyl group of the nucleoside attached to the G bead and the 5’ to 3’ linkage is formed . The unbound base and by-products are washed out, the tetrazole is reconstituted, and the process continues………… The use of tetrazole increased coupling efficiency to greater than 99% which allowed longer and longer oligonucleotides to be synthesized. Berner S, Muhlegger K, and Seliger H. (1989) Studies on the role of tetrazole in the activation of phosphoramidites. Nucleic Acids Res, 17(3): 853 864.
O O HN HN N N N
O
O
N
THPO
THPO
O O H
H
H N
H
O
H
H
H N
N
H
H
H
H
N
H
O
N
N
N
O
P
N
O
P N
O
N NH
C
N
N
C
N
N
N
N H 2
HO O
O
H
H
O
OH
H
H
HN
N
O
N
O
THPO
O
O H
H
O
H
N NH H
H
HN N
N
NH2
O
N
P
O H
H
O
OH
O
N O C
H
O THPO
N
N
O
NH
H
H
O
H
H N
H
N
N
HO
N N
O
P O
H
H
O
OH
H C N
N
NH 2
Step 1 : Tetrazole attack O O HN HN N N N N
O
O THPO
THPO
O O H
H
H N H
H
N
N
H N
H
H
O
H
H
H
H
O
N N
N
P
N
O
P N
O
C
N
N
C N
Step 2 coupling the nucleotide bound to a
N
N
N
N
THPO N
H H
N THPO
P
N
O O
H H
H O
HN
P O
O
O H HN O
N
O
N
N
H H
N C
H
O
H
HO
N
N C
H
O N
O
O H N
O
N
N HN NH2
OH H
NH2 NH2
HN O
HN
N O
N
N
N
H H
OH
N
N
H
O H
H
O
N
HO
N
O
C
O
N HN
OH
O
O
O O
H
C H
N H
N
O H O
HN
H
N H
O
O
P H
O
O
P
H THP O
H
H
O H
N
N
THPO HN
N
N
N N
N
Synthesis Scale and Yield
Scale refers to the amount of starting material which is composed solely of the 3’-most nucleotide of a sequence attached to a solid used to make the oligonucleotide. Yield refers to the amount of final product recovered after all the synthesis, processing, and purification steps associated with the oligonucleotide have been completed.
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