The dipeptide ester L-Asp-L-Phe-OMe (Aspartame) is used in large amounts as a
low-calorie sweetener. One of the most economical strategies for its synthesis
involves an enzymatic step, which is run at a capacity of 2000 t/year (Scheme
3.28). Benzyloxycarbonyl-(Z)-protected L-aspartic acid is linked with L-Phe–OMe
in a thermodynamically controlled condensation reaction catalyzed by the protease
thermolysin without formation of the undesired (bitter) β-isomer. Removal of the
product via formation of an insoluble salt was used as driving force to shift the
equilibrium of the reaction in the synthetic direction [350, 351].
α-Chymotrypsin has been used for the kg-scale synthesis of the dipeptide Nbenzoyl-L-Tyr-L-Arg-NH 2 , a precursor to the powerful analgesic kyotorphin
[352]. The wide potential of enzymatic peptide synthesis is illustrated with the
chemo-enzymatic synthesis of an enkephalin derivative – (D-Ala 2 , D-Leu 5 )-enkephalin amide (Scheme 3.29) [353]. Enkephalins are pain regulators and commonly
undergo rapid enzymatic degradation in vivo, and therefore exert only limited
biological activities. However, by substitution of a D-amino acid into the sequence,
‘chirally muted’ derivatives are obtained, which can elicit long-lasting pharmacological effects [354]. The Tyr-D-Ala and Gly-Phe subunits were obtained via an
ester aminolysis and a condensation reaction using α-chymotrypsin and
thermolysin, respectively. The latter dipeptide was extended by a D-Leu unit
Table 3.7 Selectivities of proteases
Protease
Type
Specificity
Achromobacter protease
Serine protease
–Lys–X
α-Chymotrypsin, subtilisins
Serine protease
–Trp(Tyr,Phe,Leu,Met)–X
Carboxypeptidase Y
Serine protease
Nonspecific
Elastase
Serine protease
–Ala(Ser,Met,Phe)–X
Trypsin, Streptomyces griseus protease
Serine protease
–Arg(Lys)–X
Staphylococcus aureus V8 protease
Serine protease
–Glu(Asp)–X
Papain, ficin
Thiol protease
–Phe(Val,Leu)–X
Clostripain, cathepsin B
Thiol protease
–Arg–X
Cathepsin C
Thiol protease
H–X–Phe(Tyr,Arg)–X
Thermolysin, B. subtilis protease
Metalloprotease
–Phe(Gly,Leu)–Leu(Phe)
Myxobacter protease II
Metalloprotease
X–Lys
Pepsin, cathepsin D
Carboxyl protease
–Phe(Tyr,Leu)–Trp(Phe,Tyr)
X ¼ unspecified amino acid or peptide residue
Z-H 2 N-Asp-OH + 2 H 2 N-Phe-OMe
Thermolysin
Z-H 2 N-Asp-NH-Phe-OMe
H 2 N-Phe-OMe
(complex)
purification
recycling
H 2 N-Asp-NH-Phe-OMe
deprotection
Z-H 2 N-Asp-NH-Phe-OMe
Z = Ph-CH 2 -O-COAspartame
Scheme 3.28 Enzymatic synthesis of Aspartame
3.1 Enzymes in Organic Solvents
351
low-calorie sweetener. One of the most economical strategies for its synthesis
involves an enzymatic step, which is run at a capacity of 2000 t/year (Scheme
3.28). Benzyloxycarbonyl-(Z)-protected L-aspartic acid is linked with L-Phe–OMe
in a thermodynamically controlled condensation reaction catalyzed by the protease
thermolysin without formation of the undesired (bitter) β-isomer. Removal of the
product via formation of an insoluble salt was used as driving force to shift the
equilibrium of the reaction in the synthetic direction [350, 351].
α-Chymotrypsin has been used for the kg-scale synthesis of the dipeptide Nbenzoyl-L-Tyr-L-Arg-NH 2 , a precursor to the powerful analgesic kyotorphin
[352]. The wide potential of enzymatic peptide synthesis is illustrated with the
chemo-enzymatic synthesis of an enkephalin derivative – (D-Ala 2 , D-Leu 5 )-enkephalin amide (Scheme 3.29) [353]. Enkephalins are pain regulators and commonly
undergo rapid enzymatic degradation in vivo, and therefore exert only limited
biological activities. However, by substitution of a D-amino acid into the sequence,
‘chirally muted’ derivatives are obtained, which can elicit long-lasting pharmacological effects [354]. The Tyr-D-Ala and Gly-Phe subunits were obtained via an
ester aminolysis and a condensation reaction using α-chymotrypsin and
thermolysin, respectively. The latter dipeptide was extended by a D-Leu unit
Table 3.7 Selectivities of proteases
Protease
Type
Specificity
Achromobacter protease
Serine protease
–Lys–X
α-Chymotrypsin, subtilisins
Serine protease
–Trp(Tyr,Phe,Leu,Met)–X
Carboxypeptidase Y
Serine protease
Nonspecific
Elastase
Serine protease
–Ala(Ser,Met,Phe)–X
Trypsin, Streptomyces griseus protease
Serine protease
–Arg(Lys)–X
Staphylococcus aureus V8 protease
Serine protease
–Glu(Asp)–X
Papain, ficin
Thiol protease
–Phe(Val,Leu)–X
Clostripain, cathepsin B
Thiol protease
–Arg–X
Cathepsin C
Thiol protease
H–X–Phe(Tyr,Arg)–X
Thermolysin, B. subtilis protease
Metalloprotease
–Phe(Gly,Leu)–Leu(Phe)
Myxobacter protease II
Metalloprotease
X–Lys
Pepsin, cathepsin D
Carboxyl protease
–Phe(Tyr,Leu)–Trp(Phe,Tyr)
X ¼ unspecified amino acid or peptide residue
Z-H 2 N-Asp-OH + 2 H 2 N-Phe-OMe
Thermolysin
Z-H 2 N-Asp-NH-Phe-OMe
H 2 N-Phe-OMe
(complex)
purification
recycling
H 2 N-Asp-NH-Phe-OMe
deprotection
Z-H 2 N-Asp-NH-Phe-OMe
Z = Ph-CH 2 -O-COAspartame
Scheme 3.28 Enzymatic synthesis of Aspartame
3.1 Enzymes in Organic Solvents
351