Peter Kuhl

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.

Enzyme-catalyzed peptide synthesis in biphasic aqueous-organic systems

Abstract A preparative protease-catalyzed peptide synthesis in biphasic aqueous-organic systems is described. This approach using both free as well as immobilized proteases as catalysts provides high yields and includes the possibility to re-utilize the enzymes.

Thermodynamics of solid-to-solid conversion and application to enzymic peptide synthesis

Abstract Efficient enzyme-catalyzed syntheses may be carried out even when most of the starting materials and products are in the form of suspended solids. To some examples in the literature of such solid-to-solid conversions, we add the thermolysin-catalyzed reaction of X-Phe-OH (X = For, Ac, Z) with Leu-NH2 to give X-Phe-Leu-NH2. This can proceed to equilibrium yields of over 90% even in aqueous media. Thermodynamic analysis of such systems shows that solid-to-solid conversion itself will be equally favorable in any solvent at a given water activity. The reaction will normally show a “switchlike” behavior, continuing in one direction until at least one reactant has passed completely into solution. If the solid-to-solid synthesis is favorable, the equilibrium yield will be maximized by choice of a solvent in which the solubility of the starting materials is minimized. Some of the dependencies predicted by our equations can be seen in data from our own studies or in the literature. This includes a linear decrease in yield with the reciprocal of the initial amount of reactants suspended in a fixed volume.

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.

Thermolysin- and Chymotrypsin-Catalysed Peptide Synthesis in the Presence of Salt Hydrates

Abstract Thermolysin (EC 3.4.24.4.) suspended in hexane in the presence of Na 2 SO 4 •10H 2 O catalyses synthesis of N-protected dipeptide- and tripeptide amides in good yields. The influence of different mixing conditions including sonification has been investigated. Besides further results of the chymotrypsin-catalysed peptide coupling of Y-Ala-Phe-OMe (Y = Z, Boc) with Leu-NH 2 under modified reaction conditions are reported.

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.

Specificity in the hydrolysis of N-acyl-L-phenylalanine 4-nitroanilides by chymotrypsin

The affinity of N-acyl-L-phenylalanine 4-nitroanilides for chymotrypsin is enhanced as the hydrophobicity of non-amino acid residues in the P2-position of the substrates increases, whereas kcat remains nearly constant. On the other hand, if alanine or leucine is in the P2-position kcat increases with decreasing KM.

Studies on enzymatic peptide synthesis in biphasic aqueous-organic systems with product extraction

The dipeptide derivativesZ-Tyr-Leu-NH2 andMca-Tyr-Leu-NH2 were synthesized by α-chymotrypsin-catalyzed coupling reactions in solvent systems consisting of buffer and ethyl acetate. In comparison to a pure aqueous medium, in which only insignificant synthesis takes place, the product formation is greatly enhanced in a biphasic medium due to extraction of the dipeptide into the organic phase. The influence of several reaction parameters, such as buffer concentration, reaction time, volume ratio of organic and aqueous phase, and reagent concentration on the yield ofZ-Tyr-Leu-NH2 was investigated. Replacement of the hydrophobicZ-group by the more hydrophilic chloroacetyl group resulted in better dipeptide yields at higher reaction rates.ZusammenfassungDie DipeptidderivateZ-Tyr-Leu-NH2 undMca-Tyr-Leu-NH2 wurden durch α-chymotrypsin-katalysierte Kupplung in biphasischen Systemen aus Puffer und Essigsäureethylester hergestellt. Im Vergleich zu einem rein wäßrigen Medium, in dem nahezu keine Synthese erfolgt, wird in einem biphasischen System die Synthesereaktion durch Extraktion des Dipeptids in die organische Phase wesentlich begünstigt. Es wurde der Einfluß verschiedener Reaktionsparameter, wie Pufferkonzentration, Reaktionszeit, Volumenverhältnis von organischer zu wäßriger Phase und Reaktandenkonzentration auf die Ausbeute anZ-Tyr-Leu-NH2 untersucht. Ein Austausch der hydrophobenZ-Gruppe gegen die hydrophilere Chloracetylgruppe führte zu besseren Dipeptidausbeuten bei höheren Reaktionsgeschwindigkeiten.

Synthese von N-geschütztem Eledoisin (6–11)-Hexapeptid unter Verwendung von Proteasen als Biokatalysatoren

Papain and α-chymotrypsin were used for the protease-catalyzed assembly ofBoc-protected eledoisin (6−11)-hexapeptide by (2+4)- and (3+3)-segment condensation, respectively, in aqueous-organic solvent systems. As C-components, chemically synthesizedBoc-protected peptide methyl esters were employed. The nucleophilic tetrapeptide amide was prepared by papain-catalyzed (2+2)-segment coupling, while theZ-protected C-terminal dipeptide amide could be obtained by α-chymotrypsin- and thermolysin-catalyzed peptide bond formation. In addition, the influence of various reaction conditions, such as solvent composition, nucleophile concentration and reaction time, on the yield of theBoc-protected eledoisin (6−11)-hexapeptide was determined.