MODULE 3: The Nucleotides
NUCLEOTIDE CHEMISTRY
Biomedical importance:
a. energy metabolism, protein synthesis, regulation of enzyme activity, signal transduction
b. prinicipal donors and acceptors of phosphoryl groups in metabolism
c. nucleoside tri- and diphosphates (ATP, ADP) – energy transduction with metabolic interconversions and oxidative phosphorylation
d. form a portion of coenzymes with vitamins / vitamin derivatives
e. nucleosides linked to sugars or lipids -> key biosynthetic intermediates
f. UDP-glucose, UDP-galactose – sugar interconversions in biosynthesis of starch and glycogen
g. CDP-acylglycerol (nucleoside-lipid derivatives) – intermediates in biosynthesis
h. Metabolic regulation:
a. ATP-dependent phosphorylation by key metabolic enzymes
b. Allosteric regulation by ATP, AMP, CTP and control by ADP of rate of oxidative phosphorylation
i. Second messengers in hormonally regulated events: cyclic cAMP, cGMP
j. Signal transduction pathways: GTP, GDP
Heterocyclic compounds
- nitrogen containing (other than C)
- rings numbered in opposite directions (Pu – right; Py – left)
- planar – close association or “stacking” – stabilizes DNA
- oxo and amino groups of Pu and Py exhibit keto-enol and amine-imine tautomerism
Tautomerism – permitting some compounds to ‘switch’
Isomers = interconversible at equilibrium
- formal migration
- switch of single bond to double bond
1. Components of nucleoside & nucleotide
1.1. Types of bonds
1.1.1. Glycosidic bond
- nucleosides – derivatives of Pu and Py that have sugar linked to ring nitrogen
- (sugar)
o ribonucleosides – D-ribose
o deoxyribonucleosides – 2-deoxy-D-ribose
✓ linked to the heterocycle: B-N-glycosidic bond
▪ to the N-1 of pyrimidine; N-9 of purine
▪ steric hindrance (by the heterocycle) – no freedom of rotation about the bond – both (nucleosides or nucleotides)
therefore exist as syn or anti conformers – noninterconvertible; only interconverted by cleavage and reformation of the
glycosidic bond
1.1.2. Acid anhydride bond
- ligates additional phosphoryl groups to phosphoryl group of a mononucleotide = form nucleoside diphosphates and triphosphates
- high energy
Mononucleotides – nucleosides with phosphoryl group esterified to a hydroxyl group
- 3’- and 5’- nucleotides – with phosphoryl group in 3’- or 5’- hydroxyl group
- C5 – UMP, dAMP
*nucleosides and nucleotides - Syn or anti conformers – noniconvertible
- only inconverted by cleavage and reformation of the glycosidic bond
2. Few minor or modified bases and its significance
X=H X = Ribose X = ribose phosphate
Adenine Adenosine Adenosine monophosphate (AMP)
Guanine Guanosine Guanosine monophosphate (GMP)
Cytosine Cytidine Cytidine monophosphate (CMP
Uracil Uridine Uridine monophosphate (UMP)
Thymine Thymidine Thymidine monophosphate (TMP)
Modification – can generate additional structures
- 5-methylcytosine5-methylcytosine in bacterial and human DNA
- 5-hydroxymethylcytosine of bacterial and viral nucleic acids
- mono- and di-N-methylated adenine, guanine of mRNA – oligonucleotide recognition in regulating half-lives of RNA
- free nucleotides: hypoxanthine, xanthine, uric acid – intermediates in catabolism of adenine, guanine
- methylated hetercycles of plants: xanthine derivatives (coffee) [1, 3, 7 C atom], theophylline (tea), theobromine (cocoa) [3, 7 C atom]
Polynucleotides – DNA, RNA
- phosphodiester – can be formed by esterification of second hydroxyl group by 5’-phosphoryl group of mononucleotide
- 2nd hydroxyl is 3’-OH of pentose of 2nd nucleotide -> dinucleotide (pentose moieties linked by 3’,5’-phosphodiester bond – backbone of RNA, DNA)
, - Dinucleotide – elimination of H2O between 2 mononucleotides
- No biologic formation; hydrolysis is strongly favored on thermodynmic grounds
- In absence of catalysis by phosphodiesterases, hydrolysis of phosphodiester bonds of DNA occur over long periods of time
- Posttranslational modification -> additional structures
o Pseudouridine – D-ribose is linked to C5 of uracil by a carbon to carbon bond (usual β-N-glycosidic bond)
o Pseudouridylic acid – rearrangement of a UMP of a performed tRNA
o TMP (thymidine monophosphate) – methylation by S-adenosylmethionine of a UMP; contains ribose rather than deoxyribose
- Directional macromolecules
- 5’-end – free / phosphorylated 5’-hydroxyl
3. Name / enumerate the physicochemical properties of purine and pyrimidine bases and their relevance
3.1. Predominant tautomer of each base -> oxo & amino (favored by physiologic conditions)
3.2. uV absorption spectrum of base, nucleoside, nucleotide
- conjugated double bonds absorb uV light
- mutagenic effect – d/t absorption by nucleotides in DNA
- spectra – pH dependent
- pH 7.0 – all common NT absorb light at wavelength of 260nm = “absorbance at 260nm”
3.3. Differentiate solubility of uric acid from urate at neutral pH and lower pH
- depends on acidity of environment
- urates:
o seen in acidic pH >5.5
o soluble with alkali and heat
o normal in urine
- uric acid:
o seen in acidic pH <5.5
o soluble in alkali only
o if above normal value, indicates hyperuricemia
4. Differentiate ribonucleotide & deoxyribonucleotide as to:
Ribonucleotide Deoxyribonucleotide
Purine bases Adenine, guanine Adenine, guanine
Pyrimidine bases Uracil, cytosine Thymine, cytosine
Pentose sugar Ribose Deoxyribose
5. Enumerate different metabolic functions of naturally occurring nucleotides
5.1. Specific nucleotide involved
- ATP – principal biologic transducer of free energy; second messenger of cAMP
o Most abundant free nucleotide
o Mean intracellular concentration = 1mmol/L
* cAMP concentration = 2nmol/L
Precursors of nucleic acids:
- Adenosine 3’-phosphate-5’phosphosulfate – sulfate donor for sulfated proteoglycans; for sulfate conjugates of drugs
- S-adenosylmethionine – methyl group donor
- GTP – allosteric regulator; energy for protein synthesis
- cGMP – 2nd messenger in response to nitric oxide during relaxation of smooth muscle; binding of hormones to ligands
- UDP-sugar derivatives – sugar epimerization; biosynthesis of glycogen, glucosyl disaccharides, oligosaccharides of glycoproteins and proteoglycans
- UDP-glucuronic acid – forms urinary glucuronide conjugates of bilirubin and of many drugs (eg aspirin)
- CTP – biosynthesis of phosphoglycerides, sphingomyelin and other sphingosines
5.2. Relate its function to its structural feature
- high group transfer potential – acid anhydrides
- purine and pyrimidine nucleoside triphosphates -> function as group transfer reagents (most freq of the γ-phosphoryl
group)
- cleavage – acid anhydride bond
– coupled with a highly endergonic process
▪ covalent bond synthesis – eg polymerization of nucleoside triphosphates to form nucleic acid
endergonic – absorbs heat
exergonic – release heat; in hydrolysis of ATP
6. Identify and / or name some synthetic nucleoside, nucleotide analogs -> antimetabolites (chemotherapeutic agents)
6.1. Name its two general mechanisms of action
- Toxic effects:
o Inhibition of enzyme for nucleic acid synthesis or
o Incorporation into nucleic acids with resulting disruption of base-pairing; substitutes purine and pyrimidine
- Incorporated into DNA prior to cell division:
o 5-fluoro or 5-iodouracil
o 3-deoxyuridine
o 6-thioguanine
o 6-mercaptopurine
NUCLEOTIDE CHEMISTRY
Biomedical importance:
a. energy metabolism, protein synthesis, regulation of enzyme activity, signal transduction
b. prinicipal donors and acceptors of phosphoryl groups in metabolism
c. nucleoside tri- and diphosphates (ATP, ADP) – energy transduction with metabolic interconversions and oxidative phosphorylation
d. form a portion of coenzymes with vitamins / vitamin derivatives
e. nucleosides linked to sugars or lipids -> key biosynthetic intermediates
f. UDP-glucose, UDP-galactose – sugar interconversions in biosynthesis of starch and glycogen
g. CDP-acylglycerol (nucleoside-lipid derivatives) – intermediates in biosynthesis
h. Metabolic regulation:
a. ATP-dependent phosphorylation by key metabolic enzymes
b. Allosteric regulation by ATP, AMP, CTP and control by ADP of rate of oxidative phosphorylation
i. Second messengers in hormonally regulated events: cyclic cAMP, cGMP
j. Signal transduction pathways: GTP, GDP
Heterocyclic compounds
- nitrogen containing (other than C)
- rings numbered in opposite directions (Pu – right; Py – left)
- planar – close association or “stacking” – stabilizes DNA
- oxo and amino groups of Pu and Py exhibit keto-enol and amine-imine tautomerism
Tautomerism – permitting some compounds to ‘switch’
Isomers = interconversible at equilibrium
- formal migration
- switch of single bond to double bond
1. Components of nucleoside & nucleotide
1.1. Types of bonds
1.1.1. Glycosidic bond
- nucleosides – derivatives of Pu and Py that have sugar linked to ring nitrogen
- (sugar)
o ribonucleosides – D-ribose
o deoxyribonucleosides – 2-deoxy-D-ribose
✓ linked to the heterocycle: B-N-glycosidic bond
▪ to the N-1 of pyrimidine; N-9 of purine
▪ steric hindrance (by the heterocycle) – no freedom of rotation about the bond – both (nucleosides or nucleotides)
therefore exist as syn or anti conformers – noninterconvertible; only interconverted by cleavage and reformation of the
glycosidic bond
1.1.2. Acid anhydride bond
- ligates additional phosphoryl groups to phosphoryl group of a mononucleotide = form nucleoside diphosphates and triphosphates
- high energy
Mononucleotides – nucleosides with phosphoryl group esterified to a hydroxyl group
- 3’- and 5’- nucleotides – with phosphoryl group in 3’- or 5’- hydroxyl group
- C5 – UMP, dAMP
*nucleosides and nucleotides - Syn or anti conformers – noniconvertible
- only inconverted by cleavage and reformation of the glycosidic bond
2. Few minor or modified bases and its significance
X=H X = Ribose X = ribose phosphate
Adenine Adenosine Adenosine monophosphate (AMP)
Guanine Guanosine Guanosine monophosphate (GMP)
Cytosine Cytidine Cytidine monophosphate (CMP
Uracil Uridine Uridine monophosphate (UMP)
Thymine Thymidine Thymidine monophosphate (TMP)
Modification – can generate additional structures
- 5-methylcytosine5-methylcytosine in bacterial and human DNA
- 5-hydroxymethylcytosine of bacterial and viral nucleic acids
- mono- and di-N-methylated adenine, guanine of mRNA – oligonucleotide recognition in regulating half-lives of RNA
- free nucleotides: hypoxanthine, xanthine, uric acid – intermediates in catabolism of adenine, guanine
- methylated hetercycles of plants: xanthine derivatives (coffee) [1, 3, 7 C atom], theophylline (tea), theobromine (cocoa) [3, 7 C atom]
Polynucleotides – DNA, RNA
- phosphodiester – can be formed by esterification of second hydroxyl group by 5’-phosphoryl group of mononucleotide
- 2nd hydroxyl is 3’-OH of pentose of 2nd nucleotide -> dinucleotide (pentose moieties linked by 3’,5’-phosphodiester bond – backbone of RNA, DNA)
, - Dinucleotide – elimination of H2O between 2 mononucleotides
- No biologic formation; hydrolysis is strongly favored on thermodynmic grounds
- In absence of catalysis by phosphodiesterases, hydrolysis of phosphodiester bonds of DNA occur over long periods of time
- Posttranslational modification -> additional structures
o Pseudouridine – D-ribose is linked to C5 of uracil by a carbon to carbon bond (usual β-N-glycosidic bond)
o Pseudouridylic acid – rearrangement of a UMP of a performed tRNA
o TMP (thymidine monophosphate) – methylation by S-adenosylmethionine of a UMP; contains ribose rather than deoxyribose
- Directional macromolecules
- 5’-end – free / phosphorylated 5’-hydroxyl
3. Name / enumerate the physicochemical properties of purine and pyrimidine bases and their relevance
3.1. Predominant tautomer of each base -> oxo & amino (favored by physiologic conditions)
3.2. uV absorption spectrum of base, nucleoside, nucleotide
- conjugated double bonds absorb uV light
- mutagenic effect – d/t absorption by nucleotides in DNA
- spectra – pH dependent
- pH 7.0 – all common NT absorb light at wavelength of 260nm = “absorbance at 260nm”
3.3. Differentiate solubility of uric acid from urate at neutral pH and lower pH
- depends on acidity of environment
- urates:
o seen in acidic pH >5.5
o soluble with alkali and heat
o normal in urine
- uric acid:
o seen in acidic pH <5.5
o soluble in alkali only
o if above normal value, indicates hyperuricemia
4. Differentiate ribonucleotide & deoxyribonucleotide as to:
Ribonucleotide Deoxyribonucleotide
Purine bases Adenine, guanine Adenine, guanine
Pyrimidine bases Uracil, cytosine Thymine, cytosine
Pentose sugar Ribose Deoxyribose
5. Enumerate different metabolic functions of naturally occurring nucleotides
5.1. Specific nucleotide involved
- ATP – principal biologic transducer of free energy; second messenger of cAMP
o Most abundant free nucleotide
o Mean intracellular concentration = 1mmol/L
* cAMP concentration = 2nmol/L
Precursors of nucleic acids:
- Adenosine 3’-phosphate-5’phosphosulfate – sulfate donor for sulfated proteoglycans; for sulfate conjugates of drugs
- S-adenosylmethionine – methyl group donor
- GTP – allosteric regulator; energy for protein synthesis
- cGMP – 2nd messenger in response to nitric oxide during relaxation of smooth muscle; binding of hormones to ligands
- UDP-sugar derivatives – sugar epimerization; biosynthesis of glycogen, glucosyl disaccharides, oligosaccharides of glycoproteins and proteoglycans
- UDP-glucuronic acid – forms urinary glucuronide conjugates of bilirubin and of many drugs (eg aspirin)
- CTP – biosynthesis of phosphoglycerides, sphingomyelin and other sphingosines
5.2. Relate its function to its structural feature
- high group transfer potential – acid anhydrides
- purine and pyrimidine nucleoside triphosphates -> function as group transfer reagents (most freq of the γ-phosphoryl
group)
- cleavage – acid anhydride bond
– coupled with a highly endergonic process
▪ covalent bond synthesis – eg polymerization of nucleoside triphosphates to form nucleic acid
endergonic – absorbs heat
exergonic – release heat; in hydrolysis of ATP
6. Identify and / or name some synthetic nucleoside, nucleotide analogs -> antimetabolites (chemotherapeutic agents)
6.1. Name its two general mechanisms of action
- Toxic effects:
o Inhibition of enzyme for nucleic acid synthesis or
o Incorporation into nucleic acids with resulting disruption of base-pairing; substitutes purine and pyrimidine
- Incorporated into DNA prior to cell division:
o 5-fluoro or 5-iodouracil
o 3-deoxyuridine
o 6-thioguanine
o 6-mercaptopurine
