CHEE 323 J.S. Parent 1

Enzymatic Synthesis of Aspartame

Aspartame is a low-calorie sweetener whose apparent sweetness is 150- 200 times that of sucrose. It is prepared by condensation of L-aspartic acid and the methyl ester of L-phenylalanine (two amino acids).

Its sweet taste depends on: L-conformation of the two constituent amino acids presence of the methyl ester correct coupling of the amino acids.

Sweet Bitter

-L-aspartyl-L-phenylalanine methyl ester-aspartame (APM)]

H

CO2H

NHNH2

O

Ph

CO2MeH

-L-aspartyl-L-phenylalanine methyl ester

NH2H

O

NH

Ph

CO2MeHCO2H

CHEE 323 J.S. Parent 2

Industrial Enzymatic Synthesis of Aspartame

The unique regio and stereoselectivity afforded by enzymes has been exploited on an industrial scale Aspartame production.

The process employs a protease, thermolysin, to catalyze the condensation of the modified Aspand Phe).

The forward reaction is written as:

Note however, that the synthesis reaction is equilibrium limited by the reverse (hydrolysis) reaction for which proteases are known. Furthermore, the equilibrium strongly favours hydrolysis.

-L-aspartyl-L-phenylanaline methyl ester-aspartame (APM)]

H

CO2H

NHNH2

O

Ph

CO2MeH

CO2H

CO2HNH H

X

Amine-protected (X)L-aspartic acid(Z-L-Asp)

Methyl ester ofL-phenylanaline(L-PM)

thermolysin+CO2H

NHNH

O

Ph

CO2MeHH

XNH2 CO2MeH

Ph

(APM)

+ OH2

CHEE 323 J.S. Parent 3

Structural Properties of Thermolysin

Thermolysin is a metalloenzyme (316 amino acids) requiring a zinc ion and four calcium ions to maintain an active tertiary structure.

Two distinct hemispheres exist with a zinc atom located at the bottom of the cleft. Three residues (142, 146 and 166) serve as ligands for zinc. Calcium is a structural element, and is not believed to interact with the substrate at the active site.

Open circles: -carbon positions Stippled circle: zinc with its three protein ligands as broken linesSolid circles: four calcium atoms

CHEE 323 J.S. Parent 4

Chemical Properties of Thermolysin

Thermolysin is an extracellular enzyme produced by a bacterial strain that can withstand high temperatures. Hence, themolysin has a temperature stability that is superiour to most enzymes.

Thermolysin is classified as a protease, in that it catalyzes the cleavage of the peptide bonds that constitute proteins.

The term endopeptidase applies, as the internal bonds in polypeptides are susceptible to the action of thermolysin

The term neutral protease applies, as the pH optimum lies about pH 7.5

The term metalloenzyme is appropriate, given the necessity of zinc at the active site and the requirement for calcium to maintain an active tertiary structure. Chelating agents deactivate thermolysin.

Enzymes of this class demonstrate substrate specificity which requires a hydrophobic amino acid such as phenylalanine as the residue whose amido group is cleaved.

CHEE 323 J.S. Parent 5

Kinetics of the Aspartame Synthesis

The rate of APM production is first-order with respect to the total concentration of enzyme [Eo], and a bell-shaped pH-rate profile with the highest activity at pH 7.5 is observed.

Shown is a typical time course of the thermolysin catalysed condensation of N-benzyloxycarbonylaspartic acid with phenylalanine methyl ester. Initial rate measurements (from t=0 to t=10 min) as a function of reagent concentrations define the overall reaction kinetics.

[Z-L-Asp] = 1.82 x 10-2 M[L-PM] = 3.64 x 10-2 M [Eo] = 4.85 x I0-6 M pH = 6.5; 0.364 MT = 40C

CHEE 323 J.S. Parent 6

Influence of [PM] on the Condensation Rate

APM synthesis is first-order WRT phenylalananine methyl ester, with no apparent saturation behaviour that is common in enzyme-mediated reactions.

Note that the presence of D-PM has no effect on the reaction rate, and it is not found in the product.

* [L-PM] = 1.82x10-2 M with [D-PM] = 9.09x10-3 M

** [L-PM ] = 3.64 x10-2 M with [D-PM] = 1.82 x10-2 M

[Z-L-Asp] = 1.82 x 10-2 M [Eo] = 4.85 x I0-6 M

pH 6.5; 0.364 M; 40C

L-PM

D,L-PM

CHEE 323 J.S. Parent 7

Influence of [Z-L-Asp] on the Condensation Rate

A plot of [Z-L-Asp] against the APM production rate shows saturation of the rate, typical Michaelis-Menten behaviour.

Rate retardation occurs in the presence of Z-D-Asp, indicating that the enantiomer acts as a competitive inhibitor. Hence only pure L-Asp can be used in APM synthesis, while racemic mixtures of D,L-PM can be accommodated.

[L-PM] = 3.64 x 10-2 M [Eo] = 4.85 x I0-6 M pH 6.5; 0.364 M; 40C

Pure Z-L-Asp

9.1x10-3 M Z-D-Asp added

CHEE 323 J.S. Parent 8

Proposed Reaction Mechanism

Competitive inhibitors reduce the rate of product formation through binding the enzyme in an inactive form.

Often these inhibitors are structurally similar to the substrate, and therefore are capable of binding at the active site

Enzyme-bound inhibitor either lacks a needed functional group or is held in an unsuitable position for reaction.

We have seen an example of this behaviour in aspartame production, where the enantiomer of L-Asp inhibited the reaction. A plausible mechanism for this inhibition is shown below:

Note that Z-D-Asp binds thermolysin in aninactive state, thereby reducing the activeenzyme concentration and lowering the reaction rate.

Z-L-Asp+E Z-L-Asp*E

k1

k-1k2

L-PMEZ-APM +

+

r.d.s.Z-L-Asp-

Z-D-Asp*E

Z-D-Asp+k3k-3

Z-D-Asp-

CHEE 323 J.S. Parent 9

Competitive Inhibition by Z-D-Asp

From this proposed mechanism we can derive a rate expression that accounts for competitive inhibition.

r1:

r3:

r2:

Assigning r2 as the rate determining step of the process, we find the reaction velocity is:

Z-L-Asp + E Z-L-Asp*Ek1

k-1

Z-D-Asp + E Z-D-Asp*Ek-3

k2L-PM EZAPM +Z-L-Asp*E +r.d.s.

k3

]ZLAsp[K

]ZDAsp[1K

]LPM][ZLAsp[]E[kr

31

T2

CHEE 323 J.S. Parent 10

Validating the Proposed Reaction Scheme

Although more sophisticated regression techniques are available, the simplest means of testing the model is to linearize the rate expression

by inverting it:

A plot of 1/rate versus 1/[Z-L-Asp] should be linear, with a slope of K1(1+[Z-DAsp]/K3)/(k2[E]T[L-PM]) and an intercept 1/(k2[E]T[L-PM])

This is commonly referred to as a Lineweaver-Burk plot It is necessary that the data fit the rate expression, but it is

not sufficient proof that the mechanism is correct From the slope, intercept, [E]T and [L-PM], numerical

estimates of K1 and k2 can be derived.

]LPM[]E[k1

]ZLAsp[1

]LPM[]E[k

K]ZDAsp[

1K

r1

T2T2

31

]ZLAsp[K

]ZDAsp[1K

]LPM][ZLAsp[]E[kr

31

T2

CHEE 323 J.S. Parent 11

Lineweaver-Burk Plot of the Kinetic Data

Plotting the inverse of the APM production rate (moleL-1s-1) against 1/[Z-L-Asp] reveals a linear relationship

The proposed mechanism is consistent with the kinetic data, and may be correct.

From the slopes and intercepts,k2 = 2.65 L mole-1 s-1

K1 = 1.03x10-2 mole L-1

K3 = 2.35x10-2 mole L-1

Line A: no Z-D-Asp; Line B: [Z-D-Asp]=9.09x10-3 M

[L-PM] = 1.82 x 10-2 M [Eo] = 4.85 x 10-6 M pH 6.5; 0.364 M; 40C

]LPM[]E[k1

]ZLAsp[1

]LPM[]E[k

K]ZDAsp[

1K

r1

T2T2

31

CHEE 323 J.S. Parent 12

Isolation of the Aspartame Product

Proteases are recognized as catalysts for peptide bond cleavage, and using them to catalyze the reverse condensation reaction can be problematic.

The equilibrium constant derived from the Gibbs energies of the reaction components is quite small, making the conversion of a standard batch reaction equilibrium limited. LPMLAsp

OH2APM

LPMLAsp

O2HAPMAPM

]LPM[]LAsp[

]OH[]APM[

aaaa

K

2

CO2H

CO2HNH H

X

Amine-protected (X)L-aspartic acid(Z-L-Asp)

Methyl ester ofL-phenylalanine(L-PM)

thermolysin+CO2H

NHNH

O

Ph

CO2MeHH

XNH2 CO2MeH

Ph

(APM)

+ OH2

CHEE 323 J.S. Parent 13

Isolation of the Aspartame Product

Luckily APM forms, via its free side-chain carboxylic acid, a sparingly soluble addition compound with excess PM.

The synthesis can be driven using LeChatalier’s Principle by removal of the precipitation of the product.

Once isolated from the enzyme, hydrolysis of Z-APM is no longer a concern, excess PM can be removed and the product can be deprotected to yield aspartame.

CO2H

CO2HNH H

X

Amine-protected (X)L-aspartic acid

Methyl ester ofL-phenylalanine thermolysin

Methyl ester ofD-phenylalanine

+

CO2H

NHNH

O

Ph

CO2MeHH

X

NH2 CO2MeH

Ph

NH2 CO2MeH

Ph

NH2 CO2MeH

Ph

L-L dipeptide depositsas an addition compd.