Nucleic Acids for M.Sc Organic Chemistry students

1. Prepared By
Dr. Krishnaswamy. G
Faculty
DOS & R in Organic Chemistry
Tumkur University
Tumakuru
For
II M.Sc., III Semester
DOS & R in Organic Chemistry
Tumkur University
Tumakuru

2. Nucleic acids are biopolymers having 5’ and 3’ end composed of
chains of five membered rings of sugars (monosaccharide) linked to
phosphate groups via phosphodiester bonds (nucleotide subunits) and
anomeric carbon of each sugar is bonded to a nitrogen of heterocyclic
amine (organic base) through a β-glycosidic linkage.
Monosaccharide
Residue
Phosphate
Group
Organic
Base
5’
3’

3. Nucleotide triphosphate serves as substrate precursor for the
Biosynthesis of nucleic acids. The nucleotides are linked by
the nucleophilic attack of 3’-OH of one nucleotide triphosphate on the α-
phosphorus of another nucleotide.

4. Nucleic Acids = Nucleotides
Nucleotides = Sugar + Base + Phosphate
Nucleotides = Nucleoside + Phosphate

5. Nucleosides
A compound with a nitrogenous base covalentaly bonded to
D-ribose or 2’-deoxy-D-ribose via β-glycosidic bond to the C1
carbon of sugar is called nucleoside.
Riboses are five carbon sugars with an aldehyde
functional group (aldopentoses).
Nucleic acids are composed exclusively β-D stereoisomer of
ribose or 2’-deoxy-D-ribose.

6. CHO
OHH
OHH
OHH
CH2OH
1
2
3
4
5
OH
O
OHOH
HH
HH
HO
C
OHH
OHH
OH
CH2OH
1
2
3
4
5
OH
H
D-Ribose
OH
O
OHOH
HH
HH
HO
-2'-deoxy-D-Ribose
1'
2'3'
4'
5' OH
O
HOH
HH
HH
HO
1'
2'3'
4'
5'
-D-Ribose

7. N
N N
N
H
PURINE
N
N N
N
H
N
N N
N
H
NH2 O
H2N
Adenine Guanine
N
N
PYRIMIDINE
HN
N
H
HN
N
H
N
N
H
O
O
O
O O
NH2
CH3
Uracil Thymine Cytosine
Nitrogenous bases are planar, aromatic molecules that are
derivatives of purine or pyrimidine. The five most common organic bases
are

8. OH
O
OHOH
HH
HH
HO
1'
2'3'
4'
5'
N
N N
N
H
NH2
H2O
O
OHOH
HH
HH
HO
1'
2'3'
4'
5'
N
N N
N
NH2
O
OHOH
HH
HH
HO
1'
2'3'
4'
5'
N
N
N
N
H2N
syn-Adenosine anti-Adenosine
Anti conformation (favored) places bulky groups away from ribose.
Syn conformation (disfavored) places bulky groups over the ribose hence
poses steric repulsion.

9. Nucleotides
Nucleotide is nucleoside with either the 5’ or the 3’-OH
group bonded in an ester linkage to phosphoric acid.
The nucletotide where the sugar is D-ribose is called
ribonuleotide.
The nucleotide with 2’-deoxy-D-ribose is called
deoxyribonuleotide.

10. Phosphate is covalently attached to the D-ribose via
phosphate ester bonds
Phosphates are typically attached to the C5’ carbon of sugar.
In polymer, the phosphate is attached to both the C5’ and
C3’ carbons of sugar.
O
PO OH
O
hydrogen phosphate ion

11. OH
O
OHOH
HH
HH
HO
1'
2'3'
4'
5'
O
PO OH
O
H2O
O
OHOH
HH
HH
O
1'
2'3'
4'
5'
O
PO
O
Phosphoester bond
N
N
N
N
H2N
OH
O
OHOH
HH
HH
HO
1'
2'3'
4'
5'
O P
O
HO
O
H2O
O
OHO
HH
HH
HO
1'
2'3'
4'
5'
Phosphoester bond
N
N
N
N
H2N
O P
O
O

12. There are two types of nucleic acids
Deoxyribo Nucleic Acid
(DNA)
Ribo Nucleic Acid
(RNA)
DNA encodes the hereditary
details and controls the
growth and division of the
cells
The genetic information
stored in DNA is then
transcribed into RNA, and
the details in RNA are then
translated for the synthesis
of the proteins

13. Deoxyribo Nucleic Acid
(DNA)
O
O N
H
O
O N
O H
O
PO O
NH
N
N
NH
PO O
O
O N
HO
PO O
O
O N
H
N
N
N
HO
PO
O
O
N
NH2
O
NH2
O
O
NH2
O
Thymine (T)
Guanine (G)
Cytosine (C)
Adenine (A)
5' terminus
3' terminus
Phosphodiester
bond
D-Deoxyribose
β-glycosidic
linkage

14. DNA has equal amounts of purines and pyrimidines
(G+A = C+T)
Amount of A = T and amount of G = C

15. O
O N
OH
O
O N
O OH
O
PO O
NH
N
N
NH
PO O
O
O N
OHO
PO O
O
O N
OH
N
N
N
HO
PO
O
O
N
NH2
O
NH2
O
O
NH2
O
Uracil (U)
Guanine (G)
Cytosine (C)
Adenine (A)
5' terminus
3' terminus
Ribo Nucleic Acid
(RNA)
D-Ribose
Phosphodiester
bond
β-glycosidic
linkage

16. Polynucleotide Oligonucleotide
A biopolymer containing 13 or more
nucleotides
Composes of 13 or more
Comparatively large in size
Shows an infinite degree of
polymerization
Plays a role as molecules which store
genetic information in all living
organisms as well as viruses
Short DNA or RNA molecules with a
small number of nucleotides
Composes of 10 or 100 nucleotides
Short fragments of DNA or RNA
Shows a finite degree of
polymerization
Important as primers for DNA
polymerization and probes for in situ
hybridization and gene knockdown
assays

17. Stability of DNA and RNA
In RNA, the 2’-OH of ribose attacks the adjacent
phosphodiester group that leads to the cleavage of the
strand. This reaction does not take place in DNA, because it
does not have the 2’-OH group. Thus, DNA remain intact
throughout the life span of cells.

18. A protecting group (PG) is a molecular framework that is
introduced onto a specific functional group (FG) in a poly-
functional molecule to block its reactivity under reaction
conditions needed to make modifications elsewhere in the
molecule
What is a protecting group

19. Ether derivative of carbohydrates
A mild base such as silver oxide (Ag2O) is used to deprotonate all the alcohol
groups forming alkoxides which readily react with CH3I in an SN2 reaction.
Although all the ether groups look identical, one of them (on the anomeric carbon)
is part of an acetal and can be hydrolyzed back to the alcohol.
The other four, on the other hand, are very stable and just like regular ethers
can only be cleaved with strong acids such as HBr and HI.

20. Benzyl ethers are suitable since they are stable under acidic and basic
conditions and can be cleaved by catalytic hydrogenation with H2 over
Pd/C.

21. Silyl Ether derivative of carbohydrates

22. Ester derivative of carbohydrates
The OH groups of monosaccharides, can be converted into esters by reacting with
either anhydrides or acid chloride in the presence of a base. Most often, acetic
anhydride and pyridine (a base) are used for the esterification of carbohydrates.
The acetyl group is often abbreviated as Ac

23. O
HO
HO OH
N
O
HO
HO OH
LG
Base
N
Base
H
Common disconnection towards nucleosides
Formation of glycosidic bond

24. Regioselectivity of base substitution is controlled by
Neighbouring Group Participation
In case of 2-deoxyribonucleoside
(i) Use a ribonucleoside then deoxygenate
(ii) Use an alternative directing group. Eg: 3’-thiocarbamate

25. Nucleoside synthesis via N-glycosylation
All methods for N-glycosylation of nucleobases employs a reactive
(activated) glycosyl intermediate that is subjected to nucleophilic attack by
a nucleobase.
Nucleobase glycosylation
with glycosyl acetate
Nucleobase glycosylation
with halogenose

26. Fischer-Helferich
synthesis
Coupling metal salt to a C1’ halogenated sugar

27. Ether derivative of carbohydrates
4,4’-Dimethoxytrityl chloride (DMT) also used as protecting group for
hydroxyl group. This protection reaction is known as tritylation reaction.
Regioselective
tritylation of primary
alcohol

28. Detritylation - Deprotection of DMT using trichloroacetic acid

29. Oligonucleotides Synthesis
Methods of formation of internucleotide bond
Phosphotriester Approach
Phosphodiester Approach
Phosphite triester Approach
Phosphoramidite Approach
Chemical synthesis of relatively short fragments of nuclei acid with defined
chemical structure / sequence.
Enzymes synthesize DNA and RNA in 5’ to 3’ direction while in chemical
synthesis of oligonucleotide synthesis is carried out in the 3’ to 5’ direction.

30. Phosphotriester Approach
Chemical Synthesis of Oligonucleotides
Utilizes protection of the 3’ and 5’ hydroxy groups of the deoxy ribose as well as
protection of the internucleotide bond. Overall there are three protecting groups
are utilized in phosphotriester approach.
Michelson and Todd reported the synthesis of the dinucleotide in 1955.
This synthetic approach later known as the phosphotriester approach.
Step-1: Phosphorylation between 5’-O-acetyldeoxyribosethymidine and
diphenylphosphorochloridate
Step-2: 3’-O-acetyldeoxyribosethymidine phosphorylation to give dinucleotide
Where B = A,C, G or T nucleo bases;
R1, R2, R3 = protecting groups;
X = Halogen

31. O
O N
NH
O
O
O
OH
O P
O
O
Cl
O
O N
NH
O
O
O
O
O P O
Cl
Diphenylphosphoro
chloridate
5'-O-acetyl
deoxyribothymidine
O
O N
NH
O
O
O
O
O P O
Cl
O
HO N
NH
O
O
O
O
O
O N
NH
O
O
O
O
O P O
O
O
N
NH
O
O
O
O
3'-O-acetyl
deoxyribothymidine
O
HO N
NH
O
O
O
O P O
O
O
N
NH
O
O
OH
Phosphotriester Approach
Dinucleotide

32. In the 1960s, groups led by R. Letsinger and C. Reese developed a
phosphotriester approach.
Monomethoxytrityl chloride (MMT)

33. Chemical Synthesis of Oligonucleotides
Phosphodiester Approach
Utilizes protection of the 3’ and 5’ hydroxy groups of the deoxy ribose
Gobind Khorana, in 1956, accidentally discovered the phosphodiester method
for the chemical synthesis of deoxyribo-oligonucleotides
Where B = A,C, G or T nucleo bases;
R1, R2= protecting groups;
In this approach, 5’-O-trityldeoxyribosethymidine and 3’-O-
acetyldeoxyribosethymidine-5’-phosphate are reacted in the presence of
toluene-4-sulfonyl chloride (TsCl) or N1,N3-dicyclohexylcarbodiimide (DCC) in a
pyridine solution. The removal of the trityl and acetyl group yields the
dinucleotide.

34. O
O N
NH
O
O
OH
O
O N
NH
O
O
O
O
O
N
NH
O
O
O
HO P O
O
O N
NH
O
O
O
O
5'-O-trityl
deoxyribothymidine
O
HO N
NH
O
O
O
O P O
O
O
N
NH
O
O
OH
O
O
P
OH
OH
O
O
O
O
3'-O-acetyl
deoxyribothymidine-5'-phosphate
Pyridine
or
N C N
DCC
S
O
O
Cl
TsCl
AcOH/ H2O
NaOH/ H2O
Khorana’s dinucleotide synthesis via
phosphodiester approach

35. Activation of nucleotide by TsCl forms Mixed anhydride
Activation of nucleotide by DCC forms Pseudourea

36. Chemical Synthesis of Oligonucleotides
Phosphite triester Approach
Utilizes protection of the 3’ and 5’ hydroxy groups of the deoxy ribose as
well as phophorous group
Where B = A,C, G or T nucleo bases;
R1, R2, R3 = protecting groups;
X = Halogen, morpholine
Willi Bannwarth in 1985 reported a simple synthesis of phosphoramidite
dinucleotides with two different phosphorous-protecting groups for the
synthesis of 2′-oligodeoxynucleotides on a polymer support called the
“Phosphite-Triester Method.

37. Phosphoramidite chemistry, developed in the 1980s and later enhanced
with solid-phase supports and automation, is the method of choice for
DNA oligonucleotide manufacturing.
Phosphoramidite Method
McBride and
Caruthers, 1983
Phosphoramidite monomer

38. Opposite of biosynthesis,
chemical synthesis
proceeds in the 3' → 5'
direction according to the
steps outlined

39. Step 1 (Detritylation)
The cycle is initiated by removal of the 5'-DMT (4,4'-dimethoxytrityl) protecting
group of the solid-support-linked nucleoside (contains the terminal 3' base of the
oligonucleotide). The 5'-DMT prevents polymerization of the nucleoside during
functionalization of the solid support resin.
5'-DMT protecting group is removed by TCA (trichloroacetic acid) in the solvent
dichloromethane

40. Step 2 (Coupling)
Once the DMT has been removed, the free 5'-OH of the solid-support-linked
nucleoside is able to react with the next nucleoside, which is added as a
phosphoramidite monomer.
The diisopropylamino group of the phosphoramidite monomer in the solvent
acetonitrile is ‘activated’ (protonated) by the acidic catalyst ETT [5-(ethylthio)-1H-
tetrazole].

41. Step 3 (Oxidation)
The phosphite triester formed during the coupling reaction is unnatural and
unstable; therefore, it must be converted to a more stable phosphorus species prior
to the start of the next cycle. Oxidation converts the phosphite triester to the stable
phosphate triester.
Oxidation of the phosphite triester is achieved with iodine in the presence of water
and pyridine.

42. Step 4 (Capping)
Since 100% coupling efficiency is impossible, there are always some solid-support-
linked nucleosides with unreacted 5'-OH. If not blocked, these hydroxyl groups will
12react during the next cycle, and hence, lead to a missing base.

43. Deprotection of solid support
At the end of the oligonucleotide synthesis solid support is cleaved by treating with
concentrated ammonia solution. Cleavage is necessary so that the free 3’-OH group
may take part in biochemical reactions.

44. Deprotection of Organic bases
Treating with concentrated ammonia solution also deprotects the organic bases. The
protecting groups include: N(6)-benzoyl A, N(4)-benzoyl C and N(2)-isobutyryl G.
These protecting groups must be removed so that proper hydrogen bonds between
oligonucleotide and target nucleic acid may form.

45. Nucleotides with standard numbering convention

46. Deprotection of β-cyanoethyl group on oxygen of the phosphate
Treating with concentrated ammonia solution also reomoves the cyanoethyl group
via β-elimination. Hence, it is conversion of phosphate triester to phosphate diester
(phosphodiester).

47. 2’tert-butydimethylsilyl (TBDMS)
Phosphoramidites
Automated RNA synthesis on solid support use one extra 2’-hydroxy group
makes this chemistry more difficult compared to conventional DNA synthesis.
The protective group of 2’-hydroxyl in ribose must remain intact both during the
synthesis and base deprotection step.
All modern methods of RNA synthesis that employ different RNA
phosphoramidite monomers have protective groups at the 2’ hydroxyl group.
2’triisopropyl-silyl-oximethyl (TOM)
Phosphoramidites