Compound 1 displays weak fluorescence. When 150 equiv. of Cr3+ was added to the solution, a significant increase of the
fluorescence intensity at 556 nm, which was attributed to the Cr3+ induced ring-opening of the spirolactam moiety, was observed. The
significant fluorescence enhancement of up to 13-fold with a bright yellow-green emission was shown in Fig. 4, which suggested a
higher fluorescence selectivity of 1 towards Cr3+ compared to the other tested ions.
3
+
To understand the binding mode-fluorescence change relationship, the binding mechanism of compound 1 with Cr was studied.
According to the literature [9], a reasonable binding mechanism was proposed (Scheme 2). The spirolactam moiety of the rhodamine
3
+
3+
group acts as a signal switcher, when 1 binds Cr , the fluorescence-off state of 1 converts to the Cr -promoted ring-opened amide
form with a fluorescence-on state. In this work, 1 is most likely to bind Cr3+ via the hydrazide and quinoline N and O atoms like other
3
+
reported researches [10]. The fluorescence intensity at 556 nm was plotted as a concentration of Cr , and detection limit was
calculated to be 5.6×10 L/mol by using detection limit 3σ/k: Where σ is the standard deviation of blank measurement, k is the slope
between the fluorescence intensity versus Cr3+ concentration [11].
To demonstrate the feasibility of 1 for its application in in vivo imaging, Cr imaging tests were performed in C. elegans. In our
previous work, we successfully tested and evaluated the mercury probes and their fluorescence imaging applications in cells, C.
elegans and zebrafish [12]. In present study, the application of 1 in in vivo imaging was evaluated by visualizing the distribution of
-6
3
+
3
+
3+
Cr in nematodes previously incubated with various concentrations of Cr for 3 h.
-
5
C. elegans larvae at developmental stage 4 (L4) were incubated in Petri dishes filled with M9 buffer, which contained 1 (1.0 × 10
o
mol/L), at 20 C for 2 h. Almost no fluorescence could be observed in this case (Fig. 5d). Very weak fluorescence was observed in the
3
+
-5
3+
pretreated nematodes until the concentration of Cr reached 75 × 10 mol/L (Fig. 5e). With the dose of Cr was increased,
fluorescence emission became brighter accordingly. Green fluorescence emission color was mainly observed in the intestinal part of
3
+
the pretreated nematodes (Fig. 5f). It suggested that the aggregation of exogenous Cr mainly locates in the intestine of C. elegans.
These results showed that 1 is a good bonding ligand for Cr and it could be used for fluorescence imaging and monitoring
intracellular Cr in C. elegans.
. Conclusion
In conclusion, a rhodamine-based probe (1) was studied for colorimetric and fluorescent sensing of Cr in vitro and in vivo. The
3
+
3
+
3
3
+
3
+
addition of Cr induced ring-opening of the spirolactam moiety in 1 and generated a distinct “off/on” fluorescent change with a
solution color change. A selective significant fluorescence enhancement of up to 13-fold, with a bright yellow-green emission, was
3
+
only observed in the case of Cr , among all the tested ions. Compound 1 was successfully applied in the in vivo fluorescent imaging of
3
+
3+
Cr in C. elegans. The low toxicity, high penetrability and selectivity of 1 supported its further utility in Cr tests of polluted
environmental substances and tobacco samples.
4. Experimental
4.1. Reagents and chemicals
All reagents were of analytical grade or the best grade commercially available, and were put into use without further purification.
Deionized water was used throughout. Tris-HCl buffer solutions (0.01 mol/L, pH 7.4) were prepared in deionized water. Analyte
+
+
+
+
2+
2+
2+
2+
2+
2+
3+
2+
3+
solutions of the perchlorate of Na , K , Li , Ag , Co , Ni , Cu , Zn , Pb , Cd , Fe , Hg and Cr were prepared by dissolving the
salts in distilled water to final concentrations of 0.1 mol/L.
4
.2 Synthesis of compound 5
A solution of 2-aminopyridine (188 mg, 2 mmol) and 0.3 mL trimethylaminein was stirred for 30 min at C in CH
chloroacetyl chloride (0.175 mL, 2.2 mmol) was added to the solution, and the mixture was stirred for 3 h at room temperature. After
the reaction, the solution was extracted with H O (5 mL× 3), then the combined organic layer was concentrated under reduced pressure.
The crude product was purified by column chromatography with CH Cl /EtOAC (v/v, 3:1) to get the target compound 5 (270 mg,
): δ 8.87 (s, 1H), 8.33-8.32 (d, 1H, J = 5), 8.21-8.18 (d, 1H, J = 15), 7.77-7.72 (m, 1H),7.09-7.13 (m,
o
2
2
Cl . Then
2
2
2
1
7
1
8.5%). H NMR (500 MHz, CDCl
H), 4.21 (s, 2H).
3
4.3 Synthesis of compound 1
A solution of compound 4 [6] (584 mg, 1 mmol), K
stirred for 1 h. Then compound 5 (205 mg, 1.2 mmol) was added to the stirred solution and the mixture was refluxed for 12 h under N
atmosphere. The solution was then cooled to 0 ℃ and adjusted to neutral condition with 1 mol/L NH Cl. The solution was extracted
with CH Cl (10 mL × 3), then the combined organic layer was concentrated under reduced pressure. The crude product was purified
by column chromatography with CH /EtOAC (v/v, 3:1) to afford the target product 1 (430 mg, 60%). H NMR (500 MHz, DMSO-
2
CO
3
(276 mg, 2 mmol) and NaI (84 mg, 0.56 mmol) in 30 mL acetone was
2
4
2
2
1
2
Cl
): δ 10.72 (s, 1H), 8.76 (s, 1H), 8.32-8.28 (m, 2H), 7.98-7.96 (d, 1H, J = 10 Hz), 7.90-7.88 (d, 1H, J = 10 Hz), 7.62 (s, 1H), 7.57 (s,
H), 7.50-7.48 (m, 3H), 7.21 (s, 1H), 7.06- 7.05 (d, 1H, J = 5 Hz), 6.35 (s, 2H), 6.26 (s, 2H), 5.08-5.06 (t, 2H, J = 6.67 Hz), 5.03 (s,
2
d
1
2
6
1
3
H), 3.12-3.09 (m, 4H), 1.83 (s, 6H), 1.20-1.16 (m, 6H). C NMR (125 MHz, DMSO-d ): δ 165.19, 152.34, 151.84, 147.96, 141.75,
6
Li, Xue-Mei
