Chem 27 - Exam 1 Review
Wednesday Feb. 22, 2006
Science Center Hall D
K.C. O’Brien
Carol Fang
Walter Kowtoniuk
Outline of Topics
• 1) Conformational Analysis of amino acids
• 2) Protein Folding
• 3) Edman Degradation(-like) chemistry
• 4) Cyanogen bromide(-like) chemistry
• 5) Peptide Coupling/Synthesis
• 6) Biosynthesis of Proteins
Conformational Analysis
K.C. O’Brien
Amino Acid Structure
•Amino acids are chiral molecules
•Stereochemistry at a-carbon always
as shown (R group coming out)
O
-
NH3+
O
•All natural amino acids have S
configuration, except cysteine
•pKa’s: NH3+ is about 9
COO- is about 2.2
H R
•Hydrophobic, polar and charged side
chains
Staggered vs. Eclipsed Conformation
H
HH
H
H
H
H
+3.0 kcal/mol
H
H
H
H
H
staggered
eclipsed
• Hyperconjugation sC-H-> s*C-H
• Newman projections help visualize interactions
Gauche interactions
CH3
CH3
H
H
H
CH3
H
H
+0.9 kcal/mol
H
H
CH3
anti
H
gauche
Cyclohexane Chair Conformations
H
H
H
H3C
H
H3C
CH3
CH3
• Ring flip changes groups from axial to equatorial
• Lower energy conformation has large groups
equatorial
• A values are used to quantify the energy difference
between the axial and equatorial positions
Syn-pentane Interaction
H's cause
steric strain
• Syn-pentane > 3.7
kcal /mol
• 1,3-diaxial groups
generate a syn
pentane interaction
H
H
H
H
H
H
A1,3 Strain
• H is in the same plane as
double bond
• If R=R’=R”=Me, A1,3=3.5
kcal/mol
• Minimize A1,3 in amide bonds
R'
R"
H
H
H
H
H
H
3
H
2
1
R
H
H
Template Projection
of
Amino
Acids
H
H
N
H
N
H
O
O
H
N
R1 H
H
R1
O
R2
•
•
•
•
R3
R1
R2
N
H
H
O
H
Amino acid template projection is based on cyclohexane chair structure
Add up gauche and syn-pentane interactions to find the lowest energy conformation
R1>R2>R3 is a good place to start, but consider other conformations
Make sure you don’t invert the stereochemistry of the amino acid or its side chain!!!!
Protein Folding: Hydrogen Bonds
R
O :
H
N
R
R
R
• 1-4 kcal/mol
• Directionality is
important
• N-H-----O=C
• Stabilize a-helices,
b-sheets and turns
Protein Folding: a-helix
• stabilized by hydrogen bonding
• 3.6 amino acids per turn
Protein Folding: b-sheet
• NH’s of one strand H-bond to C=O of next strand
• R groups alternate on opposite sides of the plane
Protein Folding: b-turn
• C=O and N-H are 10
atoms apart
• Changes the
direction of the main
chain
Protein Folding: Electrostatic Interactions
NH2
N
H
O
N
H
R
O-
R
• Between oppositely
charged amino
acids
• Most important in
the interior of the
protein
• Neutralizes charges
Protein Folding:
Hydrophobic Interactions:
• Hydrophobic amino acids
pack into the interior of the
protein
• Folding increases the
disorder of the solvent
• Positive DH is overcome by
positive DS
Disulfide Bonds:
• Dihedral angle 90o
• ns donates into s*S-R
• Two Cys oxidized to
form a disulfide bond
Edman Degradation
Carol Fang
S
R1
O
S
H
N
C
O
H
N
N
N
Ph
O
Ph
H2N
N
R1
H
R2
H
H
A
O
Nucleophilic Amine (primary and secondary)
R2
B
HN
S
1
Ph
S
4
N
2
N
H
O
5
3
R1
1
A
N
H
H
H
N
4
N
2
O
5
3
Ph
H
R1
H
O
R2
E and Nu are 5 atoms apart
HN
HN
S
1
Ph
2
N
H
O
5
4
N
3
H
S
H
O
S
H
N
H
N
R1
B
B
S
O
Ph
N
N
R2
H
N
H
H
N
H
H2N
S
O
Ph
R1
H
H
A
H2N
Ph
R1
Rotatable bond
R1
O
+
Ph
N
N
R1
H
Thiazolinone Derivative
Kinetic product
NH2
O
New N-terminal
H+
O
S
S
Ph
O H
OH
S
Ph
N
H
N
N
H
R1
OH
Ph
N
N
H
R1
B
N
R1
H2O
H
A
OH
S
Ph
N
H
N
Enol Formation
R1
Pre-note
H+
S
O
Ph
N
H
N
Ph
R1
+
Potential racemization
S
O
O
N
S
N
H
R1
Ph
N
H
N
R1
PTH, to be detected by HPLC
Thermodynamic Product
Frame of Reaction
When racemization is taken care of
R1
O
H
N
H3N
H2N
O
R2
Ph
O
+
R2
O
N
S
N
H
R1
Brain teasers:
a)
a peptide is not reactive to Edman Degradation
D05
Cyclic peptide
No nucleophilic amine
b) After a round of Edman degradation, only one fragment is obtained
Breaking the peptide bond does not break the molecule
Presence of Nu amine; Cyclic
D10, D12
c) After a round of Edman degradation, two PTH products are obtained
2 Nu amines at both ends / 1 PTH end and 1 Nu amine end
D10, D12
d) Bicyclic PTH product from Edman Degradation
A ring before Edman degradation
c) Special case: Lysine
A more protonated amine
D02, D04
D09
Cyanogen Bromide Cleavage
N
Me
Me
S
Br
C
N
Me
CN
C
S
S
Br
-Br
1
- MeSCN
1
O
2
2
H
N
N
H
N
H
O
H
N
3
N
H
4
O5
3
4
NH
O5
Nucleophilic S
N
H
Nu and E 5 atoms apart
H2O
Rotatable bond
O
O
+ H N
2
N
H
N
H
O
O
HN
O
NH2
N
H
H
H
BH
Met (C)
NH
O
BH
N cleaved
H
H
A
Brain teasers:
1) A peptide gives only one fragment after CNBr cleavage
a) A cyclic peptide
b) C-terminal Methionine
2) It is known that a peptide has n Met. It gives n pieces of
fragments
3) How about (n+1) fragments?
How this reacts with CNBr? (2004 Exam 1)
O
NH2
H
N
H3N
N
H
N
H
O
S
NH
O
O
O
H
OH
H
N
N
O
N
O
O
H
OH
NH2
Why S / C=O combo can be so
different in these two reactions?
Edman Degradation
S
CNBr Cleavage
S
O
O
C=S bond, S is Nucleophilic
3 C-S bond, S has an extra
Covalent bond; adjacent C
is ready for SN2
Peptide Syntheses
Walter Kowtoniuk
Amide Bond Synthesis
- Synthesis of an amide bond using the corresponding
carboxylic acid and amine.
- Use DCC to both activate the acid and serve as a dehydrating
agent
O
H
N
O
C
H
O
R
N
H
N
N
H
C
N
R
O
NH2
R2
R2
C
N
H
C
N
H
Amide Bond Synthesis
B-
O
O
H
N
C
H
N
N
H
O
C
N
O
C
R
R
N
H
N
C
H
H
N
N
C
C
O
C
C
H
O
H
N
H
N
C
R
NH2
H
R
N
O
H
C
O
N
H
N
R2
C
N
H2N
R
A
N
O
H
O
N
N
H
A
H
O
H
R
B-
R2
O
R2
N
H
H
C
N
H
Amide Bond Synthesis
O
H
N
O
R2
C
N
H
R
N
H
C
N
H
R2
C
N
H
H
B-
O
H
N
R
Protecting Groups
Why do we need them?
Protecting Groups
Lecture Notes pg33
Protecting Groups
• t-Boc Synthesis
H3C
O
CH3 O
Cl
H3C
H3C
H3C
H3C
Cl
OH
O
CH3 O
CH3 O
Cl
R
H3C
H3C
R
H2N
O
H3C
H3C
N
H
H
B-
R
O
N
H
• t-Boc Deprotection
H+
CH3 O
H3 C
H3C
CH3 O
R
O
N
H
H
TFA
H3C
H3C
CH3
R
O
N
H
O
CH3
H
H+
E1
CH3
(gas)
H2 C
R
O
H2C
CH3
H
N
H
R
CO2
H2 N
Protecting Groups
• Cbz follows the same mechanism as
shown for t-Boc
OH
O
Cl
Cl
Cl
O
O
R
O
O
N
H
or
O
O
R
N
H
R
O
TFA
CO2
R
H2C
NH2
Et3Si-H
H3C
O
O
Protecting Groups
• Ts synthesis
H2N
O
H2N
C
NH2
CH3
O
C
OH
NH2
HN
O
F
HN
CH3
O
S
N
O
CH3
B-
H2N
F-
O
C
HF
OH
O
O
NH2
HN
S
N
NH2
O
• Ts Deprotection
H2N
OH
S
N
H
H
C
O
HN
O
NH2
O
OH
Cl S
HN
H2N
C
O
OH
NH
O
CH3
F
S
NH
O
CH3
Protecting Groups
• DNP synthesis
O
O
OH
H2N
O2N
NO2
OH
O
H2 N
H2N
NO2
HN
HN
N
Cl
N
NO2
• DNP deprotection
O
OH
HS
H2N
O
N
NO2
OH
H2N
NO2
HN
N
NO2
OH
H2N
NO2
HN
NO2
HN
N
Cl
O
OH
HN
NO2
S
H
B-
N
C to N Directionality
why not N to C?
BH
N
R2
R2
N
R1
N
H
N
H
Cy
HN
O
O
O
H
O
Cy
N
H
R1
O
A
R2
N
N
H
H
R1
O
O
R2
N
N
H
R1
H
O
O
B-
Solid Phase Peptide
Synthesis
Solid Phase Peptide
Synthesis
Peptide Fragment Coupling
• Thioester
BO
O
R
S
N
H
O
R
R
O
O
R
S
S
H
N
H
O
R
N
H
O
Br
O
N
H
N
H
O
O
• True coupling
O
O
R
SH
N
H
H2N
R
H+
SH
O
R
H N
H
N
H
N
H
O
O
H
O
O
H
N
S
O
R
H S
BO
H N
R
H
H
N
S
N
H
O
O
O
O
BO
O
R
H
H
N
R
N
H
O
S
H+
O
R
O
R
HN
N
H
H
N
O
HS
R
N
H
O
Determining Yield
• Synthesizing a 100mer
requires 99rxns, thus n=99
• If we factor in the initial
coupling to the solid
phase, the 100mer
requires 100rxns, thus
n=100
• For convergent synthesis
we are concerned with the
longest linear sequence of
steps. In this case the yield
of each individual reaction
is multiplied
Translation
Biological Carbonyl Activation
Biological Carbonyl Activation
Biological Carbonyl Activation
Ribosome
Role of A2486