Synthesis of dipeptides by suspension-to-suspension conversion via thermolysin catalysis: from analytical to preparative scale

Abstract

When using proteases in direct reversal of their normal hydrolytic function, the equilibrium position is very important in limiting the attainable yield in equilibrium-controlled enzymic peptide synthesis. Analysis of the equilibrium position reveals a favourable shift towards the peptide product if starting materials are largely undissolved in the reaction medium and the product precipitates. This approach enabled us to obtain high peptide yields in thermolysin-catalysed reactions in high-density aqueous media with an equimolar supply of substrates. The easy scale-up (up to mol-scale) of this approach is demonstrated by two examples. Z-His-Phe-NH2 and Z-Asp-Phe-OMe, precursors for cyclo-[-His-Phe-] and the low-calorie sweetener Aspartame, respectively, were synthesized in preparative yields of 84-88%.

Kinetics of enzymatic solid-to-solid peptide synthesis: Synthesis ofZ-aspartame and control of acid-base conditions by using inorganic salts

“…The use of NaOH to neutralize the hydrochloride of Phe-OMe in the solid-to-solid synthesis of Z-aspartame was first reported by Eichhorn et al (1995Eichhorn et al ( , 1997. However, we expected that an "excess" of basic salt could further improve acid-base conditions in the liquid phase, and Figure 1 shows this to be correct.…”

Kinetics of enzymatic solid-to-solid peptide synthesis: Synthesis ofZ-aspartame and control of acid-base conditions by using inorganic salts

“…The use of NaOH to neutralize the hydrochloride of Phe-OMe in the solid-to-solid synthesis of Z-aspartame was first reported by Eichhorn et al (1995Eichhorn et al ( , 1997. However, we expected that an "excess" of basic salt could further improve acid-base conditions in the liquid phase, and Figure 1 shows this to be correct.…”

Role of Thermolysin in Catalytic-Controlled Self-Assembly of Fmoc-Dipeptides

“…Additionally, the hydrophobicity of terminal residues could affect the binding capability of peptide sequences at the catalytic site of thermolysin, and the subsequent catalytic efficiency. 40,41 Further, thermolysin has a preference for a particular substrate pattern, in which a hydrophilic amino acid must be in the first residue and hydrophobic amino acid in the second residue. Finally, based on the bidirectional catalytic actions of thermolysin pertaining to hydrolysis and condensation, we designed a two-step reaction achieved by the modification of the Fmoc-dipeptide sequence, for instance, from Fmoc-YL to Fmoc-YY-NH 2 to create gel-sol-gel transition hydrogel films in this research.…”

“…According to recent reports, an amino acid amide derivative exhibited a more hydrophobic nature, 40 and thermolysin could couple a carboxy component to an amino acid amide at about one order of magnitude faster than to the corresponding amino acid. 41 Therefore, we designed leucine amide derivative (L-NH 2 ) and leucine methyl ester (L-OMe) and used in combination with Fmoc-X (where "X"= Y, L, T, and S; S is serine) to explore whether thermolysin could trigger the condensation reaction of such amino acid derivatives (Figure 3a) . Our results demonstrated that various Fmoc-XL derivatives could form different combinations of Fmoc-X and L-NH 2 /L-OMe from each other in HPLC profiles (Supporting Information Figure S3c and d), and illustrated that the binding affinity between the thermolysin binding pocket and L-NH 2 was enhanced when L-COOH was modified with -NH 2 to generate L-CONH 2 .…”

Protease Catalyzed In Situ C-Terminal Modification of Oligoglutamate

“…Furthermore, thermolysincatalyzed coupling of H 2 N-(OdC)-His-NH 2 (R-carboxyl amide derivative of H) with Z-Phe-OH resulted in no dipeptide formation. 39 In an effort to understand what structural features of these natural amino acids resulted in their inability to react as amine nucleophiles during oligo(γ-L-Et-Glu) synthesis, further studies were performed with both F and H analogs. L-Phenylmethanamine (BzA) is similar to F but lacks an Rcarboxyl group.…”

“…Specifically, carboxypeptidase-Y catalyzed reaction of Bz-Ala-(CO)-OMe with F and H gave dipeptide in 25 and 15% yield, respectively. Furthermore, thermolysin-catalyzed coupling of H 2 N-(OC)-His-NH 2 (α-carboxyl amide derivative of H ) with Z-Phe-OH resulted in no dipeptide formation …”

Kinetics of enzymatic solid-to-solid peptide synthesis: Synthesis ofZ-aspartame and control of acid-base conditions by using inorganic salts

“…The use of NaOH to neutralize the hydrochloride of Phe-OMe in the solid-to-solid synthesis of Z-aspartame was first reported by Eichhorn et al (1995Eichhorn et al ( , 1997. However, we expected that an "excess" of basic salt could further improve acid-base conditions in the liquid phase, and Figure 1 shows this to be correct.…”

Role of Thermolysin in Catalytic-Controlled Self-Assembly of Fmoc-Dipeptides

“…Additionally, the hydrophobicity of terminal residues could affect the binding capability of peptide sequences at the catalytic site of thermolysin, and the subsequent catalytic efficiency. 40,41 Further, thermolysin has a preference for a particular substrate pattern, in which a hydrophilic amino acid must be in the first residue and hydrophobic amino acid in the second residue. Finally, based on the bidirectional catalytic actions of thermolysin pertaining to hydrolysis and condensation, we designed a two-step reaction achieved by the modification of the Fmoc-dipeptide sequence, for instance, from Fmoc-YL to Fmoc-YY-NH 2 to create gel-sol-gel transition hydrogel films in this research.…”

“…According to recent reports, an amino acid amide derivative exhibited a more hydrophobic nature, 40 and thermolysin could couple a carboxy component to an amino acid amide at about one order of magnitude faster than to the corresponding amino acid. 41 Therefore, we designed leucine amide derivative (L-NH 2 ) and leucine methyl ester (L-OMe) and used in combination with Fmoc-X (where "X"= Y, L, T, and S; S is serine) to explore whether thermolysin could trigger the condensation reaction of such amino acid derivatives (Figure 3a) . Our results demonstrated that various Fmoc-XL derivatives could form different combinations of Fmoc-X and L-NH 2 /L-OMe from each other in HPLC profiles (Supporting Information Figure S3c and d), and illustrated that the binding affinity between the thermolysin binding pocket and L-NH 2 was enhanced when L-COOH was modified with -NH 2 to generate L-CONH 2 .…”

Protease Catalyzed In Situ C-Terminal Modification of Oligoglutamate

“…Furthermore, thermolysincatalyzed coupling of H 2 N-(OdC)-His-NH 2 (R-carboxyl amide derivative of H) with Z-Phe-OH resulted in no dipeptide formation. 39 In an effort to understand what structural features of these natural amino acids resulted in their inability to react as amine nucleophiles during oligo(γ-L-Et-Glu) synthesis, further studies were performed with both F and H analogs. L-Phenylmethanamine (BzA) is similar to F but lacks an Rcarboxyl group.…”

“…Specifically, carboxypeptidase-Y catalyzed reaction of Bz-Ala-(CO)-OMe with F and H gave dipeptide in 25 and 15% yield, respectively. Furthermore, thermolysin-catalyzed coupling of H 2 N-(OC)-His-NH 2 (α-carboxyl amide derivative of H ) with Z-Phe-OH resulted in no dipeptide formation …”

Kinetics of enzymatic solid-to-solid peptide synthesis: Synthesis ofZ-aspartame and control of acid-base conditions by using inorganic salts

“…The use of NaOH to neutralize the hydrochloride of Phe-OMe in the solid-to-solid synthesis of Z-aspartame was first reported by Eichhorn et al (1995Eichhorn et al ( , 1997. However, we expected that an "excess" of basic salt could further improve acid-base conditions in the liquid phase, and Figure 1 shows this to be correct.…”

Role of Thermolysin in Catalytic-Controlled Self-Assembly of Fmoc-Dipeptides

“…Additionally, the hydrophobicity of terminal residues could affect the binding capability of peptide sequences at the catalytic site of thermolysin, and the subsequent catalytic efficiency. 40,41 Further, thermolysin has a preference for a particular substrate pattern, in which a hydrophilic amino acid must be in the first residue and hydrophobic amino acid in the second residue. Finally, based on the bidirectional catalytic actions of thermolysin pertaining to hydrolysis and condensation, we designed a two-step reaction achieved by the modification of the Fmoc-dipeptide sequence, for instance, from Fmoc-YL to Fmoc-YY-NH 2 to create gel-sol-gel transition hydrogel films in this research.…”

“…According to recent reports, an amino acid amide derivative exhibited a more hydrophobic nature, 40 and thermolysin could couple a carboxy component to an amino acid amide at about one order of magnitude faster than to the corresponding amino acid. 41 Therefore, we designed leucine amide derivative (L-NH 2 ) and leucine methyl ester (L-OMe) and used in combination with Fmoc-X (where "X"= Y, L, T, and S; S is serine) to explore whether thermolysin could trigger the condensation reaction of such amino acid derivatives (Figure 3a) . Our results demonstrated that various Fmoc-XL derivatives could form different combinations of Fmoc-X and L-NH 2 /L-OMe from each other in HPLC profiles (Supporting Information Figure S3c and d), and illustrated that the binding affinity between the thermolysin binding pocket and L-NH 2 was enhanced when L-COOH was modified with -NH 2 to generate L-CONH 2 .…”

Protease Catalyzed In Situ C-Terminal Modification of Oligoglutamate

“…Furthermore, thermolysincatalyzed coupling of H 2 N-(OdC)-His-NH 2 (R-carboxyl amide derivative of H) with Z-Phe-OH resulted in no dipeptide formation. 39 In an effort to understand what structural features of these natural amino acids resulted in their inability to react as amine nucleophiles during oligo(γ-L-Et-Glu) synthesis, further studies were performed with both F and H analogs. L-Phenylmethanamine (BzA) is similar to F but lacks an Rcarboxyl group.…”

“…Specifically, carboxypeptidase-Y catalyzed reaction of Bz-Ala-(CO)-OMe with F and H gave dipeptide in 25 and 15% yield, respectively. Furthermore, thermolysin-catalyzed coupling of H 2 N-(OC)-His-NH 2 (α-carboxyl amide derivative of H ) with Z-Phe-OH resulted in no dipeptide formation …”