nucleotide chemistry & metabolism will help to students to gain knowledge about molecular basics & drugs used in certain cancer therapies , viral disorders etc.

1. Chemistry, Metabolism, Biological
& Clinical significance
Basant Joshi
Assistant Professor
NUCLEOTIDES
1

2.  Nucleic acids are macromolecules
present in all living cells in combination
with proteins to from nucleoproteins.
The protein is usually basic in nature,
eg. Protamines & histones conatining
high conc. Of basic amino acids.
 Nucleic acids are polymers of
nucleoside monophosphates.
4/24/20202

3. Composition of nucleotides:-
 Made up of 3 components
1. Nitrogenous base (purine or
pyrimidine)
2. Pentose sugar (ribose or deoxyribose)
3. Phosphate group esterified to the
sugar
4/24/20203

4. NUCLEOTIDES &NUCLEOTIDES
COMPONENTS
4/24/20204

5. NUCLEOTIDES- Components
PHOSPHATE
PENTOSE SUGAR
NITROGENOUS
BASE
4/24/20205

6. MAJOR NITROGENOUS BASES
 PURINES-
 Adenine (A) : 6-aminopurine
 Guanine (G): 2-amino 6-oxypurine
4/24/20206

7.  PYRIMIDINES-
 Cytosine (C): 2-oxy 4-
aminopyrimidine
 Uracil (U) : 2,4- dioxypyrimidine
 Thymine (T) : 2,4- dioxy 5-
methylpyrimidine
4/24/20207

8. 4/24/20208

9. MAJOR NITROGENOUS BASES
9

10. Minor bases found in nucleic
acids
Unusual / rare/ modified
bases found in DNA and
RNA
N6-methyl adenine
N7-methyl guanine
5-methyl cytosine
N4-acetyl cytosine
tRNA- about 10-20% of
total nucleotides in tRNA
contain minor bases
Thymine
Dihydrouracil
Pseudouracil
Hypoxanthine
Uric acid (2,6,8
trioxopurine)
4/24/202010

11. Properties of bases
 Tautomerization of bases:
Keto/Lactam & Enol/Lactim
froms
 The keto form predominates
at physiological pH
 Purine and pyrimidine bases
absorb uv light
 Strong absorbance of UV
light at 260 nm
4/24/202011

12. NUCLEOSIDE
A nucleoside = Nitrogenous base
in β-N-glycosidic linkage with a
pentose
4/24/202012

13. β-N-glycosidic linkage joins N-9 of the purine
base (or) N-1 of the pyrimidine base with C-1’
of pentose
•The atoms of the base in nucleoside are given cardinal numbers
• The carbon atoms of the sugar are given primed numbers13

14. NOMENCLATURE OF
NUCLEOSIDES
 Ribonucleosides
Purine nucleosides end with “-osine”
Adenine + Ribose Adenosine
Guanine + Ribose Guanosine
Hypoxanthine + Ribose Inosine
Xanthine + Ribose Xanthosine
Pyrimidine nucleosides end with “-idine”
Uracil + Ribose Uridine
Cytosine + Ribose Cytidine
4/24/202014

15. Nomenclature of nucleosids………..
 Deoxyribonucleosides
 Deoxynucleosides are denoted by adding
the prefix “d”- before the nucleoside—
Adenine + deoxyribose  Deoxyadenosine (d-Adenosine)
Guanine + deoxyribose  Deoxyguanosine (d-Guanosine)
Cytosine + deoxyribose  Deoxycytidine (d-Cytidine)
Thymine + deoxyribose  Deoxythymidine (d-thymidine)
4/24/202015

16. NUCLEOTIDES
Nucleotides are phosphate esters of
nucleosides
1. Nucleoside monophosphate
(NMP):
Nucleoside with one
phosphate group attached
Nucleoside-5’-
monophosphate
Eg: Adenosine-5’-
monophosphate/ (5’-AMP)
Nucleoside-3’-
monophosphate
Eg: Adenosine-3’- 4/24/202016

17. NOMENCLATURE OF NMPs
(NMP= Nucleoside + phosphate)
Ribonucleotides
Adenosine + Pi Adenosine monophosphate
(AMP/ Adenylate/ Adenylic acid)
Guanosine + Pi Guanosine monophosphate
(GMP/ Guanylic acid)
Cytidine + Pi Cytidine monophosphate
(CMP/ Cytidylic acid)
Uridine + Pi Uridine monophosphate
(UMP/ Uridylic acid)
Inosine + Pi Inosine monophosphate
(IMP/ Inosinic acid)
Xanthosine + Pi Xanthosine monophosphate
(XMP)
4/24/202017

18. NOMENCLATURE OF NMPs
(NMP= Nucleoside + phosphate)
Deoxyribonucleotides
dAdenosine + Pi dAMP
dGuanosine + Pi dGMP
dCytidine + Pi dCMP
dThymidine + Pi dTMP
4/24/202018

19. 4/24/202019
D-Ribose and 2’-Deoxyribose
*Lacks a 2’-OH group

20. HIGHER NUCLEOTIDES
 Nucleoside
diphosphate
s (NDP)
 Nucleoside
triphosphat
es (NTP)
The high energy
anhydride
bonds (~)
20

21. NUCLEOTIDES-
NOMENCLATURE
Ribonucleoside phosphates
Nucleoside NMP NDP NTP
Adenosine AMP ADP ATP
Guanosine GMP GDP GTP
Cytidine CMP CDP CTP
Uridine UMP UDP UTP
Deoxyribonucleoside phosphates
Nucleoside NMP NDP NTP
dAdenosine dAMP dADP dATP
dGuanosine dGMP dGDP dGTP
dCytidine dCMP dCDP dCTP
dThymidine dTMP dTDP dTTP
21

22. FUNCTIONS OF NUCLEOTIDES
They supply monomeric units of
nucleic acids
Nucleotides play important role as
energy currency in the cells
ATP is central to energy metabolism,
GTP drives protein synthesis.
CTP drives lipid synthesis.
UTP drives carbohydrate metabolism
4/24/202022

23. Functions of nucleotides………
 Nucleotides such as cyclic adenosine
monophosphate (cAMP) & cyclic
guanosine monophosphate (cGMP) serve
as second messengers in signal
transduction pathways
 Nucleotides serve as carriers of activated
intermediates in biosynthetic reactions
 UDP Glucose in glycogen synthesis
 UDP Galactose in synthesis of ceramide
 CTP Choline in phospholipid synthesis 4/24/202023

24. Functions of nucleotides………
Nucleotides act as allosteric
modulators of metabolic pathways
 Enzyme: Phosphofructokinase of glycolysis
has
 AMP as positive modulator
 ATP as negative modulator
4/24/202024

25. Functions of nucleotides………
Nucleotides are structural
components of several essential
coenzymes CoA, FAD, NAD+, NADP+
CoA
25

26. Functions of nucleotides……….
 S-adenosylmethionine (SAM): Active
methionine- serves as a methyl donor in
methylation reactions
Methionine
Ribose
Adenine
26

27. Functions of nucleotides……….
 Phosphoadenosine
phosphosulphate
(PAPS): Active sulphate-
acts as a sulphate group
donor for the formation
of—
Sulphated
mucopolysaccharides
Sulphatides &
In detoxication reactions 4/24/202027

28. SYNTHETIC NUCLEOTIDE
ANALOGUES
These compounds have structural
similarity with the bases of nucleic
acids
These are widely used for the
treatment of cancer and viral
diseases
Interfere with the synthesis of nucleic
acids (or)
Inhibit certain vital enzyme reactions in4/24/202028

29. SYNTHETIC NUCLEOTIDE
ANALOGUES
 6-mercaptopurine- in acute leukemia
 5-fluorouracil- cancer chemotherapy
 Aza thiopurine- used as an immunosuppressive
agent
 Vidarabine (Adenine arabinoside)- treatment of
herpes virus infection
 Cytarabine (cytosine arabinoside)- cancer
chemotherapy, antiviral agent
 AZT (azidothymidine) and ddI (dideoxyinosine)-
interfere with the replication of HIV-Treatment of
AIDS
 Allopurinol- has structural similarity to 4/24/202029

30. 6-Mercaptopurine 5- Fluorouracil
8-Azaguanine
4/24/202030

31. Allopurinol structural analogue of Hypoxanthine
Act as Xanthine oxidase inhibitor
4/24/202031

32. 6-mercaptopurine (6MP)
 6mp inhibits conversion of IMP to adenine &
guanine nucleotides. That are building blocks for
RNA & DNA.
 Nucleotide formation:-6 mp converted to
nucleotide analog, 6 mp ribose phosphate (6 thio
inosinic acid or TIMP)
 Inhibition of purine synthesis:- TIMP can
inhibit the 1st step of denovo purine synthesis.
 Incorporation into nucleic acid:- TIMP is
converted to thioguanine
monophosphate(TGMP), which after
phosphorylation to di , triphosphate can be
incorporated into RNA. 4/24/202032

33. 5 fluorouracil
 Is not a active species
 Must converted into active metabolite by cellular
enzyme
 Into 5 fluoro uridine triphosphate(fUTP) & 5 fluoro
deoxy uridine monophosphate(fdUMP)
 fdUMP is a potent & specific inhibitor of
thymidylate synthase
 In presence of H4folate, fdUMP & thymidylate
synthase, a ternary complex is formed that results
in covalent binding of fdUMP to thymidylate
synthase.
 This inhibit dTMP synthesis & leads a 4/24/202033

34. Cytosine arabinoside
 Must be metabolized to cytosine arabinoside 5’
triphosphate (araCTP)
 araCTP competes with dCTP in the DNA
polymerase reaction
 & araCMP is incorporated into DNA
 This inhibits synthesis of growing DNA strands.
 Ribose is replaced by arabinose.
4/24/202034

35. Methotrexate (MTX)
 A antifolate, interfere with formation of H4 folate
from H2 folate or H2 folate from folate by
inhibition of H2 folate reductase.
 A close structural analog of folic acid.
 MTX and folate differ at C-4 where an amino
group relaces a hydroxyl group &
 N-10, where a methyl group replaces a hydrogen
atom
 MTX specifically inhibits H2 folate reductase
4/24/202035

36. 4/24/202036

37. Hydroxyurea
 Inhibits ribonucleotide reductase to
block reduction of CDP, UDP, GDP &
ADP to corresponding
deoxyribonucleoside diphoaphates.
Tiazofurin
 Is converted by cellular enzymes to
NAD+ analog, thizofurin adenine
dinucleotide (TAD)
 TAD inhibits IMP dehydrogenase,
the rate limiting enzyme in GTP 4/24/202037

38. Glutamine antagonists
 Amidation reactions of denovo purine nucleotide
(N-3 & N-9), synthesis of GMP from IMP,
formation of cytosolic carbamoyl phosphate,
synthesis of CTP from UTP and synthesis of
NAD+
 Azaserine and diazo oxo norleucine(DON)
competitively inhibit glutamyl amidotransferase
4/24/202038

39. Purine & pyrimidine analogs as
antiviral agents
 Acyclovir (acycloguanosine) a purine analog.
 Is activated to monophosphate by specific HSV
thymidine kinase encoded by the HSV genome
 Which catalyze phosphorylation of acycloguanosine
 The host cellular thymidine kinase cannot utilize
acyclovir as a substrate
 Acycloguanosine monophosphate is then
phosphorylated by cellular enzyme to di & tri
phosphate forms
 Acycloganosine triphosphate serves as a substrate
for the HSV specific DNA polymerase & is
incorporated into growing viral DNA chain 4/24/202039

40. 4/24/202040

41. AZT (azido deoxythymidine) or
zidovudine
 Is phosphoraylated by cellular kinase to AZT
triphosphate
 Which blocks HIV replication by inhibiting HIV
DNA polymerase (an RNA-dependent
polymerase)
 Selectivity of AZT for HIV infected versus
uninfected cells occurs because DNA polymerase
from HIV is at least 100 fold more sensitive to
AZT triphosphate than is host cell DNA
dependent DNA polymerase. 4/24/202041

42. Nucleic acid
metabolism
Purine & Pyrimidine
4/24/202042

43. Synthesis of purines
 Three processes that contribute to
purine nucleotide biosynthesis are in
order of decreasing importance:-
1. Synthesis from amphibolic
intermediates (denovo)
2. Phosphoribosylation of purines
3. Phosphorylation of purine nucleosides
4/24/202043

44.  11 enzymes catalyzed the reactions that convert
ribose 5 phosphate to inosine monophosphate
(IMP), first intermediate formed in the denovo
pathway of purines.
 5 phosphoribosyl 5 pyrophosphate (PRPP) is
required for purine & pyrimidine synthesis
 PRPP is an intermediate in the purine salvage
pathway & biosynthesis of NAD+ & NADP+.
 Two parent purine nucleotides of nucleic acids
are:- 4/24/202044

45.  Purine nucleotides can be synthesized by two
pathways:-
1. Denovo pathway (new synthesis from
amphibolic intermediates-----amphibolic
pathway- a group of metabolic reactions
providing small metabolites.
2. Salvage pathway
 By phosphoribosylation of free purine bases &
 By phosphorylation of purine nucleosides
4/24/202045

46. Denovo Pathway (Purine)
 In denovo pathway, purine ring is assembled on
ribose 5 phosphate from a variety of precursors.
 Major site is liver
 Pathway operates in cytoplasm
 Enzymes catalyzing the reactions are existing as
a multienzyme complex in eukaryotic cells, this
arrangement increases the efficiency of the
pathway. 4/24/202046

47. 4/24/202047

48. 4/24/202048

49. Conversion of IMP to AMP &
GMP
 Both adenosine and guanosine monophosphates
are produced from IMP, using nitrogen of
asparate & glutamine respectively
4/24/202049

50. 4/24/202050

51. Regulation of denovo synthesis of
purines
Controlled by:-
Concentration of PRPP
Feedback regulation at several sites
Conc. Of PRPP:-
 Depends on the rate of its
 Synthesis utilization & degradation
The rate of PRPP synthesis depends on:-
 Avaliability of ribose 5-P
 Activity of PRPP synthetase 4/24/202051

52. 4/24/202052

53. Salvage pathway of purine
 Provides purine nucleotides for tissues, incapable
of their biosynthesis by denovo pathway.
Ex. Human brain has low level of PRPP
amidotransferase
RBCs & polymorphonuclear leucocytes cannot
synthesized 5 phosphoribosylamine.
The pathway involved in the conversion of purines,
purine ribonucleoside and purine
deoxyribonucleosides to mononucleotides is called
salvage pathway (means property saved from loss
or danger)
4/24/202053

54. Salvage Reaction by
Phosphoribosylation of Purine Bases
 Adenine Phosphoribosyl Transferase
(APRTase) catalyzes the formation of
adenylate. (AMP)
 Hypoxanthine Guanine Phosphoribosyl
Transferase (HGPRTase) catalyzes the
formation of IMP &GMP. 4/24/202054

55. 4/24/202055

56. Phosphorylation of purine
nucleotide
 2nd salvage mechanism involves direct
phosphorylation by ATP by kinase.
1. Adenosine --------AMP
2. Guanosine---------GMP
4/24/202056

57. Synthesis of
deoxyribonucleotides
4/24/202057

58. Catabolism
of Purine
4/24/202058

59. Significance of uric acid
 Uric acid plays a beneficial role as a potent
antioxidant.
 It is effective scavenger of free radicals.
4/24/202059

60.  Normal serum uric acid
 Male:- 3.5-7.2mg/dl
 Female:- 2.6-6mg/dl
 Urine:-500-700mg/day
4/24/202060

61. 4/24/202061
Applied
 The most common disorder is elevation of
uric acid level in blood –
HYPERURICEMIA.
 GOUT
 LESCH-NYHAN SYNDROME

62. Catabolism of purines, adenine &
guanine produces uric acid.
At physiological pH uric acid is mostly
ionized & present in plasma as
sodium urate.
4/24/202062

63.  Uric acid is 2,6,8 trihydroxypurine or 2,6,8
trioxopurine
 It acts like a diabasic acid & can form mono & di
sodium salts depending on pH. These salts are
deposited in the joints causing arthritis (gout)
4/24/202063

64. 4/24/202064
GOUT
 Uric acid & urates are insoluble molecules, precipitate out of
aqueous solutions such as urine or synovial fluids, resulting
inflammation, consequence of this is the condition Gout.
 At 30 ͦC, solubility of uric acid is lowered to 4.5mg/dl. So uric
acid is deposited in cooler areas of the body to cause Tophi.
Seen in distal joints of foot.
 Increased deposition causes deposition of uric acid crystal in
the urinary tract leading to calculi or stone formation with
renal damage.


65. Primary Hyperuricemia
 About 10% gout are idiopathic
 Shows familial incidence
 1:500
4/24/202065

66. Causes of primary hyperuricemia
 Over activity of PRPP synthetase:-Abnormal
PRPP synthetase – the low km & Vmax of the
enzyme is high Resistant to feed back inhibition,
leading to increased production of PRPP.
 Deficiency of salvage pathway enzyme
(HGPRTase)
 5 PHOSPHORIBOSYL AMIDO
TRANSFERASE –is active, but not sensitive to
feedback regulation by inhibitory nucleotides so
overproduction of purines.
4/24/202066

67. 4/24/202067
Secondary HYPERURICEMIA
 Increased production of uric acid –may be due to
increased turnover rate of nucleic acids as found in –
 Rapidly growing malignant tissue e.g leukemias ,
polycythemia.
 Pts undergoing radiotherapy and chemotherapy
(tumor necrosis syndrome)
 Tisssue damage due to trauma and raised rate of
catabolism as in case of starvation.
 Psoriasis
 Alcoholism
 GLUCOSE 6 PHOSPHATASE DEFICIENCY.

68. 4/24/202068
 REDUCED EXCRETION RATE –AS IN
 Renal failure
 Treatment with thiazide diuretics which
inhibit tubular secretion of uric acid
 Lactic acidosis

69. 4/24/202069
CLINICAL FEATURES OF GOUT
 Attack may be precipitated by high purine diet
and increased intake of alcohol.
 Gouty arthritis affects the first
metatarsophalageal joint and other joints too.
 Synovial fluid shows birefringent monosodium
urate crastals
 In chronic cases –tophi

70. 4/24/202070
Treatment
 Decrease intake of purine diet & restrict
alcohol
 Allopurinol
 Uricosuric drugs – decrease the
reabsorption of uric acid. eg probenecid,
sulfinpyrazone

71. 4/24/202071
HYPOURICEMIA
 ADENOSINE DEAMINASE DFICIENCY
 XANTHINE OXIDASE DEFICIENCY

72. 4/24/202072
 Lesch Nyhan Syndrome
 It is X-linked disorder .
 Incidence is 1 in 10000 males.
 Due to complete deficiency of HGPRTase
 CLINICAL FEATURES :- neurological symptoms
 Mental Retadation
 Self mutilation
 Nephrolitiasis
 Hyperuricemia
 Dystonic movement
 Kelley seegmiller syndrome :- >1.5-2% partial
deficiency of HGPRTase

73.  Associated with defects in ADA & purine
nucleoside phosphorylase(PNP) respectively.
 These enzymes are involves in degradative
pathways leading to formation of uric acid.
 Substrate for ADA- adenosine & deoxyadenosine
 Substrate for PNP-inosine, guanosine,
deoxyinosine,& deoxyguanine
4/24/202073
IMMUNODEFICIENCY DISEASE

74. 4/24/202074
 SCID (Severe Combined Immunodeficiency)-
involves both T and B cells deficiency and is
due to adenosine deaminase deficiency. Here
circulating lymphocytes are decreased and there
is failure to mount an immune response.
 Nucleoside phosporylase deficiency – T cell
deficiency only with normal B cell function.
 Both are autosomal recessive disorders.
 SCID is fatal often by 18 month of age.

75.  ADA is associated with SCID involving both T-cell
& B-cell functions.
Lack of enzyme due to:-
 In ADA deficient patients, intracellular conc. of
dATP & SAH are increased.
 High conc. of dATP inhibit ribonucleotide
reductase activity leads to inhibit DNA synthesis
 deadenosine inactivates SAH hydrolase leading
to decreased SAM for methylation of bases in
RNA & DNA
 Increased conc. of adenosine results in increased
cAMP 4/24/202075

76. Treatment
 Blood transfusion
 Bone marrow transplantation
 Enzyme replacement therapy with ADA-
polyethylene glycol conjugate(ADA-PEG)
 ADA is First disorder to be treated by gene
therapy
4/24/202076

77. Pyrimidine synthesis
 Pyrimidine nucleotides are:- CMP, UMP, TMP
 Unlike the synthesis of purine nucleotide, six
membered pyrimidine ring is made first and then
attached to ribose phosphate, which is donated by
PRPP.
 Precursors for denovo synthesis of pyrimidine:-
 Glutamine provides N3
 Aspartic acid provides C4, C5,C6 & N1
 CO2 provides C2
4/24/202077

78. Sources of Pyrimidine Ring
4/24/202078

79. 4/24/202079

80. 4/24/202080

81. 4/24/202081

82. REGULATION OF PYRIMIDINES
4/24/202082

83. Catabolism of Pyrimidine
4/24/202083

84. 4/24/202084
CLINICAL MANIFESTATION
 HEREDITARY OROTIC ACIDURIA –
 Type I – Defective enzymes are OROTIDYL
PHOSPHORIBOSYLTRANSFERASE and
OROTIDYL DECARBOXYLASE.
 Type II- Only OROTIDYL DECARBOXYLASE
deficiency.
 The disorder is characterised by large urinary
excretion of orotic acid.
 Children usually presents with anemia and growth
retardation .Due to deficient pyrimidine synthesis ,
DNA and RNA synthesis do not keep pace with cell
proliferation specially the dividing cells

85. 4/24/202085
 Treatment – diet rich in uridine
 SECONDARY OROTIC ACIDURIA/ REYE’S
SYNDROME- It is due to defective ORNITHINE
TRANSCARBAMYLASE of Urea cycle, due to
which the enzyme diffuses out into cytosol which
leads to orotic aciduria.
 There is associated increase in blood ammonia and
amino acid (glutamine) level which are distinguishing
features.

86. THANK YOU
4/24/202086