Table 1 Fluorescence characteristics of reagents 1–3 and their oxidised derivatives 19–39; maximum absorption wavelength (lab), maximum
emission wavelength (lem), fluorescence quantum yield (W), extinction coefficient (e), Stokes shift (Dn) and fluorescence enhancement (FE) equal to
the W value of the derivative divided by that of the reagent
Reagents
ab/nm
Oxidised derivatives
Solvents
l
l
em/nm
W
lab/nm
e/104 M21 cm21
l
em/nm
W
Dn/103 cm21
FE
36
1, 19
Acetonitrile
Methanol
Methanol : water 5 1 : 1
Water
Acetonitrile
Methanol
Water
Acetonitrile
Methanol
Water
388
385
385
480
470
—
—
510
—
535
492
0.0090
0.0064
364
363
360
360
384
385
389
382
383
385
d
1.01
1.06
0.86
0.96
0.87
0.95
0.77
0.82
0.91
0.85
471
476
492
496
495
494
520
495
497
521
0.32
0.35
0.21
0.11
6.2
6.5
7.5
7.6
5.8
5.7
6.5
6.0
6.0
6.8
55
.420
—
a
(,0.0005)b
—
0.0031
(,0.0005)b
0.0038
0.0072
(,0.0005)b
0.0005
c
—
c
c
d
2, 29
3, 39
a
401
400
424
389
388
424
0.32
103
.64
6
58
.90
78
a
0.032
0.022
0.42
0.045
0.039
a
—
518
b
c
Not detected. Estimated from the detection limit. Insoluble in water. Cannot be calculated.
tris(2-methylphenyl)phosphine took place chemoselectively to
afford the desired 2 in 32% yield. In the same way, the
corresponding diethylphosphine 3 was obtained from f in 35%
yield. This is the first report of the direct introduction of a
phosphine unit to the benzofurazan skeleton. The oxidised
derivatives 19–39 were prepared by oxidation with tert-butyl
hydroperoxide (Scheme 2).
could be the most effective for the detection of hydroperoxides
under water-rich conditions.
Having successfully obtained the desired properties, we then
examined the utility of 1 as a representative of the reagents. As
shown in Fig. 1, a stronger fluorescence signal was produced by
higher concentrations of hydroperoxide (a and b), and a linear
relationship was obtained between the fluorescence intensity and
the concentration of the hydroperoxide (c). The detection limit
(S/N 5 3) of hydroperoxide was 1 mM in this system, which
overruns the limits of conventional reagents (DPPP: 12 mM, NBD-
DPP: 9 mM) under comparable conditions. By using the donor–
acceptor choices from this design method,9 much improved
fluorescent derivatisation reagents 1–3 are available.
Next, the fluorescence characteristics of the reagents 1–3 and
their derivatives 19–39 were investigated in polar environments so
as to mimic bio-relevant systems. The data are summarised in
Table 1. As expected, fluorescence enhancement (FE) was
observed when comparing reagents 1–3 and the oxidised
derivatives 19–39. High FE values were obtained from 1–3
(max . 420) (cf. those in DPPP and NBD-DPP: y30)
showing the success of our strategy. The Stokes shifts of 19–
39(¢5700 cm21) were larger than those of the derivatives of
conventional reagents (¡2700 cm21). Furthermore, 19–39 showed
longer emission wavelengths (471–521 nm) than the derivative of
DPPP (y387 nm). These results indicated that reagents 1–3 have
advantages in terms of avoiding interference from real samples. In
water, the oxidised derivative 19 had the highest W value (W 5 0.11)
among the three oxidised derivatives, suggesting that reagent 1
We thank Prof. T. Fukuyama (The University of Tokyo) for his
valuable comments and discussions.
Maki Onoda,*ab Hidetoshi Tokuyama,a Seiichi Uchiyama,c
Ken-ichi Mawatari,b Tomofumi Santa,a Kiyoko Kaneko,b Kazuhiro Imaia
and Kazuya Nakagomib
aGraduate School of Pharmaceutical Sciences, The University of Tokyo,
7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
bDepartment of Analytical Chemistry, School of Pharmaceutical
Sciences, Teikyo University, Kanagawa 199-0195, Japan.
E-mail: m-onoda@pharm.teikyo-u.ac.jp; Fax: +81 426 85 3717;
Tel: +81 426 85 3717
cSchool of Chemistry, Queen’s University, Belfast, Northern Ireland, UK
BT9 5AG
Notes and references
1 V. Valls, C. Peiro, P. Mun˜iz and G. T. Saez, Process Biochem.,
2005, 40, 903; M. Hashimoto, M. H. Shahdat, T. Shimada,
H. Yamasaki, Y. Fujii, Y. Ishibashi and O. Shido, Exp. Gerontol.,
2001, 37, 89.
2 Z. Feng, W. Hu and M. Tang, Proc. Natl. Acad. Sci. USA, 2004, 101,
8598; D. A. Butterfield and C. M. Lauderback, Free Radical Biol. Med.,
2002, 32, 1050.
3 M. C. Dobarganes and J. Velasco, Eur. J. Lipid Sci. Technol., 2002, 104,
420; R. A. Wheatley, Trends Anal. Chem., 2000, 19, 617.
4 A. P. de Silva, H. Q. N. Gunaratne, T. Gunnlaugsson, A. J. M. Huxley,
C. P. McCoy, J. T. Rademacher and T. E. Rice, Chem. Rev., 1997, 97,
1515.
5 K. Akasaka and H. Ohrui, J. Chromatogr., A, 2000, 881, 159.
6 M. Onoda, S. Uchiyama, A. Endo, H. Tokuyama, T. Santa and
K. Imai, Org. Lett., 2003, 5, 1459.
7 R. A. Bissell, A. P. de Silva, H. Q. N. Gunaratne, P. L. M. Lynch,
G. E. M. Maguire and K. R. A. S. Sandanayake, Chem. Soc. Rev.,
1992, 21, 187; M. Onoda, S. Uchiyama, T. Santa and K. Imai,
Anal. Chem., 2002, 74, 4089; S. Kenmoku, Y. Urano, K. Kanda,
Fig. 1 (a) Fluorescence spectra of the reaction mixtures of 1 (100 mM)
and cumene hydroperoxide (0, 1.25, 2.5, 5, 10, 15 mM) exposed at 60 uC
for 90 min and then diluted in methanol–water (1 : 1). The excitation
wavelength was 360 nm. (b) Fluorescent images of 1 before (left) and after
(right) treatment with hydroperoxide. (c) Relationship between the
fluorescence intensity (F.I.) at 492 nm and the concentration of
hydroperoxide.
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Chem. Commun., 2005, 1848–1850 | 1849