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Organic Organic Chemistry Chemistry
William H. Brown &William H. Brown &
Christopher S. FooteChristopher S. Foote
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NucleicNucleicAcidsAcids
Chapter 28Chapter 28
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Nucleic AcidsNucleic Acids Nucleic acid:Nucleic acid: a biopolymer containing three
types of monomer units• heterocyclic aromatic amine bases derived from
purine and pyrimidine• the monosaccharides D-ribose or 2-deoxy-D-ribose• phosphoric acid
Following are names and one-letter abbreviations for the heterocyclic aromatic amine bases most common to nucleic acids
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Purine/Pyrimidine BasesPurine/Pyrimidine Bases
HN
N
O
O
H
N
N
NH2
O
H
HN
N
O
O
H
CH3N
N
HN
N N
N
O
HH2N
N
N N
N
NH2
H
N
N N
N
H
Uracil (U) Thymine (T) Cytosine (C)Pyrimidine
1
2
34
5
6
Guanine (G)Adenine (A)Purine
1
2
34
56 7
8
9
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NucleosidesNucleosides Nucleoside:Nucleoside: a building block of nucleic acids,
consisting of D-ribose or 2-deoxy-D-ribose bonded to a heterocyclic aromatic amine base by a -glycosidic bond
anomericcarbon
a - -N glycosidicbond
HH
H
H
OHOCH2
HO OH
O
O
HN
N
Uridine
β-D-riboside
uracil
1'
2'3'
4'
5'
1
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NucleotidesNucleotides Nucleotide:Nucleotide: a nucleoside in which a molecule of
phosphoric acid is esterified with an -OH of the monosaccharide,
N
NN
N
NH2
O
OHOH
HH
H
CH2
H
OP
O-
O-O
5'
Adenosine 5'-monophosphate(AMP)
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NucleotidesNucleotidesExample 28.1Example 28.1 identify these nucleotides
NO
N
O
HH
H
H
HOH
-O-P-O-P-O-CH2
NH2
(b)(a)-O -O
OO
O
HH
H
H
OH
HOCH2
HN
N N
NO
H2N
PO-
-O O
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Acyclovir & AZTAcyclovir & AZT
Azidothymidine (AZT) Acyclovir (drawn to show itsstructural relationship to 2-deoxyguanosine
O
O
N
HN
HOC
HN3
H
H
HH
O
CH3
O
H HHH
HOC
H2N
HN
N
O
N
N
H2H2
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DNA - 1° StructureDNA - 1° Structure Deoxyribonucleic acids (DNA)
• a backbone of alternating units of 2-deoxy-D-ribose and phosphate in which the 3’-OH of one 2-deoxy-D-ribose is joined by a phosphodiester bond to the 5’-OH of another 2-deoxy-D-ribose unit
Primary Structure:Primary Structure: the sequence of bases along the pentose-phosphodiester backbone of a DNA molecule (or an RNA molecule) • read from the 5’ end to the 3’ end
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DNA - 1° StructureDNA - 1° Structure
NO
HN
-O-P-O-CH2
O
HH
OH
HH
H
OCH3
O=P O CH2
O
-O
O-
NH
NN
N
O
NH2
O
HH
O
HH
H
5'
3'
phosphorylated5' end
free 3' end
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DNA - 2° StructureDNA - 2° Structure Secondary structure:Secondary structure: the ordered arrangement
of nucleic acid strands The double helix model of DNA 2° structure was
proposed by James Watson and Francis Crick in 1953
Double helix:Double helix: a type of 2° structure of DNA molecules in which two antiparallel polynucleotide strands are coiled in a right-handed manner about the same axis
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T-A Base PairingT-A Base Pairing A major factor stabilizing the double helix
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C-G Base PairingC-G Base Pairing
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Forms of DNAForms of DNA B-DNA
• the predominant form in dilute aqueous solution• a right-handed helix• 2000 pm thick with 3400 pm per ten base pairs• minor groove of 1200pm and major groove of 2200 pm
A-DNA• a right-handed helix, but thicker than B-DNA• 2900 pm per 10 base pairs
Z-DNA• a left-handed double helix
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DNA - 3° StructureDNA - 3° Structure Tertiary structure:Tertiary structure: the three-dimensional
arrangement of all atoms of nucleic acid, commonly referred as supercoiling
Circular DNA:Circular DNA: a type of double-stranded DNA in which the 5’ and 3’ ends of each stand are joined by phosphodiester bonds (Fig 28.10)
Histone:Histone: a protein, particularly rich in the basic amino acids lysine and arginine, that is found associated with DNA molecules
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DNA - 3° StructureDNA - 3° Structure Chromatin:Chromatin: consists of DNA molecules wound
around particles of histones in a beadlike structure• further coiling produces the dense chromatin found in
nuclei of plant and animal cells
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Ribonucleic Acids (RNA)Ribonucleic Acids (RNA) RNA are similar to DNA in that they, too, consist
of long, unbranched chains of nucleotides joined by phosphodiester groups between the 3’-OH of one pentose and the 5’-OH of the next. However,• the pentose unit in RNA is -D-ribose rather than -
2-deoxy-D-ribose• the pyrimidine bases in RNA are uracil and cytosine
rather than thymine and cytosine• RNA is single stranded rather than double stranded
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RNARNA RNA molecules are classified according to their
structure and function
Ribosomal RNA (rRNA):Ribosomal RNA (rRNA): a ribonucleic acid found in ribosomes, the site of protein synthesis
Molecular WeightRange (g/mol)
Number ofNucleotides
Percentageof Cell RNA
mRNA 25,000 - 1,000,000 75 - 3,000 2tRNA 23,000 - 30,000 73 - 94 16rRNA 35,000 - 1,100,000 120 - 2904 82
Type
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RNARNA Transfer RNA (tRNA):Transfer RNA (tRNA): a ribonucleic acid that
carries a specific amino acid to the site of protein synthesis on ribosomes
OBase
OHO
CH
RNH3
+
tRNA-O-P-O-CH2
amino acid, boundas an ester to itsspecific tRNA
HH H
H
C=O
O
O-
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RNARNA Messenger RNA (mRNA):Messenger RNA (mRNA): a ribonucleic acid that
carries coded genetic information from DNA to the ribosomes for the synthesis of proteins• present in cells in relatively small amounts and very
short-lived• single stranded• their synthesis is directed by information encoded on
DNA• a complementary strand of mRNA is synthesized
along one strand of an unwound DNA, starting from the 3’ end
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RNARNA• the synthesis of mRNA from DNA is called
transcription
3'-A-G-C-C-A-T-G-T-G-A-C-C-5'
5'-U-C-G-G-U-A-C-A-C-U-G-G-3'
DNA template
mRNA
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UUU PheUUC PheUUA LeuUUG Leu
UCU SerUCC SerUCA SerUCG Ser
UAU TyrUAC TyrUAA StopUAG Stop
UGU CysUGC CysUGA StopUGG Trp
GUCAG
CUU LeuCUC LeuCUA LeuCUG Leu
CCU ProCCC ProCCA ProCCG Pro
CAU HisCAC HisCAA GlnCAG Gln
CGU ArgCGC ArgCGA ArgCGG Arg
UCAG
G
AUU IleAUC IleAUA IleAUG* Met
ACU ThrACC ThrACA ThrACG Thr
AAU AsnAAC AsnAAA LysAAG Lys
AGU SerAGC SerAGA ArgAGG Arg
UCAG
UCAG
GUU ValGUC ValGUA ValGUG Val
GCU AlaGCC AlaGCA AlaGCG Ala
GAU AspGAC AspGAA GluGAG Glu
GGU GlyGGC GlyGGA GlyGGG Gly
*AUG also serves as the principal initiation codon.
U
U C A
C
A
The Genetic CodeThe Genetic Code
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The Genetic CodeThe Genetic Code Properties of the Code
• only 61 triplets code for amino acids; the remaining 3 (UAA, UAG, and UGA) signal chain termination
• the code is degenerate, which means that several amino acids are coded for by more than one triplet. Leu, Ser, and Arg, for example, are each coded for by six triplets
• for the 15 amino acids coded for by 2, 3, or 4 triplets, it is only the third letter of the codon that varies. Gly, for example, is coded for by GGA, GGG, GGC, and GGU
• there is no ambiguity in the code; each triplet codes for one and only one amino acid
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Sequencing DNASequencing DNA Restriction endonuclease:Restriction endonuclease: an enzyme that
catalyzes hydrolysis of a particular phosphodiester bond within a DNA strand• over 1000 endonucleases have been isolated and their
specificities determined• typically they recognize a set sequence of nucleotides
and cleave the DNA at or near that particular sequence
• EcoRI from E. coli, for example, cleaves as shown
5' G-A-A-T-T-C---3'EcoRI
5' G + 5'-A-A-T-T-C---3'
cleavage here
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Sequencing DNASequencing DNA• following are several more examples of
endonucleases and their specificities
AluI AG CT
BalI TGG CCA
FnuDII CG CG
HeaIII GG CC
Not I GC GGCCGC
Mbol GATC
SacI GAGCT C
Enzyme EnzymeRecognitionSequence
RecognitionSequence
HpaII C CGG
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Sequencing DNASequencing DNA Polyacrylamide gel electrophoresis:Polyacrylamide gel electrophoresis: a technique
so sensitive that it is possible to separate nucleic acid fragments differing from one another in only a single nucleotide• Maxam-Gilbert method:Maxam-Gilbert method: a method developed by Allan
Maxam and Walter Gilbert; depends on base-specific chemical cleavage
• Dideoxy chain termination method:Dideoxy chain termination method: developed by Frederick Sanger
• Gilbert and Sanger shared the 1980 Nobel prize for biochemistry for their “development of chemical and biochemical analysis of DNA structure.”
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Replication in VitroReplication in Vitro• During replication, the sequence of nucleotides in one
strand is copied as a complementary strand to form the second strand of a double-stranded DNA
• Synthesis is catalyzed by DNA polymerase• DNA polymerase will catalyze synthesis in vitro using
single-stranded DNA as a template, provided that (1) the four deoxynucleotide triphosphate (dNTP) monomers and (2) a primer are present
• Primer:Primer: an oligonucleotide capable of forming a short section of double-stranded DNA (dsDNA) by base-pairing with its complement on a single-stranded DNA (ssDNA)
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Replication in VitroReplication in Vitro• Because a new DNA strand grows from its 5' to 3' end,
the primer must have a free 3'-OH group to which the first nucleotide of the growing chain is added
5' Single-stranded DNAT C A A C G T C TA G A
TCAGPrimer 5'
3'
3'HO
OH
direction of synthesiscatalyzed by DNA polymerase
dATP, dTTP, dCTP, dGTP
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Dideoxy Chain TerminationDideoxy Chain Termination• the key to the chain termination method is addition to
the synthesizing medium of a 2’,3’-dideoxynucleotide triphosphate (ddNTP)
• because a ddNTP has no 3’-OH, chain synthesis is terminated where a ddNTP becomes incorporated
-O-P-O-P-O-P-O-CH2
O-
O
O- O-
O
H
Base
H H
H HO
H
A 2',3'-dideoxynucleoside triphosphate (ddNTP)
O
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Dideoxy Chain TerminationDideoxy Chain TerminationIn this method, the following are mixed• single-stranded DNA of unknown sequence and
primer; then divided into four reaction mixtures
To each reaction mixture is then added• the four dNTP, one of which is labeled in the 5’ end
with phosphorus-32
• DNA polymerase• one of the four ddNTPs
15P32
16S32 + Beta particle+ Gamma rays
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Dideoxy Chain TerminationDideoxy Chain Terminationafter gel electrophoresis of each reaction mixture• a piece of film is placed over the gel• gamma rays released by P-32 darken the film and
create a pattern of the resolved oligonucleotide• the base sequence of the complement to the original
strand is read directly from bottom to top of the developed film
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Dideoxy Chain TerminationDideoxy Chain Termination
A G C T
Larger fragments
Smaller fragments
3'
5'
A
G
T
T
G
C
T
A
Thissequenceis the complementof the DNAtemplate
Polyacrylamide gel electrophoresis
If the complement of the DNA template is5'-A-T-C-G-T-T-G-A-3'Then the original DNA template must be5'-T-C-A-A-C-G-A-T-3'
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Prob 28.8Prob 28.8Draw structural formulas for the enethiol (the sulfur equivalent of an enol) forms of each antimetabolite used in the treatment of certain types of cancer.
6
6-Mercaptopurine6-Thioguanine
6HN
N
S
N
N
H
N
H
N
H2N
S
N
HN
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Prob 28.9Prob 28.9Draw two additional tautomers for cytosine and three additional tautomers for thymine.
Thymine (T)Cytosine (C)
N
N
H
O
NH2 O
O
H
HN
N
CH3
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Nucleic AcidsNucleic Acids
End Chapter 28End Chapter 28