proteins- david klein Flashcards
(318 cards)
1
Q
Structure of α-Amino Acids
A
An amino acid contains two key functional groups:
Amino group (–NH₂)
Carboxylic acid group (–COOH)
These two groups can be separated by various numbers of carbon atoms, but the most biologically important amino acids are those where:
The amino group is attached to the α-carbon, which is the carbon adjacent to the carboxyl group.
➤ α-Amino Acids:
Defined by the structure:
H
2
N
–
C
H
(
R
)
–
C
O
O
H
H
2
N–CH(R)–COOH
The α-carbon:
Is bonded to:
An amino group (–NH₂)
A hydrogen (H)
A carboxyl group (–COOH)
A side chain group (R)
Becomes a chiral center if R ≠ H.
2
Q
Peptide Bonds (Amide Linkages)
A
Amino acids join to form peptides or proteins through amide bonds called peptide bonds.
This occurs via a condensation reaction:
The –NH₂ group of one amino acid reacts with the –COOH group of another.
A molecule of water (H₂O) is eliminated.
A peptide bond is formed:
–
C
O
–
N
H
–
–CO–NH–
This reaction links amino acids into long chains (polypeptides).
3
Q
Peptides
A
A peptide is a short chain of amino acids linked by peptide bonds (amide linkages).
Naming is based on the number of amino acids:
Dipeptide: 2 amino acids
Tripeptide: 3 amino acids
Tetrapeptide: 4 amino acids
And so on…
4
Q
Polypeptides vs. Proteins
When the chain grows longer:
A
Chains with fewer than 40–50 amino acids are typically called polypeptides.
Chains with more than 50 amino acids are usually considered proteins.
5
Q
Proteins and Their Roles
Proteins are biologically essential macromolecules.
A
They perform a variety of structural, regulatory, transport, and catalytic functions.
Enzymes are a class of proteins that:
Act as biological catalysts
Speed up chemical reactions in cells
Are crucial for metabolism and life processes
🧠 It is estimated that the human body uses more than 50,000 enzymes to carry out life functions efficiently.
6
Q
What is a peptide?
A
A: A short chain of amino acids linked by peptide (amide) bonds.
7
Q
How many amino acids make up a dipeptide?
A
A: 2
8
Q
How many amino acids make up a tripeptide?
A
A: 3
9
Q
What kind of bond joins amino acids in peptides?
A
A: A peptide bond (–CO–NH–)
10
Q
What is the difference between a polypeptide and a protein?
A
A: A polypeptide has fewer than 40–50 amino acids, while a protein is a longer chain (usually >50 amino acids).
11
Q
What are enzymes?
A
A: Proteins that function as biological catalysts to speed up chemical reactions in cells.
12
Q
At what point is a peptide typically considered a protein?
A
A: When it has more than 50 amino acids.
13
Q
How many enzymes are estimated
A
to be needed by the human body?
A: Over 50,000
14
Q
What are some functions of proteins in the body?
A
A: Catalysis (enzymes), structure (e.g., collagen), transport (e.g., hemoglobin), signaling (e.g., hormones).
15
Q
Not All Amino Acids Are in Proteins
A
While 20 standard amino acids are commonly found in proteins, other amino acids exist in the body that:
Are biologically important
Do not get incorporated into proteins
Serve specific physiological roles
16
Q
GABA (γ-Aminobutyric Acid)
A
Structure: NH₂–CH₂–CH₂–CH₂–COOH
Classification: γ-amino acid (amino group is on the γ-carbon—3 carbons away from the carboxyl group)
Function:
Acts as a neurotransmitter
Found in the brain
Inhibitory — reduces neuronal excitability
Not used in proteins
17
Q
Thyroxine
A
Structure: A complex derivative of tyrosine with iodine atoms
Classification: A modified amino acid
Function:
A hormone secreted by the thyroid gland
Regulates metabolism, growth, and development
Not incorporated into proteins directly
18
Q
Are all amino acids found in proteins?
A
A
A: No, some amino acids (like GABA and thyroxine) are not found in proteins but still serve important functions in the body.
19
Q
What does GABA stand for?
A
A: γ-Aminobutyric acid
20
Q
What is the role of GABA in the body?
A
A: It acts as an inhibitory neurotransmitter in the brain.
21
Q
Why is GABA classified as a γ-amino acid?
A
A: Because the amino group is on the γ-carbon (3 carbons away from the carboxyl group).
22
Q
Is GABA incorporated into proteins?
A
A: No
23
Q
What is thyroxine?
A
A: A hormone derived from tyrosine, produced by the thyroid gland.
24
Q
What is the biological role of thyroxine?
A
A: It regulates metabolism, growth, and development.
25
Is thyroxine a standard amino acid?
A: No, it is a modified amino acid, not found in proteins.
26
Essential vs. Non-Essential Amino Acids
Total amino acids in proteins: 20 standard L-amino acids.
The human body can synthesize 10 of them. These are called non-essential amino acids because they don’t need to come from the diet.
The other 10 must be obtained from food and are called essential amino acids.
27
Essential Amino Acids:
Isoleucine
Leucine
Methionine
Phenylalanine
Threonine
Tryptophan
Valine
Arginine (conditionally essential)
Histidine
Lysine
28
Complete vs. Incomplete Proteins
Complete proteins contain all 10 essential amino acids.
Sources: Meat, fish, milk, eggs
Incomplete proteins are deficient in one or more essential amino acids.
Examples:
Rice: low in lysine and threonine
Corn: low in lysine and tryptophan
Beans & peas: low in methionine
29
Nutritional Considerations
Meat eaters get all essential amino acids from animal products.
Vegetarians can meet amino acid needs through milk and eggs.
Vegans, who consume no animal products, must carefully combine plant sources (like rice + beans) to obtain a full set of amino acids.
30
Deficiency Risk
Inadequate intake of essential amino acids can cause diseases, impair growth, and reduce protein synthesis.
Proper diet planning is critical for vegetarians and especially vegans.
31
What are essential amino acids?
A: Amino acids the body cannot synthesize and must be obtained from the diet.
32
How many amino acids are essential for humans?
A: 10, including arginine which is conditionally essential.
33
What are non-essential amino acids?
A: Amino acids the body can synthesize on its own.
34
What is a complete protein?
A: A protein source that contains all 10 essential amino acids.
35
Give examples of complete protein sources.
A: Meat, fish, milk, eggs.
36
What is an incomplete protein?
A: A protein source lacking one or more essential amino acids.
37
What essential amino acids is rice low in?
A: Lysine and threonine
38
What essential amino acids is corn low in?
A: Lysine and tryptophan
39
What is missing in beans and peas?
A: Methionine
40
How can vegetarians meet their amino acid needs?
A: Through milk and eggs (complete animal-derived proteins).
41
What must vegans do to get all essential amino acids?
A: Combine complementary plant proteins, like rice and beans.
42
What happens with inadequate intake of essential amino acids?
A: It can lead to diseases and impaired protein function and growth.
43
pH 1 Condition – Fully Protonated Form
At very low pH (around 1), the solution is highly acidic.
The amino acid exists in a fully protonated form:
The carboxyl group (–COOH) is protonated.
The amino group (–NH₃⁺) is also protonated (as an ammonium ion).
The molecule carries a net positive charge due to the –NH₃⁺ group.
44
Deprotonation and pKa Values
Each ionizable proton has its own pKa value — a measure of how easily it donates a proton.
🔹 pKa₁ (Carboxylic acid group)
–COOH → –COO⁻ + H⁺
Typically around 2
This is deprotonated first as the pH rises.
🔹 pKa₂ (Amino group)
–NH₃⁺ → –NH₂ + H⁺
Typically around 9–10
This deprotonates after the carboxyl group.
45
Additional pKa Values
Some amino acids have acidic or basic side chains (like aspartic acid, lysine, histidine).
These side chains can also ionize, contributing a third pKa.
46
Important Notes
Order of deprotonation:
Carboxylic acid group (more acidic)
Ammonium group (less acidic)
(If present) Ionizable side chain
This behavior governs:
The zwitterion form
Isoelectric point (pI)
Charge at different pH values
47
What is pKa?
A: It is the pH at which 50% of a functional group is deprotonated.
48
What happens to an amino acid at very low pH (~1)?
A: Both –COOH and –NH₃⁺ groups are fully protonated; the molecule has a positive charge.
49
What is pKa₁ in amino acids?
A: The pKa of the carboxyl group, usually around 2.
50
What is pKa₂ in amino acids?
A: The pKa of the ammonium group (–NH₃⁺), usually around 9–10.
51
Which functional group deprotonates first as pH increases?
A: The carboxylic acid group (–COOH).
52
At what pH does the amino group typically deprotonate?
A: Around pH 9–10
53
Do all amino acids have only two pKa values?
A: No — amino acids with acidic or basic side chains have a third pKa.
54
Give an example of an amino acid with three pKa values.
A: Aspartic acid (acidic side chain), lysine (basic side chain)
55
What charge does the amino acid carry after losing only its carboxylic proton?
A: Neutral (zwitterion) — COO⁻ and NH₃⁺ are both present.
56
What is the net charge after both groups are deprotonated?
A: Negative (–1) — COO⁻ and NH₂
57
Understanding pKa and Charge States of Amino Acids
🔹 Carboxylic Acid Group (–COOH)
Typical pKa₁ range: ~2–3
For example, alanine has pKa₁ = 2.34.
At pH = pKa (e.g., 2.34):
50% of the group is in the uncharged form –COOH
50% is in the anionic form –COO⁻
At pH < 2.34 (acidic): The group stays protonated (–COOH)
At pH > 2.34: The group loses H⁺ → becomes –COO⁻
✅ At physiological pH (~7.4), the carboxylic acid is deprotonated (–COO⁻)
58
Understanding pKa and Charge States of Amino Acids
Ammonium Group (–NH₃⁺)
Typical pKa₂ range: ~9–10
Alanine: pKa₂ = 9.69
At pH = 9.69:
50% exists as –NH₂ (uncharged)
50% as –NH₃⁺ (charged)
At pH < 9.69: The group is protonated as –NH₃⁺
At pH > 9.69: The group loses H⁺ and becomes –NH₂
✅ At physiological pH, the group is protonated (–NH₃⁺)
59
What is the typical pKa of the –COOH group in amino acids?
A: Around 2–3
60
At what pH is –COOH 50% deprotonated and 50% protonated?
A: At its pKa, e.g., 2.34 for alanine
61
What form of the carboxyl group predominates at physiological pH (~7.4)?
A: Deprotonated form (–COO⁻)
62
What form dominates at pH below the pKa?
A: The protonated form (–COOH)
63
What is the typical pKa of the –NH₃⁺ group?
A: Around 9–10 (e.g., 9.69 for alanine)
64
At what pH does –NH₃⁺ exist 50/50 with –NH₂?
A: At its pKa, e.g., 9.69
65
What form of the amino group predominates at physiological pH?
A: Protonated form (–NH₃⁺)
66
What happens to the amino group above its pKa?
A: It deprotonates to form –NH₂
67
What is the overall charge of an amino acid at physiological pH?
A: 0 — exists as a zwitterion with both +1 (NH₃⁺) and –1 (COO⁻)
68
Which group is deprotonated first as pH rises?
A: The carboxyl group (–COOH)
69
🌟 Zwitterion Form of Amino Acids
A zwitterion is a molecule that contains both positive and negative charges, yet is electrically neutral overall.
For amino acids at physiological pH (~7.4):
The carboxyl group is deprotonated → –COO⁻
The amino group is protonated → –NH₃⁺
Thus, the amino acid:
Has internal charge separation (like a salt)
Is referred to as a zwitterionic species
69
Physical Properties of Zwitterions
Due to charge separation, amino acids:
Are highly soluble in water
Exhibit salt-like properties
Have high melting points
70
Amphoteric Nature of Amino Acids
Amino acids are amphoteric, meaning they can act as both acids and bases, depending on the environment.
➤ Acting as an Acid (Reaction with a Base):
The –NH₃⁺ group donates a proton:
–
𝑁
𝐻
3
+
+
𝑂
𝐻
−
→
–
𝑁
𝐻
2
+
𝐻
2
𝑂
–NH
3
+
+OH
−
→–NH
2
+H
2
O
This occurs in basic conditions
➤ Acting as a Base (Reaction with an Acid):
The –COO⁻ group accepts a proton:
–
𝐶
𝑂
𝑂
−
+
𝐻
3
𝑂
+
→
–
𝐶
𝑂
𝑂
𝐻
+
𝐻
2
𝑂
–COO
−
+H
3
O
+
→–COOH+H
2
O
This occurs in acidic conditions
71
What is a zwitterion?
A: A molecule with both a positive and negative charge, but an overall neutral charge.
72
What groups are ionized in a zwitterionic amino acid?
A: –NH₃⁺ (amino group) and –COO⁻ (carboxyl group)
73
What form do amino acids take at physiological pH?
A: Zwitterionic form
74
Why are amino acids highly water-soluble?
A: Because they exist as ionic zwitterions in water.
75
Why do amino acids have high melting points?
A: Due to internal salt-like interactions in the zwitterionic structure.
76
What does it mean that amino acids are amphoteric?
A: They can act as both acids and bases.
77
What happens when a zwitterion reacts with a base like OH⁻?
A: The –NH₃⁺ group loses a proton → becomes –NH₂
78
What happens when a zwitterion reacts with an acid like H₃O⁺?
A: The –COO⁻ group gains a proton → becomes –COOH
79
What does the zwitterion become when it donates a proton?
A: A conjugate base form (–NH₂)
80
What does the zwitterion become when it accepts a proton?
A: A conjugate acid form (–COOH)
81
What is the Isoelectric Point?
The isoelectric point (pI) is the pH at which an amino acid exists predominantly as a zwitterion — i.e., it has no net charge.
At this point:
The positive and negative charges within the molecule balance each other out.
The amino acid is least soluble in water and will not migrate in an electric field.
82
Amino Acids Without Ionizable Side Chains
For amino acids that lack an acidic or basic side chain (e.g., alanine), the pI is calculated by taking the average of the two main pKa values:
pKa₁: For the carboxylic acid (–COOH)
pKa₂: For the ammonium ion (–NH₃⁺)
✅ Example: Alanine
pKa₁ = 2.34 (–COOH)
pKa₂ = 9.69 (–NH₃⁺)
pI = (2.34 + 9.69)/2 = 6.02
82
Amino Acids With Acidic or Basic Side Chains
For these amino acids, the pI is calculated from the two closest pKa values that flank the neutral zwitterion form:
✅ Example: Lysine (Basic Side Chain)
pKa₁ (α-NH₃⁺) = 8.95
pKa₂ (side chain –NH₃⁺) = 10.53
pI = (8.95 + 10.53)/2 = 9.74
Lysine's zwitterion exists between the two cationic forms, so use the two highest pKa values (both basic)
83
Why is the pI important?
A: At the pI, the amino acid has lowest solubility and no net movement in an electric field.
84
At pH below pI, what is the net charge of the amino acid?
A: Positive
85
At pH above pI, what is the net charge?
A: Negative
86
When is the amino acid least soluble in water?
A: At its isoelectric point (pI)
87
What is Electrophoresis?
Electrophoresis separates amino acids in a mixture based on differences in their isoelectric points (pI).
A sample is applied to a buffered medium (like paper or gel), and an electric field is applied across it.
Amino acids migrate depending on their net charge at the pH of the buffer.
88
Detection of Amino Acids – Ninhydrin Test
Amino acids are colorless, so a detection method is needed.
Ninhydrin is a reagent that reacts with primary amines in amino acids to form a deep purple-colored product.
The reaction releases H₂O, CO₂, and an aldehyde (RCHO) as by-products.
Proline, a secondary amine, reacts differently (gives a yellow product).
The number of purple spots = number of primary amino acids in the mixture.
88
Migration Behavior
General Rule:
pH < pI → Amino acid is positively charged → Migrates toward cathode (–)
pH > pI → Amino acid is negatively charged → Migrates toward anode (+)
pH = pI → Amino acid is neutral (zwitterion) → No migration
89
electrophoresis is for analytical use only
Analytical Use Only
Electrophoresis is ideal for analytical purposes, i.e., detecting how many different amino acids are present.
It’s not suitable for isolating large amounts of amino acids.
For full separation and collection, methods like column chromatography are used.
90
What principle is electrophoresis based on?
A: The difference in pI values of amino acids.
91
What happens when pH = pI?
A: The amino acid is a zwitterion and does not migrate.
92
What is the net charge of an amino acid when pH < pI?
A: Positive — migrates toward the cathode (–)
93
What is the net charge when pH > pI?
A: Negative — migrates toward the anode (+)
94
What happens to lysine (pI = 9.74) at pH 6?
A: It is positively charged and moves to the cathode (–)
95
What happens to glutamic acid (pI = 3.22) at pH 6?
A: It is negatively charged and moves to the anode (+)
96
What happens to alanine (pI = 6.02) at pH 6?
A: It is zwitterionic, so it does not migrate
97
What reagent is used to detect amino acids after electrophoresis?
A: Ninhydrin
98
What color appears when ninhydrin reacts with a primary amino acid?
A: Purple
99
What does the number of purple spots indicate?
A: The number of primary amino acids present
100
Which amino acid gives a different color with ninhydrin and why?
A: Proline, because it’s a secondary amine; gives a yellow product
101
What is electrophoresis mainly used for in amino acid analysis?
A: Identifying and counting different amino acids in a mixture
102
Can electrophoresis be used to isolate amino acids in bulk?
A: No, it is not suitable for large-scale separation
103
What technique is used for preparative amino acid separation?
A: Column chromatography
104
Latent Fingerprints and Amino Acids
Latent fingerprints are invisible prints left on surfaces by residues from the skin — mainly sweat (~99% water).
Sweat contains trace organic compounds, including amino acids.
These amino acids:
Are present in small amounts
Are chemically stable over time
Allow detection long after the print is made
105
Ninhydrin Reaction
Ninhydrin is a reagent that reacts with amino acids, specifically primary amines.
When ninhydrin contacts the amino acids in the fingerprint residue, it forms a fluorescent purple compound.
This reaction reveals the pattern of the fingerprint.
The product is the same purple compound formed during amino acid analysis.
Application Procedure
A solution of ninhydrin is sprayed on the suspected surface.
Mild heating is applied to accelerate the reaction.
Within a few minutes to hours:
The purple prints appear.
These images are then photographed or analyzed.
⚠️ Limitations
Background staining may occur, reducing image contrast.
Light fading may affect the longevity of the image.
Development can be slow (up to 2 weeks) if not accelerated by heat
106
Ninhydrin Analogues
Over the years, modified versions (analogues) of ninhydrin have been developed.
These analogues:
Aim to improve contrast, sensitivity, or development speed.
Include additional functional groups like Cl or OMe (methoxy).
But most are not widely adopted due to:
High cost
Only slight improvements
107
Amino Acid Synthesis via α-Haloacids
This is one of the oldest and most classical methods of synthesizing α-amino acids, especially racemic mixtures. It is a two-step process:
Step 1: α-Halogenation of a Carboxylic Acid
This uses the Hell–Volhard–Zelinsky (HVZ) Reaction.
Reagents:
Br₂ + PBr₃ (to convert the –COOH into an acyl bromide and enable α-enolization)
H₂O (to hydrolyze back to the acid)
Reaction:
R–CH₂–COOH
→
𝐵
𝑟
2
,
𝑃
𝐵
𝑟
3
R–CH(Br)–COOH
R–CH₂–COOH
Br
2
,PBr
3
R–CH(Br)–COOH
The result is a racemic α-bromo acid (a mixture of R and S enantiomers).
Step 2: Substitution with Ammonia (SN2 Reaction)
The α-bromo group is replaced with an amino group (–NH₂) using excess NH₃.
This is a nucleophilic substitution (SN2 mechanism).
Reaction:
R–CH(Br)–COOH
→
𝑒
𝑥
𝑐
𝑒
𝑠
𝑠
𝑁
𝐻
3
R–CH(NH₂)–COOH
R–CH(Br)–COOH
excessNH
3
R–CH(NH₂)–COOH
Final product: Racemic α-amino acid.
⚠️ Special Note on Polyalkylation
In general, ammonia reacting with alkyl halides leads to polyalkylation (multiple substitutions).
But in this reaction:
The α-halo acid is sterically hindered.
That prevents multiple alkylations, so only one NH₂ is added.
108
What classic method is used to synthesize α-amino acids from carboxylic acids?
A: The Hell–Volhard–Zelinsky (HVZ) reaction followed by SN2 substitution with ammonia.
109
What type of amino acid mixture is produced using this method?
A: A racemic mixture (50:50 R and S enantiomers).
110
What reagents are used to brominate the α-carbon of a carboxylic acid?
A: Br₂ + PBr₃, followed by H₂O
111
What reagent is used to substitute Br with NH₂?
A: Excess NH₃
112
What kind of mechanism is used to replace Br with NH₂?
A: SN2 nucleophilic substitution
113
What structural feature is introduced at the α-carbon during Amino Acid Synthesis via α-Haloacids?
A: A new chiral center, leading to R and S enantiomers
114
What functional groups are present in the final α-amino acid? Amino Acid Synthesis via α-Haloacids
A: An amino group (–NH₂) at the α-carbon, and a carboxylic acid (–COOH)
115
Amino Acid Synthesis via the Amidomalonate Synthesis
Background
This method is based on a clever adaptation of the malonic ester synthesis, which produces substituted carboxylic acids.
The starting material is diethyl acetamidomalonate — a malonic ester with an amide group (–NHAc) already built in.
116
Steps of the Reaction Amino Acid Synthesis via the Amidomalonate Synthesis
There are 3 main steps, similar to malonic ester synthesis
Step 1: Deprotonation
Reagent: NaOEt (a strong base)
The base removes the acidic proton from the α-carbon (between the two esters).
EtOOC–CH(NHAc)–COOEt
→
NaOEt
Carbanion
EtOOC–CH(NHAc)–COOEt
NaOEt
Carbanion
Step 2: Alkylation
Reagent: Alkyl halide (R–X)
The carbanion attacks the alkyl halide (SN2), forming a new C–C bond at the α-carbon.
Carbanion
+
𝑅
–
𝑋
→
EtOOC–CH(R)(NHAc)–COOEt
Carbanion+R–X→EtOOC–CH(R)(NHAc)–COOEt
Step 3: Hydrolysis and Decarboxylation
Reagents: H₃O⁺, Heat
Both ester groups are hydrolyzed to carboxylic acids.
One COOH group is decarboxylated (lost as CO₂).
The amide is also hydrolyzed to a primary amine (–NH₂).
EtOOC–CH(R)(NHAc)–COOEt
→
H₃O⁺, Heat
H₂N–CH(R)–COOH
EtOOC–CH(R)(NHAc)–COOEt
H₃O⁺, Heat
H₂N–CH(R)–COOH
✅ Result: A racemic α-amino acid
💡 Key Insight
The identity of the final amino acid is determined by the alkyl halide used in step 2.
For example, using benzyl bromide gives phenylalanine.
117
What is the starting material in amidomalonate synthesis?
A: Diethyl acetamidomalonate
118
What is the purpose of the amidomalonate synthesis?
A: To synthesize racemic α-amino acids
119
What functional group in the starting material will eventually become the amino group?
A: The amide group (–NHAc)
120
What happens in Step 1 of amidomalonate synthesis?
A: Deprotonation at the α-carbon using NaOEt
121
What happens in Step 2 amidomalonate synthesis?
A: Alkylation with an alkyl halide (R–X) via an SN2 mechanism
122
What happens in Step 3. amidomalonate synthesis?
Hydrolysis of esters and amide, followed by decarboxylation of one COOH group
123
Is the product optically active? amidomalonate synthesis
A: No — it's a racemic mixture
124
What determines the identity of the final amino acid? amidomalonate synthesis
A: The R group introduced by the alkyl halide in Step 2
125
STRECKER SYNTHESIS OVERVIEW
Purpose:
To synthesize racemic α-amino acids from aldehydes in two steps:
Formation of an α-amino nitrile
Hydrolysis of the nitrile to a carboxylic acid
Reagents:
NH₄Cl (provides NH₃)
NaCN (source of nucleophilic cyanide)
H₃O⁺ (acidic hydrolysis)
General Reaction: Aldehyde → α-amino nitrile → α-amino acid
RCHO → RCH(NH₂)CN → RCH(NH₃⁺)COO⁻
126
DETAILED MECHANISM STRECKER SYNTHESIS
Step 1: Imine Formation
Nucleophilic attack: NH₃ attacks the carbonyl carbon of the aldehyde.
Proton transfers create a stable imine intermediate.
Step 2: Nitrile Formation
Cyanide ion (⁻C≡N) attacks the imine carbon.
This gives an α-amino nitrile.
Step 3: Hydrolysis of the nitrile
Under acidic conditions, the nitrile undergoes hydrolysis.
Forms a carboxylic acid group → yielding a racemic α-amino acid.
127
What is the starting material for the Strecker synthesis?
A: An aldehyde.
128
What type of amino acid does Strecker synthesis produce?
A: A racemic mixture of α-amino acids (both D and L forms).
129
Which nucleophile attacks the aldehyde in Strecker synthesis?
A: Ammonia (NH₃), forming an imine.
130
What is the role of NaCN in the Strecker synthesis?
A: Provides cyanide ion (⁻C≡N) for nucleophilic attack on the imine.
131
What intermediate is formed after the cyanide attack?
A: α-Amino nitrile.
132
How is the nitrile converted into a carboxylic acid?
A: By acidic hydrolysis using H₃O⁺ and heat.
133
What determines the identity of the amino acid formed?
A: The structure of the starting aldehyde.
134
Why is the product racemic?
(strecker synthesis )
A: The carbon becomes a chiral center, and the reaction is not stereoselective.
135
Enantioselective Synthesis of L-Amino Acids
When amino acids are synthesized in the laboratory through most common methods (e.g., Strecker synthesis, reductive amination, or malonic ester synthesis), the result is usually a racemic mixture of both enantiomers (D and L forms). However, in biological systems, only L-amino acids are used to build proteins. Therefore, synthesizing optically active (chiral) L-amino acids requires one of two strategies:
Resolution of racemic mixtures (less efficient because half of the material is wasted).
Enantioselective synthesis (preferred due to higher efficiency and reduced waste).
136
Enantioselective Synthesis Example: Knowles' Procedure
Enantioselective Synthesis Example: Knowles' Procedure
The strategy used by Knowles involves asymmetric hydrogenation of a prochiral double bond.
A chiral catalyst is employed to reduce the double bond in a way that produces one enantiomer preferentially, such as the (S)-enantiomer of L-dopa.
Example Reaction:
A substrate containing a C=C bond next to a carbonyl group undergoes asymmetric hydrogenation using a chiral ruthenium catalyst complexed with (R)-BINAP.
This results in the (S)-enantiomer of an amino acid (e.g., L-dopa).
Why BINAP?
BINAP is a chiral phosphine ligand.
When bound to Ru, it gives a chiral environment that biases the face of the double bond being attacked during hydrogenation.
This bias allows for control over stereochemistry, often yielding >99% enantiomeric excess (%ee).
137
What is the main advantage of enantioselective synthesis over resolution of racemic mixtures?
A: It avoids waste by producing only one enantiomer directly, making the process more efficient.
138
What type of catalyst is used in asymmetric hydrogenation to synthesize L-amino acids?
A: A chiral metal catalyst, often ruthenium complexed with BINAP.
139
What is BINAP?
A: (R)-BINAP is a chiral phosphine ligand used in asymmetric catalysis to control enantioselectivity.
140
What product is formed by asymmetric hydrogenation in the synthesis of L-dopa?
A: (S)-3,4-Dihydroxyphenylalanine (L-dopa).
141
What does "% ee" stand for and what does it measure?
A: Enantiomeric excess; it measures the purity of one enantiomer over the other in a chiral product.
142
What role does the chiral catalyst play in the hydrogenation process?
A: It ensures that hydrogen is added to one face of the double bond preferentially, leading to one enantiomer.
143
Which amino acid was synthesized with 99% ee using (R)-Ru(BINAP)Cl2?
A: D-Phenylalanine.
144
Why is enantioselective synthesis important in pharmaceutical and biological chemistry?
A: Because only one enantiomer (usually L-form) is biologically active and functional in proteins.
145
What type of molecule is glutathione?
A: A tripeptide made from glutamic acid, cysteine, and glycine.
145
What bond links amino acids in peptides?
A: Peptide bond (amide linkage) formed via condensation (dehydration synthesis).
146
Which three amino acids make up glutathione?
A: Glutamic acid (Glu), cysteine (Cys), and glycine (Gly).
147
Which functional groups participate in peptide bond formation?
A: The amino group (-NH₂) of one amino acid and the carboxyl group (-COOH) of another.
148
What is the structure of a peptide bond?
A: -CO–NH- (a carbonyl group linked to a nitrogen via a single bond).
149
What type of reaction forms a peptide bond?
A: A condensation reaction (water is removed).
150
What is the directionality of a peptide chain?
A: From the N-terminus (free –NH₂) to the C-terminus (free –COOH).
151
Sequence/order matters
– Ala-Gly ≠ Gly-Ala. They are constitutional isomers (same atoms, different structure).
152
Key Points:
Peptides are written and drawn from N-terminus (amino end) to C-terminus (carboxyl end).
The N-terminus always has a free NH₂ group, and the C-terminus has a free COOH group.
153
What makes Ala-Gly and Gly-Ala constitutional isomers?
A: They have the same molecular formula but different sequence and structure due to different peptide bond orientations.
153
What happens during peptide bond formation?
A: A water molecule is lost (condensation), and a new C–N bond (peptide bond) forms between amino acids.
154
What does the term “amino acid residue” refer to in peptides?
A: An amino acid unit that has been incorporated into a peptide chain, missing a hydrogen from the amino group or an OH from the carboxyl group.
155
N Terminus (Amino End):
Located at the left end.
Contains a free –NH₂ group.
Starting point for peptide notation.
156
C Terminus (Carboxyl End):
Located at the right end.
Contains a free –COOH group (shown as –OH after bond formation).
End point of the peptide sequence.
157
Peptide Bond Locations:
Between C=O of one amino acid and NH of the next.
Each peptide bond forms through condensation, releasing H₂O.
158
What is the repeating unit in a peptide backbone?
A: –NH–CHR–CO–
159
What does “R” represent in the peptide structure?
A: The side chain specific to each amino acid.
160
Amino Acid Abbreviations:
Amino acids can be abbreviated using one-letter or three-letter codes.
Example: Glycine = Gly, Alanine = Ala.
161
Directionality and Naming:
Peptide sequences are always written left to right, from the N terminus to the C terminus.
In the decapeptide (10 amino acids), Ala is the N terminus, and Leu is the C terminus.
162
One N terminus and One C terminus:
All linear (unbranched) peptides have exactly one free amino group (N terminus) and one free carboxyl group (C terminus).
163
How many termini does a linear peptide have?
A: One N terminus and one C terminus.
164
Peptide Bonds Are Amides:
Peptide bonds are a type of amide linkage formed between the carboxyl group of one amino acid and the amino group of another.
Due to resonance, the C–N bond in the amide has partial double-bond character, making it planar and rigid.
165
Resonance in Amides:
The lone pair on the nitrogen can delocalize, forming a resonance structure with the adjacent carbonyl group.
This delocalization gives the C–N bond partial π-bond character, restricting rotation.
165
Hybridization and Planarity:
The nitrogen is sp² hybridized, making the entire peptide bond planar.
166
Conformations – s-cis vs s-trans:
The peptide bond can adopt either:
s-trans: More stable due to less steric hindrance.
s-cis: Less stable due to steric clash between groups.
Energy barrier to rotation: ~80 kJ/mol, indicating restricted rotation.
167
Restricted vs Free Rotation:
Only peptide bonds (C–N amide) are rigid.
The σ bonds adjacent to the α-carbons can freely rotate, giving flexibility to the polypeptide backbone.
168
Structural Implication:
Though each peptide unit is planar, the entire polypeptide is not; it can adopt various conformations (e.g., α-helix, β-sheet).
169
What type of bond is a peptide bond?
A: An amide bond formed between the carboxyl of one amino acid and the amino group of another.
170
Why is the peptide bond planar?
A: Due to resonance, the C–N bond has partial double-bond character, restricting rotation and making it planar.
170
What structural feature results from restricted rotation in peptide bonds?
A: Each peptide unit is planar, influencing the overall 3D shape of proteins.
170
Can a polypeptide chain be fully planar?
A: No, despite each peptide bond being planar, the entire chain is flexible due to rotatable σ bonds.
171
Thiol Group and Disulfide Formation:
Thiol group (–SH) is found in cysteine, the only amino acid with this functional group.
Two thiol groups can undergo oxidation to form a disulfide bond (–S–S–).
This process is reversible via reduction, which breaks the disulfide bond.
172
chemical Reaction: - disulphide formation
2 R–SH → R–S–S–R (disulfide) + 2 H⁺ + 2 e⁻
Cysteine + Cysteine (oxidation) → Cystine (a disulfide-linked dimer)
172
Role in Peptides/Proteins:
Disulfide bridges are covalent bonds that stabilize protein tertiary and quaternary structures.
They affect the folding, stability, and function of proteins.
173
Intrastrand vs Interstrand Disulfide Bonds:
Intrastrand: Disulfide bond between cysteine residues within the same peptide strand.
Interstrand: Disulfide bond between cysteine residues in different peptide chains.
174
Which amino acid contains a thiol group?
A: Cysteine.
175
What bond forms between two cysteine residues upon oxidation?
A: A disulfide bond (–S–S–).
176
What is the product formed when two cysteines are oxidized?
A: Cystine (a disulfide-linked dimer).
177
What type of reaction forms a disulfide bond?
A: Oxidation.
178
Edman degradation
Edman degradation is a stepwise method for sequencing peptides, removing one amino acid at a time from the N-terminus.
178
What type of reaction breaks a disulfide bond?
A: Reduction.
179
Reagents Involved in Edman degradation
Reagents Involved:
Phenyl isothiocyanate (Ph–N=C=S): Reacts with the N-terminal amino group.
Trifluoroacetic acid (CF₃CO₂H): Cleaves the N-terminal residue and forms a stable derivative.
180
Process Overview: Edman degradation
The free N-terminal –NH₂ group attacks the carbon in phenyl isothiocyanate.
This forms a cyclic intermediate (via nucleophilic attack).
Acid treatment (TFA) cleaves the peptide, releasing a PTH–amino acid.
This derivative is analyzed to identify the specific amino acid.
The shortened peptide is then subjected to another round of Edman degradation.
181
Product: edman degradation
Product:
The N-terminal amino acid is released as a PTH derivative (phenylthiohydantoin), which is stable and easy to identify using chromatography or spectroscopy.
182
Mechanism Highlights: edman degradation
Nucleophilic attack forms an initial intermediate.
Intramolecular ring closure and rearrangement.
Proton transfers and cleavage under acidic conditions (loss of amino acid as PTH).
Automation:
Edman degradation has been automated and can sequence up to 50 residues
183
How does the N-terminal amino group participate in the reaction?
A: It acts as a nucleophile and attacks phenyl isothiocyanate.
184
What happens after the PTH-amino acid is removed?
A: The shortened peptide undergoes the same process again to remove the next residue.
185
What analytical techniques are used to identify the PTH derivative?
A: Chromatography or spectroscopy (e.g., HPLC, UV).
186
How many amino acids can automated Edman degradation sequence accurately?
A: Up to 50 residues.
187
Why is Edman degradation a preferred method for sequencing short peptides?
A: It’s non-destructive to the rest of the chain and provides stepwise identification.
188
Analysis: Enzymatic Cleavage in Protein Sequencing
Purpose
For peptides >50 residues, Edman degradation becomes impractical due to:
Side product accumulation
Low yield
Sequence complexity
Solution: Use enzymes (peptidases) to cleave large peptides into smaller, manageable fragments, which are then sequenced individually.
189
Trypsin
Cleaves after basic amino acids:
Arginine (Arg, R)
Lysine (Lys, K)
Cleavage occurs on the C-terminal side of these residues.
Example:
Ala–Phe–Lys | Pro–Met–Tyr–Gly–Arg | Ser–Trp–Leu–His
→ Cleaves after Lys and Arg.
190
Chymotrypsin
Cleaves after aromatic amino acids:
Phenylalanine (Phe, F)
Tyrosine (Tyr, Y)
Tryptophan (Trp, W)
Also cleaves on the C-terminal side.
Example:
Ala–Phe | Lys–Pro–Met–Tyr | Gly–Arg–Ser–Trp | Leu–His
→ Cleaves after Phe, Tyr, and Trp.
191
Why is Edman degradation not ideal for peptides over 50 residues?
A: It becomes inefficient due to side reactions, product buildup, and low yield.
192
What is the purpose of enzymatic cleavage in sequencing large peptides?
A: To break the peptide into smaller, sequenceable fragments.
193
What type of enzymes are used to cleave peptides at specific locations?
A: Peptidases (proteolytic enzymes).
194
What does trypsin specifically cleave after?
A: Arginine (Arg, R) and Lysine (Lys, K).
195
Does trypsin cleave before or after the target amino acid?
A: After (on the C-terminal side).
196
What amino acids does chymotrypsin cleave after?
A: Phenylalanine (Phe, F), Tyrosine (Tyr, Y), and Tryptophan (Trp, W).
196
In the sequence Ala–Phe–Lys–Pro–Met–Tyr–Gly–Arg–Ser–Trp–Leu–His, how many fragments will trypsin produce?
A: Three fragments (after Lys and Arg).
197
How many fragments will chymotrypsin produce from the same sequence?
A: Four fragments (after Phe, Tyr, and Trp).
198
What is the advantage of using different enzymes to cleave the same peptide?
A: It produces overlapping fragments for accurate reconstruction of the full sequence.
199
After enzymatic cleavage, how is sequencing completed?
A: Each fragment is sequenced (e.g., by Edman degradation), and the full sequence is reconstructed.
200
Analysis: Peptide Synthesis Using DCC (Dicyclohexylcarbodiimide)
Goal
To chemically synthesize a peptide bond between a carboxylic acid group and an amino group using DCC as a coupling reagent.
🔹 Key Reagent
DCC (Dicyclohexylcarbodiimide)
Structure: R–N=C=N–R (with R = cyclohexyl)
Function: Activates the carboxyl group of one amino acid to make it more reactive toward nucleophilic attack by an amine.
201
Step-by-Step Mechanism Overview Peptide Synthesis Using DCC (Dicyclohexylcarbodiimide)
Step-by-Step Mechanism Overview
Proton Transfer (Activation):
DCC abstracts a proton from the carboxylic acid group of one amino acid → forms a carboxylate anion.
Nucleophilic Attack (Activation Complex Formation):
The carboxylate anion attacks the central carbon of DCC.
This forms a reactive O-acylisourea intermediate, which is highly susceptible to nucleophilic attack.
Second Nucleophilic Attack (Peptide Bond Formation):
The amine group of another amino acid attacks the carbonyl carbon of the intermediate.
This results in peptide bond formation.
Leaving Group Departure and Product Formation:
DCC is expelled as dicyclohexylurea (DCU).
Final product is a peptide bond (amide linkage) between the two amino acids.
🔹 Important Notes
This is a non-biological peptide synthesis, used in lab settings.
Avoids the use of enzymes.
DCC is not used in living systems due to its toxicity and harsh conditions.
202
What functional groups must react to form a peptide bond?
A: A carboxylic acid group and an amino group.
203
What is the role of DCC in peptide synthesis?
A: It activates the carboxyl group of one amino acid to make it reactive toward the amine group of another.
204
What is the full name of DCC?
A: Dicyclohexylcarbodiimide.
205
What intermediate is formed when DCC reacts with the carboxylate group?
A: An O-acylisourea intermediate.
205
What does the nucleophilic amine attack in the reaction mechanism?
A: The activated carbonyl carbon of the O-acylisourea intermediate.
206
What is the byproduct of peptide bond formation via DCC?
A: Dicyclohexylurea (DCU).
207
Is DCC-mediated peptide synthesis used in biological systems?
A: No, it is a chemical method used in laboratory settings.
208
What drives the formation of the peptide bond in this reaction?
A: The high reactivity of the O-acylisourea intermediate and the nucleophilic amine.
209
Why is proton transfer necessary at the beginning of the reaction?
A: To deprotonate the carboxylic acid and form a reactive carboxylate nucleophil
210
What type of bond is formed at the end of the reaction?
A: A peptide bond (amide bond).
211
Problem: Lack of Regioselectivity in DCC-Mediated Peptide Synthesis
When coupling two different amino acids (e.g., R₁ and R₂) with DCC, up to four different dipeptides can form due to random reaction of both –NH₂ and –COOH groups.
This lack of selectivity can result in undesired products.
🔹 Solution: Protecting Groups
To prevent uncontrolled coupling:
Protect the amino group (–NH₂) of one amino acid.
Protect the carboxyl group (–COOH) of the other.
Then couple selectively using DCC.
Finally, remove the protecting groups to yield the desired dipeptide with correct orientation.
212
Protecting the Amino Group
A carbamate is formed to protect the amine.
Most common protecting group: Boc (tert-butoxycarbonyl).
Installed using:
Di-tert-butyl dicarbonate (Boc₂O).
🔹 Mechanism: Boc Installation
Nucleophilic Attack:
The amine group attacks the carbonyl carbon in Boc₂O.
Intermediate Formation:
A tetrahedral intermediate is formed with a positively charged nitrogen.
Proton Transfer:
The ammonium intermediate is deprotonated.
Loss of Leaving Group:
The resonance-stabilized tert-butyl carbonate is expelled.
Result:
A Boc-protected amino acid, with the amine converted to a carbamate (less nucleophilic).
213
What issue arises when using DCC to couple two unprotected amino acids?
A: Lack of regioselectivity leads to the formation of multiple dipeptide products.
214
How many dipeptides can form when coupling two different amino acids with DCC unselectively?
A: Four different dipeptides.
215
How can regioselectivity be achieved in peptide synthesis?
A: By protecting the amino group of one amino acid and the carboxyl group of the other.
216
What is a protecting group?
A: A chemical group added to a functional group to temporarily block its reactivity.
217
What is the Boc group?
A: Tert-butoxycarbonyl; a common protecting group for amino groups.
218
What reagent is used to install the Boc group?
A: Di-tert-butyl dicarbonate (Boc₂O).
219
What type of group does Boc convert the amino group into?
A: A carbamate, which is less nucleophilic.
220
Why is a carbamate less nucleophilic than a free amine?
A: Because its lone pair is delocalized by the adjacent carbonyl group.
221
What step in Boc installation involves proton removal?
A: Proton transfer from the ammonium intermediate to yield the neutral carbamate.
222
What happens after peptide bond formation using protected amino acids?
A: The protecting groups are removed to yield the final peptide.
223
Analysis: Removal of Boc and Use of Fmoc Protecting Groups
Boc Group Removal (tert-Butoxycarbonyl)
Boc is a widely used protecting group for amino groups because it is easily removed under acidic conditions, e.g., trifluoroacetic acid (CF₃COOH).
✅ Mechanism of Boc Removal (Mechanism 25.5):
Protonation of the carbonyl oxygen by CF₃COOH.
Loss of a leaving group: Carbamic acid group departs, leaving a carbocation.
Gas evolution:
Isobutylene and carbon dioxide are released.
These drive the reaction to completion (nearly 100% yield).
Result: The free amine (NH₂) is regenerated.
🔹 Fmoc Protecting Group (Fluorenylmethyloxycarbonyl)
Alternative to Boc; used when acidic deprotection is not ideal.
Installed using 9-fluorenylmethyl chloroformate.
Removed under basic conditions (e.g., piperidine).
✅ Fmoc Removal (Described Mechanism):
Involves:
Deprotonation of the benzylic hydrogen (aromatic stabilization).
Formation of a stabilized aromatic anion.
Loss of CO₂, which drives deprotection.
Fmoc chemistry allows orthogonal protection strategies in peptide synthesis (acid/base separation).
224
Boc Group Removal (tert-Butoxycarbonyl)
Boc is a widely used protecting group for amino groups because it is easily removed under acidic conditions, e.g., trifluoroacetic acid (CF₃COOH).
✅ Mechanism of Boc Removal (Mechanism 25.5):
Protonation of the carbonyl oxygen by CF₃COOH.
Loss of a leaving group: Carbamic acid group departs, leaving a carbocation.
Gas evolution:
Isobutylene and carbon dioxide are released.
These drive the reaction to completion (nearly 100% yield).
Result: The free amine (NH₂) is regenerated.
🔹 Fmoc Protecting Group (Fluorenylmethyloxycarbonyl)
Alternative to Boc; used when acidic deprotection is not ideal.
Installed using 9-fluorenylmethyl chloroformate.
Removed under basic conditions (e.g., piperidine).
✅ Fmoc Removal (Described Mechanism):
Involves:
Deprotonation of the benzylic hydrogen (aromatic stabilization).
Formation of a stabilized aromatic anion.
Loss of CO₂, which drives deprotection.
Fmoc chemistry allows orthogonal protection strategies in peptide synthesis (acid/base separation).
224
What two gases are produced during Boc removal?
A: Isobutylene and carbon dioxide (CO₂)
225
What type of conditions are required for Boc removal?
A: Acidic conditions.
225
What drives the Boc deprotection reaction to completion?
A: Evolution of gases (CO₂ and isobutylene).
226
What kind of group is Boc?
A: A carbamate-based protecting group.
227
What is the byproduct when the Boc group leaves?
A: A carbocation and CO₂.
227
What is Fmoc short for?
A:
Fluorenylmethyloxycarbonyl.
228
Under what condition is the Fmoc group removed?
A: Basic conditions (e.g., piperidine).
229
What stabilizes the anion formed during Fmoc removal?
A: Aromaticity of the fluorenyl system.
230
Why use both Boc and Fmoc strategies in synthesis?
A: To allow orthogonal protection—Boc removed under acid, Fmoc under base.
230
Fmoc Group Removal (Mechanism 25.6)
Reagent used: Piperidine (a strong base)
Mechanism Summary:
Piperidine deprotonates the benzylic position on the Fmoc group.
This generates an aromatic, resonance-stabilized anion.
Carbon dioxide (CO₂) is expelled.
The resulting products are:
Free amine (NH₂–R)
Fmoc fragment (fluorene derivative)
CO₂
This deprotection is mild and base-compatible, making it orthogonal to acid-labile groups like Boc.
230
Protecting the Carboxylic Acid Group
🧪 Method:
Convert –COOH into an ester (e.g., methyl ester or benzyl ester).
Esterification: Alcohol (ROH) + acid catalyst (H⁺) + amino acid → ester-protected amino acid
🔄 Deprotection methods:
Hydrolysis:
With NaOH/H₂O to regenerate the carboxylate (–COO⁻)
Hydrogenolysis (for benzyl esters):
H₂, Pt catalyst
Acid cleavage:
HBr in acetic acid
231
What reagent is commonly used to remove the Fmoc protecting group?
A: Piperidine.
232
What drives the Fmoc deprotection to completion?
A: Formation of an aromatic anion and loss of CO₂.
233
What type of chemical intermediate is formed during Fmoc removal?
A: An aromatic, resonance-stabilized anion.
234
What are the products of Fmoc removal?
A: Free amine, fluorenyl compound, and CO₂.
235
How is a carboxylic acid group protected in amino acid synthesis?
A: By converting it to an ester.
236
What alcohol is commonly used to form a methyl ester?
A: Methanol (MeOH).
237
What alcohol is used to make benzyl esters?
A: Benzyl alcohol (PhCH₂OH).
238
How can ester protecting groups be removed?
A: With aqueous base (e.g., NaOH/H₂O).
239
How are benzyl esters specifically removed?
A: Via hydrogenolysis (H₂, Pt) or HBr in acetic acid.
240
Why are protecting groups used in peptide synthesis?
A: To prevent unwanted side reactions and ensure regioselectivity.
241
Analysis: Preparing a Dipeptide (Ala–Gly)
🔹 Objective
To synthesize a specific dipeptide (Ala–Gly) selectively, avoiding undesired isomers or side products.
🔹 Steps Involved
Step 1: Protect Functional Groups
Ala (Alanine):
Its amino group is protected with a Boc group → Boc–Ala
Gly (Glycine):
Its carboxylic acid group is protected as a methyl ester using acidic methanol → Gly–OCH₃
Step 2: Coupling with DCC
Boc–Ala and Gly–OCH₃ are coupled using DCC.
This forms a protected dipeptide:
Boc–Ala–Gly–OCH₃
Step 3: Deprotection
CF₃COOH removes the Boc group from the N-terminus.
NaOH/H₂O hydrolyzes the methyl ester, regenerating the C-terminal carboxylic acid.
✅ Final Product: Ala–Gly (with correct orientation, no scrambling).
🔹 Key Concepts
Regioselectivity is achieved by orthogonal protection.
Protecting groups prevent uncontrolled coupling during DCC-mediated synthesis.
This method is scalable for tripeptides and tetrapeptides.
242
What is the first step in synthesizing Ala–Gly?
A: Protect the amino group of Ala with Boc and the carboxyl group of Gly as a methyl ester.
243
What reagent is used to install the Boc group on Ala?
A: Di-tert-butyl dicarbonate (Boc₂O).
244
What reagent is used to convert Gly into its methyl ester?
A: Methanol and acid (H⁺).
245
What reagent is used to couple protected amino acids?
A: Dicyclohexylcarbodiimide (DCC).
246
What is the structure of the intermediate product before deprotection?
A: Boc–Ala–Gly–OCH₃.
247
How is the Boc group removed?
A: Using trifluoroacetic acid (CF₃COOH).
248
How is the methyl ester removed?
A: With NaOH and H₂O.
249
What is the final product after deprotection in this synthesis?
A: Ala–Gly (a dipeptide).
250
Why are protecting groups used in peptide synthesis?
A: To control regioselectivity and avoid formation of undesired dipeptides.
251
Can this method be extended to tripeptides and tetrapeptides?
A: Yes, using similar protection and coupling steps.
252
Analysis: Merrifield Synthesis (Solid-Phase Peptide Synthesis) Why Merrifield Synthesis?
For longer peptides, solution-phase synthesis is inefficient due to:
Complex purification
Accumulation of side products
Solid-phase synthesis eliminates this by tethering the growing peptide to an insoluble resin.
253
Analysis: Merrifield Synthesis (Solid-Phase Peptide Synthesis) Core Concept
Invented by R. Bruce Merrifield (Rockefeller University)
Peptide is anchored to a solid polymer (resin), allowing:
Easy washing/removal of reagents and by-products
Repetition of coupling cycles without isolation
254
Analysis: Merrifield Synthesis (Solid-Phase Peptide Synthesis) Step-by-Step Summary
Attachment of First Amino Acid
A Boc-protected amino acid reacts with the chloromethyl-polystyrene resin via SN2 reaction.
This attaches the C-terminus of the amino acid to the polymer.
Deprotection
The Boc group is removed using acid (e.g., TFA), exposing the amino group.
Coupling Next Residue
A second Boc-protected amino acid is coupled using DCC.
Cycle Repeats
The process (deprotect–couple–wash) repeats to build a long peptide chain.
Cleavage from Resin
Once synthesis is complete, peptide is cleaved off the resin using hydrofluoric acid (HF).
255
Analysis: Merrifield Synthesis (Solid-Phase Peptide Synthesis)Historical Significance
Merrifield synthesized a 128-residue ribonuclease protein using this method.
The method required 369 steps over 6 weeks.
Earned Merrifield the 1984 Nobel Prize in Chemistry.
Modern versions are fully automated via peptide synthesizers.
256
What is the main advantage of Merrifield synthesis over traditional peptide synthesis?
A: It simplifies purification by tethering the peptide to an insoluble solid phase, allowing washing between steps.
257
What type of polymer is commonly used in Merrifield synthesis?
A: Chloromethyl-polystyrene resin.
258
What type of reaction is used to attach the first amino acid to the resin?
A: SN2 reaction.
259
What protecting group is used on the amino acids during Merrifield synthesis?
A: Boc (tert-butoxycarbonyl).
260
What reagent is used to couple Boc-protected amino acids in Merrifield synthesis?
A: Dicyclohexylcarbodiimide (DCC).
261
What acid is typically used to remove the peptide from the resin after synthesis?
A: Hydrofluoric acid (HF).
262
How is excess reagent and by-products removed during Merrifield synthesis?
A: They are washed away while the peptide remains attached to the solid support.
263
Who invented solid-phase peptide synthesis?
A: R. Bruce Merrifield.
264
What major award did Merrifield receive for this work?
A: The 1984 Nobel Prize in Chemistry.
265
What modern tool automates Merrifield synthesis today?
A: Peptide synthesizer machines.
266
Proteins are described across four hierarchical levels:
Primary
Secondary
Tertiary
Quaternary
267
Primary StructurePrimary Structurev
The primary structure is the linear sequence of amino acids in the polypeptide chain.
In human insulin, there are two chains:
Chain A: 21 amino acids
Chain B: 30 amino acids
These chains are:
Connected via disulfide bridges (S–S)
Each chain has N-terminus (start) and C-terminus (end)
Disulfide Bridges:
Exist within Chain A
Exist between Chains A and B
Created via oxidation of cysteine residues
268
Secondary Structure
Refers to localized 3D structures formed by the backbone:
Arises due to planarity and restricted rotation of peptide bonds
Common motifs include:
α-helices
β-sheets
269
What is the primary structure of a protein?
A: The sequence of amino acid residues in the polypeptide chain.
270
How many chains are in human insulin?
A: Two chains – Chain A (21 residues) and Chain B (30 residues).
271
What links the two chains of insulin?
A: Disulfide bridges (S–S bonds).
272
Which amino acid forms disulfide bridges?
A: Cysteine.
273
Give two examples of secondary structure elements.
A: α-helices and β-sheets.
273
What defines the secondary structure of a protein?
A: Localized 3D folding patterns caused by restricted rotation around peptide bonds.
274
What Is Secondary Structure?
The secondary structure refers to the local 3D conformations of amino acid residues in a polypeptide.
It arises due to hydrogen bonding between the C=O and N–H groups of the peptide backbone.
Each peptide bond is planar due to partial double bond character, restricting rotation and enforcing flat geometry between atoms.
274
Two Main Secondary Structures
✅ 1. α-Helix
A right-handed spiral.
Every C=O forms a hydrogen bond with the N–H of the residue four positions ahead.
R groups point outward from the helix.
Proline cannot participate (no N–H proton).
Found in hair (α-keratin) and in both chains of insulin.
✅ 2. β-Pleated Sheet
Formed by hydrogen bonding between neighboring strands.
R groups alternate above and below the sheet.
Found in fibroin (silk) and spider webs.
Can be parallel or antiparallel.
275
α-Helix
A right-handed spiral.
Every C=O forms a hydrogen bond with the N–H of the residue four positions ahead.
R groups point outward from the helix.
Proline cannot participate (no N–H proton).
Found in hair (α-keratin) and in both chains of insulin.
276
β-Pleated Sheet
Formed by hydrogen bonding between neighboring strands.
R groups alternate above and below the sheet.
Found in fibroin (silk) and spider webs.
Can be parallel or antiparallel.
277
What causes the formation of protein secondary structure?
A: Hydrogen bonding between backbone C=O and N–H groups.
278
Why is the peptide bond planar?
A: Due to partial double bond character between the C and N atoms.
279
What are the two major types of secondary structure?
A: α-Helix and β-Pleated Sheet.
280
What is the hydrogen bonding pattern in an α-helix?
A: The C=O of one residue bonds to the N–H four residues ahead.
281
Can proline participate in α-helices? Why or why not?
A: No; it lacks an N–H proton for hydrogen bonding.
282
Where do side chains (R groups) point in an α-helix?
A: Outward from the helix.
283
How are β-sheets stabilized?
A: By hydrogen bonding between neighboring strands
284
In β-sheets, how are R groups arranged?
A: Alternating above and below the plane.
285
What kind of structural protein is primarily α-helical?
A: α-Keratin (in hair).
285
What structural protein is mainly composed of β-sheets?
A: Fibroin (in silk).
286
Why are standard amino acids called α-amino acids?
A: Because their amino group (–NH₂) is attached to the α-carbon, which is the carbon directly next to the carboxylic acid group (–COOH).
Would you like a diagram to visualize it?
286
What regions of insulin show α-helical structure?
A: Chain A: Gly-1 to Ser-12; Chain B: Gly-8 to Cys-19.
286
In the context of an amino acid:
A bond-line structure of an amino acid shows:
In the context of an amino acid:
A bond-line structure of an amino acid shows:
Carbon atoms as vertices (corners) or line ends (unless labeled otherwise).
Hydrogen atoms attached to carbon are not shown (unless it's important).
Functional groups like –NH₂ and –COOH are often drawn in full or partially shown, depending on clarity.
287
Amino acids are colorless, so a detection technique is necessary in order to visualize the location of the various spots. The most common method involves treating the filter paper or gel with a
solution containing ninhydrin followed by heating in an oven. Ninhydrin reacts with amino acids to
produce a purple product.
The nitrogen atom of the amino acid is ultimately incorporated into the purple product, and the rest
of the amino acid is degraded into a few by-products (water, carbon dioxide, and an aldehyde). The
purple compound is obtained regardless of the identity of the amino acid, provided that the amino
acid is primary (i.e., not proline). The number of purple spots indicates the number of different kinds
of amino acids present.
Electrophoresis cannot be used to separate large quantities of amino acids. It is used just as an analytical method for determining the number of amino acids in a mixture. In order to actually separate an
entire mixture of amino acids, other laboratory techniques are used, such as column chromatography
288
sweat is primarily composed of ?
Sweat is comprised primarily of
water (99%)
289
Sweat is comprised primarily of
water (99%), but it also contains a variety o
Sweat is comprised primarily of
water (99%), but it also contains a variety of organic compounds,
including amino acids.These amino acids are present only in
very small concentrations, but they are relatively stable over
long periods of time. When treated with ninhydrin, a reaction
occurs between the amino acids and the ninhydrin, forming a
fluorescent purple image.
290
One of the oldest methods for preparing racemic mixtures of α-amino acids involves the use
of the Hell–Volhard–Zelinsky reaction (Section 21.2) to functionalize the α position of a
carboxylic acid.
we saw that polyalkylation is often unavoidable when ammonia is treated with an
alkyl halide. However, in this case, polyalkylation is not a problem because the alkyl halide is fairly
large and steric hindrance prevents subsequent alkylations.
291
Identify the reagents necessary to make amino acids using a Hell–Volhard–Zelinsky reaction:
292
Amino Acid Synthesis via the Amidomalonate Synthesis
293
Racemic α-amino acids can also be prepared from aldehydes via a two-step process called the Strecker
synthesis
293
dentify the reagents necessary to make amino acids using a Strecker synthesis:
1) NH4Cl, NaCN. 2) H3O+
294
The essential amino acids are
isoleucine, leucine, methionine,
phenylalanine, threonine, tryptophan, valine, arginine, histidine,
and lysine
295
