chem 27 - exam 1 review wednesday feb. 22, 2006 science center hall d k.c. o’brien carol fang...
TRANSCRIPT
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Chem 27 - Exam 1 Review
Wednesday Feb. 22, 2006Science Center Hall D
K.C. O’Brien
Carol Fang
Walter Kowtoniuk
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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
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Conformational Analysis
K.C. O’Brien
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Amino Acid Structure
-ONH3
+
O
RH
•Amino acids are chiral molecules
•Stereochemistry at -carbon always as shown (R group coming out)
•All natural amino acids have S configuration, except cysteine
•pKa’s: NH3+ is about 9
COO- is about 2.2
•Hydrophobic, polar and charged side chains
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Staggered vs. Eclipsed Conformation
HH
H
H
H H
H
HH
H
HH
+3.0 kcal/mol
staggeredeclipsed
• Hyperconjugation C-HC-H• Newman projections help visualize interactions
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Gauche interactions
CH3
H H
CH3
HH
+0.9 kcal/mol
anti gauche
H
H CH3
CH3
HH
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Cyclohexane Chair Conformations
H
H3C
H
H3C
CH3
H
H
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
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Syn-pentane Interaction
• Syn-pentane > 3.7 kcal /mol
• 1,3-diaxial groups generate a syn pentane interaction H
H
H
H
H
H
H's cause steric strain
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A1,3 Strain
H RR"R'
12
3
HHH
H
HHH
H
• 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
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Template Projection of Amino Acids
NH
H
O
NH
H
O
NH R1
H
R2
O
R1 R3
NH H
H
HR1 O
R2
• 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!!!!
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Protein Folding: Hydrogen Bonds
O H N
R
RR
R
:
• 1-4 kcal/mol• Directionality is
important• N-H-----O=C• Stabilize -helices,
-sheets and turns
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Protein Folding: -helix
• stabilized by hydrogen bonding• 3.6 amino acids per turn
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Protein Folding: -sheet
• NH’s of one strand H-bond to C=O of next strand• R groups alternate on opposite sides of the plane
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Protein Folding: -turn
• C=O and N-H are 10 atoms apart
• Changes the direction of the main chain
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Protein Folding: Electrostatic Interactions
O
O-R
N
NR
H
NH2H• Between oppositely
charged amino acids• Most important in
the interior of the protein
• Neutralizes charges
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Protein Folding:
Hydrophobic Interactions:
• Hydrophobic amino acids pack into the interior of the protein
• Folding increases the disorder of the solvent
• Positive H is overcome by positive S
Disulfide Bonds:
• Dihedral angle 90o
• ns donates into *S-R
• Two Cys oxidized to form a disulfide bond
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Edman Degradation
Carol Fang
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N
S
NH
Ph
O
H2N
H
R1 N
S
NH
Ph
R1
O
+ NH2
O
R2
Nucleophilic Amine (primary and secondary)
E and Nu are 5 atoms apartRotatable bond
Thiazolinone DerivativeKinetic product New N-terminal
S
C
N
Ph
H2N
HN
R1
O R2
O
N
HN
R1
O R2
O
N
S
Ph
A H
H
H
B
N
HN
R1
O R2
O
NH
S
Ph
H
1
2
34
5NN
H
S
Ph
H
1
23
4
HN
R1
5O
H A
NNH
S
Ph
H
1
23
4
HN
R1
5O
H
N
S
NH
Ph
O
HN
H
H
H A
R1 N
S
NH
Ph
O
H2N
H
R1
B B
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Enol Formation
Potential racemization
PTH, to be detected by HPLCThermodynamic Product
Pre-note
N
S
NH
Ph
R1
O H+
N
S
NH
Ph
R1
OH
N
S
NH
Ph
R1
OH
H+
N
S
NH
Ph
R1
O
+
N
S
NH
Ph
R1
O
H2O
N
S
NH
Ph
R1
O
OH
HB
H A
NH
NO
SR1
Ph
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Frame of Reaction
H2N
HN
R1
O R2
O
H3N
R2
O
NH
NO
S
R1
Ph
+
When racemization is taken care of
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Brain teasers:
a) a peptide is not reactive to Edman Degradation
b) After a round of Edman degradation, only one fragment is obtained c) After a round of Edman degradation, two PTH products are obtained
d) Bicyclic PTH product from Edman Degradation
c) Special case: Lysine
No nucleophilic amine Cyclic peptide D05
Breaking the peptide bond does not break the molecule
Presence of Nu amine; Cyclic D10, D12
2 Nu amines at both ends / 1 PTH end and 1 Nu amine endD10, D12
A ring before Edman degradationD02, D04
A more protonated amine D09
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Cyanogen Bromide Cleavage
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NH
HN
O
S
Me
Br C N
NH
HN
O
S
MeC
N
Br
NH
O
NH
S
MeCN
- MeSCNO
HNNH
H2O
O
ONH
NH
H A
HHBH
O
ONH2
NH
HBH
O
NH
O
+ H2N
1
34
5
2
12
34
5
-Br
Nucleophilic SNu and E 5 atoms apartRotatable bond
Met (C) N cleaved
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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?
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H3N
NH
HN
NH
NH2
O
OS
NN
O
O
O H
H
HN
O
OH
NHO
O
OH
NH2
How this reacts with CNBr? (2004 Exam 1)
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Why S / C=O combo can be so different in these two
reactions?
S
O
S
O
Edman Degradation CNBr Cleavage
C=S bond, S is Nucleophilic3 C-S bond, S has an extra Covalent bond; adjacent C is ready for SN2
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Peptide Syntheses
Walter Kowtoniuk
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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
HN C
R
O
O
H
N
C
N
R2
NH2
HN C
R
NH
O
R2
NH
CNH
O
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Amide Bond Synthesis
HN C
R
O
O
H
N
C
N
HA
B-
HN C
R
O
O
N
C
N
H
HN C
R
O
O
NC
N
H
HA
HN C
R
O
O
NC
N
H
H
R2
NH2
HN C
R
O
O
NH
CNH
R2N
HH
B-
HN C
R
O
O
NC
N
H
H
R2
H2N
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Amide Bond Synthesis
HN C
R
O
O
NH
CNH
R2N
HH
B-
HN C
R
O
R2NH
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Protecting GroupsWhy do we need them?
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Protecting Groups
Lecture Notes pg33
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Protecting Groups
• t-Boc Synthesis
• t-Boc Deprotection
H3C
H3C
H3C
OH
O
Cl
Cl
H3CH3C
CH3
O
O
ClR
NH3C
H3C
CH3
O
O
H HH2N
R
B-
RNHH3C
H3C
CH3
O
O
RNHH3C
H3C
CH3
O
OTFA
H+
RNHH3C
H3C
CH3
O
OH
RNHCH3
H2C
CH3
O
OH
H+
RH2N
CO2
(gas)
HE1
CH3H2C
CH3
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Protecting Groups
• Cbz follows the same mechanism as shown for t-Boc
Cl Cl
O OH
OCl
O
ONH
O
R
or
OO
O
R
ONH
O
R TFA
H2C
NH2R
CO2 H3CEt3Si-H
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Protecting Groups
• Ts synthesis
• Ts Deprotection
H2NC
OH
O
HNNH2
NH CH3S
O
Cl
O
H2NC
OH
O
HNN
NH2
CH3
S
O
OH
B-
H2NC
OH
O
HNN
NH2
CH3
S
O
O
H2NC
OH
O
HNN
NH2
CH3
S
O
OH
F
HFF-
H2NC
OH
O
HNNH
NH2CH3S
O
O
F
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Protecting Groups
• DNP synthesis
• DNP deprotection
H2N
OHO
HNN
NO2
NO2
HS H2N
OHO
HNN
NO2
NO2S
H2N
OHO
HNN
H
B-
H2N
OHO
HNN Cl
O2N NO2
H2N
OHO
HNN
NO2
NO2Cl
H2N
OHO
HNN
NO2
NO2
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C to N Directionalitywhy not N to C?
NH
O
R2
N
O O
R1
HN
NH
Cy
Cy
B-
H
NH
O
R2
N
O
R1
H
B-
NH
O
R2
N
O
R1NH
O
R2
N
O
R1
H
HA
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Solid Phase Peptide Synthesis
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Solid Phase Peptide Synthesis
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Peptide Fragment Coupling
• Thioester
• True coupling
O
NH
O
RO
NH
O
RSH
O
NH
O
RSH2N
SHO
HN
O
R
N
O
NH
O
R
HH
H+
O
NH
O
RHN
HS
O
NH
O
RS
O
HN
O
R
NHH
SH
H
B-
O
NH
O
R
SO
HN
O
RN
H H
H+
B-
O
NH
O
RO
NH
O
RS
Br
B-
H
O
NH
O
RS
O
NH
O
RS
O
NH
O
R
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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
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Translation
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Biological Carbonyl Activation
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Biological Carbonyl Activation
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Biological Carbonyl Activation
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Ribosome
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Role of A2486