NUCLEIC ACID

Nucleotides

These are the monomer units or building blocks of nucleic acids. They serve multiple

additional functions. For example, they form a part of many coenzymes and serve as donors

of phosphoryl groups (e.g. ATP or GTP).

PURINES, PYRIMIDINES, NUCLEOSIDES AND NUCLEOTIDES

Purines and pyrimidines are nitrogen-containing heterocycles, cyclic compounds whose rings

contain both carbon and other elements (hetero atoms). Note that their six-atom rings are

numbered in opposite direction (Figure 1). The planar character of purines and pyrimidines

facilitates their close allocation or stacking which stabilizes double stranded DNA. The Oxo

and amino groups of purines and pyrimidines exhibit heto-enol amine-imine tautomerism

(Figure 2), but physiologic conditions strongly favour the amino and oxo forms.

Figure 1: Purine and Pyrimidine atoms are numbered according to the international system.

Nucleosides and Nucleotides

Nucleosides

These are derivatives of purines and pyrimidines that have a sugar linked to a ring nitrogen

numerals with a prime e.g. 21 or 31 to differentiate it from that of the heterocyclic base. For

example, the sugar in ribonucleosides is D-ribose, and in that of deoxyribonucleosides is 2-

deoxy-D-ribose. The sugar is linked to the heterocyclic base via a Beta-N-glycosidic bond,

always to N-1 of a pyrimidine or to N-9 of a purine (Figure 3)

 Figure 2: Tautomerism of the oxo and amino functional group of purines and pyrimidines

Figure 3: Ribonucleosides, drawn as the Syn conformers.

Nucleotides

Mononucleotides are nucleosides with a phosphoryl group esterified to hydroxy group of the

sugar. For example, 3- and 5- nucleotides are nucleosides with a phosphoryl group on the 31-
or 51-hydroxy group of the sugar, respectively. Since most nucleotid76es are 51- is usually

omitted from their names. Additional phosphoryl groups linked by acid anhydride bonds to

the phosphoryl group of mononucleotide form nucleoside diphosphates and triphosphates

(Fig 4)

Figure 4: ATP, its di, tri and monophosphate.

Since steric hindrance by the base restricts rotation about the β-N-glycosidic bond of

nucleosides and nucleotides both therefore exist as syn or anti conformers (Figure 5). Both

conformers occur in nature but anti conformers predominate. The table below contain the

major purines and pyrimidines and their nucleosides and nucleotides derivatives. The

following abbreviations are used for bases Adenine (A), guanine (G), cytosine (C), thymine

(T) and uracil (U), whether free or present in nucleosides or nucleotides. The prefix d’ is

(deoxy) indicates that the sugar is 2i- deoxy-D-ribose (e.g. dGTP). (Figure 6)
 Figure 5: The Syn and anti-conformers of adenosine differ with respect to orientation about
N-glycosidic bond.

Table 1: Bases nucleosides, and nucleotides

Base Formula Base (X =H) Nucleosides X= Nucleotide, where
Ribose or X= Ribose phosphate
Deoxyribose
Adenine (A) Adenosine (A) Adenosine
monophosphate

Guanine (G) Guanosine (G) Guanosine

monophosphate
(GMP)

Cytosine (C) Cytidine (C) Cytidine

monophosphate
(CMP)

Uracil (U) Uridine (U) Uridine

monophosphate

Thymine Thymidine Thymidine

monophosphate
(TMP)
 Figure 6: AMP, dAMP, UMP, dTMP

Additional Bases in Nucleic Acids.

Small quantities of additional purines and pyrimidine occur in DNA and RNA’s. Examples

are 5-methylcytosine of bacterial and human DNA, 5-hydroxymethylcytosine of bacterial and

viral nucleic acids and mono- and di-N-methylated adenine and guanine of mammalian

messenger RNAs (Figure 7)

Figure 7: 4 common naturally occurring pyrimidines and purines

Physiological Functions of Nucleotides

They take part in reactions that fulfill physiological functions such as proteins synthesis

nucleic acid synthesis, regulatory cascades and signal transduction pathways.

ATP serves as transducer of free energy adenosine 31-phosphate-51-phosphosulphate donor

for sulphated proteoglycans and for sulphate conjugates of drugs and methyl group donor to

5-adenosylmethionine (Figure 11).

Figure 9: c AMP, 31,51-cyclic AMP. And c GMP

Figure 10: Adenine 31-phosphate-51-phosphosulphate.

Figure 11: S-Adenosylmethionine

GTP serves as allosteric regulator as an energy source for protein synthesis, and cGMP serves

as second messenger in response to nitric oxide (NO) during relaxation of smooth muscles.

UDP-sugar derivatives participate in sugar epimerization and biosynthesis of glycogen,

glycosyldisaccharides and the oligosaccharides of glycoproteins and proteoglycans: UDP-

glucuronic acid forms. The urinary glucuronide conjugates of bilirubin and of drugs such as

aspirin.

CTP participates in biosynthesis of phosphoglycerides, sphingomyelin and other substituted

sphingosines. Finally, many coenzymes incorporate nucleotides as well as structures similar

to purines and pyrimidines nucleotides.

Properties of nucleotides

(1) They are more water soluble than their corresponding bases.

(2) They are relatively strong acids

(3) 5-phosphate group is relatively stable to acid hydrolysis; however, 5-positions can be

easily hydrolysed by 51- nucleotilase.

(4) Tender components containing either the purine or pyrimidine bases can be easily

detected because of strong absorption of uv light by these compound. Their mode of

absorption obeys Beer Lamberts law and uv maximum for the based is about 260 nm

In general, purines bases nucleosides and nucleotides have stronger absorption at 260 nm

than the pyrimidines and their derivatives. This differences can be used for the analysis of

their compounds both quantitatively and qualitatively. For example, if cytosine is

delaminated, a marked shift is caused in λmax from 271 to 262 nm. (Figure 12)
 λ 271 nm λ 262 nm

Figure 12

Synthetic Nucleotides Analogs-Their uses in Chemotherapy

Synthetic analogs of purines, pyrimidines nucleosides and nucleotides altered in either the
heterocyclic ring or the sugar moiety have a numerous clinical application in medicine.
They perform their functions by inhibiting enzymes essential of nucleic acids synthesis or
their incorporating into nucleic acid resulting disruption of base pairing. Examples of
synthetic pyrimidine and purine analogs used by oncologists are 5-fluro-, or any 5-
iodouracil, 3-deoxyuridene, 6-thioguarines and 6-mercaptopurine, 5- or 6- azauridine or
5- or 6- cytidine, and 8-azaguanine (Figure 13). They are incorporated into DNA prior to
cell division. for example, allopurinol an analog of purine is used in the treatment of
hyperuricemia and gout where it inhibits purine biosynthesis and xanthine oxidase
activity. Cytarabine is used in chemotherapy of cancer. Azathioprine which is catabolized
to 6-mercaptopurine is used during organ transplantation to supposes immunologic
rejection.

Figure 13: Selected synthetic pyrimidine and purine analogs

POLYNUCLEOTIDES

The 51-phosphoryl group of a mononucleotide can esterify a second – OH group, forming

a phosphodiester. Most commonly, this second –OH groups is the 31 OH of the pentose of

a second nucleotide. This leads to the formation of dinucleotide in which a sugar moieties
are linked by a 31 51 phosphodiester bond to form backbone of RNA and DNA. RNA’s

are far less stable than DNA since the 21- OH groups of RNA (absent from DNA)

functions as a nucleophile during hydrolysis of 31,51-phosphodiester bond.

The primary structure of polynucleotides

The base sequence that is primary structure of polynucleotide can be represented as given

below. The phosphodiester bond is represented by P or p, bases by simple letter and

pentose by a vertical line.

Here all the phosphodiester bonds are 5 31, a more compact notation is possible. This

representation indicates that 51- hydroxy but not 31- hydroxy is phosphorylated. The most

compact representation shows only the base sequence with the 51-end on the left and the

31-end on the right. The phosphoryl group are assumed but not shown.

GGATCA

Nucleic Acid Structure and Function

Biomedical important

The discovery that genetic information is codded along the length of a polymeric

molecule made up of four types of monomeric units was a major scientific breakthrough

in the twenty century. This molecule is called DNA and is organized into genes, the

fundamental units of genetic information’s. The basic information’s pathway i.e. DNA

directs the synthesis of RNA which in turn direct the synthesis of protein has been
elucidated. Genes do not function autonomously but their replication and function are

controlled by various gene products, often in collaboration with components of various

signal transduction pathways knowledge of the structure and functions of nucleic acids is

essential in understanding genetics and many aspects of pathophysiology as well as the

genetic basic dieses

DNA contains the genetic information

The evidence that DNA contained the genetic information was first made in 1944 by

Avery, MacLeod, and McCarty. All the experiment conducted revealed DNA to be donor

of genetic information. DNA contain four Deoxynucleotides. The monomeric units of

DNA are deoxyadenylate, deoxyguanylate, deoxycytidylate and thymidylate. These

monomeric units of DNA are held in polymeric form by 3,51-phosphodiester bridges

constituting single strand (Figure 14). The information content of DNA (the genetic code)

resides in the sequence in which these monomers- purine and pyrimidine

deoxyribonucleosides are ordered. The polymers possess polarity one end had a 51-

hydroxyl or phosphate terminal while the other has 31-phosphate or hydroxy terminal.

Since the genetic information resides in the order of the monomeric units within the

polymers, hence there must be a mechanism of reproducing or replicating this specific

information with a high degree of fidelity. X-ray analysis of DNA revealed that the

concentration of deoxyadenosine (A) nucleotide equals that of thymidine (T) nucleotide

(A = T), while the concentration of deoxyguanosine (G) nucleotide equals that of

deoxycytidine (C) nucleotide (G = C), this revelation led to the proposal of a model of a

double stranded DNA molecule (Figure 15). The two strands of this double-stranded helix

are held by hydrogen bonds between the purine and pyrimidine bases of the respective

linear molecules. The pairing between the purine and pyrimidine nucleotide on the

opposite stands are very specific and depended upon hydrogen bonding of A with T and
G with C (figure 16). DNA is right handed because the double helix is restricted to the

following A&T only and G&C only.

This pairing explains the earlier observation that in a double stranded DNA molecule A =

T and G = C. the two strands of the double helical molecule, each of which possess a

polarity are anti-parallel i.e. one strand runs in 51 to 31 direction and other 31 to 51

direction.

O
N
NH
G
N N
5 1 NH2

O O
H H N
P C
H O O
H O N
P NH NH2
T
O
O H H O N O N
N
H A
H P
N N
O
H H O
H
O H O
P H H
H
HO H
31
O

Figure 14: Segment of one strand of a DNA molecule in which purine and pyrimidine

bases guanine (G), cytosine (C), thymine (T), and adenine (A) are held together by a

phosphodiester back bone between 21-deoxyribosyl nucleotide attached to the

nucleobases by a N-glycosidic bond.

 Figure 15: A diagrammatic representation of the Watson and Crick model of the double

helical structure of the B form of DNA. The horizontal arrow indicates the width of the

double helical helix and the vertical arrows indicates the distance spanned by one

complete turn of the double helix (34 Å). One turn of B- DNA includes ten base pairs, so

the rise in 3.4 Å per bp the central axis of the double helix is indicated by the vertical rod.

The short arrows designate the polarity of the anti-parallel strands. The major and minor

grooves are depicted. (A) adenine (C) cytosine. G guanine, T, thymine; P phosphate; S,

sugar (deoxy ribose)

O H
N N
H H
N N N N
O H N
H
thymine adenosine

Figure 16: Bases pairing between deoxyadenosine and thymidine involves the formation of
two hydrogen bonds. Three such bonds are formed between deoxycytidine and
deoxyguanosine. The broken line represents hydrogen bonds.
This is analogous to two parallel streets, each running one way but carrying traffic in

opposite direction. In the double stranded DNA molecules, the genetic information

resides in the sequence of nucleotides one strand, the template strand. This template

strand is copied during nucleic acid synthesis. It is sometimes referred to as the

noncoding strand. The opposite strand is considered the coding strand because it matches

the RNA transcript that encoded the protein.

Two strands are held together by hydrogen bonds between opposing bases. As seen in

figure 16, three hydrogen bonds hold the deoxyguanosine nucleotide to the deoxycytidine

nucleotide, whereas the other pair A-T pair is held together by two hydrogen bonds. Thus,

G-C bond are more resistant to denaturation or melting than A-T rich regions.

Structural analysis of DNA by denaturation (melting)

The double stranded structure of DNA can be separated into two strands (melted) in

solution by increasing the temperature or decreasing the salt concentration, this lead to the

pulling apart of the two stacks of bases while the bases are still connected in the polymer

by phosphodiester back bone. This denaturation of the DNA molecule leads to an increase

in the optical absorbance of the purine and pyrimidine bases- a phenomenon referred to as

hyperchromicity of denaturation. The double stranded DNA molecules exhibit properties

of rigid rod and in solution is a viscous material that loses its viscosity upon denaturation.

The separation of the strands of a given DNA molecule over a temperature range. The

mid-point is called the melting temperature (Tm). it is affected by the base compositions

of the DNA and the salt concentration of the solution. DNA and the salt concentration of

the solution. DNA that is rich in G-C pair melts at a higher temperature than those rich in

A-T pairs. A tenfold increase in monovalent cation concentration increases the Tm by

16.6 o C on the other hand destabilizes hydrogen bonding between bases, hence lowering

the Tm. This allow the strands of DNA or DNA-RNA hybrids to be separated at much

lower temperature there by reducing phosphodiester bond breakage that occurs at high

temperatures.

DNA Function as a template for Replication and Transcription

The genetic information stored in the nucleotide sequence if DNA serves two purposes:

(1) It is the source of information for the synthesis of all pattern molecules of the cell and

organisms and

(2) It provides the information inherited by daughter cells or offspring’s. Both functions

given above require that the DNA molecule serves as a template in the first case for

the transcription of the information into RNA and in the second case for the

replication of the information into daughter DNA molecules.

The complementary of double stranded of DNA suggest that replication of the DNA

molecule occurs in a semi conservative manner. Thus when each strand of the double

stranded parental DNA molecule separated to form its compliment during replication

each serve as a template on which a Nero-complementary strand is synthesized (see

figure 4). The two newly formed double stranded daughter DNA molecule each

containing one strand (but complementary rather than identical) from the parent

double-stranded DNA molecule (see figure 5). Each daughter cell contains DNA

molecule with information identical to that which the parent possessed yet in each

daughter cell the DNA molecule of the parent cell has been only semi conserved.

The chemical Nature of RNA differs from that DNA

RNA is a polymer of purine and pyrimidine ribonucleotides linked together by 31,51-

phosphodiester bridges analogues to those DNA. It shares many features with DNA

but it possesses served specific differences.

1 The sugar moiety in RNA to which the phosphate and bases are attached is ribose

rather than 21-deoxyribose of DNA

2 The pyrimidine components of RNA differ from those of DNA. For examples DNA

contains A, G, T and C while RNTA contains A G C t that instead of thymine, RNA

contains ribonucleotide of uracil.

3 RNA exists as a single strand rather than double strand in DNA. However, given

proper complementary base sequence with opposite polarity, the single strand of RNA

is capable of folding back itself like hairpin and these acquiring double-stranded

characteristics

4 Since the RNA is a single strand its G does not necessary equal its C content nor

does it’s A equal it content.

5 RNA can be hydrolysed by alkali to 2131 cyclic diesters Fig 17 mononucleotide

compounds that cannot be formed from alkali-treated DNA because of the absence of

a 21-hydroxyl group. The alkali lability at RNA is useful both diagnostically and

analytically.

Figure 17: Adenosine-21,31-cyclic phosphoric acid

RNA and Protein Synthesis

RNA and Protein Synthesis

The cytoplasmic RNA molecules that serve as templates for protein synthesis (i.e.

those tracts transfer genetic information from DNA to the protein synthesizing

machinery are called messenger RNAs, (mRNA). Many other cytoplasmic RNA

molecules (ribosomal RNAs, i.e. mRNAs) have structural roles wherein they

contribute to the formation and function of ribosomes (the organelle machinery

involves in protein synthesis) or serve as adapter molecules for the translation of RNA

information into specific sequences of polymerized amino acids

Hydrolysis of Polynucleotides

Nucleic acids can be hydrolysed by treatment with enzyme, acid or alkali. It is

possible to identify nucleic acids (RNA or DNA) in the base of their sensitively to

above regents.

(a) Acid and base hydrolysis

In the case of DNA, gently hydrolyses with 0.1M HCl at pH 3.0 causes selective

hydrolytic removal of its purine bases without affecting the N-glycosidic bond of

pyrimidine bases or phosphodiesters bonds of the back bone. The resulting DNA

derivatives devoid of the purine bases is called apurinic acid. Selective removal of

pyrimidine bases by different chemical conditions produces a pyrymidinic acid.

Heating of DNA at 110 degrees in 1.0 M HCl for 1 hr gives purine and pyrimidine

bases, deoxyribose and phosphate. DNA is not hydrolysed by bases (0.3M NaOH),

but RNA does. Research shown that OH group of 21 of D-ribose is required for

alkaline hydrolysis of RNA. The presence of base attacks the P-O-C2 and P-O-C3

linkages equally yielding an equimolar mixture of 2131-nucleoside monophosphate.

The cyclic monophosphate appears to be intermediate in alkaline hydrolysis of RNA

and Since DNA has no 21-OH group hence cannot form 21 and 31-cyclic

monophosphate hence cannot be hydrolysed by pancreatic ribonuclease.

 THE SYNTHESES OF SOME PURINES

1. Uric acid

This acid is a derivatives of purine found in the human urine and dropping of some

birds. Its molecular formula is C5H4N4O3. Its oxidation with HNO3 gives equal

amount of alloxan and urea.

Proof of the structure of alloxan (C4H2N2O4)

Hydrolysis with alkali

Since alloxan has no free NH2 and –COOH groups, the product of alkali hydrolysis

above shows that it is mesoxaly urea. This was confirmed by the reaction between

mexooxalic acid

When uric acid is oxidized in aqueous solution of lead dioxide, allantoin and

cabondioxide are obtained

Structure of allantoin in (C4H6N4O3)

i. Alkali hydrolysis of allantoin gives:

ii. Its oxidation with HNO3 gives:

iii. Hydrolysis of parabanic acid gives:

The structure of parabanic acid was confirmed by its synthesis from the condensation

reaction between oxaly chloride and urea.

The above information showed that allantoin contains parabanic acid joined to a

molecule of urea. The point of the attachment is obtained by the following

experimental evidence:

Reduction with HI
 iv. Controlled hydrolysis of hydantoin gives:

This shows that hydantoin is glycollylurea. The synthesis of hydantoin by West (1918) has

been used to confirm its structure thus:

HCl H
H N
O KCN O N O O
heat
H2N HN
OH AcOH NH2 COOH
hydantoin
glycine

v. It can also be prepared from the reaction between bromoacetylbromide and urea:

vi. All the above reaction and the reactions at allantoin will account for its structure.

vii. The oxidation of uric acid gives allantoin where uric acid lost one carbon atom as CO2.

To know the structure of uric acid we need to fix the one carbon back. The structure given to
uric acid must also include alloxan skeleton since its oxidation with HNO3 above gave

alloxan:

Studies by other workers e.g. Fischer (1884) supported Medicus formula while Fittig formula

was shown to be untenable. The proof of the Medicus formula was achieved by the synthesis

of uric acid. Three methods are given below.

(1) Behrend and Roosen (1888) synthesis of uric acid

viii. The reduction section led to the conversion of some aminouracil into hydroxyuracil.

Nitrous acid was used to convert 5-aminouracil into 5-hydroxyuracil.

(2) Fischer (1885) synthesis of uric acid