Download CM3004 Amino Acids

CM3004 Amino Acids
Proteins:
- Greek word “proteios” meaning first, are primary constituents of
living matter
- Have high molecular weights
- Biological polymers
- Long chains of amino acids bonded to each other by amide bonds
(peptide bonds)
Amide bond – Between carboxylic acid group of one amino acid and the
amino group in another amino acid.
Portion of a protein molecule
H
N
R1
O
C
H
C
H
N
R2
O
C
H
C
H
N
R3
O
C
H
C
Amide bond
CH3
H2N
C
H
CO2H
Alanine
-amino acid
Primary structure: Sequence of amino acids
along protein chain
Polypeptide: Chains of <50 amino acids
Proteins: Structural e.g Keratin
Hormonal e.g. Insulin
Enzymes – Biological catalysts
Proteins – Linear polyamides
Building blocks – 20 amino acids
- Amino acids
L- Amino acids S configuration
H
H2N
R

CO2H
Stereogenic centre (except glycine)
All are primary amines except proline
Isoleucine + Threonine - 2nd stereogenic centre (single diastereomers in nature)
H3C
H
H
H3N
H3C
Et
CO2
Isoleucine
H
H3N
H
OH
CO2
Threonine
10 Essential - Isoleucine, Leucine, Methionine, Phenylalanine, Threonine, Tryptophan,
Valine, Arginine, Histidine, Lysine
Amino acids are difunctional molecules: Acid/Base in the same molecule.
Therefore we can have intramolecular proton transfer – zwitterion ion.
Predominant form:
R
H3N
C
H
CO2
Evidence - Physical Properties
Soluble in H2O
Crystalline
Non-volatile
High mp
High dipole moment
Internal Salt
Amino Acids are amphoteric (can act as an acid/base)
Acid
H3N
C
H
R
R
R
CO2H
H3N
C
H
CO2
Base
Cation
H2N
C
H
CO2
Anion
Acidity/Basicity depndent on nature of R- classified as acidic, neutral or basic
Low pH: Acidic solution (source of H+). Amino acid protonated: exists as cation.
High pH: Basic solution. Amino acid deprotonated: exists as anion.
Isoelectric point: pH at which amino acid exists as neutral, dipolar zwitterion.
Balance between anionic and cationic forms.
Neutral amino acids: pH 5.0-6.5 Isoelectric point
Acidic amino acids: Low isoelectric point- suppresses dissociation of CO2H side chain
eg Aspartic acid: pH 3.0
Basic amino acids: High isoelectric point- suppresses protonation of extra amino group
eg Lysine: pH 9.7
Can use varying acidity/basicity to separate and characterize amino acid
mixture - Electrophoresis
Synthesis of Amino Acids
(i)
Amination (ammonolysis of -haloacids)
-Bromination of carboxylic acids:
Hell-Volhard-Zelinskii (HVZ) reaction
O
O
PBr 3
Br
OH
O
OH
Br2
Br
Br
+ HBr
Br
Acid bromide enol
H2O
O
O
NH3
OH
OH
Br
-Haloacid
NH 2
Valine
Disadvantage- Yields tend to be low as the N is still nucleophilic.
This leads to di- and tri-substituted amines as by-products.
Also get the racemic product
(ii)
Gabriel Synthesis
O
O
CO2 Et
KOH
NH
N
K
+
Br
CO2 Et
O
O
Phthalimide
O
O
CO2 Et
N
CO2 Et
1. NaOEt
CH 2CH2 SCH 3
CO2 Et
N
2. ClCH2CH2SCH3
SN2 substitution
O
CO2 Et
O
Hydrolysis
CO2H
CH 2CH2 SCH 3
+
H3 N
+
CO2
CO2
CO2H
Methionine
Advantages: Mono-substitution only at N and can use a range of
electrophiles.
Disadvantages: Racemic product
(iii)
Amidomalonic Ester Synthesis
CO2 Et
CO2 Et
H 3 C(H2 C) 3
O
N
CO2 Et
O
N
0-20 oC
HO
CO2 Et
H2SO4
H2
Ni
O
O
CO2 Et
Ph
CO 2Et
Cl
Ph
H2 N
N-protected reduces
nucleophilicity
N
H
CO 2Et
CO2 Et
Acidic proton
NaOEt
Br
O
HCl
CO2 Et
Leucine
R,S

Ph
N
H
CO2 Et
Can use a variety of electrophiles, however racemic product is formed.
Similar strategy to Gabriel above – protect N, activate -C to substitution
followed by hydrolysis and decarboxylation.
(iv)
Strecker Synthesis (1850)
CN
O
H3O+
NH3
Leucine
H
NH 2
HCN
R,S
-aminonitrile
Can use NH4Cl/NaCN in H2O
Mechanism:
O
O
H
NH3
OH
NH 3
H
H
NH 2
NH 2
H
CN
CN
H3O+
Leucine
NH 2
R,S
-aminonitrile
To obtain enantiopure amino acid
- Resolution techniques
- Natural sources (S series only)
Resolution of R,S Amino Acids
Amino acids produced by the methods described above are produced as
racemic forms, ie equal mixture of S and R products.
The exception to this is glycine which has no stereogenic centre.
To obtain the naturally occurring L-amino acid (S configuration), this
racemic mixture must be resolved into pure enantiomers, S and R.
(i)
Chemical Methods of Resolution
R amine
R acid + S acid
(racemic mixture)
R,R salt + S,R salt
(diastereomers)
separate by fractional
crystallisation
R acid
R,R salt
S,R salt
HCl
HCl
R amine.HCl
S acid
R amine.HCl
(ii) Enzymatic methods of resolution
Enzymes: biological catalysts. React selectively with one
enantiomer of R,S mixture
R
H
C
CO2H
(CH3CO) 2O
R
NH2
H
C
CO2H
HN
CH3
S,R (racemic mixture)
O
S,R (racemic mixture)
Carboxypeptidase
H2O
N-acetyl S amido acid selectively
hydrolysed by carboxypeptidase
leading to desired S amino acid.
Products easily separated by chemical means.
R
H
C
CO2H
NH2
S amino acid
+
R
H
C
CO2H
HN
CH3
O
R amido acid
Reactivity of Amino Acids
(i) Esterif ication
H3N
C
H
CO2
MeOH
HCl
Leucine
HO2C
H3N
C
H
H2N
Cl
C
H
CO2CH3
PhH2CO2C
CO2
Glutamic acid
PhCH2OH
TsOH
H3N
C
H
CO2CH2Ph
Note- side chain reaction also
Can remove esters by hydrolysis for benzyl esters - can also use hydrogenolysis
Pd/H2 (neutral conditions)
Often used as protecting group f or C-terminal
(ii) Acylation
O
R
H3N
C
H
Ac2O
or
PhCOCl
CO2
R1
C
R
H
N
R1 = CH3/Ph
C
H
CO2H
More common- carbamate/urethane formation
E.g.
O
PhH2C
O
C
R
Cl
+
H2N
C
H
Benzyl chloroformate
Benzyloxycarbonyl chloride
Maintain pH just above
pKa of N
O
R
(i) pH ~ 9
PhH2C
(ii) H+
O
CO2 Na
H
N
C
C
H
CO2H
Carbobenzoxy derivative (Cbz)
or N-Benzyloxycarbonyl
R
O
(H3C)3C
O
C
O
+
H2N
C
H
Maintain pH just above
pKa of N
2
t-Butyl di-carbonate
O
(i) pH ~ 9
(ii) H+
CO2 Na
(H3C)3C
O
C
R
H
N
C
H
N-t-Butoxycarbonyl
Boc derivative
CO2H
Urethane Protecting Groups
O
BOC
O
N
H
R
O
O
Cbz
N
H
R
N
H
R
O
Fmoc
O
O
O
R
O
C
H
N
R
1
H+
H2O
- Removes nucleophilicity of N
- Widely used as N-terminal protecting groups
- Can be easily cleaved under conditions which
do not cleave peptide bonds
R-OH + HO
NHR1
Decarboxylation
R1-NH2 + CO2
O
PhH2C
O
C
R
H
N
C
H
HBr
CO2H
PhCH2Br + CO2 +
AcOH
R
Carbobenzoxy derivative (Cbz)
or N-Benzyloxycarbonyl
H2N
C
H
CO2H
R
or
H2
PhCH3 + CO2 + H2N
Pd
C
H
CO2H
R
H3N
O
O
C
C
H
CO2
R
H
N
C
H
R
CO2H
TFA
H2N
C
H
CO2H
TFA (Trif luoroacetic acid) - strong acid
cleaves t-butyl esters
O
O
H
C
R
H
N
O
C
H
CO2H
+
HO
C
NH
Carbamic acid
- CO2
R
H 2N
C
H
CO2H
Racemisation
- Can occur via enolate formation
O
R1
OH
H
N
R1
X
H
H
N
X
R
R
AA - free or in peptide
Achiral
For amino acid racemisation
O
20 oC, pH 7 t1/2 ~ 200,000 yrs
100 oC
~ 600 days
o
200 C
~ 2 hours
Also- presence of strong base greatly
accelerates process
e.g. Serine in a peptide chain t1/2 pH, 25oC 6h
R1
H
N
X
R
H
Rate-depends on groups on N/C terminals and on side chain
Another mechanism with activated acyl derivatives - azlactone or oxazolone f ormation
O
O
H
N
X
H
R
X-good leaving group (necessary f or peptide f ormation)
May be in a peptide
O
X
O
O
O
R
N
N
H
H
R
H
B
O
O
OH
O
N
N
H
Achiral
R
R
O
O
O
N
R
H-X
N
H
R
Aromatic
C
H
X
O
Racemised or epimerised product
N.B. Acylating agents used in peptide synthesis must avoid azlactone formation
-Major advantage of urethane
Diketopiperazine Formation
On esterif ication amino acid esters are stored as HCl salt.
To isolate the free amino acid ester, dissolve in H2O, raise pH, and extract.
However can't store in this form as they are unstable and lose alcohols
-Diketopiperazines
R
H2N
R
C
H
- CH3OH
CO2CH3
O
H2N
H3CO
R
C
H3CO2C
C
H
C
H
H
C
C
NH
O
NH2
R
- CH3OH
Formation of dipeptide -slow
Cyclisation - fast
R
O
2 CH3OH
HN
+
NH
O
R
Ph
O
H2N
C
H
C
O
H
N
C
H
CO2CH3
HN
1-2 h
20 oC
- CH3OH
NH
O
Also with dipeptide esters
Ph