Industrial production of Aspartame
Mastercourse “Sweeteners”, Univ. of Amsterdam
January 18, 2006
Theo Sonke / Hans Schoemaker
DSM Research B.V., Geleen
Advanced Synthesis, Catalysis & Development
[email protected] / [email protected]
Mastercourse Sweeteners – January 18, 2006
Contents
• Introduction high-intensity sweeteners, Aspartame
• Chemical process
• Enzymatic process
• background of enzymatic peptide synthesis
• HSC plant in Geleen, The Netherlands
• DSM R&D results on improved “chemical” process
Mastercourse Sweeteners – January 18, 2006
1
Aspartame and other high-intensity sweeteners
O
HOOC
H2N
N
H
H
N
SO2
NH
C
S
O
O
O
Alitame (2000)
HO2C
CH3
O
Saccharin (300)
NH
NH
CO2CH3
H
N
SO2
- +
C
NK
SO3H
O
Acesulfame-K (200)
HOCH2
O
Cl
ClCH2
O
OH
Neotame (8000)
O
O
CH2Cl
HO
OH
OH
Sucralose (500)
Mastercourse Sweeteners – January 18, 2006
Cyclamate (30)
HO2C
NH
NH2
CO2CH3
Aspartame (200)
2
Aspartame - Holland Sweetener Company (HSC)
OH
NH2
H
N
O
O
•
•
•
•
O
O
L-α-Asp -L-PheOMe
150-200x sweeter than sucrose, other isomers: bitter, non-sweet
Splits into Asp, Phe and methanol in gastrointestinal tract
Use: approx. 70% in US, of which >70% in beverages
History:
1965 Discovered by G.D. Searle (Dr. Schlatter)
Searle/Monsanto further develop product
1971 Lab scale R&D starts at DSM Research
1975 Tosoh (Japan) starts R&D on Aspartame
1981 Definitive FDA approval; NutraSweet starts production
1985 HSC founded (50/50 joint venture DSM/Tosoh)
1988 Start of production in HSC plant Geleen
1992 NutraSweet US patent expiration
Mastercourse Sweeteners – January 18, 2006
3
Market situation
4
• Artificial sweetener, low calorie value; no bitter after-taste
• Consisting only of natural components
• Cost benefit: competitive with sugar
• Aspartame is applied in:
•
soft drinks/fruit juices
- dairy
•
table tops
- confectionary
•
pharmaceutical products
• Production site: Geleen (NL); Annual production: > 3000 mt/y
• Other Aspartame producers: NutraSweet (US, Korea, 6000 mt/y) and
Ajinomoto (JP, F, 6000 mt/y), various Chinese
• First commercial process (NS/Ajinomoto); chemical with Z-protection
Mastercourse Sweeteners – January 18, 2006
HSC products
•
Granular (Pearl 700)
for bulk application:
i.e. beverages
•
Fine granular (Powder 200)
for table tops
•
Powder (Fine Grade)
i.e. for pharmaceuticals
and chewing gum
Mastercourse Sweeteners – January 18, 2006
5
Aspartame: stability profile at 25°C
300
t 1/2 = 260
250
t1/2 days (25°C)
6
t 1/2 = 242
200
150
t 1/2 = 116
100
t 1/2 = 82
t 1/2 = 86
50
t 1/2 = 12
1
2
3
5
4
pH
Mastercourse Sweeteners – January 18, 2006
6
7
8
Industrial synthesis of Aspartame
7
•
Raw materials: L-phenylalanine (L-Phe), L-aspartic acid (L-Asp), methanol
•
1 specific peptide bond to be made; methyl ester on 1 specific position
Mastercourse Sweeteners – January 18, 2006
Industrial synthesis of dipeptides
R1
R1
R2
OH
H2 N
+
OH
H2 N
O
-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
O
H
N
H2 N
O
8
O
OH
R2
Peptide synthesis: basic concept
O
H2N
9
O
H2N
OH
R1
OH
R2
Protection
O
protection HN
Protection
OH
R1
Activation
O
protection HN
O
H2N
X
R1
Coupling & Deprotection
O
H2N
NH
R1
Mastercourse Sweeteners – January 18, 2006
R2
OH
O
protection
R2
NutraSweet/Ajinomoto “Formyl” process to APM
10
L-Asp
O
O
HCO2H
Ac2O
HO2C
O
NH
O
N
H
HCl/MeOH
CO2H
HO2C
H2O
N
H
NH3Cl
CO2H
O
H
O
For-α-Asp-Phe (~ 80%)
NH
O
toluene/
acetic acid
O
H
HO2C
O
For-L-Asp=O
H
O
OH
NH2
L-Phe
NH
HO2C
N
H
NH3Cl
O
CO2CH3
CH3O2C
N
H
NH3Cl
CO2H
α-APM.HCl (~ 50%)
O
N
H
CO2H
O
For-β-Asp-Phe (~ 20%)
CH3O2C
4 β-isomers
N
H
NH3Cl
CO2CH3
• Advantages: cheap protection and coupling
• Disadvantages: difficult deprotection (1-3 d, only 50% yield), large L-Asp/L-Phe recycles,
final neutralisation crystallization required
Mastercourse Sweeteners – January 18, 2006
DSM/Tosoh chemo-enzymatic process
L-Asp
OCH3
NH
O
HCl/CH3OH
OH
NH2
NH2
O
Z-L-Asp
O
O
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
O
O
O
Z-APM . D-PheOMe
• Advantages:
Z-APM
Hydrogenolysis HO C
2
NH2
N
H
CO2CH3
APM
DL-Phe can be used, 100% α-isomer formed, no recycles (only D-Phe
racemization), no neutralisation crystallization
• Disadvantage: less cheap Z-protection, enzyme required
Mastercourse Sweeteners – January 18, 2006
Biocatalytic key-step in HSC process
L-PheOMe
D-PheOMe
O
H2N
12
O
H2N
O
O
Thermolysin
Z
H
N
HO
N
H
O
O
O
O
HO
O
Z-APM.D-PheOMe
OH
O
HN
Z-L-Asp
Mastercourse Sweeteners – January 18, 2006
Z
•
Regioselective
•
Stereoselective
•
Precipitation with D-PheOMe: > 90% yield
Enzymatic peptide synthesis:
kinetic versus thermodynamic approach
Kinetic
Thermodynamic
O
O
R1
X
R1
XH
R2-NH2
R2-NH3
R
ENZYME
H2O
O
O
R1
NHR2
Aminolysis
Mastercourse Sweeteners – January 18, 2006
R1
O
OH
O
O
Hydrolysis
13
R1
O
Thermodynamically controlled peptide synthesis (1)
O
R1
OH
+ H2N R2
pKa1
14
O
Enzyme
R1
N
H
R2
+ H2O
pKa2
O
R1
O
H3N R2
pKa1 and pKa2 values are crucial
[R1-CO-NH-R2]
K=
[R1-COOH] [H2N-R2]
∆pKa should be as low as possible
∆pKa = 1-2: thermodynamic coupling possible
(if solubility of product much lower than substrates)
Mastercourse Sweeteners – January 18, 2006
Thermodynamically controlled peptide synthesis (2)
15
Influence of pH on % active reactants
O
R
O
pKa = 3
OH
R'
-
NH3
+
pKa = 8
R'
NH2
O
R
Optimal reaction pH
around (pKa1 + pKa2)/2
100
Dipeptide (%)
Percentage
80
60
40
Equilibrium
20
Time
0
1
2
3
4
5
6
7
pH
Mastercourse Sweeteners – January 18, 2006
8
9
10
Thermodynamically controlled peptide synthesis (3)
16
HSC case
O
HO
OH
O
HN
Z
Thermolysin
+
O
H 2N
Z-L-Asp
L-PheOMe
Z
H
N
HO
O
O
O
N
H
+
O
O
H 2N
O
O
D-PheOMe
Z-APM
O
HO
OH
O
HN
Z-L-Asp
+
Thermolysin
Z
O
H2N
L-PheOMe
O
Z
H
N
HO
O
•
•
•
•
O
+
N
H
O
O
H2N
O
O
D-PheOMe
O
Z-APM
Z
O
H
N
O
N
H
O
O
Z
NH3+ -O
H
N
O
N
H
O
O
O
Z-APM.D-PheOMe
pKa of α-COOH of Z-Asp = 3
pKa of amino group of L-PheOMe = 7
Æ equilibrium unfavourable (< 5% to Z-APM)
But: precipitation occurs of Z-APM.D-PheOMe complex (very low solubility)
Æ enzymatic equilibrium pulled to synthetic side
Æ conversion to Z-APM > 90%
L-Phe instead of L-PheOMe: pKa = 9 and no precipitation Æ impossible
O
NH3+
-
O
O
Mastercourse Sweeteners – January 18, 2006
O
Z-APM.D-PheOMe
Thermodynamically controlled peptide synthesis (4)
O
R1
OH
+ H2 N R 2
O
Enzyme
R1
N
H
R2
+ H 2O
Advantageous for yield:
• ∆pKa as low as possible, preferably < 2
• Substrate solubility as high as possible, product solubility as low as possible
• crystallization or complexation (as in HSC case)
Advantages
• No by-products
• Easy Down Stream Processing
(DSP)
Disadvantages
• Usually not possible
• Effective substrate concentration low
Î large enzyme amount required
In HSC process disadvantages have been eliminated:
• Possible due to effective complexation Î > 90% conversion
• Thermolysin extremely active enzyme, can be recycled
Mastercourse Sweeteners – January 18, 2006
17
Kinetically controlled peptide synthesis (1)
enzyme
O
R1
HX
R1
X
O
R2 NH2 enzyme
O
18
R1
enzyme
N
H
R2
H2O
X = OR (esters)
or
X = NHR (amides)
enzyme
O
Dipeptide (%)
R1
Æ Synthesis/hydrolysis ratio crucial factor
OH
Kinetic
Equilibrium
Thermodynamic
Mastercourse Sweeteners – January 18, 2006
Time
Kinetically controlled peptide synthesis (2)
Advantages
• Conversion often higher
• Reaction at higher pH (typically 7-9)
• much faster reaction
(more neutral nucleophile)
• 10-100 x less enzyme
• mostly possible
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
In HSC process this is disadvantageous:
• Preparation of (activated) Z-Asp-α-methyl ester difficult
O
HN
Z
and therefore expensive
• Thermolysin not suitable; other enzymes require organic solvent and give
lower conversions than with thermodynamic coupling
Mastercourse Sweeteners – January 18, 2006
OCH3
HSC vs. NutraSweet Process
20
HSC
NutraSweet
Raw materials
flexibility in L or DL-Phe
(even in L or DL-Asp)
L-Asp and L-Phe required
Protective group
less cheap Z-group
cheap formyl group
α/β ratio
100:0
80:20
Recycles
only Phe racemization
(in case of DL-Phe as
feedstock)
wrong α- and all β-products
Suggested further reading:
Oyama, K., in: Chirality in Industry, A.N. Collins (Ed.), John Wiley & Sons Ltd., 1992, 237-247.
Mastercourse Sweeteners – January 18, 2006
Thermolysin (1): general
•
•
•
•
•
•
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
70°C
Ca4
Ca2
Ca1
Ca3
Zn
Lys 316
Ile 1
Mastercourse Sweeteners – January 18, 2006
Thermolysin (2): 3D-structure of complex with Z-APM
Mastercourse Sweeteners – January 18, 2006
22
100
50
0
23
4
Relative Activity (%)
Relative Activity (%)
Thermolysin (3): influence of pH and T
5
7
pH
Mastercourse Sweeteners – January 18, 2006
9
11
3
2
1
0
30
50
70
Temperature (°C)
90
Thermolysin (4): influence of CaCl2 and NaCl
95
10
8
V x 105 (M·min-1)
T50 (°C)
85
75
65
0.1
1
10
100
[CaCl2] (mM)
6
4
2
0
0
1
2
3 4
[NaCl] (M)
Reactions with Thermolysin must contain NaCl and CaCl2
Thermolysin storage in presence of CaCl2
Mastercourse Sweeteners – January 18, 2006
24
Holland Sweetener Company (HSC) plant
Mastercourse Sweeteners – January 18, 2006
25
Block diagram HSC process
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
Flowchart HSC process
27
aspartame
synthesis
aspartame
purification
raw materials production raw materials & material aids
delivery
drying
Packaging of
aspartame
aspartame
crystallization
sieving
warehouse
temperature and
humidity
Mastercourse Sweeteners – January 18, 2006
distribution
Appendix: optimized Formyl process (DSM)
28
• N-Formyl protective group: very cheap to introduce, but chemical cleavage by
acidic hydrolysis leads to ester hydrolysis and partial peptide bond cleavage Î
mild enzymatic cleavage possible ?
• PDF (Peptide Deformylase) identified
• Role in nature:
SCH3
O
H
N
H
SCH3
RNA
NH polypeptide
PDF
O
RNA
NH polypeptide
H2N
MAP
H2N polypeptide CO2H
O
• Eubacterial protein synthesis always starts with N-formylated tRNAfMet initiator
• Smooth (over-)expression in E. coli; efficient purification by affinity chrom.
(Met-Lys-Sepharose, F-)
Mastercourse Sweeteners – January 18, 2006
Application of PDF in chemical peptide synthesis
29
Example:
H
O
N
H
H
N
O
O
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
(S,S)/(R,S) 94:6 (ee = 88%)
(S,S)/(R,S) 99.5:0.5 (ee = 99%)
• 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-α-Asp-PheOMe is deformylated, For-β-Asp-PheOMe not at all !
Æ improved “chemical” Formyl process for Aspartame
Mastercourse Sweeteners – January 18, 2006
Improved Aspartame process
30
L-Asp
O
HCO2H
Ac2O
HO2C
O
NH
O
N
H
O
CO2CH3
-O C
2
O
H
O
NH3
For-α-Asp-Phe (~ 80%)
NH
H
H
O
OCH3
NH2
L-PheOMe
pH = 5.6
O
NH
HO2C
N
H
CO2CH3
CO2CH3
O
H
O
N
H
α-APM (> 90%, non-isolated
> 70% isolated, purity > 99%)
PDF
toluene/
acetic acid
O
+
HO2C
NH
O
N
H
CO2CH3
For-β-Asp-Phe (~ 20%)
Process combines best of both processes:
•
No Z-protection needed as in HSC process
•
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