台灣大學開放式課程

有機化學乙
蔡蘊明 教授

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Chapter 24 Amino acids and proteins

※ Introduction

Proteins
polyamides with α-amino acids as monomer
(~20 different α-amino acids)

C N
O H
amide linkage

Primary structure
the sequence of α-amino acids
further folding  secondary and tertiary structures
 Most natural α-amino acids are L form

COOH cf.
CHO
H2N H HO H
R CH2OH
L-α-amino acid L-glyceraldehyde

Exception:
CH2COOH
Glycine (甘胺酸)
NH2
20 α-Amino acids for protein synthesis
8 α-amino acids are essential ─ acquired from diet

Five sub-groups
R: neutral
H2N COOH H2N COOH H2N COOH N COOH
H
glycine alanine valine proline

H2N COOH H2N COOH H2N COOH

leucine isoleucine phenylalanine
(F)

NH
CONH2
CONH2
H2N COOH H2N COOH H2N COOH
tryptophan asparagine glutamine
(W) (N) (Q)
 OH
-OH
OH * OH

H2N COOH H2N COOH H2N COOH

serine threonine tyrosine
(Y)
-S SH
S

H2N COOH H2N COOH

cysteine methionine

COOH
-COOH COOH

H2N COOH H2N COOH

aspartic acid glutamic acid
(D) (E)

basic N
H
N N NH2
NH2
H
NH
H2N COOH H2N COOH H2N COOH
histidine arginine lysine
(R) (K)
※ The dipolar ion structure

H O
R C C O Amphoteric – both an acid and base
NH3

H O Ka1 H O Ka2 H O
R C C OH R C C O R C C O
NH3 NH3 NH2

+ H+ + H+
P Z N
(positive) (neutral) (negative)
[Z][H+] [N][H+]
Ka1 = Ka2 =
[P] [Z]
 [N][H+]
Ka2 = Titration curve
[Z]
pH
When [Z] = [N]
 Ka2 = [H+]
 pKa2 = pH

[Z][H+]
Ka1 =
[P] pI
When [Z] = [P]
 Ka1 = [H+]
 pKa1 = pH

0.5 1.0 1.5

equivalent of base (HO−)
★ Isoelectric point
H O Ka1 H O Ka2 H O
R C C OH R C C O R C C O
NH3 NH3 NH2

+ + H+ + + H+
[Z][H ] [N][H ]
Ka1 = Ka2 =
[P] [Z]
[Z] + [N]
+
pKa1 = −log[H ] − log pKa2 = −log[H ] − log
[P] [Z]
[N]
pKa1 + pKa2 = 2pH − log
[P]
When [N] = [P]  pKa1 + pKa2 = 2pH
The net charge of amino acid is neutral
The isoelectric point
(the dipolar ion has the highest concentration)
pKa1 + pKa2
At this point pH = = pI
2
＊ different amino acid has different pI
 In general:
When R is neutral
O pKa1 ~2-3 O pKa2 ~9-10 O
H H H
R C C OH R C C O R C C O
NH3 NH3 NH2

+ H+ + H+

pI ~6
When R is acidic
+ H+
H O pKa1 = 2.09 H O pKa2 = 3.86 H O
−
HO2CH2C C C OH HO2CH2C C C O O2CH2C C C O
NH3 NH3 NH3
+
+ H
aspartic acid pKa3 = 9.82
pI = 2.87 H O
−
O2CH2C C C O
NH2
+ H+
 When R is basic
+ H+
H O pKa1 = 2.18 H O pKa2 = 8.95 H O
H2C(H2C)3 C C OH H2C(H2C)3 C C O H2C(H2C)3 C C O
H3N NH3 H3N NH3 H3N NH2

lysine + H+
pKa3 = 10.53

H O
H2C(H2C)3 C C O
pI = 9.74
H2N NH2
 Electrophoresis

lys gly asp

9.74 6.0 2.87

pI
※ Lab synthesis
◎ From potassium phthalimide
O O
CO2Et 82-85% CO2Et
NK + Br N
CO2Et CO2Et
O O

NaOEt
ClCH2CH2SCH3

CO2− −
O
H CO 2 E SCH3
NaOH
N C CH2CH2SCH3 N
CO2− E
O O
E = CO2Et
HCl 84-85%
CO2H
CH3SCH2CH2CHCO2−
+ CO2 +
NH3
CO2H
◎ Strecker synthesis
O H3O+
+ NH3 + HCN RCHCN RCHCO2−
R H ∆ NH3
NH2

Mechanism:

O O OH −H2O
+ NH3 RCHNH3 RCHNH2 RCH=NH
R H
CN−

RHC NH2 H+ RHC NH

CN CN
※ Resolution of D,L-amino acids

 An enzymatic method

D,L-amino acid D,L-N-acylamino acid

deacylase
(hydrolyze L form only)
L-amino acid + D-N-acylamino acid

separate
※ Biosynthesis of amino acids

 Reductive amination
NADH + H2O
+
HOOCCH2CH2CCO2H + H + NH3 HOOCCH2CH2CHCO2−
O NAD+ NH3
α-ketoglutaric acid L-glutamic acid
from carbohydrate

 Transamination
transaminase
HOOCCH2CH2CHCO2− + HOOCCH2CCO2H HOOCCH2CH2CCO2H
NH3 O O
oxaloacetic acid +
L-glutamic acid
HOOCCH2CHCO2H
NH3

L-aspartic acid
※ Determination of protein sequence

 Nomenclature
Proteins and polypeptides
polyamides with MW < 10000  polypeptides
polyamides with MW > 10000  proteins

dipeptide
O O
H2NCH2 C NHCH C OH

H3C CH3
amino end glycylvaline carboxy end
(N-terminal) (Gly•Val) (C-terminal)
 O O
H2NCH C NHCH2 C OH valylglycine
(Val•Gly)
H3C CH3

O O O
H2NCH2 C NHCH C NHCH C OH glycylvalylphenylalanine
H3C CH3
H2C (Gly•Val•Phe)

O O
H3N CH C NHCH C OCH3
H2C H2C
CO2−

aspartylphenylalanine methyl ester

(aspartam; NeutraSweet) – 一種代糖
 Detection
6 M HCl
protein individual amino acids
24 h

amino acid analyzer

(using cation-exchange resin)

SO3− Different amino acids are

RCHCOOH
absorbed differently
NH3
SO3−  separation
 O O
OH + H2O
O
OH − H2O
O O

ninhydrin indane-1,2,3-trione

−
O O
RCHCO2−
ninhydrin + N + RCHO
NH3 + CO2
O O
purple color
Mechanism:
H+
O O O H
RCHCO2− − H2O O
O + N CH
NH3
R
O O

O OH
O H2O H
NH2 + N C
R H
R
O O
O O O− O

N N + H+

O O O O
※ Sequence determination

Covalent structure of protein – primary structure

The amino acid sequence

Composition information exact number of amino acids

MW information can be determined

terminal residue analysis

 Sequence determination
partial hydrolysis
◎ Terminal residue analysis
 Sanger method
HCO3−
O O
O2N F + H3N CH C NHCH C
R R'
NO2

2,4-dinitrofluorobenzene O O
(DNFB) O2 N N CH C NHCH C
H R R'
NO2

H3O+

O
O2 N N CH C OH + H3NCHCO2−
H R R'
NO2 mixture of
amino acids
Separate and identified
 Edman method
HO−, pH 9
O O
N C S + H3N CH C NHCH C
R R'
phenyl isothiocyanate
S O O
N C N CH C NHCH C
H H R R'

H+

Ph O O
N rearrange H O
S + H3N C C
S N R N R R'
N
H H H
one amino
phenylthiohydantoin
acid less

partial hydrolysis occurs

can not repeat too many times
(60 AA is about the limit)
 Carboxypeptidase method
Hydrolyze C-terminal one by one
Follow the progress of growing amino acids
Becomes more complicate as the time goes

◎ Partial hydrolysis
Use dilute acids or enzyme
 cut protein into smaller fragments
 identify each fragment
Enzyme cuts at specific site
例 trypsin cleaves carboxyl side of arg, lys
with extra amino group

chymotrypsin cleaves carboxyl side of phe, tyr, trp

with aryl side chain
Examples:
A pentapeptide
Val2, Leu, His, Phe

Sanger method: N-terminal  Val

Carboxypeptidase: C-teminal  Leu

Val-(Val, His, Phe)-Leu

Partial hydrolysis
Val•His + His•Val + Val•Phe + Phe•Leu

Ans:
Val•His
His•Val Val•His•Val•Phe•Leu
Val•Phe
Phe•Leu
※ Protein and polypeptide synthesis

O 1) SOCl2 O O
H3C CHC O− H3C CHC NHCH2CO2− + H3C CHC NHCHCO2−
2) H2NCH2CO2− NH3 CH3
NH3 NH3

Ala AlaGly AlaAla

+ AlaAlaAla + AlaAlaGly + ..........

A mixture is obtained

Solution:
protection
◎ Protection
−
OH H
+ H2N-R O N R + Cl−
O Cl 25 oC
O O
benzyl chloroformate
AcOH
(cold) HBr H2/Pd

CH2 + CO2 CH3 + CO2

Br
+ H2N-R + H2N-R
 H
O O base O N R
+ H2N-R + OH
25 oC
O O

di-t-butyl carbonate
t-butyloxycarbonyl
(BOC group)

HCl or TFA
o
in acetic acid, 25 C

+ CO2 + H2N-R
◎ Activation of carboxyl group

O O O
1) Et3N
Z N CH C OH Z N CH C O C OEt
H R 2) O H R
a mixed anhydride
EtO Cl
ethyl chloroformate
O
H3N CH C O−
R'

O O
+ CO2
Z N CH C N CH C OH
H R H R' + C2H5OH
◎ Synthesis
O − O
O OH
H3C CHC O− + H3C CHC OH
Cl OCH2Ph 25 oC
NH3 NH
O (Z-Ala)
OBn

1) Et3N
2) O
Cl OEt
O
(H3C)2HCH2C CH C O− O O
O
NH3 H3C CHC O C OEt
H3C CHC NHCHCO2H
NH
NH O
O
OBn
OBn

Z-Ala-Leu H2/Pd
O
H3C CHC NHCHCO2− + CO2
NH3 + toluene

Ala-Leu
polystyrene ★ Merrifield method – polymer-bonded synthesis
O O
CH2Cl + HO C CHNH C O
R
base
O O
CH2O C CHNH C O
CF3CO2H/CH2Cl2 R

O
CH2O C CHNH2 O O
HO C CHNH C O
R
DCC R'

O O O
CH2O C CHNH C CHNH C O
CF3CO2H/CH2Cl2 R R'

repeat
O O O
CH2Br HO C CHNH C CHNH C CHNH etc.
HBr
R R' R"
CF3CO2H
Successful for the synthesis of ribonuclease
─ an 124 amino acid residues protein
─ overall yield: 17%
─ average yield for each step: 99% (in six weeks)
※ Protein structure
 Primary structure – the sequence

 Secondary structure: α-helix and β-sheet

H O H R H O
N C N C
N C
R H H O R H

transoid
 β structure
H O H R H O H R
N C N C
N C N C
R H H O R H H O
bad interaction
R H O H R H O H
if planar C N C N
C N C N
O H H R O H H R

R
H H
C N
O OR
exists as R C N H
H H N C
H N O
β-pleated sheet O N
C
O R
H
H
C H O
R N
C H
to avoid steric H
O
N C
H
C H OR
interaction N
H
α structure: helical

helical axis

O
3.6 amino acids per turn
H

N
Tertiary structure
overall three dimensional structure
determined by stability

Hydrophilic part sticking outside

Lipophilic part sticking inside
Disulfide bonding
Salt bridges