Download Chapter 28. Heterocycles and Nucleic Acids

Chapter 28
Biomolecules: Heterocycles
and Nucleic Acids
Based on McMurry’s Organic Chemistry,
6th edition
©2003 Ronald Kluger
Department of Chemistry
University of Toronto
Heterocycles
 Cyclic organic compounds are carbocycles or heterocycles
Carbocycle rings contain only carbon atoms
 Heterocycle rings atoms in addition to carbon (N,S,O are
common)
 Heterocycles include many important natural materials as well
as pharmaceuticals

Based on McMurry, Organic Chemistry, Chapter
28, 6th edition, (c) 2003
2
28.1 Five-Membered Unsaturated
Heterocycles
 Pyrrole, furan, and thiophene are common five-
membered unsaturated heterocycles
 Each has two double bonds and N, O, or S
Based on McMurry, Organic Chemistry, Chapter
28, 6th edition, (c) 2003
3
Pyrrole
 Commercially from coal tar or by treatment of
furan with ammonia over an alumina catalyst
at 400°C.
Based on McMurry, Organic Chemistry, Chapter
28, 6th edition, (c) 2003
4
Furan
 Made commercially by extrusion of CO from
furfural, which is produced from sugars
Based on McMurry, Organic Chemistry, Chapter
28, 6th edition, (c) 2003
5
Thiophene
 From coal tar or by cyclization of butane or
butadiene with sulfur at 600°C
Based on McMurry, Organic Chemistry, Chapter
28, 6th edition, (c) 2003
6
Unusual Reactivity
 Pyrrole is an amine but it is not basic
 Pyrrole, furan, and thiophene are conjugated
dienes but they undergo electrophilic
substitution (rather than addition)
Based on McMurry, Organic Chemistry, Chapter
28, 6th edition, (c) 2003
7
28.2 Structures of Pyrrole, Furan, and
Thiophene
 Pyrrole, furan, and thiophene are aromatic
(Six  electrons in a cyclic conjugated system
of overlapping p orbitals)
 In pyrrole  electrons come from C atoms and
lone pair on sp2-N
Based on McMurry, Organic Chemistry, Chapter
28, 6th edition, (c) 2003
8
Why Pyrrole is Not a Base
 The nitrogen lone pair is a part of the aromatic sextet,
protonation on nitrogen destroys the aromaticity,
giving its conjugate acid a very low pKa (0.4)
 The carbon atoms of pyrrole are more electron-rich
and more nucleophilic than typical double-bond
carbons (see comparison with cyclopentadiene)
Based on McMurry, Organic Chemistry, Chapter
28, 6th edition, (c) 2003
9
28.3 Electrophilic Substitution Reactions of Pyrrole,
Furan, and Thiophene
 The heterocycles are more reactive toward
electrophiles than benzene
Based on McMurry, Organic Chemistry, Chapter
28, 6th edition, (c) 2003
10
Position of Substitution
 Electrophilic substitution normally occurs at C2, the
position next to the heteroatom, giving more stable
intermediate
Based on McMurry, Organic Chemistry, Chapter
28, 6th edition, (c) 2003
11
28.4 Pyridine, a Six-Membered
Heterocycle
 Nitrogen-containing heterocyclic analog of benzene
 Lone pair of electrons on N not part occupies an sp2
orbital in the plane of the ring and is not involved in
bonding (Figure 28.3).
Based on McMurry, Organic Chemistry, Chapter
28, 6th edition, (c) 2003
12
Electronic structure of pyridine
 Pyridine is a stronger base than pyrrole but a weaker
base than alkylamines
 The sp2-hybridized N holds the lone-pair electrons
more tightly than the sp3-hybridized nitrogen in an
alkylamine
Based on McMurry, Organic Chemistry, Chapter
28, 6th edition, (c) 2003
13
28.5 Electrophilic Substitution of
Pyridine
 The pyridine ring undergoes electrophilic aromatic
substitution reactions with great difficulty, under
drastic conditions
Based on McMurry, Organic Chemistry, Chapter
28, 6th edition, (c) 2003
14
Low Reactivity of Pyridine
 Complex between ring nitrogen and incoming
electrophile deactivates ring with positive charge
 Electron-withdrawing nitrogen atom deactivates
causes a dipole making positively polarized C’s poor
Lewis bases
Based on McMurry, Organic Chemistry, Chapter
28, 6th edition, (c) 2003
15
28.6 Nucleophilic Substitution of
Pyridine
 2- and 4-substituted (but not 3-substituted)
halopyridines readily undergo nucleophilic aromatic
substitution
Based on McMurry, Organic Chemistry, Chapter
28, 6th edition, (c) 2003
16
Mechanism of Nucleophilic
Substitution on Pyridine
 Reaction occurs by addition of the nucleophile to the
C=N bond, followed by loss of halide ion
Based on McMurry, Organic Chemistry, Chapter
28, 6th edition, (c) 2003
17
Addition-Elimination
 Addition favored by ability of the electronegative
nitrogen to stabilize the anionic intermediate
 Leaving group is then expelled
Based on McMurry, Organic Chemistry, Chapter
28, 6th edition, (c) 2003
18
28.7 Fused-Ring Heterocycles
 Quinoline, isoquinoline, and indole are fused-ring
heterocycles, containing both a benzene ring and a
heterocyclic aromatic ring
Based on McMurry, Organic Chemistry, Chapter
28, 6th edition, (c) 2003
19
Quinoline and Isoquinoline
 Quinoline and isoquinoline have pyridine-like nitrogen
atoms, and undergo electrophilic substitutions
 Reaction is on the benzene ring rather than on the
pyridine ring
Based on McMurry, Organic Chemistry, Chapter
28, 6th edition, (c) 2003
20
Indole
 Has pyrrole-like nitrogen (nonbasic)
 Undergoes electrophilic substitution at C3 of the
electron-rich pyrrole
Based on McMurry, Organic Chemistry, Chapter
28, 6th edition, (c) 2003
21
Purine and Pyrimidine
 Pyrimidine contains two pyridine-like nitrogens in a
six-membered aromatic ring
 Purine has 4 N’s in a fused-ring structure. Three are
basic like pyridine-like and one is like that in pyrrole
Based on McMurry, Organic Chemistry, Chapter
28, 6th edition, (c) 2003
22
28.8 Nucleic Acids and Nucleotides
 Deoxyribonucleic acid (DNA) and ribonucleic acid
(RNA), are the chemical carriers of genetic
information
 Nucleic acids are biopolymers made of nucleotides,
aldopentoses linked to a purine or pyrimidine and a
phosphate
Based on McMurry, Organic Chemistry, Chapter
28, 6th edition, (c) 2003
23
Sugars in DNA and RNA
 RNA is derived from ribose
 DNA is from 2-deoxyribose

(the ' is used to refer to positions on the sugar
portion of a nucleotide)
Based on McMurry, Organic Chemistry, Chapter
28, 6th edition, (c) 2003
24
Heterocycles in DNA and RNA
 Adenine, guanine, cytosine and thymine are in DNA
 RNA contains uracil rather than thymine
Based on McMurry, Organic Chemistry, Chapter
28, 6th edition, (c) 2003
25
Nucleotides
 In DNA and RNA the heterocycle is bonded to C1 of
the sugar and the phosphate is bonded to C5 (and
connected to 3’ of the next unit)
Based on McMurry, Organic Chemistry, Chapter
28, 6th edition, (c) 2003
26
The Deoxyribonucleotides
Based on McMurry, Organic Chemistry, Chapter
28, 6th edition, (c) 2003
27
The Ribonucleotides
Based on McMurry, Organic Chemistry, Chapter
28, 6th edition, (c) 2003
28
28.9 Structure of Nucleic Acids
 Nucleotides join together in DNA and RNA by as
phosphate between the 5-on one nucleotide and the
3 on another
 One end of the nucleic acid polymer has a free
hydroxyl at C3 (the 3 end), and the other end has a
phosphate at C5 (the 5 end).
Based on McMurry, Organic Chemistry, Chapter
28, 6th edition, (c) 2003
29
Generalized Structure of DNA
Based on McMurry, Organic Chemistry, Chapter
28, 6th edition, (c) 2003
30
Nucleic Acid Sequences
 Differences arise from the sequence of bases
on the individual nucleotides
Based on McMurry, Organic Chemistry, Chapter
28, 6th edition, (c) 2003
31
Describing a Sequence
 Chain is described from 5 end, identifying the bases
in order of occurrence, using the abbreviations A for
adenosine, G for guanosine, C for cytidine, and T for
thymine (or U for uracil in RNA)
 A typical sequence is written as TAGGCT
Based on McMurry, Organic Chemistry, Chapter
28, 6th edition, (c) 2003
32
28.10 Base Pairing in DNA: The
Watson–Crick Model
 In 1953 Watson and Crick noted that DNA consists of
two polynucleotide strands, running in opposite
directions and coiled around each other in a double
helix
 Strands are held together by hydrogen bonds
between specific pairs of bases
 Adenine (A) and thymine (T) form strong hydrogen
bonds to each other but not to C or G
 (G) and cytosine (C) form strong hydrogen bonds to
each other but not to A or T
Based on McMurry, Organic Chemistry, Chapter
28, 6th edition, (c) 2003
33
H-Bonds in DNA
 The G-C base pair involves three H-bonds
Based on McMurry, Organic Chemistry, Chapter
28, 6th edition, (c) 2003
34
A-T Base Pairing
 Involves two H-bonds
Based on McMurry, Organic Chemistry, Chapter
28, 6th edition, (c) 2003
35
The Difference in the Strands
 The strands of DNA are complementary
because of H-bonding
 Whenever a G occurs in one strand, a C
occurs opposite it in the other strand
 When an A occurs in one strand, a T occurs in
the other
Based on McMurry, Organic Chemistry, Chapter
28, 6th edition, (c) 2003
36
Grooves
 The strands of the DNA
double helix create two
continuous grooves (major
and minor)
 The sugar–phosphate
backbone runs along the
outside of the helix, and the
amine bases hydrogen bond
to one another on the inside
 The major groove is slightly
deeper than the minor
groove, and both are lined by
potential hydrogen bond
donors and acceptors.
Based on McMurry, Organic Chemistry, Chapter
28, 6th edition, (c) 2003
37
28.11 Nucleic Acids and Heredity
 Processes in the transfer of genetic information:
 Replication: identical copies of DNA are made
 Transcription: genetic messages are read and carried out of the
cell nucleus to the ribosomes, where protein synthesis occurs.
 Translation: genetic messages are decoded to make proteins.
Based on McMurry, Organic Chemistry, Chapter
28, 6th edition, (c) 2003
38
28.12 Replication of DNA
 Begins with a partial unwinding of the double helix,
exposing the recognition site on the bases
 Activated forms of the complementary nucleotides (A
with T and G with C) associate two new strands begin
to grow
Based on McMurry, Organic Chemistry, Chapter
28, 6th edition, (c) 2003
39
The Replication Process
 Addition takes place 5  3, catalyzed by DNA
polymerase
 Each nucleotide is joined as a 5-nucleoside
triphosphate that adds a nucleotide to the free 3hydroxyl group of the growing chain
Based on McMurry, Organic Chemistry, Chapter
28, 6th edition, (c) 2003
40
28.13 Structure and Synthesis of RNA:
Transcription
 RNA contains ribose rather than deoxyribose and
uracil rather than thymine
 There are three major kinds of RNA - each of which
serves a specific function
 They are much smaller molecules than DNA and are
usually single-stranded
Based on McMurry, Organic Chemistry, Chapter
28, 6th edition, (c) 2003
41
Messenger RNA (mRNA)
 Its sequence is copied from genetic DNA
 It travels to ribsosomes, small granular particles in
the cytoplasm of a cell where protein synthesis takes
place
Based on McMurry, Organic Chemistry, Chapter
28, 6th edition, (c) 2003
42
Ribosomal RNA (rRNA)
 Ribosomes are a complex of proteins and rRNA
 The synthesis of proteins from amino acids and ATP
occurs in the ribosome
 The rRNA provides both structure and catalysis
Based on McMurry, Organic Chemistry, Chapter
28, 6th edition, (c) 2003
43
Transfer RNA (tRNA)
 Transports amino acids to the ribosomes where they
are joined together to make proteins
 There is a specific tRNA for each amino acid
 Recognition of the tRNA at the anti-codon
communicates which amino acid is attached
Based on McMurry, Organic Chemistry, Chapter
28, 6th edition, (c) 2003
44
Transcription Process
 Several turns of the DNA double helix unwind,
exposing the bases of the two strands
 Ribonucleotides line up in the proper order by
hydrogen bonding to their complementary bases on
DNA
 Bonds form in the 5  3 direction,
Based on McMurry, Organic Chemistry, Chapter
28, 6th edition, (c) 2003
45
Transcription of RNA from DNA
 Only one of the two DNA strands is transcribed into
mRNA
 The strand that contains the gene is the coding or
sense strand
 The strand that gets transcribed is the template or
antisense strand
 The RNA molecule produced during transcription is a
copy of the coding strand (with U in place of T)
Based on McMurry, Organic Chemistry, Chapter
28, 6th edition, (c) 2003
46
Mechanism of Transcription
 DNA contains promoter sites that are 10 to 35 base
pairs upstream from the beginning of the coding
region and signal the beginning of a gene
 There are other base sequences near the end of the
gene that signal a stop
 Genes are not necessarily continuous, beginning
gene in a section of DNA (an exon) and then resume
farther down the chain in another exon, with an intron
between that is removed from the mRNA
Based on McMurry, Organic Chemistry, Chapter
28, 6th edition, (c) 2003
47
28.14 RNA and Protein Biosynthesis:
Translation
 RNA directs biosynthesis of peptides and proteins
which is catalyzed by mRNA in ribosomes, where
mRNA acts as a template to pass on the genetic
information transcribed from DNA
 The ribonucleotide sequence in mRNA forms a
message that determines the order in which different
amino acid residues are to be joined
 Codons are sequences of three ribonucleotides that
specify a particular amino acid
 For example, UUC on mRNA is a codon that directs
incorporation of phenylalanine into the growing
protein
Based on McMurry, Organic Chemistry, Chapter
28, 6th edition, (c) 2003
48
Codon Assignments of Base
Triplets
Based on McMurry, Organic Chemistry, Chapter
28, 6th edition, (c) 2003
49
The Parts of Transfer RNA
 There are 61 different tRNAs, one for each of the 61
codons that specifies an amino acid
 tRNA has 70-100 ribonucleotides and is bonded to a
specific amino acid by an ester linkage through the 3
hydroxyl on ribose at the 3 end of the tRNA
 Each tRNA has a segment called an anticodon, a
sequence of three ribonucleotides complementary to
the codon sequence
Based on McMurry, Organic Chemistry, Chapter
28, 6th edition, (c) 2003
50
The Structure of tRNA
Based on McMurry, Organic Chemistry, Chapter
28, 6th edition, (c) 2003
51
Processing Aminoacyl tRNA
 As each codon on mRNA is read, tRNAs bring amino
acids as esters for transfer to the growing peptide
 When synthesis of the proper protein is completed, a
"stop" codon signals the end and the protein is
released from the ribosome
Based on McMurry, Organic Chemistry, Chapter
28, 6th edition, (c) 2003
52
28.15 DNA Sequencing
 The order of the bases along DNA contains the




genetic inheritance.
Determination of the sequence is based on chemical
reactions rather than physical analysis
DNA is cleaved at specific sequences by restriction
endonucleases
For example, the restriction enzyme AluI cleaves
between G and C in the four-base sequence AG-CT
Note that the sequence is identical to that of its
complement, (3)-TC-GA-(5)
Other restriction enzymes produce other cuts
permitting partially overlapping sequences of small
pieces to be produced for analysis
Based on McMurry, Organic Chemistry, Chapter
28, 6th edition, (c) 2003
53
Analytical Methods
 The Maxam–Gilbert method uses organic chemistry
to cleave phosphate linkages at with specificity for
the adjoining heterocycle
 The Sanger dideoxy method uses enzymatic
reactions
 The Sanger method is now widely used and
automated, even in the sequencing of genomes
Based on McMurry, Organic Chemistry, Chapter
28, 6th edition, (c) 2003
54
The Sanger Dideoxy Method
 The fragment to be sequenced is combined with:


A small piece of DNA (primer), whose sequence
is complementary to that on the 3 end of the
restriction fragment
The four 2-deoxyribonucleoside triphosphates
(dNTPs)
Based on McMurry, Organic Chemistry, Chapter
28, 6th edition, (c) 2003
55
The Dideoxy Nucleotides
 The solution also contains small amounts of the four
2,3-dideoxyribonucleoside triphosphates (ddNTPs)
 Each is modified with a different fluorescent dye
molecule
Based on McMurry, Organic Chemistry, Chapter
28, 6th edition, (c) 2003
56
The Dideoxy Method - Growing the and
Stopping the Copied Chains
 DNA polymerase is added and a strand of DNA
complementary to the restriction fragment begins to
grow from the end of the primer
 Whenever a dideoxyribonucleotide is incorporated,
chain extension cannot continue
Based on McMurry, Organic Chemistry, Chapter
28, 6th edition, (c) 2003
57
Dideoxy Method - Analysis
 The product is a mixture of dideoxy-terminated DNA
fragments with fluorescent tags
 These are separated according to weight by
electrophoresis and identified by their specific
fluorescence
Based on McMurry, Organic Chemistry, Chapter
28, 6th edition, (c) 2003
58
28.16 DNA Synthesis
 DNA synthesizers use a solid-phase method starting
with an attached, protected nucleotide
 Subsequent protected nucleotides are added and
coupled
 After the final nucleotide has been added, the
protecting groups are removed and the synthetic
DNA is cleaved from the solid support
 The bases are protected from reacting
Based on McMurry, Organic Chemistry, Chapter
28, 6th edition, (c) 2003
59
DNA Synthesis: Attachment
 Attachment of a protected deoxynucleoside to a
polymeric or silicate support as an ester of the 3
OH group of the deoxynucleoside
 The 5 OH group on the sugar is protected as its pdimethoxytrityl (DMT) ether
Based on McMurry, Organic Chemistry, Chapter
28, 6th edition, (c) 2003
60
DNA Synthesis: DMT Removal
 Removal of the DMT protecting group by treatment
with a moderately weak acid
Based on McMurry, Organic Chemistry, Chapter
28, 6th edition, (c) 2003
61
DNA Synthesis: Coupling
 The polymer-bound (protected) deoxynucleoside
reacts with a protected deoxynucleoside containing a
phosphoramidite group at its 3 position, catalyzed by
tetrazole, a reactive heterocycle
Based on McMurry, Organic Chemistry, Chapter
28, 6th edition, (c) 2003
62
DNA Synthesis: Oxidation and
Cycling
 Phosphite is oxidized to phosphate by I2
 The cycle is repeated until the sequence is complete
Based on McMurry, Organic Chemistry, Chapter
28, 6th edition, (c) 2003
63
DNA Synthesis: Clean-up
 All protecting groups are removed and the product is
released from the support by treatment with aqueous
NH3
Based on McMurry, Organic Chemistry, Chapter
28, 6th edition, (c) 2003
64
28.17 The Polymerase Chain Reaction
(PCR)
 Copies DNA molecules by unwinding the double




helix and copying each strand using enzymes
The new double helices are unwound and copied
again
The enzyme is selected to be fast, accurate and
heat-stable (to survive the unwinding)
Each cycle doubles the amount of material
This is exponential template-driven organic
synthesis
Based on McMurry, Organic Chemistry, Chapter
28, 6th edition, (c) 2003
65
PCR: Heating and Reaction
 The subject DNA is heated (to separate strands) with



Taq polymerase (enyzme) and Mg2+
Deoxynucleotide triphosphates
Two, oligonucleotide primers, each
complementary to the sequence at the end of one
of the target DNA segments
Based on McMurry, Organic Chemistry, Chapter
28, 6th edition, (c) 2003
66
PCR: Annealing and Growing
 Temperature is reduced to 37 to 50°C,
allowing the primers to form H-bonds to their
complementary sequence at the end of each
target strand
Based on McMurry, Organic Chemistry, Chapter
28, 6th edition, (c) 2003
67
PCR: Taq Polymerase
 The temperature is then raised to 72°C, and Taq
polymerase catalyzes the addition of further
nucleotides to the two primed DNA strands
Based on McMurry, Organic Chemistry, Chapter
28, 6th edition, (c) 2003
68
PCR: Growing More Chains
 Repeating the denature–anneal–synthesize cycle a
second time yields four DNA copies, a third time
yields eight copies, in an exponential series.
 PCR has been automated, and 30 or so cycles can
be carried out in an hour
Based on McMurry, Organic Chemistry, Chapter
28, 6th edition, (c) 2003
69