Hans-Dieter Jakubke

Proteolytically stable peptides by incorporation of α-Tfm amino acids

A series of model peptides containing α‐trifluoromethyl‐substituted amino acids in five different positions relative to the predominant cleavage site of the serine protease α‐chymotrypsin was synthesized by solution methods to investigate the influence of α‐Tfm substitution on the proteolytic stability of peptides. Proteolysis studies demonstrated absolute stability of peptides substituted in the P1 position and still considerable proteolytic stability for peptides substituted at the P2 and P′2 positions compared with the corresponding unsubstituted model peptide. Comparison with peptides containing the fluorine‐free disubstituted amino acid α‐aminoisobutyric acid allowed to separate electronic from steric effects. Furthermore, the absolute configuration of the α‐Tfm‐substituted amino acid was found to exert considerable effects on the proteolytic stability, especially in P′1 substituted peptides. Investigations of this phenomenon using empirical force field calculations revealed that in the (S,R,S)‐diasteromer the steric constraints exhibited by the α‐Tfm group can be outweighed by an advantageous interaction of the fluorine atoms with the serine side chain of the enzyme. In contrast, a favourable interaction between substrate and enzyme is impossible for the (S,S,S)‐diastereomer.

Chymotrypsin suspended in organic solvents with salt hydrates is a good catalyst for peptide synthesis from mainly undissolved reactants

Abstract Chymotrypsin powder suspended in organic solvents in the presence of Na 2 CO 3 .10H 2 O catalyses peptide synthesis from X-Ala-Phe-OMe and Leu-NH 2 (X = Boc or Z). The reaction proceeds best in the most non-polar solvents, such as hexane, despite the fact that both reactants and products remain largely undissolved. Leu-NH 2 .HCl can be used in place of the free base.

Penicillin acylase-catalyzed protection and deprotection of amino groups as a promising approach in enzymatic peptide synthesis.

Penicillin acylase from E. coli is able to catalyze both the introduction and the removal of the phenylacetyl group. We have established that phenylacetyl derivatives of amino acids and peptides can be used in protease‐catalyzed peptide synthesis. Here the synthesis of leucine‐enkephalin using enzymes for N‐terminal amino group protection, peptide bond formation and deprotection is described.

Peptide synthesis by chymotrypsin in frozen solutions. Free amino acids as nucleophiles.

Nucleophilic efficiency of free amino acids in chymotrypsin‐catalyzed acyl transfer in ice at −18°C using ethyl esters of N‐maleyl‐l‐tyrosine and l‐tyrosine as the acyl group donors has been studied. Although the amino acids did not act as acyl acceptors in liquid water, the high yields of peptides were obtained in frozen solutions at pH 10.5 (before freezing). The efficiency of amino acids in the formation of the corresponding dipeptides depended on the substrate used, and decreased in the order Ser,Thr,Gln>Lys>Cit>Ala>Gly>Asn>Arg>Glu>Val>Orn>Asp (with no peptide formed with His, Leu, He and Pro) for N‐maleyl‐l‐tyrosine ethyl ester and Ser>Lys>Orn>Arg,Cit>Gln>Thr>Asn>Ala>Gly (with no peptide formed with Glu, Val, Asp, His, Leu, Ile and Pro) for l‐tyrosine ethyl ester.Nucleophilic efficiency of free amino acids in chymotrypsin-catalyzed acyl transfer in ice at −18°C using ethyl esters of N-maleyl-l-tyrosine and l-tyrosine as the acyl group donors has been studied. Although the amino acids did not act as acyl acceptors in liquid water, the high yields of peptides were obtained in frozen solutions at pH 10.5 (before freezing). The efficiency of amino acids in the formation of the corresponding dipeptides depended on the substrate used, and decreased in the order Ser,Thr,Gln>Lys>Cit>Ala>Gly>Asn>Arg>Glu>Val>Orn>Asp (with no peptide formed with His, Leu, He and Pro) for N-maleyl-l-tyrosine ethyl ester and Ser>Lys>Orn>Arg,Cit>Gln>Thr>Asn>Ala>Gly (with no peptide formed with Glu, Val, Asp, His, Leu, Ile and Pro) for l-tyrosine ethyl ester.

On the use of carboxamidomethyl esters in the protease-catalyzed peptide synthesis

Carboxamidomethyl esters (CAM esters) ofZ- andBoc-protected alanine and phenylalanine were prepared in order to investigate their usefulness as substrates for α-chymotrypsin- and papain-catalyzed hydrolysis and peptide synthesis reactions. The easy removal of theCAM-C-protecting group under mild conditions and dependent on the enzyme specificity was demonstrated. Examples are given for the protease-catalyzed synthesis of various peptide derivatives usingCAM esters as C- and N-components in aqueous-organic media. Comparatively short reaction times were observed.ZusammenfassungEs wurden die Carboxamidomethylester (CAM-Ester) vonZ- undBoc-geschütztem Alanin und Phenylalanin hergestellt mit dem Ziel, ihre Eignung als Substrate für α-chymotrypsin- und papainkatalysierte Hydrolyse- sowie Peptidsynthesereaktionen zu untersuchen. Die leichte, unter milden Bedingungen und in Abhängigkeit von der Enzymspezifität erfolgende Abspaltung derCAM-C-Schutzgruppe wurde nachgewiesen. An Beispielen wird die proteasekatalysierte Synthese verschiedener Peptidderivate unter Verwendung vonCAM-Estern als C-und N-Komponenten in wäßrig-organischen Medien belegt. Die für die Umsetzungen benötigten Reaktionszeiten sind vergleichsweise gering.

Peptide synthesis by means of immobilized enzymes

Abstractα-Chymotrypsin covalently bound to silica, enzacryl AA, and enzacryl AH catalyzes peptide bond formation between N-protected dipeptide methyl esters and H-Leu-NH2 with results similar to those with the free enzyme. The influence of water-miscible and water-immiscible cosolvents, of the supports, and of the structure of the substrates is shown to be of importance for the ease of the chymotrypsin-medicated coupling reactions. The best yields were obtained using biphasic aqueous-organic solvent mixtures, silica-bound chymotrypsin, and substrates with leucine in the P2-position. The yields of the syntheses are discussed in terms of the reactivity of substrates with similar structure in enzymatic hydrolyses. All the immobilized chymotrypsin preparations could be re-utilized successfully for further couplings.ZusammenfassungKovalent an Kieselgel, Enzacryl AA und Enzacryl AH gebundenes α-Chymotrypsin katalysiert die Peptidbindungsknüpfung zwischen N-geschützten Dipeptid-Methylestern und H-Leu-NH2 mit ähnlichen Ergebnissen wie das freie Enzym. Es wird gezeigt, daß mit Wasser mischbare und nichtmischbare Lösungsmittel, die Trägermaterialien sowie die Struktur der Substrate den Verlauf der Chymotrypsin-katalysierten Kupplungsreaktionen beeinflussen. Die besten Ausbeuten wurden in wäßrig-organischen Zweiphasen-systemen mit Kieselgel-gebundenem Chymotrypsin und mit Substraten mit Leucin in P2-Position erhalten. Die Syntheseausbeuten werden in bezug auf die Reaktivität von Substraten ähnlicher Struktur bei der enzymatischen Hydrolyse diskutiert. Alle immobilisierten Chymotrypsin-Präparationen konnten mit Erfolg für weitere Kupplungsreaktionen wiedergenutzt werden.

Single-step synthesis of kyotorphin in frozen solutions by chymotrypsin

Abstract A novel method of Kyotorphin (H-Tyr-Arg-OH) synthesis without protecting and deprotecting procedures is described -α-chymotrypsin-catalyzed aminolysis of H-Tyr-OEt by H-Arg-OH in frozen mixtures. In this one-step process yields of the peptide product above 80% have been obtained.

Modelluntersuchungen zur papainkatalysierten Peptidsynthese im wrig-organischen Zweiphasensystem@@@Model studies on papain-catalyzed peptide synthesis in a biphasic aqueous-organic system

The usefulness of biphasic aqueous-organic solvent systems for pepsin-catalyzed synthesis of model peptidesZ-X-Phe-Phe-OMe (X=Ala, Gln, Leu) has been demonstrated by coupling the correspondingZ-X-Phe-OH with H-Phe-OMe. The influence of various organic solvents on pepsin activity was examined. Some examples are given for the influence of nucleophile and enzyme concentration, bufferpH and organic solvent portion on product yield. Tetrachloromethane and mixtures of ethyl acetate/n-hexane proved to be especially useful allowing syntheses in good yields and within comparatively short reaction times of 2–6 hours.

Modelluntersuchungen zur papainkatalysierten Peptidsynthese im wäßrig-organischen Zweiphasensystem

Several model peptides have been synthesized enzymatically using papain as a catalyst in biphasic aqueous-organic systems. The effect of different cosolvents,pH, buffer concentration, and reaction time on the papaincatalyzed synthesis was examined. A comparison of the results obtained indicates that water-immiscible organic solvents provide higher yields than methanol in papain mediated peptide synthesis with carboxyl components in the carboxyl free form. Furthermore, it could be established that papaincatalyzed peptide synthesis can be considerably speeded up by employing acyl peptide esters instead of acyl peptides. The former should promote the rapid formation of the acyl-enzyme intermediate.

Enzymatic peptide synthesis in frozen aqueous systems: Use of Nα-unprotected unusual acyl donors

Abstract α-Chymotrypsin (EC 3.4.21.1) was used for catalyzing the reaction of various N α -unprotected non-coded phenylalanine ester derivatives with H-Leu-NH 2 and H-Arg-NH 2 in frozen aqueous solution at −15 °C. Compared with reactions at room temperature, a significant yield increasing effect could be established. The kinetic parameters of ester hydrolysis show that most of the unusual acyl donors (compared with the coded phenylalanine methyl ester) are well accepted substrates for α-chymotrypsin.