FIELD OF THE INVE TION

Fluorescent probes selectively chelating Sn ²⁺ .

BACKGROUND OF THE INVENTION .

Stannous (Sn ²⁺ ) is added to toothpaste to prevent dental plaque and oral disease. Sn ²⁺ is found to effectively inhibit certain bacteria that can lead to tooth decay in human interproximal dental plaque. More recently, there has been increasing interest in the biological roles of Sn ²⁺ because tin is an essential trace mineral for humans and is found in the greatest amount in the adrenal gland, liver, brain, spleen and thyroid gland. There is some evidence that tin is involved in growth factors and cancer prevention. Deficiency of tin may result in poor growth and hearing loss, but excess tin accumulation can negatively affect respiratory and digestive systems. However, studies of the physiological role and bacteriostatic mechanism of tin ion are restricted by the lack of versatile Sn ²⁺ detection methods applicable to living cells - either eukaryotic or prokaryotic.

There is a need for chemical probe that is highly selective for Sn ²⁺ in the presence of various metal ions and will exhibit high fluorescence upon Sn ²⁺ chelation. There is a further need for such probe for use in living cells. There is yet a further need for a probe that minimizes background noise while providing high fluoresce intensity in living cells consistent where Sn ²⁺ is found in the cell.

It is an advantage is to have a probe that works well in pH conditions in an organelle that plays a role in Sn accumulation in the cell.

It is a further advantage to have a probe that is relatively easy and simple to synthesize.

SUMMARY OF THE INVENTION

The present invention address this need by the surprising discovery of a Sn ⁺ fluorescent probe containing rhodamine B derivative moiety as fluorophore, linked via amide moiety to a carbazate group. The use of this class of probe compounds is demonstrated as an imaging probe for monitoring Sn ²⁺ in living cells to study the physiological function of Sn ²⁺ in biological systems. This class of compounds is particularly useful given the additional surprising discovery that lysosomes appear to be an organelle where Sn concentrations are found. And given the rather acidic microenvironment of this organelle, the probes of the present invention exhibit high fluorescent intensity and yet minimizes background noise, compared to other probes that are otherwise subject to low pH induced fluoresce. In other words, a comparative probe, given the acidity of the lysosome, leads to undesirably induce fluorescence emission (thereby generating background noise).

A first aspect of the invention provides for a compound having the following Formula (I):

Formula (I)

wherein Ri is unsubstituted, branched or unbranched, alkyl, alkenyl, or alkynyl; and wherein R ₂ , R ₃ , R ₄ , R ₅ , R6, and R ₇ are each independently a hydrocarbyl; or an optical isomer, diastereomer or enantiomer for Formula (I), or a salt thereof.

In one embodiment, Ri is unsubstituted, branched or unbranched, Ci-Cio alkyl, preferably Ci-Cg alkyl. In another embodiment, Ri is selected from the group consisting of methyl, ethyl, propyl, butyl, isobutyl, pentanyl, and hexanyl, preferably isobutyl.

In one embodiment, R ₂ , R ₃ , R4, R5, R5, and R ₇ are each independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroakyl, heteroalkenyl, heteroalkynyl, heterocycloalkyl, heterocycloalkenyl, heteroaryl, and wherein the aforementioned may be substituted or unsubstituted. In another embodiment, R ₂ , R ₃ , R4, R ₅ , R ₆ , and R ₇ are each independently selected from H, C ₁ -C ₁₀ alkyl, alkenyl, or alkynyl, and wherein the aforementioned may be substituted or unsubstituted, preferably unsubstituted. In yet still another embodiment, R ₂ and R ₇ are hydrogen, and/or R ₃ , R4, R5, and R ₆ are each independently selected from hydrogen, or Ci to C ₅ alkyl, branched or unbranched, preferably unsubstituted Ci to C ₅ alkyl. In yet still another embodiment, R ₃ , R ₄ , R ₅ , and 5, are each independently selected from unsubstituted Ci to C ₃ alkyl. Another aspect of the invention provides a compound of Formula (II):

Formula (II) wherein Ri is unsubstituted, branched or unbranched, C ₁ -C ₁₂ alkyl or alkenyl; or an optical isomer, diastereomer or enantiomer for Formula (I), or a salt thereof. In one embodiment, Ri is an unsubstituted C ₁ -C ₁₀ alkyl, preferably Ri is an unsubstituted, branched or unbranched, Ci-C ₈ alkyl. In yet still another embodiment, Ri is selected from the group consisting of methyl, ethyl, propyl, butyl, isobutyl, pentanyl, and hexanyl, preferably isobutyl.

In another aspect of the invention, a compound according to Formula (I) or (II) is provided, wherein the compound is selected from the group consisting of:

(a) Tert-butoxy-carboxamide, N-[3',6'-bis(diethylamino)-3-oxospiro

[lH-isoindole-l,9 ^* -[9H]xanthen]-H)-yl]- ;

(b) Tert-butoxy-carboxamide,N-[3',6'-bis(dimethylamino )))-3 -oxospiro [ 1 H-isoindole- 1,9'-

[9H]xanthen]-2(3H)-yl]-;

(c) Methoxy-carboxamide,N-[3',6'-bis(diethylamino )))-3 -oxospiro [ 1 H-isoindole- 1,9'- [9H]xanthen]-2(3H)-yl]-;

(d) Ethoxy-carboxamide,N-[3',6'-bis(diethylamino )))-3 -oxospiro [ 1 H-isoindole- 1,9'- [9H]xanthen]-2(3H)-yl]-; and

(e) Methoxy-carboxamide,N-[3',6'-bis(dimethylamino )))-3 -oxospiro [ 1 H-isoindole- 1,9'- [9H]xanthen]-2(3H)-yl]-; and

(f) Ethoxy-carboxamide,N-[3',6'-bis(dimethylamino )))-3 -oxospiro [ 1 H-isoindole- 1,9'- [9H]xanthen]-2(3H)-yl]-. In yet another embodiment, the compound is selected from: N-(3',6'-bis(diethylamino)-3- oxospiro[isoindoline-l,9'-xanthen]-2-yl)propionamide; N-(3',6'-bis(diethylamino)-3- oxospiro[isoindoline- 1 ,9'-xanthen]-2-yl)butyramide; and N-(3',6'-bis(diethylamino)-3- oxospiro[isoindoline- 1 ,9'-xanthen]-2-yl)pentanamide.

Yet still another aspect of the invention provides for a method of detecting fluorescence in a biological cell comprising the steps: (a) incubating the biological cell with a compound described above (e.g., a compound of Formula (I) or Formula (II), or preferred or alternative compound embodiments within said Formulas (I) or (II)); (b) shining excitation light to the incubated cell , preferably wherein the shined light has wavelength of at least from 520 to 580 nm, alternatively at 560 nm; and (c) detecting light emission from the compound from 560 to 660 nm. In one embodiment, the method further comprises subjecting the biological cell to Sn ²⁺ , alternatively wherein the biological cell is selected from an oral epithelial cell or Streptococcus genus of bacterium, alternatively wherein the biological cell is any eukaryotic cell. In another embodiment, the light emission detection is at a lysosome organelle of eukaryotic cell. In yet still another embodiment, the method is conducted with at least one specific compound previously described above or herein.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1 - 5 are provided. Figure 1 (a) and (c) is fluorescence spectra of comparative Compound Rl and inventive Compound R2 upon addition of Sn ²⁺ ; (b) and (d) is fluorescent spectra of Compounds Rl and R2 upon various metal ions with an excitation of 560 nm, respectively.

Figure 2 shows dependence of fluorescence at 580 nm of comparative Compound Rl and inventive Compound R2 (10 μΜ) at different pH range. Excitation is at 560 nm.

Figure 3 are CLSM images of KB cells, (al-cl) and (a2-c2) Cells are separately incubated with 10μΜ comparative Compound Rl and inventive Compound R2 for 30 min, (dl-fl) and (d2- f2) followed incubated with 50μΜ SnF ₂ for 30 min. Emission is collected in red channel at 560- 660 nm (bl, el, b2, e2); al, dl, a2, d2 are bright field images andcl, fl, c2, f2 are overlay images, respectively (λεχ = 543 nm).

Figure 4 are CLSM images of KB cells, (al- dl) Cells are incubated with 10 μΜ Rl and

ΙμΜ Lyso Tracker Green for 30 min; (a2-d2) successively incubated with 10 μΜ R2, 50μΜ SnF _2i ΙμΜ Lyso Tracker Green each for 20 min. Emission is collected in red channel (bl, b2) at 560- 660 nm (λεχ = 543 nm) or in green channel at 500-540 nm (λεχ = 488 nm); al, a2, are bright field images and dl, d2 are overlay images, respectively.

Figure 5 is CLSM images of KB cells, (al- dl) Cells are successively incubated with 50μΜ SnF _{2 ,} 10 μΜ of comparative Compound Rl and ΙμΜ LysoTracker Green each for 20 min; (a2-d2) successively incubated with 50μΜ SnF ₂ , 10μΜ of inventive Compound R2 and ΙμΜ LysoTracker Green each for 20 min. Emission is collected in red channel (bl, b2) at 560-660 nm (λεχ = 543 nm) or in green channel at 500-540 nm (λεχ = 488 nm); al, a2, are bright field images and dl, d2 are overlay images, respectively. DETAILED DESCRIPTION OF THE INVENTION

Definitions:

For purposes of the present invention the term "hydrocarbyl" is defined herein as any organic unit or moiety which is comprised of carbon atoms and hydrogen atoms. Included within the term hydrocarbyl are heterocycles. Non- limiting examples of various unsubstituted non- heterocyclic hydrocarbyl units include pentyl, 3-ethyloctanyl, 1,3-dimethylphenyl, cyclohexyl, cis-3-hexyl, 7, 7-dimethylbicyclo[2.2.1]-heptan-l-yl, and napth-2-yl. Included with the definition of "hydrocarbyl" are the aromatic (aryl) and non-aromatic carbocyclic rings. The term "heterocycle" includes both aromatic (heteroaryl) and non-aromatic heterocyclic rings.

The term "substituted" is used throughout the specification. The term "substituted" is defined herein as "encompassing moieties or units which can replace a hydrogen atom, two hydrogen atoms, or three hydrogen atoms of a hydrocarbyl moiety. Also substituted can include replacement of hydrogen atoms on two adjacent carbons to form a new moiety or unit." For example, a substituted unit that requires a single hydrogen atom replacement includes halogen, hydroxyl, and the like. A two hydrogen atom replacement includes carbonyl, oximino, and the like. A two hydrogen atom replacement from adjacent carbon atoms includes epoxy, and the like. Three hydrogen replacement includes cyano, and the like. An epoxide unit is an example of a substituted unit which requires replacement of a hydrogen atom on adjacent carbons. The term "substituted" is used through the present specification to indicate that a hydrocarbyl moiety, inter alia, aromatic ring, alkyl chain, can have one or more of the hydrogen atoms replaced by a substituent. When a moiety is described a "substituted" any number of the hydrogen atoms may be replaced. For example, 4-hydroxyphenyl is a "substituted aromatic carbocyclic ring," (N, N- dimethyl-5-amino)octanyl is a "substituted Cg alkyl unit, 3-guanidinopropyl is a "substituted C ₃ alkyl unit," and 2-carboxypyridinyl is a "substituted heteroaryl unit". In one embodiment, wherein R ₂ , R3, R ₄ , R5, R ₆ , and R ₇ are each independently selected from the group consisting of H, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroakyl, heteroalkenyl, heteroalkynyl, heterocycloalkyl, heterocycloalkenyl, heteroaryl, substituted, and wherein the aforementioned may be substituted or unsubstituted. These terms are well known in the art. For a detailed definition, see U.S. Pat. No. 6,919,346 B2 at column 2, line 61 to column 9, line 53, incorporated herein by reference.

Synthesis path of Tert-butoxy-carboxamide,N-[3 ^, ,6 ^, -bis(diethylamino)-3-oxospiro[lH-isoindole- 1.9'-r9H1xanthen1-HVvn- (herein after "Compound R2" or simply "R2"

Compound R2, an exemplary compound of the present invention, is rhodamine B derivative moiety linked via amide moiety to tert-butyl carbazate group. The synthesis path of this compound is provided.

¾ H c s U it \ /

HiN~ H _? · & ^■ ·>() Η·ι „ A ><■

The synthesis of Compound R2 is described. Unless otherwise noted, materials were obtained from commercial suppliers and were used without further purification. Rhodamine (95%) is obtained from Sinopharm Chemical Reagent Co., Ltd. (Shanghai). Other chemicals are provided from Shanghai No. 1 chemical reagent. Flash chromatography is carried out on silica gel (200-300 mesh). The 1H NMR (500 MHz) and ¹³ C NMR (125 MHz) spectra is recorded on a Bruker DRX-500 spectrometer. Proton chemical shifts are reported in parts per million downfield from tetramethylsilane (TMS). HRMS is recorded on LTQ-Orbitrap mass spectrometer (ThermoFIsher, San Jose, CA). Melting points are determined on a hot-plate melting point apparatus XT4-100A and uncorrected. UV-Vis spectra are recorded on a Shimadzu UV-2250 spectrophotometer. Fluorescence spectra are recorded on an Edinburgh FLS-920 spectrophotometer. All pH measurements are made with a model Mettler -Toledo meter.

The synthesis of chemical intermediate M2 (of the schematic above) is described. Hydrazine monohydrate (5.2 g, 100.0 mmol) is stirred in 20 mL of isopropanol at 0 °C for 15 min, and treated dropwise with a solution of Boc ₂ 0 (10.0 g, 45.8 mmol) in 10 mL of isopropanol. The reaction turns cloudy upon addition and stirring is continued at room temperature for 20 min. The solvent is removed by rotary evaporation and the residue is dissolved in DCM and dried over MgS0 ₄ . The DCM is removed by rotary evaporation and the remaining liquid is distilled under reduced pressure to obtain t-butyl carbazate (M2) as a white solid, ^l U NMR (400 MHz, CDC1 ₃ ) δ 6.42 (s, 1H), 3.60 (s, 2H), 1.37 (s, 9H).

Synthesis of chemical intermediate R4 of the above scheme is described. A solution of rhodamine B (442 mg, 1 mmol) in Cl ₂ SO (10 mL) is kept at room temperature overnight. The reaction mixture is evaporated under vacuum and co-evaporated with anhydrous CH ₂ C1 ₂ (3 x 15 mL) to give rhodamine B acid chloride (R4). The crude acid chloride is dissolved in anhydrous CH ₂ C1 ₂ (10 mL) and added dropwise to a solution of Boc-NH-NH ₂ (132 mg, 1 mmol) and Et ₃ N (200 mL, 2 mmol) in anhydrous CH ₂ C1 ₂ (15 mL). The reaction mixture is kept at room temperature for 10 min. Evaporation of the solvent yielded a crude that is purified by column chromatography using petroleum ether/ethyl acetate (3/1 , v/v) to give R2 as a white solid (0.26 g, 50% yield). 1H NMR (500 MHz, CDC1 ₃ ) δ 7.96 (d, J = 7.6 Hz, 1H), 7.57 - 7.44 (m, 2H), 7.16 (d, J = 7.5 Hz, 1H), 6.52 (s, 2H), 6.38 (d, J = 1.8 Hz, 2H), 6.28 (d, J = 8.4 Hz, 2H), 3.34 (q, J = 7.0 Hz, 8H), 1.61 (s, 4H), 1.26 (s, 9H), 1.16 (d, J = 14.1 Hz, 12H). ¹³ C NMR (125 MHz, CDC1 ₃ ) δ 168.92, 153.56, 152.87, 148.92, 132.58, 131.45, 129.05, 128.32, 123.80, 123.09, 108.24, 105.41 , 97.68, 59.49, 57.65, 54.48, 44.13, 39.49, 12.31. HRMS calc. for C ₃₃ H ₄ iN ₄ 0 ₄ ⁺ (M+H ⁺ ): 557.3122, found: 557.31 1 1.

Comparative Example - Compound Rl or "Rl"

Comparative compound Rl , outside the scope of the present invention, is compared to inventive Compound R2. Compound Rl is: Spiro[lH-isoindole-l ,9'-[9H]xanthen]-3(2H)-one, 2-[2-[bis(2-hydroxyethyl)amino]amino]ethyl]-3 ',6'-bis(diethylamino), and has the CAS Registry Number of 1217892-36-8 (C ₃₄ , H ₄₄ , N ₄ , 0 ₄ ). The structure of Compound Rl and its synthesis is provided herein:

Synthesis of Compound Rl

The synthesis of comparative Compound Rl is described. Synthesis of intermediate R5 is described: Rhodamine B hydrochloride (5.0 g, 10.4 mmol) and ethylenediamine (12.5 g, 208.8 mmol) is dissolved in EtOH (50 mL) and refluxed for 12 h. Most of solvent is removed by evaporation, and the residue is dispersed in water with magnetic stirring. Then the pink precipitate appeared and is recovered by filtration, washed thoroughly with water. At last pink precipitate is washed with petroleum ether, and then dried in vacuum, yielding Compound R5 as a pink powder (3.6 g, 72% yield): 1H NMR (400 MHz, CDC1 ₃ ) δ 7.91 (d, J= 2.3 Hz, 1H), 7.45 (d, J= 2.5 Hz, 2H), 7.14 - 7.05 (m, 1H), 6.44 (s, 1H), 6.42 (s, 1H), 6.37 (d, J= 2.3 Hz, 2H), 6.28 (d, J = 2.3 Hz, 1H), 6.26 (d, J = 2.4 Hz, 1H), 3.33 (q, J= 7.0 Hz, 9H), 3.19 (t, J = 6.6 Hz, 2H), 2.40 (t, J = 6.6 Hz, 2H), 1.62 (s, 5H), 1.16 (t, J = 7.0 Hz, 13H). See Shiraishi, Y.; Miyamoto, R.; Zhang, X.; Hirai, T. Org. Lett. 2007, 9, 3921-3924.

Synthesis of Rl from R5 is described. Oxirane (0.44 g, 10.0 mmol) is added to a cooled (-

5°C) solution of R5 (0.48 g, 0.1 mmol) in dichloromethane (10 mL). The solution is stirred for 4 h at -5°C and then overnight at room temperature before being concentrated under reduced pressure. The resulted mixture is purified by column chromatography using dichloromethane/ methanol (10/1, v/v) to give Compound Rl as a white solid (0.2 g, 40% yield): 1H NMR (500 MHz, CDC1 ₃ ) <5 7.91 (d, J= 2.9 Hz, 1H), 7.46 (dd, J= 5.5, 3.1 Hz, 2H), 7.13 - 7.06 (m, 1H), 6.46 (s, 1H), 6.44 (s, 1H), 6.39 (d, J = 2.4 Hz, 2H), 6.30 (d, J = 2.5 Hz, 1H), 6.28 (d, J = 2.5 Hz, 1H), 3.54 - 3.45 (m, 4H), 3.34 (q, J = 7.0 Hz, 9H), 3.22 (t, J = 5.7 Hz, 2H), 2.55 (t, J = 7.5 Hz, 4H), 2.23 (t, J = 5.0 Hz, 2H), 1.89 (s, 2H), 1.17 (t, J = 7.0 Hz, 13H). ¹³ C NMR (125 MHz, CDC1 ₃ ) δ 153.96, 150.99, 148.86, 133.12, 129.67, 129.32, 128.31, 124.24, 123.41, 107.85, 104.52, 97.76, 44.10, 27.81, 12.59. HRMS calc. for C34H45N404 (M+H ⁺ ): 573.3435, found: 573.3463.

Derivations of Compound R2

Many further derivations from this basic molecule of Compound R2 can be made by those skilled in the art consistent with Formulas (I) and (II) by using starting materials and intermediates that are known or commercially available or by further modifying these molecules by known methods. Non-limiting examples of these compounds within the scope of Formula (I) and/or Formula (II) including the following:

(1) tert-butyl (3',6'-diamino-3-oxospiro[isoindoline-l,9'-xanthen]-2-yl)car bamate (Chemical Formula: C25H24N404), (Molecular Weight: 444.48);

(2) tert-butyl (3',6'-bis(dimethylamino)-3-oxospiro[isoindoline-l ,9'-xanthen]-2-yl)carbamate (Chemical Formula: C29H32N404), (Molecular Weight: 500.59);

(3) tert-butyl (3',6'-bis(diethylamino)-3-oxospiro[isoindoline-l ,9'-xanthen]-2-yl)carbamate (Chemical Formula: C33H40N4O4), (Molecular Weight: 556.70);

(4) tert-butyl (3',6'-bis(ethylamino)-2',7'-dimethyl-3-oxospiro

[isoindoline-l,9'-xanthen]-2-yl)carbamate (Chemical Formula: C31H36N404),

(Molecular Weight: 528.64);

(5) tert-butyl (3',6'-diamino-2',7'-dimethyl-3-oxospiro[isoindoline- 1 ,9'-xanthen]-2- yl)carbamate (Chemical Formula: C27H28N404), (Molecular Weight: 472.54);

(6) tert-butyl (3-oxo-3',6'-di(pyrrolidin- 1 -yl)spiro[isoindoline- 1 ,9'-xanthen]-2-yl)carbamate (Chemical Formula: C33H36N404), (Molecular Weight: 552.66);

(7) tert-butyl (3-oxo-3',6'-bis(phenylamino)spiro[isoindoline- 1 ,9'-xanthen]-2-yl)carbamate (Chemical Formula: C37H32N404), (Molecular Weight: 596.67);

(8) tert-butyl (3-oxo-3',6'-di(piperidin-l-yl)spiro[isoindoline-l,9'-xanthe n]-2-yl)carbamate (Chemical Formula: C35H40N4O4), (Molecular Weight: 580.72);

(9) tert-butyl (3',6'-dimorpholino-3-oxospiro[isoindoline- 1 ,9'-xanthen]-2-yl)carbamate (Chemical Formula: C33H36N406), (Molecular Weight: 584.66);

(10) tert-butyl(2',7'-dibutyl-3',6'-bis(diethylamino)

-3-oxospiro[isoindoline-l,9'-xanthen]-2-yl)carbamate (Chemical Formula: C41H56N404), (Molecular Weight: 668.91);

(11) tert-butyl (2',7'-dimethyl-3-oxo-3',6'-di(piperidin-l-yl)spiro

[isoindoline-l,9'-xanthen]-2-yl)carbamate (Chemical Formula: C37H44N404), (Molecular Weight: 608.77);

(12) tert-butyl (3-oxo- ,2',3',4',10',i ,12',13'-octahydrospiro[isoindoline-l,7'-pyrano

[2,3-f:6,5-f]diquinolin]-2-yl)carbamate (Chemical Formula: C31H32N404), (Molecular

Weight: 524.61);

(13) tert-butyl (3-oxo- ,2',3',4',8',9',10',i -octahydrospiro[isoindoline-l,6'-pyrano [3,2-g:5,6-g']diquinolin]-2-yl)carbamate (Chemical Formula: C31H32N404), (Molecular Weight: 524.61);

(14) N-(3',6'-bis(diethylamino)-3-oxospiro[isoindoline-l,9'-xanth en]-2-yl)propionamide (Chemical Formula: C31H36N403), (Molecular Weight: 512.64);

(15) N-(3',6'-bis(diethylamino)-3-oxospiro[isoindoline-l,9'-xanth en]-2-yl)butyramide

(Chemical Formula: C32H38N403), (Molecular Weight: 526.67); and

(16) N-(3',6'-bis(diethylamino)-3-oxospiro[isoindoline-l,9'-xanth en]-2-yl)pentanamide; (Chemical Formula: C33H40N4O3), (Molecular Weight: 540.70). Metal Ion Sensing:

The procedure for metal ion sensing is described. Solutions of the metal ions (10.0 mM) are prepared in deionized water. A stock solutions of Compounds Rl and R2 (0.2 mM) are each prepared in ethanol and then diluted to 20 μΜ with ethanol-water (1 : 1, v/v, pH 7.04) for spectral measurement. For titration experiments, a 2.0 mL solution of Compounds Rl and R2 (20 μΜ) are filled in a respective quartz optical cell of 1 cm optical path length. Sn ²⁺ stock solution is added into the quartz optical cell gradually by micro-pipette. Spectral data is recorded at 5 min after addition. In selectivity experiments, the test samples are prepared by placing appropriate amounts of metal ion stock into 2.0 mL solution of Rl, R2 (20 μΜ). For fluorescence measurements, excitation is provided at 560 nm, while emission is collected from 565 to 700 nm. pH titration of Compounds Rl and R2 is described. Stock solutions of Compounds Rl and

R2 are respectively added to sodium phosphate buffers of various pH to a final concentration of 10 μΜ. The fluorescence emission spectra is recorded as a function of pH using λ _βχ at 560 nm. The titration curves are plotted by fluorescence emission intensities at 580 nm versus pH.

Cell culture preparation is described. The KB cell line was provided by of Biochemistry and Cell Biology (China). Cells are grown in MEM (Modified Eagle's Medium) supplemented with 10% FBS (Fetal Bovine serum) and 5% C0 ₂ at 37 ^° C . Cell (5 10 ^"8 L ^"1 ) are plated on 18 nm glass co vers lips and allowed to adhere for 24 hours. The Streptococcus mutans (ATCC® 700610™) is prepared by inoculating the single colony from the BHI agar plate into 5 mL BHI broth and incubating at 37 C for 48 h.

Fluorescence imaging is described. Confocal fluorescence imaging is performed with an

OLYMPUS 1X81 laser scanning microscope and a 60 x oilimmersion objective lens. The microscope is equipped with multiple visible laser lines (405, 488, 543 nm). Images are collected and processed with Olympus FV10-ASW software. For fluorescence imaging of intracellular Sn ²⁺ : 10 μΜ of Compound Rl or R2 in the culture media containing 0.2% (v/v) DMSO is added to the cells. The cells are incubated at 37 °C for 30 min, and washed with PBS three times to remove the excess probe and bathed in PBS (2 mL) before imaging. After washing with PBS (2 mL x 3) to remove the excess probe, the cells are treated with 50 μΜ SnF ₂ for 30 min. Excitation of Rl or R2 loaded cells at 543 nm is carried out with a semiconductor laser, and emission is collected at 560-660 nm (single channel). 50 μΜ SnF ₂ in the culture media is added to the cells. The cells are incubated at 37 °C for 30 min, and washed with PBS three times to remove the excess SnF ₂ . After washing with PBS (2 mL ^χ 3) to remove the excess SnF ₂ , the cells are treated with 10 μΜ Rl or R2 separately for 30 min, and washed with PBS three times to remove the excess probe and bathed in PBS (2 mL) before imaging. Cell imaging is then carried out as the former.

Colocalization experiments are described. 10 μΜ of Compound Rl or R2 in the culture media containing 0.2%> (v/v) DMSO is added to the cells. The cells are incubated at 37°C for 30 min, and washed with PBS three times to remove the excess probe and bathed in PBS (2 mL) before imaging. After washing with PBS (2 mL x 3) to remove the excess probe, the cells are treated with 50 μΜ SnF ₂ at 37 °C for 30 min, and washed with PBS three times to remove the excess SnF ₂ and bathed in PBS (2 mL) before imaging. After washing with PBS (2 mL x 3) to remove the excess SnF ₂ , the cells are treated with 1.0 μΜ LysoTracker® Green DND at 37 °C for 30 min. The cells are bathed with 2 mL of PBS for fluorescence imaging equipped with the appropriate excitation and emission filters for Rl or R2 (λ _βχ = 543 nm, _em = 560-660 nm), Lysotracker Green (λ _βχ = 488 nm, λ _βπι = 500-540 nm). 50 μΜ SnF ₂ in the culture media is added to the cells. The cells are incubated at 37 °C for 30 min, and washed with PBS three times to remove the excess SnF ₂ . After washing with PBS (2 mL x 3) to remove the excess SnF ₂ , the cells are treated with 10 μΜ of Compound Rl or R2 separately for 30 min, and washed with PBS three times to remove the excess probe and bathed in PBS (2 mL) before imaging. Cell imaging is then carried out as the former. After washing with PBS (2 mL x 3) to remove the excess probes, the cells were treated with 1.0 μΜ LysoTracker® Green DND at 37 °C for 30 min. Cell imaging is then carried out as the former.

For fluorescence imaging of Sn ²⁺ in bacteria is described. Freshly diluted Streptococcus mutans (ATCC® 700610™) is sub cultured in the presence of the 10 μΜ Compound Rl or R2, separately at 37°C on a shaker bed at 400 rpm for 60 min. Then the bacteria are collected by centrifugation at 8,000 rpm for 2 min and rinsed with Saline (pH = 7.0). The process is repeated three times before imaging. After washing with Saline (2 mL x 3) to remove the excess probes, the bacteria is cultured in the presence of the 50 μΜ SnF ₂ at 37 °C on a shaker bed at 400 rpm for 60 min. Then the bacteria are collected by centrifugation at 8,000 rpm for 2 min and rinsed with Saline (pH = 7.0). The process was repeated three times before imaging. The light source is at = 543 nm provided excitation and emission are collected in the range = 560-660 nm.

Metal ion response is described. Fluorescent 'turn on' probe is conducive for detection target. The solution of Compound Rl or R2 (20 μΜ) in ethanol-water (1 : 1, v/v, PH 7.04) is non- fluorescent. With addition of Sn ²⁺ (0-20 eq) , fluorescence at 580 nm is turned on and grown drastically with an excitation of 560 nm (Fig la and lc) due to the ring open reaction of rhodamine induced by Sn ²⁺ chelating. High-level selectivity is of paramount importance for an excellent chemosensor. Compounds Rl and R2 show selectivity on sensing Sn ²⁺ . The solution of Rl and R2 (20 μΜ) in ethanol-water (1 : 1, v/v, PH 7.04) are tuning on just in the presence of Sn and Cr , while other transition and heavy metal ions such as K , Ag , Ca , Mg , Zn , Pb , Ni , Mn , Co , Cd , Hg , displayed minimal enhancement with an excitation of 560 nm (Fig lb and Id). Metal ion response of Rl and R2 are suited for detection of Sn ²⁺ in living Streptococcus mutans cells.

Figure 1 (a) and (c) show the fluorescence spectra of comparative Compound Rl and inventive Compound R2 (20 μΜ) in ethanol-water (1 : 1, v/v, pH 7.04) upon addition of 0-20 eq of Sn ²⁺ . Figure 1 (b) and (d) show the fluorescent spectra of Compounds Rl and R2 (2.0 x 10 ^~5 M) upon various metal ions (20.0 x 10 ⁵ M) in ethanol-water (1 : 1, v/v, pH 7.04) with an excitation of 560 nm.

The impact of pH values on fluorescence is described. The pirolactam ring of the rhodamine derivatives will open in a certain pH range and indicates the fluorescence of rhodamine. It is therefore necessary to check the fluorescence properties of Compounds Rl and R2 in solutions with different pH values. Furthermore, in the cell, the acidity of different organelles may vary greatly. For example, the normal pH of lysosomes is 4.5-5.5, which may induce ring opening of Rl or R2. Considering the application of Sn ²⁺ probe Rl and R2 intracellular or extracellular may be disturbed by the pH, the acid-base titration experiments are carried out by adjusting the pH with an aqueous solution of NaOH and HC1 in Phosphate- Buffered Saline (Fig.2 a and b ). The titration revealed that the pH range for inducing Compounds Rl or R2 fluorescence turning on is 2.5-6 or 2-4.5, respectively. It is predicted that Rl will be turned on by lysosomes in cell without Sn ²⁺ present. Figure 2 shows the dependence of fluorescence at 580 nm of comparative Compound Rl and inventive Compound R2 (10 μΜ) at different pH in pbs solution. Excitation was at 560 nm. Fluorescence imaging of intracellular Sn ²⁺ is described. Sn ²⁺ is usually added to toothpaste, so oral epithelial cells are most likely to come into contact with Sn ²⁺ . The Kb cells are a good candidate for explored Sn ²⁺ distribution in cell level by fluorescence imaging. Here the practical applicability of Rl and R2 as a Sn ²⁺ probe in the fluorescence imaging of living Kb cells is investigated. Firstly, the Kb cells are separately stained with 10 μΜ of Compound Rl or R2 at 37 °C for 30 min. As determined by laser scanning confocal microscopy, Rl gave fluorescence emission in a site of Kb cells without Sn ²⁺ present (fig 3 .bl); R2 gave scarcely fluorescence (fig 3. b2). This result is consistent with the pH titration experiment that lysosomes acidity may induce Rl fluorescence emission. R2 gave scarcely fluorescence due its lower pH response. This demonstrates the superiority of R2 over Rl given the greater diversity of pH environments that R2 may be used, and subsequently less background noise, particularly in more acidic environments like lysosomes.

Furthermore, when the cells are supplemented with Compound Rl or R2 in the PBS for 30 min at 37 °C and then incubated with 50 μΜ Sn ²⁺ under the same conditions, inventive Compound R2 gave a significant fluorescence increasing from the certain intracellular region (Fig 3 .c2, f2) whereas comparative Compound Rl showed slightly changing in fluorescence intensity (fig 3 .cl, fl). Accordingly, cell imaging experiment indicate that R2 is more suited for detection Sn ²⁺ at a cell level. Furthermore, Rl and R2 may be specificity targeting for lysosomes, due to Rl and R2 bear the groups similar to 'dimethylethylamino' that is the targeting anchor for lysosomes. Figure 3 shows CLSM images of KB cells (al-cl) and (a2-c2). Cells separately incubated with 10μΜ of Compounds Rl and R2 for 30 min, (dl-fl) and (d2-f2) followed incubated with 50μΜ SnF ₂ for 30 min. Emission was collected in red channel at 560-660 nm (bl, el, b2, e2); al, dl, a2, d2 are bright field images and cl, fl, c2, f2 are overlay images, respectively (λεχ = 543 nm).

Colocalization experiments are described. To probe the intracellular locations of

Compounds Rl or R2, Kb cells were co-stained with Rl and LysoTracker® Green DND or R2, Sn ²⁺ and LysoTracker® Green DND, which is a commercially available marker for lysosomes and has good separation in excitation and emission spectra with Rl and R2. As shown in Figure 4al ^~ dl, the fluorescence images between Rl and lysotracker overlapped very well. This result is consistent with the pH titration experiment and fluorescence imaging of intracellular Sn ²⁺ , that Rl accumulates in lysosomes and exhibits acidity induced fluorescence emission. Furthermore, Sn ²⁺ can also locate in lysosomes and enhance the fluorescence of Rl (fig 3 .cl, fl). Fluorescence of R2 induced by Sn ²⁺ and lysotracker overlapped very well, which indicates that R2 can detection Sn ²⁺ in lysosomes; at the same time fluorescence signal of R2 demonstrates distribution of Sn ²⁺ .

Figure 4 shows CLSM images of KB cells (al- dl). Cells are incubated with 10 μΜ of comparative Compound Rl and ΙμΜ LysoTracker Green for 30 min; (a2-d2) successively incubated with 10 μΜ of inventive Compound R2, 50μΜ SnF ₂ , ΙμΜ LysoTracker Green each for 20 min. Emission is collected in red channel (bl, b2) at 560-660 nm (λεχ = 543 nm) or in green channel at 500-540 nm (λεχ = 488 nm); al, a2, are bright field images and dl, d2 are overlay images, respectively.

Without wishing to be bound by theory, Compounds Rl and R2 may be specificity targeting for lyosomes because these compounds have a groups similar to "dimethylethylamino" which is reported as a targeting anchor for lysosomes. Therefore, it's not clear whether Sn ²⁺ is widely distributed in the cell or accumulated in lysosomes. To help answer this question, comparative colocalization experiment is carried out. Firstly, the Kb cells are stained with 50 μΜ Sn ²⁺ at 37 °C for 30 min, and then separately incubated with 10 μΜ of Compound Rl and ΙμΜ LysoTracker® Green DND or Compound R2 and 1 μΜ LysoTracker® Green DND under the same conditions. As determined by laser scanning confocal microscopy, Rl and R2 give fluorescence emission in a site of Kb cells (Fig.5 bl, b2), which overlap with LysoTracker® Green DND very well (Fig.5 dl, b2). There was no fluorescence in other site intracellular. This surprising result indicated that Sn ²⁺ is internalized into cells and leading to accumulation of the ions in lysosomes. This may be a transmission path of Sn. Accordingly, inventive Compound R2 is superior over Rl in this application. Many pharmaceutical agents, including various large and small molecules, must be delivered specifically to particular cell organelles in order to efficiently exert their therapeutic action. Such delivery is still mainly an unresolved problem, but targeting detection is helpful attempt.

Figure 5 shows CLSM images of KB cells (al- dl). Cells are successively incubated with

50μΜ SnF ₂ , 10 μΜ of comparative Compound Rl and ΙμΜ LysoTracker Green each for 20 min; (a2-d2) successively incubated with 50μΜ SnF _2, 10μΜ of inventive Compound R2 and ΙμΜ LysoTracker Green each for 20 min. Emission is collected in red channel (bl, b2) at 560-660 nm (λεχ = 543 nm) or in green channel at 500-540 nm (λεχ = 488 nm); al, a2, are bright field images and dl, d2 are overlay images, respectively.

Fluorescence imaging of Sn ²⁺ in bacteria is described. It has been reported that Sn ²⁺ can inhibit Streptococcus mutans. The practical applicability of Compounds Rl and R2 as a Sn ²⁺ probe in the fluorescence imaging of living Streptococcus mutans (ATCC® 700610™) are investigated. Firstly, the Streptococcus mutans (ATCC® 700610™) are separately stained with ΙΟμΜ Compound Rl or R2 at 37 ^° C for 60 min. As determined by laser scanning confocal microscopy, Compounds Rl and R2 gave no fluorescence emission without Sn ²⁺ present (Fig 6. bl, b2). When the Streptococcus mutans are supplemented with Rl or R2 in the PBS for 60 min at 37 ^° C and then incubated with 50 μΜ Sn ²⁺ under the same conditions, Rland R2 gave a significant fluorescence increasing (fig 6 .el, e2). The overlay of fluorescence and Brightfield images revealed that the fluorescence signals are localized in the Streptococcus mutans (ATCC® 700610™) (Figure 6.fl,f2), indicating that the Sn ²⁺ plays its physiological role within bacteria. This data provides evidence for antibacterial mechanism of Sn ²⁺ .

Figure 6 shows CLSM images of streptococcus mutans (ATCC® 700610™). (al-cl) and

(a2-c2) Cells are separately incubated with 10μΜ comparative Compound Rl and inventive Compound R2 for 30 min, (dl-fl) and (d2-f2) followed incubated with 50μΜ SnF ₂ for 30 min. Emission is collected in red channel at 560-660 nm (bl, el, b2, e2); al, dl, a2, d2 are bright field images andcl, fl, c2, f2 are overlay images, respectively (λεχ = 543 nm).

In summary, the biological application of inventive Compound R2 is demonstrated by the imaging of Sn ²⁺ in Kb cells and Streptococcus mutans (ATCC® 700610™). Furthermore compound R2 as lysosomes tracker is demonstrated by the distribution of Sn ²⁺ in cells and bacteria, which is helpful to research of pharmaceutical agents delivery and antibacterial mechanism of Sn ²⁺ .

The dimensions and values disclosed herein are not to be understood as being strictly limited to the exact numerical values recited. Instead, unless otherwise specified, each such dimension is intended to mean both the recited value and a functionally equivalent range surrounding that value. For example, a dimension disclosed as "40 mm" is intended to mean "about 40 mm."

Every document cited herein, including any cross referenced or related patent or application and any patent application or patent to which this application claims priority or benefit thereof, is hereby incorporated herein by reference in its entirety unless expressly excluded or otherwise limited. The citation of any document is not an admission that it is prior art with respect to any invention disclosed or claimed herein or that it alone, or in any combination with any other reference or references, teaches, suggests or discloses any such invention. Further, to the extent that any meaning or definition of a term in this document conflicts with any meaning or definition of the same term in a document incorporated by reference, the meaning or definition assigned to that term in this document shall govern. While particular embodiments of the present invention have been illustrated and described, it would be obvious to those skilled in the art that various other changes and modifications can be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.