Synthesis and Characterization of Model Compounds of the Lysine Tyrosyl Quinone Cofactor of Lysyl Oxidase

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Abstract
4-n-Butylamino-5-ethyl-1,2-benzoquinone (1ox) has been synthesized as a model compound for the LTQ (lysine tyrosyl quinone) cofactor of lysyl oxidase (LOX). At pH 7, 1ox has a λmax at 504 nm and exists as a neutral o-quinone in contrast to a TPQ (2,4,5-trihydroxyphenylalanine quinone) model compound, 4, which is a resonance-stabilized monoanion. Despite these structural differences 1ox and 4 have the same redox potential (ca. −180 mV vs SCE). The structure of the phenylhydrazine adduct of 1ox (2) is reported, and 2D NMR spectroscopy has been used to show that the position of nucleophilic addition is at C1. UV−vis spectroscopic pH titration of phenylhydrazine adducts of 1ox and 4, 2, and 11, respectively, reveals a similar red shift in λmax at alkaline pH with the same pKa (∼11.8). In contrast, the red shift in λmax at acidic pH conditions yields different pKa values (2.12 for 2 vs −0.28 for 11), providing a means to distinguish LTQ from TPQ. Reactions between in situ generated 4-ethyl-1,2-benzoquinone and primary amines give a mixture of products, indicating that the protein environment must play an essential role in LTQ biogenesis by directing the nucleophilic addition of the ε-amino group of a lysine residue to the C4 position of a putative dopaquinone intermediate. Characterization of a 1,6-adduct between an o-quinone and butylamine (3-n-butylamino-5-ethyl-1,2-benzoquinone, 13) confirms the assignment of LTQ as a 1,4-addition product.