Chapter 27 Amino Acids, Peptides, and Proteins

1
Chapter 27Amino Acids, Peptides, and Proteins
2
27.1Classification of Amino Acids
3
Fundamentals

• While their name implies that amino acids are
  compounds that contain an NH2 group and a CO2H
  group, these groups are actually present as NH3
  and CO2 respectively.
• They are classified as ?, ?, ?, etc. amino acids
  according the carbon that bears the nitrogen.

4
Amino Acids
an ?-amino acid that is anintermediate in the
biosynthesisof ethylene
?
a ?-amino acid that is one ofthe structural
units present incoenzyme A
?
a ?-amino acid involved inthe transmission of
nerveimpulses
?
5
The 20 Key Amino Acids

• More than 700 amino acids occur naturally, but 20
  of them are especially important.
• These 20 amino acids are the building blocks of
  proteins. All are ?-amino acids.
• They differ in respect to the group attached to
  the ? carbon.
• These 20 are listed in Table 27.1.

6
Table 27.1

• The amino acids obtained by hydrolysis of
  proteins differ in respect to R (the side chain).
• The properties of the amino acid vary as the
  structure of R varies.

7
Table 27.1

• The major differences among the side chains
  concern
• Size and shape Electronic characteristics

8
Table 27.1

• General categories of a-amino acids
• nonpolar side chains polar but nonionized side
  chains acidic side chains basic side chains

9
Table 27.1

• General categories of a-amino acids
• nonpolar side chains polar but nonionized side
  chains acidic side chains basic side chains

10
Table 27.1
Glycine
(Gly or G)

• Glycine is the simplest amino acid. It is the
  only one in the table that is achiral.
• In all of the other amino acids in the table the
  ? carbon is a chirality center.

11
Table 27.1
O
H


O
H3N
C
C
CH3
Alanine
(Ala or A)

• Alanine, valine, leucine, and isoleucine have
  alkyl groups as side chains, which are nonpolar
  and hydrophobic.

12
Table 27.1
O
H


O
H3N
C
C
CH(CH3)2
Valine
(Val or V)
13
Table 27.1
O
H


O
H3N
C
C
CH2CH(CH3)2
Leucine
(Leu or L)
14
Table 27.1
15
Table 27.1
O
H


O
H3N
C
C
CH3SCH2CH2
Methionine
(Met or M)

• The side chain in methionine is nonpolar, but the
  presence of sulfur makes it somewhat polarizable.

16
Table 27.1
Proline
(Pro or P)

• Proline is the only amino acid that contains a
  secondary amine function. Its side chain is
  nonpolar and cyclic.

17
Table 27.1
Phenylalanine
(Phe or F)

• The side chain in phenylalanine (a nonpolar amino
  acid) is a benzyl group.

18
Table 27.1

• The side chain in tryptophan (a nonpolar amino
  acid) is larger and more polarizable than the
  benzyl group of phenylalanine.

19
Table 27.1

• General categories of a-amino acids
• nonpolar side chains polar but nonionized side
  chains acidic side chains basic side chains

20
Table 27.1
O
H


O
H3N
C
C
CH2OH
Serine
(Ser or S)

• The CH2OH side chain in serine can be involved
  in hydrogen bonding.

21
Table 27.1
O
H


O
H3N
C
C
CH3CHOH
Threonine
(Thr or T)

• The side chain in threonine can be involved in
  hydrogen bonding, but is somewhat more crowded
  than in serine.

22
Table 27.1
O
H


O
H3N
C
C
CH2SH
Cysteine
(Cys or C)

• The side chains of two remote cysteines can be
  joined by forming a covalent SS bond.

23
Table 27.1
Tyrosine
(Tyr or Y)

• The side chain of tyrosine is similar to that of
  phenylalanine but can participate in hydrogen
  bonding.

24
Table 27.1
Asparagine
(Asn or N)

• The side chains of asparagine and glutamine (next
  slide) terminate in amide functions that are
  polar and can engage in hydrogen bonding.

25
Table 27.1
Glutamine
(Gln or Q)
26
Table 27.1

• General categories of a-amino acids
• nonpolar side chains polar but nonionized side
  chains acidic side chains basic side chains

27
Table 27.1
Aspartic Acid
(Asp or D)

• Aspartic acid and glutamic acid (next slide)
  exist as their conjugate bases at biological pH.
  They are negatively charged and can form ionic
  bonds with positively charged species.

28
Table 27.1
Glutamic Acid
(Glu or E)
29
Table 27.1

• General categories of a-amino acids
• nonpolar side chains polar but nonionized side
  chains acidic side chains basic side chains

30
Table 27.1
O
H


O
H3N
C
C
Lysine

(Lys or K)
CH2CH2CH2CH2NH3

• Lysine and arginine (next slide) exist as their
  conjugate acids at biological pH. They are
  positively charged and can form ionic bonds with
  negatively charged species.

31
Table 27.1
O
H


Arginine
O
H3N
C
C
(Arg or R)
CH2CH2CH2NHCNH2

NH2
32
Table 27.1
O
H


O
H3N
C
C
Histidine
(His or H)

• Histidine is a basic amino acid, but less basic
  than lysine and arginine. Histidine can interact
  with metal ions and can help move protons from
  one site to another.

33
27.2Stereochemistry of Amino Acids
34
Configuration of ?-Amino Acids

• Glycine is achiral. All of the other amino acids
  in proteins have the L-configuration at their
  ??carbon.

35
27.3Acid-Base Behavior of Amino Acids
36
Recall

• While their name implies that amino acids are
  compounds that contain an NH2 group and a CO2H
  group, these groups are actually present as NH3
  and CO2 respectively.

How do we know this?
37
Properties of Glycine

• The properties of glycine
• high melting point (when heated to 233C it
  decomposes before it melts)solubility soluble
  in water not soluble in nonpolar solvent

38
Properties of Glycine

• The properties of glycine
• high melting point (when heated to 233C it
  decomposes before it melts)solubility soluble
  in water not soluble in nonpolar solvent

more consistent with this
called a zwitterion or dipolar ion
39
Acid-Base Properties of Glycine

• The zwitterionic structure of glycine also
  follows from considering its acid-base
  properties.
• A good way to think about this is to start with
  the structure of glycine in strongly acidic
  solution, say pH 1.
• At pH 1, glycine exists in its protonated form
  (a monocation).

40
Acid-Base Properties of Glycine

• Now ask yourself "As the pH is raised, which is
  the first proton to be removed? Is it the proton
  attached to the positively charged nitrogen, or
  is it the proton of the carboxyl group?"
• You can choose between them by estimating their
  respective pKas.

41
Acid-Base Properties of Glycine

• The more acidic proton belongs to the CO2H group.
  It is the first one removed as the pH is raised.

typical carboxylic acid pKa 5
42
Acid-Base Properties of Glycine

• Therefore, the more stable neutral form of
  glycine is the zwitterion.

typical carboxylic acid pKa 5
43
Acid-Base Properties of Glycine

• The measured pKa of glycine is 2.34.
• Glycine is stronger than a typical carboxylic
  acid because the positively charged N acts as an
  electron-withdrawing, acid-strengthening
  substituent on the ? carbon.

typical carboxylic acid pKa 5
44
Acid-Base Properties of Glycine
A proton attached to N in the zwitterionic form
of nitrogen can be removed as the pH is increased
further.

• The pKa for removal of this proton is 9.60.This
  value is about the same as that for NH4 (9.3).

45
Isoelectric Point pI

• The pH at which the concentration of the
  zwitterion is a maximum is called the isoelectric
  point. Its numerical value is the average of the
  two pKas.
• The pI of glycine is 5.97.

pKa 2.34
pKa 9.60
46
Acid-Base Properties of Amino Acids

• One way in which amino acids differ is in respect
  to their acid-base properties. This is the basis
  for certain experimental methods for separating
  and identifying them.
• Just as important, the difference in acid-base
  properties among various side chains affects the
  properties of the proteins that contain them.
• Table 27.2 gives pKa and pI values for amino
  acids with neutral side chains.

47
Table 27.2 Amino Acids with Neutral Side Chains
pKa1 2.34pKa2 9.60pI 5.97
Glycine
48
Table 27.2 Amino Acids with Neutral Side Chains
pKa1 2.34pKa2 9.69pI 6.00
Alanine
49
Table 27.2 Amino Acids with Neutral Side Chains
pKa1 2.32pKa2 9.62pI 5.96
Valine
50
Table 27.2 Amino Acids with Neutral Side Chains
pKa1 2.36pKa2 9.60pI 5.98
Leucine
51
Table 27.2 Amino Acids with Neutral Side Chains
O
H
pKa1 2.36pKa2 9.60pI 5.98


H3N
O
Isoleucine
C
C
CH3CHCH2CH3
52
Table 27.2 Amino Acids with Neutral Side Chains
O
H
pKa1 2.28pKa2 9.21pI 5.74


H3N
O
Methionine
C
C
CH3SCH2CH2
53
Table 27.2 Amino Acids with Neutral Side Chains
pKa1 1.99pKa2 10.60pI 6.30
Proline
54
Table 27.2 Amino Acids with Neutral Side Chains
pKa1 1.83pKa2 9.13pI 5.48
Phenylalanine
55
Table 27.2 Amino Acids with Neutral Side Chains
pKa1 2.83pKa2 9.39pI 5.89
Tryptophan
56
Table 27.2 Amino Acids with Neutral Side Chains
O
H
pKa1 2.02pKa2 8.80pI 5.41


H3N
O
Asparagine
C
C
57
Table 27.2 Amino Acids with Neutral Side Chains
pKa1 2.17pKa2 9.13pI 5.65
Glutamine
58
Table 27.2 Amino Acids with Neutral Side Chains
pKa1 2.21pKa2 9.15pI 5.68
Serine
59
Table 27.2 Amino Acids with Neutral Side Chains
O
H
pKa1 2.09pKa2 9.10pI 5.60


H3N
O
Threonine
C
C
CH3CHOH
60
Table 27.2 Amino Acids with Neutral Side Chains
pKa1 2.20pKa2 9.11pI 5.66
Tyrosine
61
Table 27.3 Amino Acids with Neutral Side Chains
pKa1 1.96pKa2 8.18pI 5.07
Cysteine
62
Table 27.3 Amino Acids with Ionizable Side Chains
O
H
pKa1 1.88pKa2 3.65pKa3 9.60 pI 2.77


H3N
O
Aspartic acid
C
C

• For amino acids with acidic side chains, pI is
  the average of pKa1 and pKa2.

63
Table 27.3 Amino Acids with Ionizable Side Chains
O
H
pKa1 2.19pKa2 4.25pKa3 9.67 pI 3.22


H3N
O
Glutamic acid
C
C
64
Table 27.3 Amino Acids with Ionizable Side Chains
pKa1 2.18pKa2 8.95pKa3 10.53pI 9.74
Lysine

• For amino acids with basic side chains, pI is the
  average of pKa2 and pKa3.

65
Table 27.3 Amino Acids with Ionizable Side Chains
pKa1 2.17pKa2 9.04pKa3 12.48pI 10.76
Arginine
66
Table 27.3 Amino Acids with Ionizable Side Chains
pKa1 1.82pKa2 6.00pKa3 9.17 pI 7.59
Histidine
67
27.4Synthesis of Amino Acids
68
From ?-Halo Carboxylic Acids
H2O

2NH3
69
Strecker Synthesis
NH4Cl
NaCN
70
Using Diethyl Acetamidomalonate

• Can be used in the same manner as diethyl
  malonate (Section 21.7).

71
Example
1. NaOCH2CH3
2. C6H5CH2Cl
(90)
72
Example
O
O
HOCCCOH
CH2C6H5
H3N

HBr, H2O, heat
73
27.5Reactions of Amino Acids
74
Acylation of Amino Group

• The amino nitrogen of an amino acid can be
  converted to an amide with the customary
  acylating agents.

75
Esterification of Carboxyl Group

• The carboxyl group of an amino acid can be
  converted to an ester. The following illustrates
  Fischer esterification of alanine.

CH3CH2OH
HCl
76
Ninhydrin Test

• Amino acids are detected by the formation of a
  purple color on treatment with ninhydrin.

CO2
H2O
77
27.6Some Biochemical Reactionsof Amino Acids
78
Biosynthesis of L-Glutamic Acid

NH3
enzymes and reducing coenzymes

• This reaction is the biochemical analog of
  reductive amination (Section 22.10).

79
Transamination via L-Glutamic Acid
L-Glutamic acid acts as a source of the amine
group in the biochemical conversion of
?-ketoacids to other amino acids. In the
example to beshown, pyruvic acid is converted to
L-alanine.
80
Transamination via L-Glutamic Acid
enzymes

81
Mechanism
The first step is imine formation between
theamino group of L-glutamic acid and
pyruvicacid.
82
Mechanism

HO2CCH2CH2CHCO2
N

CH3CCO2
83

• Formation of the imine is followed by proton
  removal at one carbon and protonation of another
  carbon.

H
84

H
85

• Hydrolysis of the imine function gives?-keto
  glutarate and L-alanine.

86

H2O

87
Biosynthesis of L-Tyrosine
L-Tyrosine is biosynthesized from
L-phenylalanine. A key step is epoxidation of the
aromatic ring to give an arene oxide
intermediate.
88
Biosynthesis of L-Tyrosine
O2, enzyme
89
Biosynthesis of L-Tyrosine
enzyme
90
Biosynthesis of L-Tyrosine

• Conversion to L-tyrosine is one of the major
  metabolic pathways of L-phenylalanine.
• Individuals who lack the enzymes necessary to
  convert L-phenylalanine to L-tyrosine can suffer
  from PKU disease. In PKU disease,
  L-phenylalanine is diverted to a pathway leading
  to phenylpyruvic acid, which is toxic.
• Newborns are routinely tested for PKU disease.
  Treatment consists of reducing their dietary
  intake of phenylalanine-rich proteins.

91
Decarboxylation

• Decarboxylation is a common reaction of ?-amino
  acids. An example is the conversion of
  L-histidine to histamine. Antihistamines act by
  blocking the action of histamine.

92
Decarboxylation
CO2, enzymes
93
Neurotransmitters

• The chemistry of the brain and central nervous
  system is affected by neurotransmitters.
• Several important neurotransmitters are
  biosynthesized from L-tyrosine.

L-Tyrosine
94
Neurotransmitters

CO2

• The common name of this compound is L-DOPA. It
  occurs naturally in the brain. It is widely
  prescribed to reduce the symptoms of Parkinsonism.

H3N
H
H
H
HO
OH
L-3,4-Dihydroxyphenylalanine
95
Neurotransmitters
H

• Dopamine is formed by decarboxylation of L-DOPA.

H2N
H
H
H
HO
OH
Dopamine
96
Neurotransmitters
H
H2N
H
H
OH
HO
OH
Norepinephrine
97
Neurotransmitters
H
CH3NH
H
H
OH
HO
OH
Epinephrine
98
27.7Peptides
99
Peptides

• Peptides are compounds in which an amide bond
  links the amino group of one ?-amino acid and the
  carboxyl group of another.
• An amide bond of this type is often referred to
  as a peptide bond.

100
Alanine and Glycine
101
Alanylglycine

• Two ?-amino acids are joined by a peptide bond in
  alanylglycine. It is a dipeptide.

102
Alanylglycine
N-terminus
C-terminus
AlaGly
AG
103
Alanylglycine and glycylalanine are
constitutional isomers
Alanylglycine AlaGly AG
Glycylalanine GlyAla GA
104
Alanylglycine

• The peptide bond is characterized by a planar
  geometry.

105
Higher Peptides

• Peptides are classified according to the number
  of amino acids linked together.
• dipeptides, tripeptides, tetrapeptides, etc.
• Leucine enkephalin is an example of a
  pentapeptide.

106
Leucine Enkephalin
TyrGlyGlyPheLeuYGGFL
107
Oxytocin
C-terminus
N-terminus

• Oxytocin is a cyclic nonapeptide.
• Instead of having its amino acids linked in an
  extended chain, two cysteine residues are joined
  by an SS bond.

108
Oxytocin
SS bond
An SS bond between two cysteines isoften
referred to as a disulfide bridge.
109
27.8Introduction to Peptide Structure
Determination
110
Primary Structure

• The primary structure is the amino acid sequence
  plus any disulfide links.

111
Classical Strategy (Sanger)

• 1. Determine what amino acids are present and
  their molar ratios.
• 2. Cleave the peptide into smaller fragments,
  and determine the amino acid composition of these
  smaller fragments.
• 3. Identify the N-terminus and C-terminus in the
  parent peptide and in each fragment.
• 4. Organize the information so that the sequences
  of small fragments can be overlapped to reveal
  the full sequence.

112
27.9Amino Acid Analysis
113
Amino Acid Analysis

• Acid-hydrolysis of the peptide (6 M HCl, 24 hr)
  gives a mixture of amino acids.
• The mixture is separated by ion-exchange
  chromatography, which depends on the differences
  in pI among the various amino acids.
• Amino acids are detected using ninhydrin.
• Automated method requires only 10-5 to 10-7 g
  of peptide.

114
27.10Partial Hydrolysis of Proteins
115
Partial Hydrolysis of Peptides and Proteins

• Acid-hydrolysis of the peptide cleaves all of the
  peptide bonds.
• Cleaving some, but not all, of the peptide bonds
  gives smaller fragments.
• These smaller fragments are then separated and
  the amino acids present in each fragment
  determined.
• Enzyme-catalyzed cleavage is the preferred method
  for partial hydrolysis.

116
Partial Hydrolysis of Peptides and Proteins

• The enzymes that catalyze the hydrolysis of
  peptide bonds are called peptidases, proteases,
  or proteolytic enzymes.

117
Trypsin
Trypsin is selective for cleaving the peptide
bond to the carboxyl group of lysine or arginine.
118
Chymotrypsin
Chymotrypsin is selective for cleaving the
peptidebond to the carboxyl group of amino acids
withan aromatic side chain.
119
Carboxypeptidase
Carboxypeptidase is selective for cleavingthe
peptide bond to the C-terminal amino acid.
120
27.11End Group Analysis
121
End Group Analysis

• Amino sequence is ambiguous unless we know
  whether to read it left-to-right or
  right-to-left.
• We need to know what the N-terminal and
  C-terminal amino acids are.
• The C-terminal amino acid can be determined by
  carboxypeptidase-catalyzed hydrolysis.
• Several chemical methods have been developed for
  identifying the N-terminus. They depend on the
  fact that the amino N at the terminus is more
  nucleophilic than any of the amide nitrogens.

122
Sanger's Method

• The key reagent in Sanger's method for
  identifying the N-terminus is 1-fluoro-2,4-dinitro
  benzene.
• 1-Fluoro-2,4-dinitrobenzene is very reactive
  toward nucleophilic aromatic substitution
  (Section 23.5).

123
Sanger's Method

• 1-Fluoro-2,4-dinitrobenzene reacts with the amino
  nitrogen of the N-terminal amino acid.

124
Sanger's Method

• Acid hydrolysis cleaves all of the peptide bonds
  leaving a mixture of amino acids, only one of
  which (the N-terminus) bears a 2,4-DNP group.

125
27.12Insulin
126
Insulin

• Insulin is a polypeptide with 51 amino acids.
• It has two chains, called the A chain (21 amino
  acids) and the B chain (30 amino acids).
• The following describes how the amino acid
  sequence of the B chain was determined.

127
The B Chain of Bovine Insulin

• Phenylalanine (F) is the N terminus.
• Pepsin-catalyzed hydrolysis gave the four
  peptides FVNQHLCGSHL VGAL VCGERGF YTPKA

128
The B Chain of Bovine Insulin
FVNQHLCGSHL
VGAL
VCGERGF
YTPKA
129
The B Chain of Bovine Insulin

• Phenylalanine (F) is the N terminus.
• Pepsin-catalyzed hydrolysis gave the four
  peptides FVNQHLCGSHL VGAL VCGERGF YTPKA
• Overlaps between the above peptide sequences were
  found in four additional peptides SHLV LVGA AL
  T TLVC

130
The B Chain of Bovine Insulin
FVNQHLCGSHL
SHLV
LVGA
VGAL
ALT
TLVC
VCGERGF
YTPKA
131
The B Chain of Bovine Insulin

• Phenylalanine (F) is the N terminus.
• Pepsin-catalyzed hydrolysis gave the four
  peptides FVNQHLCGSHL VGAL VCGERGF YTPKA
• Overlaps between the above peptide sequences were
  found in four additional peptides SHLV LVGA AL
  T TLVC
• Trypsin-catalyzed hydrolysis gave GFFYTPK which
  completes the sequence.

132
The B Chain of Bovine Insulin
FVNQHLCGSHL
SHLV
LVGA
VGAL
ALT
TLVC
VCGERGF
GFFYTPK
YTPKA
133
The B Chain of Bovine Insulin
FVNQHLCGSHL
SHLV
LVGA
VGAL
ALT
TLVC
VCGERGF
GFFYTPK
YTPKA
FVNQHLCGSHLVGALTLVCGERGFFYTPKA
134
Insulin

• The sequence of the A chain was determined using
  the same strategy.
• Establishing the disulfide links between cysteine
  residues completed the primary structure.

135
Primary Structure of Bovine Insulin
N terminus of A chain
C terminus of A chain
N terminus of B chain
C terminus of B chain
136
27.13The Edman Degradation and Automated
Sequencing of Peptides
137
Edman Degradation

• 1. Method for determining N-terminal amino acid.
• 2. Can be done sequentially one residue at a time
  on the same sample. Usually one can determine
  the first 20 or so amino acids from the
  N-terminus by this method.
• 3. 10-10 g of sample is sufficient.
• 4. Has been automated.

138
Edman Degradation

• The key reagent in the Edman degradation is
  phenyl isothiocyanate.

139
Edman Degradation

• Phenyl isothiocyanate reacts with the amino
  nitrogen of the N-terminal amino acid.

140
Edman Degradation

141
Edman Degradation
The product is a phenylthiocarbamoyl
(PTC)derivative.

• The PTC derivative is then treated with HCl in an
  anhydrous solvent. The N-terminal amino acid is
  cleaved from the remainder of the peptide.

142
Edman Degradation
HCl

143
Edman Degradation
The product is a thiazolone. Under
the conditions of its formation, the
thiazolonerearranges to a phenylthiohydantoin
(PTH) derivative.

144
Edman Degradation

• The PTH derivative is isolated and identified.
  The remainder of the peptide is subjected to a
  second Edman degradation.

145
27.14The Strategy of Peptide Synthesis
146
General Considerations

• Making peptide bonds between amino acids is not
  difficult.
• The challenge is connecting amino acids in the
  correct sequence.
• Random peptide bond formation in a mixture of
  phenylalanine and glycine, for example, will give
  four dipeptides.
• PhePhe GlyGly PheGly GlyPhe

147
General Strategy

• 1. Limit the number of possibilities by
  "protecting" the nitrogen of one amino acid and
  the carboxyl group of the other.

148
General Strategy

• 2. Couple the two protected amino acids.

149
General Strategy

• 3. Deprotect the amino group at the N-terminus
  and the carboxyl group at the C-terminus.

Phe-Gly
150
27.15Amino Group Protection
151
Protect Amino Groups as Amides

• Amino groups are normally protected by converting
  them to amides.
• Benzyloxycarbonyl (C6H5CH2O) is a common
  protecting group. It is abbreviated as Z.
• Z-protection is carried out by treating an amino
  acid with benzyloxycarbonyl chloride.

152
Protect Amino Groups as Amides

153
Protect Amino Groups as Amides
is abbreviated as
or Z-Phe
154
Removing Z-Protection

• An advantage of the benzyloxycarbonyl protecting
  group is that it is easily removed by
• a) hydrogenolysis
• b) cleavage with HBr in acetic acid

155
Hydrogenolysis of Z-Protecting Group
156
HBr Cleavage of Z-Protecting Group
157
The tert-Butoxycarbonyl Protecting Group
158
HBr Cleavage of Boc-Protecting Group
O
NHCHCNHCH2CO2CH2CH3
(CH3)3COC
CH2C6H5
159
27.16Carboxyl Group Protection
160
Protect Carboxyl Groups as Esters

• Carboxyl groups are normally protected as esters.
• Deprotection of methyl and ethyl esters is by
  hydrolysis in base.
• Benzyl esters can be cleaved by hydrogenolysis.

161
Hydrogenolysis of Benzyl Esters
162
27.17Peptide Bond Formation
163
Forming Peptide Bonds

• The two major methods are
• 1. coupling of suitably protected amino acids
  using N,N'-dicyclohexylcarbodiimide (DCCI)
• 2. via an active ester of the N-terminal amino
  acid.

164
DCCI-Promoted Coupling

165
Mechanism of DCCI-Promoted Coupling

166
Mechanism of DCCI-Promoted Coupling

• The species formed by addition of the Z-protected
  amino acid to DCCI is similar in structure to an
  acid anhydride and acts as an acylating agent.
• Attack by the amine function of the
  carboxyl-protected amino acid on the carbonyl
  group leads to nucleophilic acyl substitution.

167
Mechanism of DCCI-Promoted Coupling
168
The Active Ester Method

• A p-nitrophenyl ester is an example of an "active
  ester."
• p-Nitrophenyl is a better leaving group than
  methyl or ethyl, and p-nitrophenyl esters are
  more reactive in nucleophilic acyl substitution.

169
The Active Ester Method

170
27.18Solid-Phase Peptide SynthesisThe
Merrifield Method
171
Solid-Phase Peptide Synthesis

• In solid-phase synthesis, the starting material
  is bonded to an inert solid support.
• Reactants are added in solution.
• Reaction occurs at the interface between the
  solid and the solution. Because the starting
  material is bonded to the solid, any product from
  the starting material remains bonded as well.
• Purification involves simply washing the
  byproducts from the solid support.

172
The Solid Support

• The solid support is a copolymer of styrene and
  divinylbenzene. It is represented above as if
  it were polystyrene. Cross-linking with
  divinylbenzene simply provides a more rigid
  polymer.

173
The Solid Support

• Treating the polymeric support with chloromethyl
  methyl ether (ClCH2OCH3) and SnCl4 places ClCH2
  side chains on some of the benzene rings.

174
The Solid Support

• The side chain chloromethyl group is a benzylic
  halide, reactive toward nucleophilic substitution
  (SN2).

175
The Solid Support

• The chloromethylated resin is treated with the
  Boc-protected C-terminal amino acid.
  Nucleophilic substitution occurs, and the
  Boc-protected amino acid is bound to the resin as
  an ester.

176
The Merrifield Procedure
177
The Merrifield Procedure

• Next, the Boc protecting group is removed with
  HCl.

178
The Merrifield Procedure

• DCCI-promoted coupling adds the second amino acid

179
The Merrifield Procedure

• Remove the Boc protecting group.

180
The Merrifield Procedure

• Add the next amino acid and repeat.

181
The Merrifield Procedure

• Remove the peptide from the resin with HBr in
  CF3CO2H

182
The Merrifield Procedure

183
The Merrifield Method

• Merrifield automated his solid-phase method.
• Synthesized a nonapeptide (bradykinin) in 1962 in
  8 days in 68 yield.
• Synthesized ribonuclease (124 amino acids) in
  1969. 369 reactions 11,391 steps
• Nobel Prize in chemistry 1984

184
27.19Secondary Structuresof Peptides and
Proteins
185
Levels of Protein Structure

• Primary structure the amino acid sequence plus
  disulfide links
• Secondary structure conformational relationship
  between nearest neighbor amino acids
• ? helix pleated ? sheet

186
Levels of Protein Structure
The ?-helix and pleated ? sheet are both
characterized by

• planar geometry of peptide bond
• anti conformation of main chain
• hydrogen bonds between NH and OC

187
Pleated ? Sheet

• Shown is a ? sheet of protein chains composed of
  alternating glycine and alanine residues.
• Adjacent chains are antiparallel.
• Hydrogen bonds between chains.
• van der Waals forces produce pleated effect.

188
Pleated ? Sheet

• ? Sheet is most commonly seen with amino acids
  having small side chains (glycine, alanine,
  serine).
• 80 of fibroin (main protein in silk) is
  repeating sequence of GlySerGlyAlaGlyAla.
• ? Sheet is flexible, but resists stretching.

189
? Helix

• Shown is an ? helix of a protein in which all of
  the amino acids are L-alanine.
• Helix is right-handed with 3.6 amino acids per
  turn.
• Hydrogen bonds are within a single chain.
• Protein of muscle (myosin) and wool (?-keratin)
  contain large regions of ?-helix. Chain can be
  stretched.

190
27.20Tertiary Structureof Peptides and Proteins
191
Tertiary Structure

• Refers to overall shape (how the chain is folded)
• Fibrous proteins (hair, tendons, wool) have
  elongated shapes
• Globular proteins are approximately spherical
• most enzymes are globular proteins
• an example is carboxypeptidase

192
Carboxypeptidase

• Carboxypeptidase is an enzyme that catalyzes the
  hydrolysis of proteins at their C-terminus.
• It is a metalloenzyme containing Zn2 at its
  active site.
• An amino acid with a positively charged side
  chain (Arg-145) is near the active site.

193
Carboxypeptidase
Disulfide bond
Zn2
Arg-145
N-terminus
C-terminus
tube model
ribbon model
194
What happens at the active site?

O
O
H2N


C

H3N
peptide
NHCHC

Arg-145
C
O
H2N
195
What happens at the active site?

O
O
H2N


C

H3N
peptide
NHCHC

Arg-145
C
O
H2N

• The peptide or protein is bound at the active
  site by electrostatic attraction between its
  negatively charged carboxylate ion and
  arginine-145.

196
What happens at the active site?

O
O
H2N


C

H3N
peptide
NHCHC

Arg-145
C
O
H2N

• Zn2 acts as a Lewis acid toward the carbonyl
  oxygen, increasing the positive character of the
  carbonyl carbon.

197
What happens at the active site?

O
O
H2N


C

H3N
peptide
NHCHC

Arg-145
C
O
H2N

• Water attacks the carbonyl carbon. Nucleophilic
  acyl substitution occurs.

198
What happens at the active site?
H2N

Arg-145
C
199
27.21Coenzymes
200
Coenzymes

• The range of chemical reactions that amino acid
  side chains can participate in is relatively
  limited.
• acid-base (transfer and accept
  protons) nucleophilic acyl substitution
• Many other biological processes, such as
  oxidation-reduction, require coenzymes,
  cofactors, or prosthetic groups in order to occur.

201
Coenzymes

• NADH, coenzyme A and coenzyme B12 are examples of
  coenzymes.
• Heme is another example.

202
Heme

• Molecule surrounding iron is a type of porphyrin.

203
Myoglobin
Heme

• Heme is the coenzyme that binds oxygen in
  myoglobin (oxygen storage in muscles) and
  hemoglobin (oxygen transport).

204
27.22Protein Quaternary StructureHemoglobin
205
Protein Quaternary Structure

• Some proteins are assemblies of two or more
  chains. The way in which these chains are
  organized is called the quaternary structure.
• Hemoglobin, for example, consists of 4 subunits.
• There are 2 ? chains (identical) and 2 ? chains
  (also identical).
• Each subunit contains one heme and each protein
  is about the size of myoglobin.

206
End of Chapter 27