Remarkable stabilization of the anionic semiquinone radical of 6-azaflavin by hydrogen bonding with a receptor in chloroform

摘要

N N N N H H N Et N Et Bu H N N H H N+ R H H N C12H25 N H H N+ H N N N N H H N Et N Et Bu N N N N N O C12H25 R O 1 R = Ph, X = I 2 R = H, X = ClO4 Xndash; 3 4 6-AzaFl R = H 6-Aza-MeFl R = Me PF6 ndash; Remarkable stabilization of the anionic semiquinone radical of 6-azaflavin by hydrogen bonding with a receptor in chloroform Takeshi Kajiki,a Hideki Moriya,a Shin-ichi Kondo,a Tatsuya Nabeshimab and Yumihiko Yano*a a Department of Chemistry, Gunma University, Kiryu, Gunma 376-8515, Japan. E-mail: yano@chem.gunma-u.ac.jp b Department of Chemistry, University of Tsukuba, Tsukuba, Ibaraki 305-8571, Japan Received (in Cambridge, UK) 5th October 1998, Accepted 29th October 1998 An anionic semiquinone radical of 6-azaflavin (6-AzaFl) was found to be stabilized by hydrogen bonding of a melamine derivative bearing an N-phenylguanidinium ion in CHCl3, but not by the correponding N-unsubstituted guanidinium ion.Flavin conenzymes such as FMN and FAD exhibit diverse functions through interactions with apoproteins, in which hydrogen bondings play important roles in the regulation of redox prperties.1 Flavin semiquinone radicals are known to be stable when bound to apoproteins, whereas non-bound semiquinone radicals are unstable due to disproportionation.2 Yoneda et al.reported that the anionic semiquinone radical of flavin 6-carboxylate is stabilized by intramolecular hydrogen bonding of the 6-CO2H group at the N(5) position even in aqueous solution.3 This suggests that the hydrogen bonding to the N(5) position is essential for stabilization of an anionic semiquinone radical of flavin.This was tested by employing 6-AzaFl and a melamine derivative bearing an N-phenylguanidinium ion 1 in CHCl3. We report herein that receptor 1 is able to stabilize the anionic semiquinone radical of 6-AzaFl in CHCl3, whereas receptor 2 is unable to stabilize it. Receptor 1 was prepared by reaction of 2-butylamino- 4-diethylamino-6-(3-aminomethyl-benzylamino)-s-triazine4 with S-methyl-N-phenylisothiouronium iodide5 in EtOH, and 3 was prepared from dodecylamine and S-methyl-N-phenylisothiouronium iodide, followed by counteranion exchange with KPF6.dagger; The pKa value of the guanidinium hydrogen of 1 was determined to be 10.7 by spectroscopic pH titration at 280 nm in buffer solutions containing 20 MeCN,Dagger; which is considered to be lower than that of 2 by at least 1ndash;2 pKa units.6sect; The binding constant of 6-AzaFlmiddot;1 was determined spectrophotometrically K = (5.3 plusmn; 0.3) 3 103 dm3 mol21 in CHCl3 as described previously.4 Despite of more acidic guanidinium hydrogen of 1, the K value of 6-AzaFlmiddot;1 is smaller than that of 6-AzaFlmiddot;2 K = (1.4 plusmn; 0.1) 3 105 dm3 mol21 in CHCl3.4 This requires an explanation, since a more acidic Hdonor is known to give a larger binding constant for H-bonded complexation.7 The thermodynamic parameters for the complex formation (DH and TDS298: 227 and 26.0 kJ mol21 for 6-AzaFlmiddot;1; 234 and 25.0 kJ mol21 for 6-AzaFlmiddot;2)para; indicated that the complex formation is mainly controlled by the enthalpy term.The 1H NMR study of the complexes implied steric hindrance for complexation of 6-AzaFl and 1. Namely, as shown in Fig. 1, the larger upfield shifts of C(7)-H of 6-AzaFl upon addition of 1 rather than 2 suggest that C(7)-H is situated in a position close enough to feel the ring current of the Nphenyl ring of 1 due to the steric hindrance between C(7)-H and the ortho-H of the N-phenyl ring.Redox potentials of 6-AzaFl were determined by cyclic voltammetry in CH2Cl2.8middot; In the absence of the receptors, 6-AzaFl showed a reversible redox couple (E1/2 = 2971 mV vs. ferrocene/ferrcenium). Upon increasing the concentration of the receptors, the redox potentials shifted in a positive direction in both receptors, finally leading to fixed potentials; E1/2 = 2738 mV for 1 (5 equiv.), and 2767 mV for 2 (3 equiv.). The shifts of the potentials due to the receptors (DE1/2) are 233 mV for 1 and 204 mV for 2, corresponding to stabilization of the 6-AzaFl radical anion by 22 and 20 kJ mol21, respectively.It should be noted that the cyclic voltammogram of 6-Aza-MeFl was not affected by addition of 1. Formation of a semiquinone radical of 6-AzaFl was detected spectrophotometrically by employing the oxidation of dithiothreitol (DTT) in CHCl3 under anaerobic conditions as shown in Fig. 2. In the presence of 2 or a mixture of 3 (K = 180 plusmn; 2 dm3 mol21) and 4 (K = 150 plusmn; 6 dm3 mol21),4 the absorption spectrum of 6-AzaFl Fig. 2(a) was changed to that of 2ereduced 6-AzaFl Fig. 2(b). On the other hand, in the presence of 1, the spectrum shown in Fig. 2(c) was observed, suggesting formation of the anionic semiquinone radical of 6-AzaFl,2,9 which was confirmed to be stable for at least 48 h. With a large Fig. 1 Changes of chemical shifts of C(7)H in CDCl3 upon addition of the receptors at 25 deg;C: (5) 1, (2) 2. Chem.Commun., 1998, 2727ndash;2728 2727N N N N N H O O C12H25 N N N Bu N N H H Et N Et N H N N H H H ndash; + excess of DTT, the spectrum shown in Fig. 2(c) changed to that shown in Fig. 2(b). The spectrum shown in Fig. 2(b) was found to give that in Fig. 2(c) after O2 bubbling only with the receptor 1, suggesting formation of the radical by coproportionation of reduced 6-AzaFl and oxidized 6-AzaFl, or direct electron transfer from the reduced 6-AzaFl to O2.2b Plots of the amount of the anion radical (absorption at 525 nm) vs.1 allowed us to calculate the binding constant as 7.7 3 105 dm3 mol21 which is much larger than that of 6-AzaFlmiddot;1 due to stronger hydrogen acceptability of the anionic radical (6-AzaFlmiddot;2) as shown in Fig. 3. Formation of 6-AzaFl radical anion in the presence of 1 was also confirmed by EPR spectroscopy in CHCl3 under anaerobic conditions (Fig. 4). Although hyperfine lines could not be obtained, a g value of 2.0040 is in reasonable agreement with those obtained for other flavin radicals.In summary, we have demonstrated that the acidity of a Hdonor of a receptor molecule plays a crucial role in the stabilization of the anionic semiquinone radical of 6-azaflavin. This is the first example showing that intermolecular hydrogen bonds are able to stabilize the anionic semiquinone radical. The receptor molecule could be regarded as an apoprotein model. Furthermore the present results are of use for understanding the functional groups at the active sites of flavoenzymes which give a stable anionic semiquinone radical.This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan. Notes and references dagger; Compound 1: Yield 54, mp 178ndash;179 deg;C (EtOHndash;diethyl ether). Satisfactory elemental analyses and 1H NMR data were obtained. Compound 3: Yield 70, mp 63ndash;65 deg;C. Receptors 2 and 4, and 6-azaflavins were supplied from our previous study (ref. 4). Dagger; MeCN was added to improve the solubility of 1. sect; The pKa for 2 could not be determined by spectroscopic pH titration because of the lack of noticeable absorption changes, but was estimated to be 12ndash;13 (ref. 6). para; The thermodynamic parameters were calculated from the following data: 6-AzaFlmiddot;1; 9.1 3 103 dm3 mol21 (10 deg;C), 4.2 3 103 (20), 3.9 3 103 (30), 2.8 3103 (40). 6-AzaFlmiddot;2; 1.5 3105 dm3 mol21(20 deg;C), 1.1 3105 (30), 7.2 3 104 (40), 5.0 3 104 (50).middot; To compare the potentials, we used conditions similar to those of ref. 8. 6-AzaFl = 1.0 3 1023 mol dm23, Bu4N+ClO42 = 0.1 mol dm23, 25 deg;C. Scan rate: 100 mV s21. 1 R. M. Burnett, G. D. Darling, S. Kendal, M. E. LwQuesen, S. G. Mayhew, W. W. Smith and M. L. Ludig, J. Biol. Chem., 1974, 149, 4383; V. Massey and P. Hemmerich, Biochem. Soc. Trans., 1980, 8, 246; V. Massey, FASEB., 1995, 9, 473. 2 (a) D. E. Edmondson and G. Tollin, Top. Curr. Chem., 1983, 108, 109; (b) F.Muuml;ller, Chemistry and Biochemistry of Flavoenzymes, ed. F. Muuml;ller, CRC Press, Boston, 1991, vol. 1, p. 23. 3 T. Akiyama, F. Simeno, M. Murakami and F. Yoneda, J. Am. Chem. Soc., 1992, 114, 6613. 4 N. Tamura, T. Kajiki, T. Nabeshima and Y. Yano, J. Chem. Soc., Chem. Commun., 1994, 2583. 5 C. R. Rasmussen, F. J. Villani, Jr., L. E. Weaner, B. E. Reynolds, A. R. Hood, L. R. Hecker, S. O. Nortey, A. Hanslin, M. J. Costanzo, E. T. Powell and A. J. Molinari, Synthesis, 1988, 456; C.R. Rasmussen, F. J. Villani, Jr., B. E. Reynolds, J. N. Plampin, A. R. Hood, L. R. Hecker, S. O. Nortey, A. Hanslin, M. J. Costanzo, R. M. Howse Jr. and A. J. Molinari, Synthesis, 1988, 460. 6 C. H. Hannon and E. V. Anslyn, Bioorganic Chemistry Frontiers; Springer-Verlag, Berlin, 1993, Vol. 3, p. 193; D. D. Perrin, Dissociation Constants of Organic Bases in Aqueous Solution, Butterworths, London, 1965, p. 445. 7 C. S.Wilcox, E. Kim, D. Romanos, L. H. Kuo, A. L. Burt and D. P. Curran, Tetrahedron, 1995, 51, 621; J. DeFord, F. Chu and E. V. Anslyn, Tetrahedron Lett., 1996, 37, 1925; C-T. Chu and J. S. Siegel, J. Am. Chem. Soc., 1994,116, 5959; K. M. Neder and H. W. Whitlock, Jr., J. Am. Chem. Soc., 1990, 112, 9412. 8 E. Breinlinger, A. Niemz and V. M. Rottelo, J. Am. Chem. Soc., 1995, 117, 5379. 9 V. Massey and G. Palmer, Biochemistry, 1966, 10, 3181; D. J. Steenkamp and M. Gallup, J. Biol. Chem., 1978, 253, 4086. Communication 8/07737A Fig. 2 Absorption spectra of 6-AzaFl in the reaction with DTT. 6-AzaFl = 5.0 3 1025 mol dm23, DTT = Bu3N = 5.0 3 1024 mol dm23 in the presence of 1 or 2 (1.0 3 1024 mol dm23) in CHCl3 at 25 deg;C under N2; (a) oxidized form, (b) reduced form, and (c) anionic semiquinone radical. Fig. 3 Structure of 6-AzaFlmiddot;2 1. Fig. 4 EPR spectrum of the radical generated by reaction of 6-AzaFl (5.0 3 1023 mol dm23) with DTT (5.0 3 1023 mol dm23) and Bu3N (5.0 3 1023 mol dm23) in the presence of 1 (5.0 3 1023 mol dm23) in CHCl3 at 25 deg;C under N2. 2728 Chem. Commun., 1998, 2727ndash;2728