N.Y. Kim et al. / Tetrahedron Letters 60 (2019) 59–62
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The rate constant of borohydride signaling by 9-AA was calculated
to be 0.356 minÀ1 from the time course plot (Fig. S4, Supplemen-
tary information).
The quantitative analytical behavior of borohydride signaling
by 9-AA was investigated by fluorescence titration (Fig. 4). As the
concentration of borohydride increased, the fluorescence intensity
at 409 nm increased steadily, while the intensity at 519 nm
decreased. The detection limit for the borohydride signaling of 9-
AA was estimated to be 7.4 lM (0.11 ppm) from the plot of the flu-
orescence intensity ratio I409/I519 vs. log[BH–4] following the litera-
ture method (Fig. S5, Supplementary data) [22]. We considered
that such high analytical sensitivity is not a critical issue for the
assay of the borohydride in many practical purposes. However,
analysis of low concentration of borohydride would be a requisite
for some applications, such as monitoring of the borohydride
decomposition in solution and determination of the reduction rate
of various organic compounds [23].
Fig. 5. Partial 1H NMR spectra of 9-AA before and after treatment with borohydride
and reference 1 in DMSO d6. [9-AA] = [1] = 5.0 Â 10–3 M. [BH4–] = 1.0 Â 10–2 M. Peaks
indicated by red and blue asterisks are due to the aldehyde and methylene protons
of 9-AA and 1, respectively.
Borohydride signaling of 9-AA was attributed to the reduction
of the aldehyde functionality of 9-AA to alcohol of 1 (Scheme 1),
as confirmed by 1H NMR measurements. For instance, upon inter-
action with 2 equiv of borohydride in deuterated DMSO, the reso-
nance of the aldehyde proton in 9-AA at 11.45 ppm (indicated by a
red asterisk) disappeared, and a new resonance ascribable to the
characteristic methylene protons of 1 at 5.43 ppm (indicated with
a blue asterisk) could be identified (Fig. 5). In the 13C NMR spec-
trum, the postulated process could be evidenced by the disappear-
ance of the aldehydic carbon resonance at 193 ppm and the
appearance of a new peak for the methylene carbon of 1 at
55.8 ppm (Fig. S6, Supplementary data). This conversion was also
confirmed by the diagnostic peak at m/z = 208.11 corresponding
to signaling product 1 (calcd. for C15H12O = 208.09) in the mass
spectrum (Fig. S7, Supplementary data).
To test the generality of the borohydride signaling based on
aldehyde-to-alcohol reduction, the fluorescence signaling proper-
ties of 1-pyrenecarboxaldehyde (1-PA) were evaluated under the
optimized conditions using phosphate buffer (Na2HPO4-NaOH buf-
fer, pH 11.0, 10 mM) containing 20% DMSO (Scheme 2). Under the
signaling conditions in alkaline aqueous solution, 1-PA exhibited
two broad absorption bands centered at 368 and 396 nm (Fig. S8,
Supplementary data). Upon treatment with borohydride, 1-PA
revealed significantly blue-shifted absorption bands at 312, 324,
and 337 nm at the expense of the original bands at 368 and
396 nm.
Scheme 2. Borohydride signaling by 1-PA.
10 mM). Upon treatment with borohydride, slightly broad but
characteristic vibronic emissions of the pyrene fluorophore
emerged between 370 nm and 395 nm (inset of Fig. 6). Under the
measurement conditions, commonly encountered metal ions and
anions gave no responses (Fig. 6 and Fig. S9, Supplementary data).
Ratiometric analysis using the emission intensity ratio of the two
characteristic wavelengths at 377 nm and 473 nm (I377/I473) for
1-PA clearly showed the borohydride selectivity of the probe over
metal ions and anions. The borohydride-selective signaling of
1-PA, too, was unaffected by the presence of background metal
ions (Fig. S10, Supplementary data) and anions (Fig. S11, Supple-
mentary data), except for Fe2 and Fe3+ . These two ions induced
somewhat reduced borohydride signals (Fig. S10a, Supplementary
data), however, such interference was readily circumvented by the
ratiometric analysis using the ratio I377/I473. As shown in Fig. S10b
In fluorescence measurement, probe 1-PA showed a broad
emission at 473 nm in phosphate buffer solution (pH 11.0,
20
150
–
[BH4
]
100
50
0
15
10
5
0
20
40
60
80
–
[BH4 ] (µM)
0
373
423
473
523
573
Fig. 6. Selective ratiometric fluorescence signaling of borohydride over other
common metal ions by 1-PA, as expressed by the ratio I377/I473. Inset: fluorescence
spectra of 1-PA in the presence of borohydride or metal ions. [1-PA] = 1.0 Â 10À5 M,
[BH–4] = [Mn + ] = 5.0 Â 10À4 M, [NaOH] = 5.0 Â 10À3 M in phosphate buffer solution
(pH 11.0, 10 mM) containing 20% (v/v) DMSO. k ex = 343 nm.
Wavelength (nm)
Fig. 4. Fluorescence titration of 9-AA with borohydride. [9-AA] = 1.0 Â 10À5 M,
[BH4–] = 0–8.0 Â 10À5 M, [NaOH] = 2.5 Â 10À3 M. k ex = 368 nm.