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
theo.sonke@dsm.com / hans.schoemaker@dsm.com

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

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Aspartame and other high-intensity sweeteners

O
HOOC
H2N

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)
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Cyclamate (30)

HO2C

NH
NH2

CO2CH3

Aspartame (200)

Aspartame - Holland Sweetener Company (HSC)

OH

NH2
H
N

O
O

L--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

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Market situation

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

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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

Aspartame: stability profile at 25C

300

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

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Industrial synthesis of Aspartame

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

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Industrial synthesis of dipeptides

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
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H
N

H2 N

OH
R2

Peptide synthesis: basic concept

O
H2N

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

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

Advantages: cheap protection and coupling

Disadvantages: difficult deprotection (1-3 d, only 50% yield), large L-Asp/L-Phe recycles,
final neutralisation crystallization required
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DSM/Tosoh chemo-enzymatic process

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

DL-Phe can be used, 100% -isomer formed, no recycles (only D-Phe

racemization), no neutralisation crystallization

Disadvantage: less cheap Z-protection, enzyme required
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Biocatalytic key-step in HSC process

L-PheOMe

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

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Regioselective

Stereoselective

Precipitation with D-PheOMe: > 90% yield

Enzymatic peptide synthesis:

kinetic versus thermodynamic approach
Kinetic

Thermodynamic

R1

R1
XH

R2-NH2

R2-NH3

ENZYME

H2O

O
R1

NHR2

Aminolysis

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R1

O
OH

Hydrolysis

13

R1

Thermodynamically controlled peptide synthesis (1)

O
R1

OH

+ H2N R2

pKa1

14

Enzyme
R1

N
H

R2

+ H2O

pKa2
O

R1

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)

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Thermodynamically controlled peptide synthesis (2)

15

Influence of pH on % active reactants

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

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10

Thermodynamically controlled peptide synthesis (3)

16

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+

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Z-APM.D-PheOMe

Thermodynamically controlled peptide synthesis (4)

O
R1

OH

+ H2 N R 2

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
large enzyme amount required

In HSC process disadvantages have been eliminated:

Possible due to effective complexation > 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

R2 NH2 enzyme

18

R1

enzyme

N
H

R2

H2O
X = OR (esters)
or
X = NHR (amides)

enzyme
O

Dipeptide (%)

R1

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--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

/ ratio

100:0

80:20

Recycles

only Phe racemization

(in case of DL-Phe as
feedstock)

wrong - and all -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
70C
Ca4
Ca2
Ca1

Ca3

Zn
Lys 316
Ile 1

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Thermolysin (2): 3D-structure of complex with Z-APM

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22

100

50

23

4
Relative Activity (%)

Relative Activity (%)

Thermolysin (3): influence of pH and T

pH

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11

3
2
1
0

30

50
70
Temperature (C)

90

Thermolysin (4): influence of CaCl2 and NaCl

95

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)

Reactions with Thermolysin must contain NaCl and CaCl2

Thermolysin storage in presence of CaCl2
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24

Holland Sweetener Company (HSC) plant

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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
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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

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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
mild enzymatic cleavage possible ?
PDF (Peptide Deformylase) identified
Role in nature:
SCH3
O
H

N
H

SCH3

RNA

NH polypeptide

PDF

RNA

NH polypeptide

H2N

MAP

H2N polypeptide CO2H

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-)
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Application of PDF in chemical peptide synthesis

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

(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--Asp-PheOMe is deformylated, For--Asp-PheOMe not at all !
improved chemical Formyl process for Aspartame
Mastercourse Sweeteners January 18, 2006

Improved Aspartame process

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

-APM (> 90%, non-isolated

> 70% isolated, purity > 99%)

PDF

toluene/
acetic acid

HO2C

NH

O
N
H

CO2CH3

For--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