The Chemical Synthesis of Model Catalysts Related to Enzymes

Author

John William Baker Hershey

Date of Award

1963

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

Thesis Advisor

Dilworth Woolley

Related Items

View more theses from the Dilworth Woolley Laboratory

Keywords

chemical synthesis, enzyme catalysis, histidine derivatives, imidazole compounds, kinetic studies, hydrolysis reactions

Abstract

The chemical synthesis of highly specialized peptides that might possess catalytic action provides one of the approaches to an understanding of the mechanism of enzyme action. The experiments reported in this thesis involve the chemical synthesis of model compounds designed to explore how trypsin and chymotrypsin may function. Since several lines of evidence indicate a catalytic role for histidine residues in these enzymes, it was proposed to prepare imidazole derivatives with carefully designed side chains which might attract and orient similarly shaped substrates. A histidine with a lysine-like side chain attached to the imidazole ring was proposed as a model catalyst to mimic the specific action of trypsin, cyclo-D-Histidyl-L-phenylalanyl was proposed for a study of chymotrypsin-like activity. A third project concerned the determination of whether, in the reaction of 2-benzylimidazole with p-nitrophenyl phenylacetate, the two benzyl groups might attract each other and cause an increase in the rate of hydrolysis of the ester. The work in this thesis involved primarily the synthesis of these model compounds. The "lysohistidine derivative" is a complicated 2-α-aminoalkyl-histidine and the classical imidazole syntheses routinely used to make 2,4-dialkylimidazoles failed when applied to this problem. The "lysohistidine derivative" with the various reactive groups protected was successfully prepared only through the development of a little-known reaction which involved the condensation of amidines with α-haloketones. The reactants required were ethyl 2-carbobenzoxamido-4-oxo-5-chlorovalerate and N^α-acetyl-N^ε-tosyl-lysine amidine, both previously unknown. The chloromethylketone was synthesized from α-ethyl carbobenzoxy-aspartic acid by conversion of the β-carboxyl group to an acid chloride and treatment with diazomethane followed by hydrogen chloride. As a pilot synthesis, the chloromethyIketone was caused to react with N-benzylphenylacetamidine to produce N^α-carbobenzoxy-1,2-dibenzylhistidine, which was purified by countercurrent distribution and silicic acid column chromatography. N^α-Acetyl-N^ε-tosyl-lysine amidine was prepared from N^α-acetyl-N^ε-tosyl-lysine by conversion of the carboxylic acid to an amide by the mixed anhydride method. Dehydrations of α-acylamino carboxamides to nitriles had never been reported in the chemical literature. The usual reagents for amide dehydrations failed; only phosphorous oxychloride in pyridine could be used successfully. The nitrile was then treated with dry hydrogen chloride in ethanol and the resulting ethyl imidate ester hydrochloride was converted into the amidine with alcoholic ammonia. It was necessary to use countercurrent distribution in order to purify the N^α-acetyl-N^ε-tosyl-lysine amidine hydrochloride. This amidine was then caused to react with ethyl 2-carbobenzox-amido-4-oxo-5-chlorovalerate and the desired pure "protected lysohistidine derivative" was obtained. Countercurrent distribution and silicic acid column chromatography were employed to obtain the material in analytically pure condition. cyclo-Histidyl-phenylalanyl was synthesized by coupling N^α, N^Im-dicarbobenzoxy-histidine with phenylalanine ethyl ester by the carbodiimide method. The carbobenzoxy groups were removed by catalytic hydrogenation and the diketopiperazine ring was caused to form in a refluxing ethanolic solution of the dipeptide ester free base. Both amino acids underwent racemization during this condensation. 2-Benzylimidazole and 2-isobutylimidazole were prepared from the corresponding imidazolines by catalytic dehydrogenation at 245°. In the course of the synthetic work, nineteen new compounds were synthesized, purified and characterized. Preliminary kinetic studies were made in order to assay the model compounds for their catalytic properties. The addition of the "protected lysohistidine derivative" to reaction solutions of N^α-acetyl-N^ε-tosyl-lysine ethyl ester caused no increase in the rate of hydrolysis. Similarly, no catalytic effect of cyclo-histidyl-phenylalanyl could be detected on the rates of hydrolysis of acetyl-phenylalanine ethyl ester and acetyl-phenylalanine p-methoxyphenyl ester. Also no specific enhancement of the rate of hydrolysis of p-nitrophenyl phenylacetate with 2-benzylimidazole could be demonstrated.

Comments

A thesis presented to the faculty of The Rockefeller University in partial fulfillment of the requirements for the degree of Doctor of Philosophy

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Recommended Citation

Hershey, John William Baker, "The Chemical Synthesis of Model Catalysts Related to Enzymes" (1963). Student Theses and Dissertations. 575.
https://digitalcommons.rockefeller.edu/student_theses_and_dissertations/575