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N
H
H
N
SO2
NH
C
O
O
Alitame (2000)
HO2C
CH3
Saccharin (300)
NH
NH
CO2CH3
H
N
SO2
- +
NK
SO3H
Acesulfame-K (200)
HOCH2
Cl
ClCH2
O
OH
Neotame (8000)
CH2Cl
HO
OH
OH
Sucralose (500)
Mastercourse Sweeteners January 18, 2006
Cyclamate (30)
HO2C
NH
NH2
CO2CH3
Aspartame (200)
NH2
H
N
O
O
L--Asp -L-PheOMe
Market situation
- dairy
table tops
- confectionary
pharmaceutical products
HSC products
t 1/2 = 260
250
t1/2 days (25C)
t 1/2 = 242
200
150
t 1/2 = 116
100
t 1/2 = 82
t 1/2 = 86
50
t 1/2 = 12
4
pH
R1
R1
R2
OH
H2 N
OH
H2 N
-H2O
Requirements
Cheap protective groups to avoid side-reactions of
amino groups (and sometimes carboxy groups)
amino acid side chains if required
Cheap activation of one carbonyl function
Mastercourse Sweeteners January 18, 2006
H
N
H2 N
OH
R2
O
H2N
OH
R1
OH
R2
Protection
O
protection HN
Protection
OH
R1
Activation
O
protection HN
O
H2N
X
R1
O
H2N
NH
R1
R2
OH
O
protection
R2
10
L-Asp
O
HCO2H
Ac2O
HO2C
NH
O
N
H
HCl/MeOH
CO2H
HO2C
H2O
N
H
NH3Cl
CO2H
O
H
For--Asp-Phe (~ 80%)
NH
O
toluene/
acetic acid
O
H
HO2C
For-L-Asp=O
H
O
OH
NH2
L-Phe
NH
HO2C
N
H
NH3Cl
O
CO2CH3
CH3O2C
N
H
NH3Cl
CO2H
-APM.HCl (~ 50%)
O
N
H
CO2H
O
For--Asp-Phe (~ 20%)
CH3O2C
4 -isomers
N
H
NH3Cl
CO2CH3
L-Asp
OCH3
NH
O
HCl/CH3OH
OH
NH2
NH2
Z-L-Asp
CO2H
HO2C
11
DL-PheOMe
DL-Phe
hydrolysis &
racemization
O
Thermolysin
H2O, pH = 6-7
OCH3
NH2
O
HO2C
NH
N
H
D-PheOMe
CO2CH3
D-PheOMe
Z-APM . D-PheOMe
Advantages:
Z-APM
Hydrogenolysis HO C
2
NH2
N
H
CO2CH3
APM
D-PheOMe
H2N
12
H2N
Thermolysin
Z
H
N
HO
N
H
O
O
O
HO
Z-APM.D-PheOMe
OH
HN
Z-L-Asp
Regioselective
Stereoselective
Thermodynamic
R1
R1
XH
R2-NH2
R2-NH3
ENZYME
H2O
O
R1
NHR2
Aminolysis
R1
O
OH
Hydrolysis
13
R1
O
R1
OH
+ H2N R2
pKa1
14
Enzyme
R1
N
H
R2
+ H2O
pKa2
O
R1
H3N R2
O
R
pKa = 3
OH
R'
NH3
pKa = 8
R'
NH2
Optimal reaction pH
around (pKa1 + pKa2)/2
100
Dipeptide (%)
Percentage
80
60
40
Equilibrium
20
Time
0
1
pH
10
HSC case
O
HO
OH
O
HN
Thermolysin
+
O
H 2N
Z-L-Asp
L-PheOMe
H
N
HO
O
N
H
H 2N
D-PheOMe
Z-APM
O
HO
OH
O
HN
Z-L-Asp
Thermolysin
H2N
L-PheOMe
H
N
HO
O
+
N
H
H2N
D-PheOMe
Z-APM
H
N
O
N
H
Z
NH3+ -O
H
N
O
N
H
O
O
O
Z-APM.D-PheOMe
pKa of -COOH of Z-Asp = 3
pKa of amino group of L-PheOMe = 7
equilibrium unfavourable (< 5% to Z-APM)
But: precipitation occurs of Z-APM.D-PheOMe complex (very low solubility)
enzymatic equilibrium pulled to synthetic side
conversion to Z-APM > 90%
L-Phe instead of L-PheOMe: pKa = 9 and no precipitation impossible
O
NH3+
Z-APM.D-PheOMe
OH
+ H2 N R 2
Enzyme
R1
N
H
R2
+ H 2O
Disadvantages
Usually not possible
Effective substrate concentration low
large enzyme amount required
17
O
R1
HX
R1
R2 NH2 enzyme
18
R1
enzyme
N
H
R2
H2O
X = OR (esters)
or
X = NHR (amides)
enzyme
O
Dipeptide (%)
R1
Kinetic
Equilibrium
Thermodynamic
Mastercourse Sweeteners January 18, 2006
Time
19
Disadvantages
Reaction to be stopped at right time
Yields on amino compound < 90%
Always by-product (hydrolysed acyl comp.)
low yield on acyl component
DSP more difficult
O
HO
OCH3
20
HSC
NutraSweet
Raw materials
flexibility in L or DL-Phe
(even in L or DL-Asp)
Protective group
/ ratio
100:0
80:20
Recycles
Source:
Molecular weight:
Amino acids:
Metal ions present:
pH optimum:
Temp. optimum:
21
Bacillus thermoproteolyticus
34,333 Da
316
1 Zn2+ (activity), 4 Ca2+ (stability)
8.0
70C
Ca4
Ca2
Ca1
Ca3
Zn
Lys 316
Ile 1
22
100
50
23
4
Relative Activity (%)
pH
11
3
2
1
0
30
50
70
Temperature (C)
90
10
8
V x 105 (Mmin-1)
T50 (C)
85
75
65
0.1
1
10
100
[CaCl2] (mM)
6
4
2
0
0
2
3 4
[NaCl] (M)
24
25
26
DL-Phe
Methanol
Esterification
Purification
Sieving
HCl
Z-Asp
Crystallization
Condensation
Thermolysin
Hydrogen
Drying
Mixing
Packaging
Hydrogenolysis
Catalyst
Aspartame
Mastercourse Sweeteners January 18, 2006
27
aspartame
synthesis
aspartame
purification
drying
Packaging of
aspartame
aspartame
crystallization
sieving
warehouse
temperature and
humidity
distribution
28
N
H
SCH3
RNA
NH polypeptide
RNA
NH polypeptide
H2N
MAP
29
Example:
H
O
N
H
H
N
N
H
CH3
PDF
pH 7.2
96% conversion
H
N
H2N
O
O
N
H
CH3
For-Leu-Tle-NHMe
H-Leu-Tle-NHMe
PDF efficient enzyme for enzymatic N-Formyl removal from di- and oligopeptides
Highly L-specific for N-terminal residue: effective and versatile d.e. upgrade
For--Asp-PheOMe is deformylated, For--Asp-PheOMe not at all !
improved chemical Formyl process for Aspartame
Mastercourse Sweeteners January 18, 2006
30
L-Asp
O
HCO2H
Ac2O
HO2C
NH
O
N
H
CO2CH3
-O C
2
O
H
NH3
For--Asp-Phe (~ 80%)
NH
H
O
OCH3
NH2
L-PheOMe
pH = 5.6
O
NH
HO2C
N
H
CO2CH3
CO2CH3
O
H
N
H
toluene/
acetic acid
HO2C
NH
O
N
H
CO2CH3
For--Asp-Phe (~ 20%)
Compared to NutraSweet process: APM yield much higher, much smaller L-Asp/L-Phe
recycles and no neutralization crystallization
Proof-of-principle delivered
Mastercourse Sweeteners January 18, 2006