Large-scale production of N,N '-diBoc-dityrosine and dityrosine by HRP-catalyzed N-Boc-L-tyrosine oxidation and one-step chromatographic purification

Chemical and Biomolecular Engineering, Yonsei University, Seoul 120-749, South Korea
PROCESS BIOCHEMISTRY (Impact Factor: 2.52). 01/2011; 46(1):142-147. DOI: 10.1016/j.procbio.2010.07.031

ABSTRACT Dityrosine (DY) can be used as a biomarker to detect oxidative protein damage and selective proteolysis. It is generally prepared by horseradish peroxidase (HRP)-catalyzed oxidation of l-tyrosine (Y) followed by multistep chromatographic separations. In this study, we present an alternative method for the preparation of DY by HRP-catalyzed synthesis of N,N′-diBoc-dityrosine (DBDY) from N-Boc-l-tyrosine (BY). The presence of the tert-butoxycarbonyl (Boc) group ensured that the fraction of further oxidized by-products (e.g., trimers and pulcherosine) was quite low. The yield of DBDY (37.5%) was comparable to that reported for DY (> 26%). DBDY could be purified by a simple one-step silica column chromatography procedure that resulted in a purity of 89.7%. DBDY is considered to be better than DY for subsequent chemical reactions (for binding to polymers, amino acids, drugs, antibodies, etc.) because such reactions can be selectively performed by using the carboxylic acid and amine groups in the following sequence: first, the carboxylic acid groups are used; next, the Boc groups are removed; and finally, the amino groups are used. To prepare DY, the Boc groups in DBDY were simply and completely removed by treatment with trifluoroacetic acid.

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    ABSTRACT: N,N'-diBoc-dityrosine (DBDY), which was synthesized by the oxidative C-C coupling of 2 N-Boc-L-tyrosine molecules, was conjugated with two isoniazid (INH) molecules. Due to the quenching effect of INH, DBDY-(INH)(2) lacks the fluorescence of DBDY. As such, it was tested for use in the detection of proteases by measuring fluorescence recovery. In this study, serine proteases (chymotrypsin, trypsin, subtilisin, and proteinase K), metalloproteases (thermolysin and carboxypeptidase A, dispase, and collagenase), aspartic proteases (pepsin and aspergillopepsin) and cysteine proteases (papain and chymopapain) were chosen. Reported optimum assay conditions were chosen for each enzyme. Only papain and chymopapain catalyzed the hydrolysis of DBDY-(INH)(2) and led to fluorescence recovery, possibly due to their extensive binding sites and the INH-mediated inhibition of metalloproteases and aspartic proteases.
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    ABSTRACT: Dityrosine (DY), which is fluorescent and biocompatible, has multiple functional groups, including two amine and two carboxylic acid groups. By conjugating PEG arms to DY, we successfully synthesized a star-shaped 4-arm PEG with two carboxylic acid and two amine groups at its four ends. Two different kinds of molecules could be further linked to this 4-arm PEG.The chemical structures and molecular weights of the intermediate 2-arm PEG and 4-arm PEG were determined by 1H NMR spectroscopy and matrix assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS).
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    ABSTRACT: The increasing number of reports for disease-related proteases has necessitated materials for the fast, sensitive, and specific assessment of protease activities. The porpose of this study is to synthesize and test a dityrosine-based substrate for the selective assay of a specific cysteine cathepsin. DBDY-(Gly-INH)(2) was synthesized from the conjugation of N,N'-diBoc-dityrosine (DBDY) with two molecules of glycine and isoniazid (INH) for this purpose. The fluorescence of DBDY (λ(ex)=284-320 nm; λ(em)=400-420 nm) disappeared due to the quenching effect of INH. However, the protease-catalyzed hydrolysis resulted in the release of INH and recovered the fluorescence of DBDY. When reacted with 13 proteases, DBDY-(Gly-INH)(2) was hydrolyzed by the cysteine proteases only. Meeting the growing need to discriminate cysteine cathepsins (e.g., cathepsins B, L, and S found at high levels in various cancers), DBDY-(Gly-INH)(2) was tested as a substrate for cathepsins B, L, and S. Only cathepsin B catalyzed the hydrolysis reaction among the three cathepsins. The reaction rate followed the Michaelis-Menten kinetics, and the K(M) and k(cat)/K(M) values were 2.88 μM and 3.87×10(3) M(-1)s(-1), which were comparable to those for the materials reported for the selective assay of cathepsin B. Considering the simple preparation of DBDY-(Gly-INH)(2), DBDY-(Gly-INH)(2) is believed to be valuable for the sensitive and selective assay of cathepsin B activity.
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