biomatrices. We now report the development of a more useful
reagent for hydroperoxides utilizing our general procedure
for designing fluorescent “off-on” reagents7,8 based on the
most favored device for fluorescence switching in recent
years, i.e., photoinduced electron transfer (PET)9,10 (so-called
“PET reagents”11).
First, we designed 1 and 3 as PET reagents for hydro-
peroxides according to the following three-step procedure
(the structures of 1 and 3, and the corresponding oxidized
derivatives, 2 and 4, are shown in Scheme 1). Tri-
zoxadiazole (NBD-NHMe), on the other hand, was chosen
as the fluorophore,13 since it strongly fluoresces and its
excitation and emission wavelengths are sufficiently long to
avoid any interference from biomatrices (Φ ) 0.38, excita-
tion ) 458 nm, and emission ) 524 nm in acetonitrile14)
(step 1). The electron-donating ability of the reactive moiety
to the fluorophore was examined by the HOMO energy and/
or the K value of the Stern-Volmer plots. The PM3/COSMO
calculation indicated that the HOMO energies of tri-
phenylphosphine (-9.488 eV) and methyldiphenylphosphine
(-9.029 eV) in acetonitrile were greater than those of the
corresponding phosphine oxides (-9.916 and -9.910 eV,
respectively). The Stern-Volmer plots for NBD-NHMe gave
relatively high K values for triphenylphosphine (67.1) and
methyldiphenylphosphine (81.6) in acetonitrile, whereas
those for the corresponding oxides were negligible, thus
showing that the oxides were unable to quench NBD-NHMe
fluorescence (step 2). These results indicate that the phos-
phine moieties possess a high electron-donating ability to
NBD-NHMe, whereas the phosphine oxide moieties do not.
Accordingly, 1 and 3 have been designed by connecting the
phosphine and NBD-NHMe moieties with a methylene
spacer (step 3).15
Scheme 1. Synthesis of the Reagent (1 and 3) and Their
Oxidized Derivatives (2 and 4)a
Compounds 1 and 3, and their oxidized products were then
synthesized in a straightforward manner as depicted in
Scheme 1. Coupling reaction of commercially available
diphenylphosphinoethylamine and NBD-F by addition-
elimination type reaction furnished 1, which was readily
oxidized by treatment with MCPBA. Preparation of 4, on
the other hand, commenced with conversion of the com-
mercially available benzoic acid derivative to its amide. After
reduction of amide to benzylamine derivative, 3 was obtained
by addition to NBD-F. The corresponding oxidized product
was prepared by oxidation with MCPBA.
Next, the fluorescence characteristics of 1-416 synthesized
according to Scheme 1 were determined. The data in
acetonitrile, methanol, and benzene are summarized in Table
1. As expected, in acetonitrile, 1 and 3 were weakly
fluorescent, whereas 2 and 4 were strongly fluorescent with
long excitation and emission wavelengths (λex ) 454-458,
λem ) 520 nm). In particular, the Φ value of 2 was quite
large (Φ ) 0.44) and 31 times greater than that of 1,
indicating that 1 would be a sensitive PET reagent for
hydroperoxides. Similar behavior of 1-4 was also observed
in methanol. In benzene, however, 3 unexpectedly fluoresced.
The high Φ value of 3 in benzene is along the lines of a
previous report17,18 in which the PET process occurred more
efficiently in polar solvents than in nonpolar solvents.
a Reagents and conditions: (i) 4-fluoro-7-nitro-2,1,3-benzoxa-
diazole (NBD-F), MeCN, rt, 30 min (37% for 1, 42% for 3); (ii)
MCPBA, MeCN, rt, 30 min (93% for 2, 91% for 4); (iii) (PNCl2)3,
benzene, rt, 1 h; (iv) aqueous NH3, MeCN, rt, 40 min (56%; two
steps); (v) LiAlH4, THF, reflux, 1 h (89%).
phenylphosphine and methyldiphenylphosphine were chosen
as the reactive moieties, since they are stable and react with
hydroperoxides to give the corresponding phosphine oxides
under mild conditions.12 4-Methylamino-7-nitro-2,1,3-ben-
(7) Uchiyama, S.; Santa, T.; Imai, K. Analyst 2000, 125, 1839.
(8) Onoda, M.; Uchiyama, S.; Santa, T.; Imai, K. Anal. Chem. 2002,
74, 4089. In this research, a new fluorescent PET reagent, 4-ethylthioacetyl-
amino-7-phenylsulfonyl-2,1,3-benzoxadiazole (EPB) was developed for
peroxyacetic acid. However, EPB was nonreactive to hydroperoxides.
(9) For recent reviews on PET reagents, see: (a) de Silva, A. P.;
Gunnlaugsson, T.; Rice, T. E. Analyst 1996, 121, 1759. (b) de Silva, A. P.;
Gunaratne, H. Q. N.; Gunnlaugsson, T.; Huxley, A. J. M.; McCoy, C. P.;
Rademacher, J. T.; Rice, T. E. Chem. ReV. 1997, 97, 1515. (c) de Silva, A.
P.; Fox, D. B.; Huxley, A. J. M.; Moody, T. S. Coordin. Chem. ReV. 2000,
205, 41. (d) de Silva, A. P.; Fox, D. B.; Moody, T. S.; Weir, S. M. TRENDS
Biotechnol. 2001, 19, 29.
(10) For recent progress, see: (a) He, H.; Mortellaro, M. A.; Leiner, M.
J. P.; Young, S. T.; Fraatz, R. J.; Tusa, J. K. Anal. Chem. 2003, 75, 549.
(b) Gunnlaugsson, T.; Nieuwenhuyzen, M.; Richard, L.; Thoss, V. J. Chem.
Soc., Perkin Trans. 2 2002, 141. (c) Turfan, B.; Akkaya, E. U. Org. Lett.
2002, 4, 2857.
(12) Cadogan, J. I. G.; Mackie, R. K. Chem. Soc. ReV. 1974, 3, 87.
(13) For a recent review on fluorescent benzoxadiazoles, see: Uchiyama,
S.; Santa, T.; Okiyama, N.; Fukushima, T.; Imai, K. Biomed. Chromatogr.
2001, 15, 295.
(14) Uchiyama, S.; Santa, T.; Imai, K. J. Chem. Soc., Perkin Trans. 2
1999, 2525.
(15) This is the first utilization of a phosphorous atom for PET reagents
despite at least two hundred previous reports on the various PET reagents.
(16) For detailed synthetic procedures and spectral data, see Supporting
Information.
(17) (a) Bissell, R. A.; de Silva, A. P.; Fernando, W. T. M. L.;
Patuwathavithana, S. T.; Samarasinghe, T. K. S. D. Tetrahedron Lett. 1991,
32, 425. (b) Poteau, X.; Brown, A. I.; Brown, R. G.; Holmes, C.; Matthew,
D. Dyes Pigm. 2000, 47, 91.
(11) de Silva, A. P.; Gunaratne, H. Q. N.; Gunnlaugsson, T. Tetrahedron
Lett. 1998, 39, 5077.
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