Table 1 Spectroscopic data of Fura-2FF-O6-benzylguanine 1 and the SNAP-tag conjugate SNAP-Fura-2FF-1
Absorption maxima
KDapp/mM Excitation maxima
Zero Ca2+ Excess Ca2+ Zero Ca2+ Excess Ca2+ Zero Ca2+ Excess Ca2+
Emission maxima
Quantum yield
Zero Ca2+ Excess Ca2+
Fura-2FF (commercial)
Fura-2FF-O6-benzylguanine 1 380 nm
SNAP-Fura-2FF-1 376 nm
365 nm
336 nm
347 nm
344 nm
3.8 6.0
3.9 2.8
4.3 1.5
365 nm
371 nm
376 nm
339 nm
352 nm
350 nm
514 nm
533 nm
525 nm
507 nm
532 nm
519 nm
0.34
0.13
0.26
0.53
0.13
0.26
oxazole 6 in 50% yield.18 Hydrogenolysis of the Fura-2FF ester
7 deprotected the benzyl ester and gave the Fura-2FF acid
derivative 8 with all BAPTA carboxylates protected as methyl
esters. For further coupling of the Fura-2FF to benzylguanine
we employed a copper-catalyzed variant of the Huisgen 1,3
dipolar cycloaddition reaction, i.e. click chemistry.19 3-Amino-1-
azidopropane was coupled to acid 8 in DMF using HOBt/EDAC
as coupling reagents giving 9 in 59% yield. Hydrolysis of methyl
ester 9 with KOH in water–MeOH followed by acidification with
1 M HCl gave the corresponding tetraacid in 97% yield. Alkylation
of this tetraacid with bromomethyl acetate in the presence of
DIPEA afforded the AM derivative 11 in 65% yield. Reaction
of 11 with the benzylguanine alkyne derivative 10 in the presence
of sodium ascorbate and copper sulfate gave the AM ester 2 in
35% yield. The non-permeable product 1 was obtained by directly
coupling the ester compound 9 with the benzylguanine alkyne 10 in
61% yield and subsequently hydrolysing the methyl esters. To verify
that 1 is a substrate of SNAP-tag, 1 was incubated with purified
SNAP-tag and the reaction monitored by gel electrophoresis
and subsequent in-gel fluorescence scanning (Fig. S2, ESI†). As
observed for various other benzylguanine derivatives,12 1 reacted
readily with SNAP-tag to form the covalent conjugate SNAP-1.
UV-Vis absorption spectroscopic properties of 1 and its cor-
responding SNAP-tag conjugate (abbreviated in the following as
SNAP-Fura-2FF-1) were examined. Like all Fura-2 derivatives,
the calcium complexes of 1 and SNAP-Fura-2FF-1 absorbed
at shorter wavelengths than the free anion form present at zero
calcium concentrations (Table 1). 1 and SNAP-Fura-2FF-1 were
red-shifted by 11–15 nm in their free anion form and by 9–11 nm in
their calcium complex form relative to unmodified Fura-2FF. This
difference probably originates from the coupling of BG to Fura-
2FF via the carboxyl group, i.e. changing the carboxylate to a
neutral amide.
1 and SNAP-Fura-2FF-1 show a strong shift of at least 19 nm in
their fluorescence excitation maxima upon calcium binding, mak-
ing them useful ratiometric calcium indicators (Fig. 2, Table 1). In
comparison to the parent compound Fura-2FF, the fluorescence
excitation and (to a lesser extent) the emission maxima of 1 and
SNAP-Fura-2FF-1 are red-shifted by 6–25 nm. For example, the
excitation maximum of the calcium-bound form is 350 nm for
SNAP-Fura-2FF-1, while it is 336 nm for Fura-2FF. Quantum
yields were obtained by comparison with the fluorescence of Fura-
2, whose quantum yield has been published.3 SNAP-Fura-2FF-1
has a higher quantum yield than 1, possibly because guanine acts
as fluorescence quencher.13
This is advantageous for measurements in cells as it would lead
to a lower signal from unreacted indicator. In the free anion
form, SNAP-Fura-2FF-1 has a quantum yield similar to Fura-
2FF, while the calcium complex of SNAP-Fura-2FF-1 has a lower
quantum yield than Fura-2FF (Table S1, ESI†).
Ideally, the excitation wavelengths used to measure calcium
concentrations with SNAP-Fura-2FF-1 should not be set at the
standard wavelengths for Fura-2FF, 340 nm and 380 nm, but at
longer wavelengths corresponding to the fact that their spectra
are red-shifted. Calculation of the difference spectra between the
free anion and the calcium complex form suggests that the biggest
fluorescence changes occur at excitation wavelengths of 337 nm
and 387 nm (Table S1, ESI†). Correspondingly, excitation filters
around 340 and 390 nm and an emission filter around 530 nm
should be best suited for measurements with SNAP-Fura-2FF-1.
The wavelength choice and the spectral width of the filters chosen
and the background subtraction make a considerable difference
in the ratio quotients. The ratio quotient is obtained by dividing
the excitation ratios measured for the calcium complex and for the
free anion (ESI†). SNAP-Fura-2FF-1 possesses a ratio quotient
of 11.7, which is about half the ratio quotient of Fura-2FF of 22.9
(Table S1, ESI†).
Fluorescence spectra were used to determine the apparent
app
dissociation constants for calcium, KDapp. The KD for SNAP-
Fura-2FF-1 is comparable to that of Fura-2FF (Table 1). This high
app
KD should make SNAP-Fura-2FF-1 an appropriate indicator
for elevated calcium concentrations as they can occur near calcium
influx sites during signaling events.
In summary, we have synthesized and characterized a derivative
of the popular ratiometric calcium indicator Fura-2FF that can be
specifically coupled to SNAP-tag. As SNAP-tag fusion proteins
can be precisely localized within cells, the indicator has the
potential for probing changes in calcium concentrations at specific
signal sites. Future experiments will focus on the use of the
indicator in living cells.
Fig. 2 Fluorescence excitation spectra of SNAP-Fura-2FF-1. Ca2+
concentrations are 0 mM (10 mM EGTA) and 1 mM. The KD of
SNAP-Fura-2FF-1 is 4.3 mM. Emission was recorded at 520 nm. The
buffer used was 30 mM MOPS/KOH, pH = 7.2, 100 mM KCl in water.
app
3400 | Org. Biomol. Chem., 2010, 8, 3398–3401
This journal is
The Royal Society of Chemistry 2010
©
Ruggiu, Agostina A.
