CM3004 Peptides
Peptides:
Amino Acid polymers linked by amide/peptide bonds
Amino Acids – residues, e.g Dipeptides
CH3
H3N
CH2OH
C
H
CO2
H3N
Alanine
C
H
C
CH2OH
H
N
C
H
CO2
CH2OH O
C
H
C
H-Ala-Ser-OH
N-terminal
Alanylserine
H3N
CO2
Serine
CH3 O
H3N
C
H
C-terminal
CH3
H
N
Serylalanine
C
H
CO2
H-Ser-Ala-OH
N-terminal
Convention: N-terminal amino acid written on left
C-terminal
Strategy for Polypeptide Synthesis
R2
R1
P
P
H
N
H
N
C
H
CO2H
+ H2N
R1
O
R2
C
H
C
H
N
C
H
CO2P
selective
deprotection
C
H
CO2P
P
H
N
R1
O
C
H
C
R2
H
N
C
H
CO2H
R3
H2N
P
H
N
R1
O
C
H
C
H
N
R2
O
C
H
C
C
H
CO2P
R3
H
N
C
H
CO2P
selective deprotection
P
H
N
R1
O
C
H
C
H
N
R2
O
C
H
C
R3
H
N
C
H
CO2H
Sequence of protection, coupling, selective deprotection, coupling etc. is very tedious!
Synthesis of Peptides
Selective Coupling
Ph
O
O
PhH2C
O
C
H
N
C
H
+
CO2H
H2N
Cbz-L-Val-OH
C
H
C
OtBu
H-L-Phe-OtBu
N-Terminal Protected
C-Terminal Protected
Protecting groups must be: (1) stable to amide formation conditions
(2) removable without cleaving amide bond
Coupling agent used: Dicyclohexylcarbodiimide (DCC)
N
C
N
DCC
Ph
O
PhH2C
O
C
O
H
N
C
H
C
+
O
H
N
C
H
N
Dicyclohexylurea
H
N
C
H
CO2tBu
Ph
O
PhH2C
O
O
H
N
C
C
H
C
H
N
C
H
CO2H
C
H
HBr
CO2tBu
AcOH
Ph
O
H2N
C
H
Br
H
N
C
H-Val-Phe-OH
Mechanism:
R
R
N
C
R1CO
+
N
N
2H
C
N
R
R
H
O
O
H
R1
R
N
C
N
H
R
O
R2
O
R
H2N
1
C
H
CO2tBu
O-Acyl isourea
activated intermediate
R2
O
R1
C
H
N
C
H
O
CO2tBu
+
R
H
N
C
H
N
R
Peptide bond- normal amide bond. There is restricted rotation due to a certain degree of
double bond character due to resonance delocalization.
Restricted rotation
H
N
H
N
CO2
H3N
H3N
O
O
Also N less basic/nucleophilic
than in free amine
Peptide Backbone Reactions
-
Reactions of polyamide chain e.g. Racemisation – Azlactone formation
Peptide side chain reactions e.g. CO2H, NH2 – usual reactivity
When peptides contains cystine residues
C
HC
O
H2
C
SH
HS
O
H2
C
NH
C
Mild oxidation
CH
NH
Reduction
(Reversible)
Thiols
C
HC
O
H2
C
S
S
NH
O
H2
C
C
CH
NH
Disulfide
Cystine Disulf ide
CO2
Can link 2 peptide chains or form a loop in a chain:
SH HS
S
S
Important in Protein Structure
Peptide Analysis
(i)
Need to know which amino acid is present, how much of each and the sequence
Amino Acid analysis: Hydrolyse the peptide – gives an amino acid mixture
which can be passed through an ion exchange column. Use pH controlled
buffers as the eluent. Each amino acid has a fixed elution rate.
Abs
Thr
Ala
Asp
Glu
Pro
Identifies AA present
t
Very sensitive - Detects down to 10-9 mol A.A.
Proline - Different adduct, yellow
- V. weak and broad peak
Proline is a 2o amino acid and reacts with only one molecule of ninhydrin to f orm the f ollowing
products:
O
O
N
O
Yellow
N
CH
O
Very weak broad band
CH
Detection - Reaction with Ninhydrin
O
O
OH
O
OH
O
O
R
Exists as hydrate
H2N
OH
O
O
R
CH + CO2
+
N
O
Only N remains f rom AA
Get same product f rom all AA
except Proline
Mixture
Ion-exchange column
AA
-
UV/vis detector
Ninhydrin
-
O
Purple
C
H
CO2H
Mechanism
O
O
OH
O
OH
O
O
R
H2N
CO2H
C
H
O
O
R
N
N
C
R
O
C
C
O
H
H
O
O
H+
+ CO2
O
O
NH2
Imine hydrolysis
N
+ RCHO
CHR
H
O
O
O
OH
O
O
O
N
N
-
H
O
Purple colour
O
O
Exists mainly as enol due to
extended conjugation
(ii) Sanger Method
- Identifies N-terminal amino acid of a polypeptide
NO2
O2N
2,4-Dinitrofluorobenzene
- susceptible to nucleophilic substitution
with strong nucleophiles e.g amines
F
R1
H2N C
H
NO2
H
N
O 2N
R1
O
C
H
C
H
N
R2
O
C
H
C
H
N
R3
O
C
H
C
Substitution at N-terminal only
NO2
Hydrolysis
O 2N
H
N
R1
O
C
H
C
OH
+ Amino acid mixture
Only the N terminal amino acid is labelled
with dinitrophenyl group (DNP)
-
Identitf y labelled AA by HPLC or other techniques
(ii)
Edman Degradation
Again from N-terminal but more usef ul as it can be repeated sequentially
- peptide sequence from N-terminal
Uses phenyl isothiocyanate PhN=C=S
S
H+
H
N
C
H2N
N
Rest of peptide
O
Ph
O
O
NH
PhHN
Ph
N
H
O
O
S
N
S
N
H
N
H
Ph
NH
S
S
PhHN
N
H
Phenylthiohydantoin
Identify by chromatography
N
H
+ Shorter Peptide Sequence
Repeat Edman Sequence – 2nd AA etc. This way we can sequence the peptide from
N-terminal
(iv) Analysis from C-terminal
- Enzyme carboxypeptidase cleaves from C-terminal
P
H
N
R1
O
C
H
C
H
N
R2
O
C
H
C
R3
H
N
C
H
CO2H
Enzyme cleaves here
P
H
N
R1
O
C
H
C
H
N
R2
O
C
H
C
R3
H3N
OH
C
H
CO2
Enzyme cleaves here next
Measure the rate of appearance of amino acid in solution - information
on peptide sequence
(v) Proteases - Specific cleavage at certain residues
E.g. Serine Proteases - Breaks peptides into smaller fragments
(vi) Selective cleavage at methionine using cyanogen bromide BrCN
O
H
N
Methionine
residue
H
C
C
H
N
R2
O
C
H
C
H
N
BrCN
O
O
SCH3
+ CH3SCN +
HN
R2
O
C
H
C
Homoserine lactone