Menadione-Mediated Phosphate Ester Hydrolysis

2009 Bulletin of the Korean Chemical Society (Print)  
O O S-En O O + -OH Reducing system or DTT 2 3 4 5 6 Scheme 1 O -OH R O R O O + R = SCH 3 (8a) R = CH 3 (8b) + R = SCH 3 (9a) R = CH 3 (9b) EtO P O OEt O -+ O 2 18-crown-6, THF O O R R = SCH 3 (7a) R = CH 3 (7b) OP(OEt) 2 O OH i) Na 2 S 2 O 3 ii) 1 eq. KH Scheme 2 Menadione (2-methyl-1,4-naphthoquinone, or vitamin K3), a synthetic congener of naturally occurring vitamin K 1 and K 2 , has increasingly been of interest given its broad range of antitumor activity in human cells. 1 Although
more » ... stress was a common mechanism of tumor growth inhibition by menadione, 2 it was reported in 1993 by Juan and Wu that menadione induces alterations in the phosphorylation status of p34 cdc2 kinase (cdk1) and the activity of protein tyrosine phosphatases (PTPases) during cell cycle progression. 3 On the basis of this result, this lab demonstrated that menadione is an inactivator of cdc25 phosphatase because cdc25 phosphatase dephosphorylates cdk at the Thr-14 and Tyr-15 residues and thus stimulates cdk activity. 4 Since then, another possible mechanism of menadione cytotoxicity has been ascribed to inactivation of cdc25 phosphatases. 5 However, some time earlier, Statdman discovered that the phosphatase derived from Clostridium Sticklandii required the presence of menadione and of added thiol for its activity. 6 The Wu group also pointed out that the p34 cdc2 activity of menadione-treated cells was increased at early recovery times for unknown reasons. 3 These observations are interesting as menadione acts as a phosphatase cofactor under certain conditions, differing from the mechanism of action of menadione as a phosphatase inactivator. Meanwhile, the mechanistic investigations of vitamin K1 have provided evidence for the production of a strong base as the outcome of oxygenation. 7 It has been suggested that the gen-dialkoxide or hydroxide ion in a hydrophobic environment might be possible candidates for the active base. At this point, it is the contention of the authors that this capacity to produce the hydroxide ion can be applied to the intriguing observations of phosphatase action. As shown in Scheme 1, addition of the thiolate group in the enzyme active site would lead directly to hydroquinone anion 2. If molecular oxygen were a constituent of the active site of the enzyme, trapping with oxygen would then lead to naphthoquinone oxide 6 formation and the production of a hydroxide ion. In the proper hydrophobic setting, the latter can be sufficient to hydrolyze an aromatic phosphate. To examine the chemical behavior of the hydroquinone anion in the presence of oxygen, first, thiomethyl vitamin K derivative 7a was prepared by treatment of menadione with methane thiol, which was then readily converted to the hydroquinone by reduction of the aqueous sodium hydrosulfite. Next, the resulting hydroquinone was treated with 1.0 eq. of potassium hydride and 18-crown-6 in THF, and the reaction mixture stirred under an atmosphere of oxygen in the presence of diethyl p-tolyl phosphate. We expected that hydrolysis of the phosphate ester would occur by the resulting hydroxide anion during oxygenation. However, treatment with oxygen did not lead to the formation of the expected hydrolysis product. The 1 H-NMR spectrum of the crude product showed small amounts of the corresponding epoxy quinine 9a; 8 no hydrolysis product was detected. In another model experiment, a tetrahydrofuran solution containing potassium hydroquinone anion 8b, from the reaction of potassium hydride and 2,3-dimethyl naphthohydroquinone, was stirred at room temperature with 18-crown-6. Syringe pump
doi:10.5012/bkcs.2009.30.8.1697 fatcat:xxk4hckq2zdwlaigyh6npz422q