Download Amino Acids, Peptides and Proteins

Amino Acids, Peptides and Proteins
1. α-Amino Acids
R H
COOH
O
H2N
H2N
H
R
OH
(S) or L amino acids
a) dipolar nature (isoelectric points)
b) synthesis (racemic)
i) from α-bromoacids
ii) Strecker synthesis from aldehydes
iii) reductive amination of α-ketoacids
iv) amidomalonate synthesis
2. Peptides (up to 50 amino acids)
R H
N-terminal
H
O
N
OH
N
H2N
O
R" H
H R’
H
C-terminal
O
a) amino acid analysis
b) sequencing
i) Edman degradation (N-terminal)
ii) carboxypeptidase (C-terminal)
c) peptide synthesis (step-by-step and solid-phase)
3. Proteins (large peptides, occasionally with something
else attached)
a) structure (primary, secondary, tertiary and
quaternary)
b) classifications
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α-Amino Acids
O
H 2N
R = side chain
~ 500 known in nature
OH
20 in humans
H R
Examples:
10 of them essential
O
O
H2N
H2N
OH
H H
OH
H
valine
neutral, bulky,
hydrophobic
proline
2o cyclic, bending in
the peptide chain
O
O
O
H
OH
H
glycine
smallest, not chiral
H2N
O
H
N
H2N
OH
H
NH2
lysine
basic, nucleophilic,
used in catalysis
OH
CO2H
aspartic acid
acidic, carboxylate available,
used in catalysis
H 2N
OH
H
cysteine
crosslinking,
catalysis
O
H2 N
histidine
basic, catalytic side chain
OH
H
HN
N
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SH
Peptide Structure Determination
1. Amino acid analysis:
a) hydrolysis with HCl/H2O
b) column chromatography
c) detection with ninhydrin
O
H2N
O
OH
O
OH
O
RCOH
H R
N
OH
+
NaOH/H2O
CO2
O
O
O
(purple)
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Peptide Structure Determination
2. The Edman degradation:
a) treatment with phenyl isothiocyanate (Ph-N=C=S)
b) mild acid hydrolysis
c) the resulting phenylthiohydantoin is identified by
chromatography
C
H2N
N
NH
SH
N
O
HN
Peptide
H R
Ph
Ph
Ph
O
S
N
Ph
O
NH
H
Peptide
Next cycle
N
O
HN
H
S
R
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OH
HN
NH
H
Ph
S
N
R
H+
Peptide
H R
S
HN
NH
R
Peptide
Peptide Structure Determination
3. C-terminal residue determination
a) enzyme (carboxypeptidase) used to hydrolyze one
amino acid at the C-terminus
b) identifaction of the amino acid
c) further hydrolysis
4. Putting together peptide (protein) structure from
fragments
Asp-Arg-Val
Arg-Val-Tyr
Val-Tyr-Ile
Ile-His-Pro
Pro-Phe
Asp-Arg-Val-Tyr-Ile-His-Pro-Phe
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(angiotensin II)
(Di)Peptide Synthesis
a) protect the amino group of amino acid 1
O
O
O
H2N
O
O
O
H R1
O
BOC N
HN
OH
H
O
OH
OH
H R1
H R1
b) protect the carboxyl group of amino acid 2
H2N
O
O
O
PhCH2OH/HCl
OH
H2N
H2N
OCH 2Ph
H R2
H R2
H R2
OR
c) couple the two amino acids using DCC
H
O
O
BOC N
+
OH
H2N
H
DCC
OCH2Ph
H R2
BOC N
H R2
H R1
O
OCH 2Ph
N
H R1
H
O
d) remove the protective groups
H
O
BOC N
H R1
O
H R2
OCH 2Ph CF COOH H2N
3
N
H
OCH2Ph
N
H R1
O
H R2
H
O
H2/Pd or NaOH
O
H2N
H R1
H R2
N
OH
O
H
Note: side-groups of same amino acids require extra protectiondeprotection!
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Peptide Synthesis
Solid-Phase Technique
1. BOC-protected amino acid is linked to the polystyrene
beads (SN2 ester bond formation)
2. The beads are washed (to remove excess reagents)
and treated with CF3COOH to remove BOC group
3. A second BOC-protected amino acid is coupled to the
first one using DCC. The beads are washed.
4. The cycle of deprotection, coupling and washing is
repeated asmany times as desired to add amino
acid units to the growing chain.
5. After the desired peptide has been made, the
treatment with anhydrous HF removes the final
BOC group and cleaves the ester bond to the
polymer
6. The peptide is purified
The yields of the reactions are critical!
For a dodecapeptide (20 aa) it requires 40 chemical steps (not counting
special treatment for some side groups).
If the yield is 90% per step the overall yield is only (0.940) 1.5%
If the yield is 99% per step the overall yield is only (0.9940) 67%
If the yield is 99.9% per step the overall yield is only (0.99940) 96%
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Protein Structure and Function
1. Primary structure
sequence of amino acids
2. Secondary structure
three-dimensional structure of segments
α-helical, β-pleated sheets)
(α
3. Tertiary structure
three-dimensional arrangements of segments
4. Quaternary structure
three-dimensional shape of several proteins in a
protein complex
-------------------------------------------------------------------------Protein denaturation
--------------------------------------------------------------------------Fibrous proteins
(insoluble)
Globular proteins
(soluble)
Simple proteins
Conjugated proteins
(carbohydrates, nucleic acids)
Enzymes:
holoenzymes = apoenzyme + cofactor (vitamin)
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