amsterdam_for_website_uva.pdf

Mastercourse Sweeteners January 18, 2006

Industrial productionIndustrial production of of AspartameAspartameMastercourse Sweeteners, Univ. of Amsterdam

January 18, 2006

Theo Sonke / Hans Schoemaker

DSM Research B.V., Geleen

Advanced Synthesis, Catalysis & [email protected] / [email protected]


1ContentsContents

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


2Aspartame and other highAspartame and other high--intensity sweetenersintensity sweeteners

HOOC NH

HN

O

OS

H2N

Alitame (2000)

CN-K+SO2

O

O

CH3

Acesulfame-K (200)

OHOCH2

ClOH

OH

OO

CH2Cl

HO OH

ClCH2

Sucralose (500)

CNH

SO2

O

Saccharin (300)

HN

SO3H

Cyclamate (30)

Aspartame (200)

HO2C NHNH2

O

CO2CH3

Neotame (8000)

HO2C NHNH

O

CO2CH3


3Aspartame Aspartame -- Holland Sweetener Company (HSC)Holland Sweetener Company (HSC)

O

HN

O

OH

OO

NH2

LL----AspAsp--LL--PheOMePheOMe

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 product1971 Lab scale R&D starts at DSM Research1975 Tosoh (Japan) starts R&D on Aspartame1981 Definitive FDA approval; NutraSweet starts production1985 HSC founded (50/50 joint venture DSM/Tosoh)1988 Start of production in HSC plant Geleen1992 NutraSweet US patent expiration


4Market situationMarket 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


5HSC HSC productsproducts

Granular (Pearl 700)for bulk application:i.e. beverages

Fine granular (Powder 200)for table tops

Powder (Fine Grade)i.e. for pharmaceuticalsand chewing gum


6Aspartame: stability profile at 25Aspartame: stability profile at 25CC

1 2 3 4 5 6 7 8

300

250

200

150

100

50

pH

t

1

2

/

d

a

y

s

(

2

5

C

)

t /1 2 = 260

t /1 2 = 116

t /1 2 = 86

t /1 2 = 12

t /1 2 = 242

t /1 2 = 82


7IndustrialIndustrial synthesissynthesis of of AspartameAspartame

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


8

H2NOH

O

R1

H2NOH

O

R2

H2N

HN

O

R1

+ -H2O OH

R2

O

Industrial synthesis of Industrial synthesis of dipeptidesdipeptides

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


9Peptide synthesis: basic conceptPeptide synthesis: basic concept

O

OHH2N

R1

H2N NH

R1

R2

OHO

O

O

OHH2N

R2

O

OHHN

R1

O

XHN

O

protectionH2N

R2

protection

protection

R1

Protection

Protection

Activation

Coupling & Deprotection


10NutraSweetNutraSweet//AjinomotoAjinomoto FormylFormyl process to process to APMAPM

Advantages: cheap protection and coupling Disadvantages: difficult deprotection (1-3 d, only 50% yield), large L-Asp/L-Phe recycles,

final neutralisation crystallization required

CO2HNH

O

HO2CNH

HO

For--Asp-Phe (~ 80%)

H

NHO

OH

O

NH2

For-L-Asp=O

L-Asp

OO

O

HCO2HAc2O

L-Phe

toluene/acetic acid

CO2HNH

O

HO2C

NHH

O


HCl/MeOHH2O

CO2HNH

O

HO2CNH3Cl

CO2CH3NH

O

HO2CNH3Cl

CO2HNH

O

CH3O2CNH3Cl

CO2CH3NH

O

CH3O2CNH3Cl

-APM.HCl (~ 50%)

4 -isomers


11

Advantages: DL-Phe can be used, 100% -isomer formed, no recycles (only D-Phe racemization), no neutralisation crystallization

Disadvantage: less cheap Z-protection, enzyme required

DSM/Tosoh chemoDSM/Tosoh chemo--enzymatic processenzymatic process

CO2CH3NH

O

HO2CNH

OO

Z-APM . D-PheOMe

D-PheOMe

O

NHO

HO2CCO2H

OCH3

O

NH2

OCH3

O

NH2

CO2CH3NH

HO2C

O

NH2APM

OH

O

NH2

DL-PheZ-L-Asp

D-PheOMe

DL-PheOMe

Z-APMHydrogenolysis

hydrolysis &racemization

ThermolysinH2O, pH = 6-7

HCl/CH3OHL-Asp


12BiocatalyticBiocatalytic keykey--step in HSC processstep in HSC process

H2NO

O

H2NO

O

HN

OHHO

O

O

NH

O

O

O

HN

Z

Z

O

HO

Thermolysin

D-PheOMe L-PheOMe

Z-L-Asp

Z-APM.D-PheOMe

Regioselective Stereoselective Precipitation with D-PheOMe: > 90% yield


13

O

R1 X

O

R1 OH

O

R1 O

O

R1 NHR2

O

R1 O

O

R ENZYMEH2OR2-NH2

Kinetic Thermodynamic

Aminolysis Hydrolysis

R2-NH3

XH

Enzymatic peptide synthesis: Enzymatic peptide synthesis: kinetic versus thermodynamic approachkinetic versus thermodynamic approach


14

pKa should be as low as possiblepKa = 1-2: thermodynamic coupling possible(if solubility of product much lower than substrates)

ThermodynamicThermodynamicallyally controlled peptide synthesis (1)controlled peptide synthesis (1)

pKa1 and pKa2 values are crucial

K = [R1-CO-NH-R2]

[R1-COOH] [H2N-R2]

R1 OH

O

R1 NHR2

O

R1 O

O

H2OH2N R2

H3N R2

+ +Enzyme

pKa2pKa1


15

D

i

p

e

p

t

i

d

e

(

%

)

Time

Equilibrium

R OH

O

R O-

OR' NH2R' NH3

+pKa = 8pKa = 3

Thermodynamically controlled peptide synthesis (2) Thermodynamically controlled peptide synthesis (2) Influence of pH on % active reactantsInfluence of pH on % active reactants

0

20

40

60

80

100

1 2 3 4 5 6 7 8 9 10

pH

P

e

r

c

e

n

t

a

g

e

Optimal reaction pHaround (pKa1 + pKa2)/2


16Thermodynamically controlled peptide synthesis (3) Thermodynamically controlled peptide synthesis (3) HSC caseHSC case

H2NO

O

H2NO

O

HN

OHHO

O

O

NH

O

O

O

HN

ZZ

O

HO

Thermolysin

D-PheOMeL-PheOMe

Z-L-Asp

Z-APM.D-PheOMe

+

Z-APM

+

NH

O

O

O

HN

Z

O

-ONH3+O

O

H2NO

O

H2NO

O

HNOH

HOO

O

NH

OO

O

HN

ZZ

O

HO

Thermolysin

D-PheOMeL-PheOMe

Z-L-Asp

Z-APM.D-PheOMe

+

Z-APM

+

NH

OO

O

HN

Z

O

-ONH3+O

O

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


17ThermodynamicThermodynamicallyally controlled peptide synthesis (4)controlled peptide synthesis (4)

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

R1 OH

O

R1 NHR2

OH2OH2N R2+ +

EnzymeEnzyme


18

R1 X R1 enzyme R1 NHR2

R1 OH

O O

O

Oenzyme

enzyme

enzymeHX

H2O

R2 NH2

Kinetically controlled peptide synthesis (1)Kinetically controlled peptide synthesis (1)

X = OR (esters)or

X = NHR (amides) Synthesis/hydrolysis ratio crucial factor

D

i

p

e

p

t

i

d

e

(

%

)

Time

Equilibrium

Kinetic

Thermodynamic


19Kinetically controlled peptide synthesis (2)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

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

In HSC process this is disadvantageous: Preparation of (activated) Z-Asp--methyl ester difficult

and therefore expensive Thermolysin not suitable; other enzymes require organic solvent and give

lower conversions than with thermodynamic coupling

HN

OCH3HO

O

OZ


20HSC vs. NutraSweet ProcessHSC vs. NutraSweet Process

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

Recyclesonly 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.


21ThermolysinThermolysin (1): general(1): general

Source: Bacillus thermoproteolyticus Molecular weight: 34,333 Da Amino acids: 316 Metal ions present: 1 Zn2+ (activity), 4 Ca2+ (stability) pH optimum: 8.0 Temp. optimum: 70C

Ca1Ca2

Ca4

Ca3Zn

Ile 1

Lys 316


22ThermolysinThermolysin (2): 3D(2): 3D--structure of complex with Zstructure of complex with Z--APMAPM


23ThermolysinThermolysin (3): influence of pH and T(3): influence of pH and T

5 7 9 110

50

100

pH

R

e

l

a

t

i

v

e

A

c

t

i

v

i

t

y

(

%

)

5 7 9 110

50

100

pH

R

e

l

a

t

i

v

e

A

c

t

i

v

i

t

y

(

%

)

30 50 70 900

2

4

Temperature (C)

R

e

l

a

t

i

v

e

A

c

t

i

v

i

t

y

(

%

)

1

3

30 50 70 900

2

4

Temperature (C)

R

e

l

a

t

i

v

e

A

c

t

i

v

i

t

y

(

%

)

1

3


24ThermolysinThermolysin (4): influence of CaCl(4): influence of CaCl22 and and NaClNaCl

1 2 3 400

2

4

6

8

10

[NaCl] (M)

V

x

1

0

5

(

M

m

i

n

-

1

)

1 2 3 400

2

4

6

8

10

[NaCl] (M)

V

x

1

0

5

(

M

m

i

n

-

1

)

0.1 1 10 100[CaCl2] (mM)

T

5

0

(

C

)

65

75

85

95

0.1 1 10 100[CaCl2] (mM)

T

5

0

(

C

)

65

75

85

95

Reactions with Thermolysin must contain NaCl and CaCl2Thermolysin storage in presence of CaCl2


25Holland Sweetener Company (HSC) plantHolland Sweetener Company (HSC) plant


26

Thermolysin

Z-AspCondensation

BlockBlock diagram HSC diagram HSC processprocess

Purification

Drying

Crystallization

Aspartame

Sieving

Mixing

Packaging

DL-Phe

Methanol

HCl

Esterification

HydrogenolysisHydrogen

Catalyst


27Flowchart HSC Flowchart HSC processprocess

aspartamesynthesis

aspartamepurification

drying aspartamecrystallization

warehouse

raw materials production raw materials & material aids delivery

sieving

temperature andhumidity

Packaging ofaspartame

distribution


28Appendix: Appendix: optimizedoptimized FormylFormyl processprocess (DSM)(DSM)

N-Formyl protective group: very cheap to introduce, but chemical cleavage byacidic hydrolysis leads to ester hydrolysis and partial peptide bond cleavage mild enzymatic cleavage possible ?

PDF (Peptide Deformylase) identified Role in nature:

NH

NH

O

SCH3

H

Opolypeptide

RNA

H2NNH

O

SCH3

polypeptide

RNA

PDF MAPH2N 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-)


29ApplicationApplication of PDF in of PDF in chemicalchemical peptide peptide synthesissynthesis

O NH

HN

NH

CH3

O

O

H

H2NHN

NH

CH3

O

OpH 7.2

96% conversion

PDF

For-Leu-Tle-NHMe

(S,S)/(R,S) 94:6 (ee = 88%)

H-Leu-Tle-NHMe

(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

Example:


30ImprovedImproved AspartameAspartame processprocess

CO2CH3NH

O

HO2CNH

HO


H

NHO

OCH3

O

NH2

L-Asp

OO

O

HCO2HAc2O

L-PheOMe

toluene/acetic acid

CO2CH3NH

O

HO2C

NHH

O


CO2CH3NH

O

-O2CNH3+

CO2CH3NH

O

HO2C

NHH

O

-APM (> 90%, non-isolated > 70% isolated, purity > 99%)PDF

pH = 5.6

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-Pherecycles and no neutralization crystallization

Proof-of-principle delivered

amsterdam_for_website_uva.pdf

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