Mimicking the Reaction of Phenylalanine Ammonia Lyase by a Synthetic Model

Abstract

Dedicated to Professor Albert Eschenmoser on the occasion of his 75th birthday Phenylalanine and histidine ammonia lyases (PAL and HAL) catalyze the reversible conversion of aamino acids to the corresponding acrylic acids by elimination of ammonia. The prosthetic group 3,5-dihydro-5-methylidene-4H-imidazol-4-one (MIO) at the active site of both enzymes supposedly undergoes an electrophilic attack at the aromatic nucleus in the first step of the mechanism of action. Since no chemical analogy existed for such an electrophile-assisted elimination, we synthesized model compounds, some portion of which mimicked the essentials of the substrate phenylalanine and another portion the electrophilic Michael acceptor in a sterically appropriate distance. The first model, (AE)-rel-(1R,2S,3S)-3-[1-methylidene-2-oxo-2-(pyrrolidin-1-yl)ethyl]-2-phenylcyclohexanamine (7) did not react under Friedel-Crafts conditions in the expected way (Scheme 2). The second model compound (AE)-2-rel-(1R,2S,3S)-3-(dimethylamino)-2-(3-methoxyphenyl)cyclohexyl]prop-2-enal (12) with a more nucleophilic methoxyphenyl and a more electrophilic a,b-unsaturated carbonyl moiety, underwent an intramolecular Friedel-Crafts-type substitution, but no elimination of the dimethylamino group (Scheme 4). The third model compound, (AE)-g-[(dimethylamino)methyl]-3-methoxy-2,4,6-trimethyl-a-methylidenebenzenebutanal (25) eliminated dimethylamine upon treatment with Lewis acids and subsequent hydrolysis of the intermediate (Scheme 6). When the 3-methoxy-2,4,6-trimethylphenyl moiety of 25 was replaced by the 2,4,6-trimethyl-3-nitrophenyl group, no elimination product could be observed (Scheme 7).

Friedel–Crafts‐Type Mechanism for the Enzymatic Elimination of Ammonia from Histidine and Phenylalanine

“…Evidence for the active role of the phenyl ring in the process was provided by deactivating the phenyl ring, either by removing the methoxy group or by exchanging it for a nitro group. In these cases no elimination products were detected 62…”

“…More recently, the chemical feasibility of the electrophile‐assisted elimination of ammonia was demonstrated with a chemical model 62. One portion of the model compound mimicked the essential parts of the substrate (phenylalanine), and another portion mimicked the electrophilic Michael acceptor (MIO) in a sterically appropriate position (Scheme ).…”

“…Therefore, we had to take a further measure to mimic this “negative catalysis”. When we substituted the hydrogen atoms on the phenyl ring for methyl groups (Scheme ) the completion of the Friedel–Crafts substitution was prevented, and we could isolate the desired elimination product 62…”

Enzymatische Eliminierung von Ammoniak aus Histidin und Phenylalanin: der Friedel‐Crafts‐ähnliche Mechanismus

“…Die chemische Plausibilität der elektrophil unterstützten Eliminierung von Ammoniak wurde anhand eines chemischen Modells untermauert 62. Einige Teile der Modellverbindung imitierten den wesentlichen Teil des Substrates, andere den elektrophilen Michael‐Acceptor (MIO) in einer sterisch günstigen Stellung (Schema ).…”

The Interaction of Nitrophenylalanines with Wild Type and Mutant 4‐Methylideneimidazole‐5‐one‐less Phenylalanine Ammonia Lyase

“…Although some of the results support such a mechanism, chemical analogies with the Friedel–Crafts‐like intermediate are also possible;6 kinetic data of the interaction of various heteroaryl alanines with PAL from parsley,7 and more recent works, especially X‐ray structures of ammonia lyases with bonded substrates and inhibitors, seem to favor a mechanism in which the α‐amino group of the substrate is bound to MIO 8. However, it is known that the enzymes may not crystallize in their active conformation.…”