Nature and Position of Functional Group on Thiopurine Substrates Influence Activity of Xanthine Oxidase - Enzymatic Reaction Pathways of 6-Mercaptopurine and 2-Mercaptopurine Are Different

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Hemlata Tamta^1*, Sukirti Kalra¹, Ramasamy Thilagavathi², Asit K. Chakraborti², and Anup K. Mukhopadhyay¹

¹Department of Biotechnology and ²Department of Medicinal Chemistry, National Institute of Pharmaceutical Education and Research, Sector 67, S.A.S Nagar, Punjab, India-160062; fax: 0091-172-214-692; E-mail: hemu_28@yahoo.com; skalra11@yahoo.com

^* To whom correspondence should be addressed.

Received August 3, 2006; Revision received September 4, 2006

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Xanthine oxidase-catalyzed hydroxylation reactions of the anticancer drug 6-mercaptopurine (6-MP) and its analog 2-mercaptopurine (2-MP) as well as 6-thioxanthine (6-TX) and 2-thioxanthine (2-TX) have been studied using UV-spectroscopy, high pressure liquid chromatography, photodiode array, and liquid chromatography-based mass spectral analysis. It is shown that 6-MP and 2-MP are oxidatively hydroxylated through different pathways. Enzymatic hydroxylation of 6-MP forms 6-thiouric acid in two steps involving 6-TX as the intermediate, whereas 2-MP is converted to 8-hydroxy-2-mercaptopurine as the expected end product in one step. Surprisingly, in contrast to the other thiopurines, enzymatic hydroxylation of 2-MP showed a unique hyperchromic effect at 264 nm as the reaction proceeded. However, when 2-TX is used as the substrate, it is hydroxylated to 2-thiouric acid. The enzymatic hydroxylation of 2-MP is considerably faster than that of 6-MP, while 6-TX and 2-TX show similar rates under identical reaction conditions. The reason why 2-MP is a better substrate than 6-MP and how the chemical nature and position of the functional groups present on the thiopurine substrates influence xanthine oxidase activity are discussed.

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KEY WORDS: xanthine oxidase, 2-mercaptopurine, 6-mercaptopurine, 2-thioxanthine, 6-thioxanthine, 8-hydroxy-2-mercaptopurine, 2-thiouric acid, 6-thiouic acid

DOI: 10.1134/S000629790702006X

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