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.
Ph
O
Ph
CO2H
NH
H2N
NH
H
H
H2N
CO2Me
O
H
H
CO2Me
CO2H
-L-aspartyl-L-phenylalanine methyl ester -L-aspartyl-L-phenylalanine methyl ester
-aspartame (APM)]
Sweet
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Bitter
J.S. Parent
1
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 Asp
and Phe).
The forward reaction is written as:
CO2H
X
N
H
H
CO2H
Amine-protected (X)
L-aspartic acid
(Z-L-Asp)
Ph
CO2H
H2N
NH
H
H
CO2Me
O
-L-aspartyl-L-phenylanaline methyl ester
-aspartame (APM)]
Ph
+
CO2H
H2N
H
CO2Me
thermolysin
Methyl ester of
L-phenylanaline
(L-PM)
X
N
HH
NH
H
Ph
+
OH2
CO2Me
O
(APM)
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.
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J.S. Parent
2
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 lines
Solid circles: four calcium atoms
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J.S. Parent
3
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.
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J.S. Parent
4
Kinetics of the Aspartame Synthesis
The rate of APM production is firstorder 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; m= 0.364 M
T = 40C
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J.S. Parent
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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] =
[D-PM] =
1.82x10-2 M with
9.09x10-3 M
** [L-PM ] =
[D-PM] =
3.64 x10-2 M with
1.82 x10-2 M
L-PM
D,L-PM
[Z-L-Asp] = 1.82 x 10-2 M
[Eo] =
4.85 x I0-6 M
pH 6.5; m 0.364 M; 40C
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J.S. Parent
6
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.
Pure Z-L-Asp
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.
9.1x10-3 M Z-D-Asp added
[L-PM] = 3.64 x 10-2 M
[Eo] =
4.85 x I0-6 M
pH 6.5; m 0.364 M; 40C
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J.S. Parent
7
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:
+ Z-L-Asp k1
E
-
Z-D-Asp
k-3
-
Z-L-Asp k-1
+ Z-D-Asp
Z-D-Asp*E
CHEE 323
Z-L-Asp*E
k3
+ L-PM
k2 r.d.s.
Z-APM
+
Note that Z-D-Asp binds thermolysin in an
inactive state, thereby reducing the active
enzyme concentration and lowering the
reaction rate.
J.S. Parent
8
E
Competitive Inhibition by Z-D-Asp
From this proposed mechanism we can derive a rate expression
that accounts for competitive inhibition.
r 1:
r 3:
r 2:
Z-L-Asp
Z-D-Asp
Z-L-Asp*E
+
+
+
k1
E
Z-L-Asp*E
k-1
k3
E
Z-D-Asp*E
k-3
L-PM
k2
ZAPM
r.d.s.
+
E
Assigning r2 as the rate determining step of the process, we find
the reaction velocity is:
r=
CHEE 323
k 2 [E]T [ ZLAsp][LPM]
 [ ZDAsp] 
  [ ZLAsp]
K11 
K3 

J.S. Parent
9
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
k 2 [E]T [ ZLAsp][LPM]
r=
 [ ZDAsp] 
  [ ZLAsp]
K11 
K3 

by inverting it:
 [ ZDAsp] 

K 1 1 
K3 
1
1
1
= 

r
k 2 [E] T [LPM] [ ZLAsp] k 2 [E]T [LPM]
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.
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10
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
 [ ZDAsp] 

K 1 1 
K3 
1
1
1
= 

r
k 2 [E] T [LPM] [ ZLAsp] k 2 [E]T [LPM]
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; m 0.364 M; 40C
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J.S. Parent
11
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.
CO2H
X
N
H
H
CO2H
Amine-protected (X)
L-aspartic acid
(Z-L-Asp)
Ph
+
CO2H
H2N
H
CO2Me
thermolysin
Methyl ester of
L-phenylalanine
(L-PM)
X
N
HH
NH
H
Ph
+
CO2Me
O
(APM)
a APM aH2O
The equilibrium constant derived from K
=
APM
the Gibbs energies of the reaction
aLAsp aLPM
components is quite small, making the
[ APM] APM [H2O] H2O
conversion of a standard batch reaction
=
[LAsp] LAsp [LPM] LPM
equilibrium limited.
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J.S. Parent
12
OH2
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.
Ph
H2N
CO2H
X
N
H
H
CO2H
Amine-protected (X)
L-aspartic acid
+
CO2H
H
CO2Me
Methyl ester of
L-phenylalanine
X
thermolysin
NH
H
N
HH
CO2Me
O
Ph
Ph
H2N
H2N
H CO2Me
Methyl ester of
D-phenylalanine
CHEE 323
Ph
J.S. Parent
H
CO2Me
L-L dipeptide deposits
as an addition compd.
13