Meso-Substituent-Directed Aggregation Behavior and Water Solubility

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Meso-Substituent-Directed Aggregation Behavior and Water Solubility: Directly Functionalizing of Methine Chain in Thiazole Orange and Biological Applications in Aqueous Buffer Lanying Wang, Wenxia Lin, Wei Sun, Mengqi Yan, Junlong Zhao, Li Guan, Wenting Deng, and Yongqiang Zhang J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.8b03122 • Publication Date (Web): 05 Mar 2019 Downloaded from http://pubs.acs.org on March 11, 2019

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The Journal of Organic Chemistry

Meso-Substituent-Directed Aggregation Behavior and Water Solubility: Directly Functionalizing of Methine Chain in Thiazole Orange and Biological Applications in Aqueous Buffer Lanying Wang,*,†,§ Wenxia Lin,†,§ Wei Sun,† Mengqi Yan,† Junlong Zhao,*,† Li Guan,‡ Wenting Deng,† and Yongqiang Zhang,† †Key

Laboratory of Synthetic and Natural Functional Molecule Chemistry, Ministry of

Education, National Demonstration Center for Experimental Chemistry Education, College of Chemistry and Materials Science, Northwest University, Xi’an 710127, P. R. China. ‡School

of Science, Xi’an University of Architecture and Technology, Xi’an 710055, P. R.

China

ABSTRACT: A new strategy is presented to preclude aggregation and enhance water solubility of cyanine dyes. Namely, a heteroatom-containing substituent, for distorting molecular plane and increasing interaction with water molecules, is introduced to the methine chain of 2-thiazole orange (1, a monocyanine) via one-step, and 2-thiazole orange derivatives (2a-g) are 1

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prepared accordingly. X-ray crystal structures show that the molecular plane of 2a-g is drastically twisted, which reduces intermolecular π−π stacking. The derivatives 2a-g exhibit good to excellent water solubility and can be dissolved in aqueous phosphate buffered saline (PBS) at concentrations suitable for biomedical applications. No aggregation in aqueous PBS, relatively high molar extinction coefficients and low solvatochromism of 2a-g are reflected by UV−vis spectra. 2b shows fast response and high selectivity for biothiols (Cys, Hcy and GSH) in aqueous PBS, and is further employed to detect endogenous biothiols with decent biocompatibility as demonstrated by live cell fluorescence imaging. INTRODUCTION Functionalization of cyanine dyes has drawn researchers’ attention for hundreds of years as it helps in tuning their hydrophilicity and property with respect to light and electron transfer induced processes to widen the scope of their applications.1-4 Long wavelength cyanine dyes show advantages in biology and photomedicine owing to minimum photo-damage to biological samples, deep tissue penetration, and minimum background from cellular autofluorescence.5-7 The cyanine dyes with high water solubility and little H-aggregating in water embody many desirable qualities, i.e., they fluoresce brightly in biological tissues in benign medium without organic solvent.8 Typically, water solubility of cyanine dyes increases through introduction of carboxyl or sulfonic groups to their aromatic heterocycles,9 whilst their the absorption maxima increase along with elongating conjugated chain.10 Then, their H-aggregation is precluded by incorporation of bulky groups into the 2


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aromatic heterocycles.8,11 However, these types of functionalization proceed via multi-steps with complicated synthetic operation and limited scope of substrates. Herein, based on our previous work,12,13 the reaction between the methine chain of 2-thiazole orange (1) and several electrophiles is investigated to directly functionalize its methine chain. Specifically, the heteroatom-containing substituent (including Cl, Br, I, O or S) is directly introduced to the methine chain of 1 to produce 2-thiazole orange derivatives (2a-g) in one step (Scheme 1). The structure, water solubility, spectral properties, and applications of 2a-g are investigated thereafter. Present strategy has the advantages of efficient one-step reaction, simple operating procedures and mild conditions. As expected, the introduction of the heteroatom-containing substituent to the methine chain of 1 not only distorts molecule plane of 2a-g to make π−π stacking unfavorable, but also increases interaction with water molecules. These hence prevent their H-aggregation in water and increase water solubility simultaneously. Effective detection of biothiols via naked eyes can be achieved by using 2b. It is futher employed to identify endogenous biothiols through live cell fluorescence imaging. Essentially, these types of bio-application proceed in aqueous PBS, however, most of reported biothiols detecting are accomplished by organic solvent as cosolvent.14

3



IIV

S I

N CH3

N CH3

Page 4 of 33

S I

N CH3 R

N CH3

2a-g

1

a: R = Cl; b: R = Br; c: R = I; d: R = 2-thienylbenzyl; e: R = 3,4-methylenedioxybenzyl; f: R = SCH3; g: R = CHO I : AlCl3/H2O2, Br2 or ICl/NEt3, R = Cl, Br, I II: ArCH2OH/AlCl3, R = CH2Ar

III : DMSO/Py/SO3, R = SCH3 IV : DMF/SOCl2, R = CHO

Scheme 1. Synthesis of 2a-g via route I, II, III, or IV RESULTS AND DISCUSSIONS Synthesis of 2a-g. Derivatives 2a-c are obtained from 1 via reacting with NCS, NBS and NIS, respectively, at 25 °C in dichloromethane (DCM). The reaction is monitored by on-line TLC and UV−vis. Analysis shows product 2a is formed during the reaction, yet returns to reactant in the separation step. In contrast, 2b and 2c are obtained in 68% and 51% yields, respectively, after 3 h reaction. In order to achieve 2a, chloride reagent is changed to AlCl3/H2O2. In this reaction, AlCl3/H2O2 is in-situ oxidized to generate electrophilic intermediate, and 2a is obtained in 53% yield in DCM at 25 °C after 4 h. Essentially, as displayed in Table 1, 2b (87% yield) and 2c (72% yield) are obtained under catalyst Et3N in DCM at 25 °C after 3 h when Br2 and ICl are employed as halogenating reagents, respectively. However, this protocol is not suitable when using I2 as electrophilic reagent. Moreover, the structures of 2a and 2b are verified by single-crystal X-ray diffraction (Figures 1A and 1B). The reaction between 1 and 2-thiophene methanol is carried out using anhydrous SnCl4 as a catalyst in DCM under reflux, or in dimethylformamide (DMF) at 80, 110 °C, or reflux, but no reactions occur. When the catalyst is switched to AlCl3, the reactions at 80 and 110 4


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°C in DMF for 1 h offer 2d in yields of 43% and 55%, respectively, whereas excellent yield (81%) is obtained when the reaction is carried out under solvent-free conditions at 110 °C after 2 h. Under similar process, 2e in 45% yield is obtained by reaction between 1 and piperonyl alcohol, and 67% yield is achieved after 5 h. When using sulfonating reagent Py/SO3, 2f in yield of 10-61% is obtained accidentally in DMSO at 110 °C after 2-10 h. Its structure is determined on the basis of single-crystal X-ray diffraction analysis (Figure 1C). A Vilsmeier reaction on the methine chain of 1 using DMF as formylation reagent under catalyst SOCl2 allows for facile installation of a formyl group and delivers 2g in 41% and 72% yields at 80 °C for 2 and 5 h, respectively. The above reactions proceed efficiently under mild conditions, and the products are readily purified by column chromatography and confirmed by 1H NMR,

13C{1H}

NMR,

HRMS and Elemental analyses. Optimized conditions and yields for the synthesis of 2a-g (Scheme 1) are summarized in Table 1. It is worth noting that the heteroatom-containing substituent on the methine chain of 2-thiazole orange is comprehensively useful and provides opportunities for further reaction.

Table 1. Optimal Conditions and Yields for the Synthesis of 2a-g dyes

reagent

cat.

solvent

temp/°C

time/h

yield/%a

2a

AlCl3/H2O2

-

DCM

25

4

53

2b

Br2

Et3N

DCM

25

3

87

2c

ICl

Et3N

DCM

25

3

72

2d

2-thiophene methanol

AlCl3

-

110

2

81 5



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2e

piperonyl alcohol

AlCl3

-

110

5

67

2f

DMSO/Py/SO3

-

DMSO

110

10

61

2g

DMF

SOCl2

DMF

80

5

72

aYield

after separation of column layer.

Crystal analysis. The crystals of 2a, 2b and 2f are obtained by solvent evaporation or diffusion. 2a is crystallized in the orthorhombic space group Pbca, whilst 2b and 2f are classified in the triclinic space group P-1 (Table S1, Supporting Information (SI)). The crystal structure analysis of 1 (Figure 1D and Figure S1D, SI)12 demonstrates that two aromatic heterocycles (benzothiazole ring and quinoline ring) and central methine are almost coplanar with dihedral angle of 8.26°, indicating its tendency to plane-to-plane stacking. In stark contrast, after introducing Cl, Br or SCH3 to the methine chain of 1 (Figures 1A-C and Figures S1A-C, SI), the molecular planes of 2a, 2b and 2f are drastically twisted. In details, the benzothiazole rings and quinoline rings in 2a, 2b and 2f are oriented in different planes with dihedral angle of 131.06, 119.17, and 120.95°, separately. In this situation, π−π stacking of molecules becomes unfavorable. The average distances of C(9)-Cl(1) in 2a, C(8)-Br(1) in 2b and C(10)-S(2) in 2f are 1.760, 1.918 and 1.756 Å, respectively. These are shorter than normal C-Cl, C-Br or C-S single bond, reflecting p-π conjugation between Cl, Br or S and methine chain. Thus, the lone-pair electron on Cl, Br or S becomes delocalizated.

6


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Figure 1. Crystal structures of 2a (A), 2b (B), 2f (C) and 1 (D). The packing of crystals for 2a, 2b and 2f are displayed in Figure 2 and Figure S2 (SI). Figure 2D indicates that the molecules of 1 are stacked nearly parallel to each other via π−π interactions with face-to-face distances of 3.68 Å.12 Thus, it is prone to aggregate in a parallel

way

(plane-to-plane

stacking)

to

form

a

sandwich-type

arrangement

(H-aggregates).15-17 In the contrary, benzothiazole ring planes are parallel to each other in the crystal packing of 2a and 2b (Figures 2A and 2B), whilst quinoline ring plane is parallel to each other in 2f (Figure 2C). Among these, intermolecular π−π interaction is significantly reduced, and corresponding aggregation is retarded. The molecules of 2a, 2b and 2f are arranged in a head-to-tail orientation in the same plane, while in the neighboring plane the parallel units are arranged with face-to-face distances of 3.76 Å, 3.54 Å, and 3.57 Å, respectively. Thus, it is reasonable to assume that introducing a substituent to the methine chain of 1 results in non-planar structure of 2a-g as well as their unfavorable stacking and aggregation.

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Figure 2. Crystal stacking of 2a (A), 2b (B), 2f (C) and 1 (D).

Solubility analysis. To evaluate the effect of the heteroatom-containing substituent on water solubility, the measurement is carried out in aqueous PBS (10 mM, pH 7.4) at room temperature for 2a-g and 1. As shown in Table 2, good to superior enhancement with respect to water solubility is achieved. In details, the water solubility of 2a-c increases nearly 40 times in comparison with parent 1, and ca. 3-5 times increase is found for 2d-f. Especially, 1300 times enhancement is obtained for 2g. The increase of water solubility owing to the fact that the meso-substituent renders the dye molecules twisted, retards their aggregates via stacking, and exposes the molecular surfaces to water molecules. Additionally, dipolar or hydrogen bond interactions between the heteroatom-containing groups and water molecules account for the enhancement. As a consequence, their biomedical applications in aqueous PBS become favorable.

8


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Table 2. The Solubility of 2a-g and 1 in Aqueous PBS dyes

solubility/mg·mL-1

dyes

solubility/mg·mL-1

1

0.03 ± 0.01

2d

0.09 ± 0.01

2a

1.32 ± 0.02

2e

0.13 ± 0.02

2b

1.14 ± 0.01

2f

0.15 ± 0.01

2c

1.07 ± 0.04

2g

38.83 ± 0.50

Spectroscopic characterization. The effects of the heteroatom-containing substituents on their spectroscopic properties are evaluated, and the UV−vis absorption spectra of 2a-g and 1 in four different solvents (MeOH, PBS, DMSO and 90% glycerol-water) are displayed in Figure 3 and Table 3. Dyes 2a-g exhibit a single peak in the visible region, demonstrating that they exist in monomer form (M) and no aggregation is observed in PBS. For comparison, a shoulder band at shorter wavelength emerges in the spectrum of 1, an indicattion of H-aggregates. Due to the introduced meso-substituent, the molecules of 2a-g are nonplanar, whilst the molecule of 1 is almost planar. The formation of H-aggregation is derived from the good planarity of molecule. Then, meso-substituent also affects spectral properties of 2a-g, i.e., bathochromic shifts of 64-32 nm are found in 2a-f with respect to absorption maxima (λmax) when comparing with parent 1 in four solvents. This is mainly due to the p-π conjugation between halogen (or sulfur) and methine chain as well as the σ−π hyperconjugation between methene on heterocyclic benzyl and methine chain. The introduction of an electron-withdrawing formyl group to the meso position of 1 brings about a hypsochromic shift of 2g (30-41 nm) in four solvents. This behavior agrees well with 9



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previously reported meso-CN-4-thiazole orange, i.e., a hypsochromic shift of ca. 16 nm occurs when an electron-withdrawing cyano group is introduced at the meso position of 4-thiazole orange.3b Moreover, the solvatochromism is insignificant for 2a-g from MeOH to DMSO.

Table 3. The Spectroscopic Data of 2a-g and 1 in Different Solvents MeOH dyes

PBS

ε×10-4 λmax/nm

/M-1·cm-1

DMSO

ε×10-4 λmax/nm

/M-1·cm-1

90% glycerol

ε×10-4 λmax/nm

/M-1·cm-1

ε×10-4 λmax/nm

/M-1·cm-1

Stocks λem/nm

shift/nm

1

481

5.06

478

4.51

488

4.81

486

4.94

544

58

2a

536

3.66

534

4.19

540

4.21

541

3.48

602

61

2b

545

3.21

541

3.02

548

2.42

549

2.33

601

52

2c

537

4.50

534

4.33

540

4.21

541

4.00

593

52

2d

520

2.96

518

4.33

524

4.21

522

3.12

588

66

2e

523

2.89

518

3.32

525

3.03

525

2.91

592

67

2f

520

3.09

514

3.18

520

3.18

520

2.85

600

80

2g

451

1.25

437

1.14

454

1.25

447

1.04

530

83

10


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Figure 3. UV-vis spectra recorded for 1 or 2a-g (10 μM) in PBS.

The fluorescence properties of 2a-g are investigated in a viscous solvent (90% glycerol-water) (Figure 4A and Table 3) because of their low background fluorescence in a nonviscous solvent (Figure 4B). The emission maximum (λem) is affected by the substituent in the meso position of 1. Compared to parent 1, the λem of 2a-f red-shifts 44-58 nm, while λem of 2g blue-shifts 14 nm. It is worth mentioning that the fluorescence intensity of 2g is stronger than those of 2a-f in 90% glycerol-water (up to 10-fold). This can be explained by forming hydrogen bonds between glycerol and formyl group of 2g, which prevents molecules from vibrating or rotating to dissipate absorbed energy. In addition, the Stokes shift of 2g is larger than that of the parent 1, reaching 83 nm.

11



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Figure 4. Fluorescence spectra recorded for 1 (2 μM) or 2a-g (10 μM) in 90% glycerol-water (A); 2g in MeOH, PBS (10 mM, pH 7.4) or 90% glycerol-water (B).

Application research. High water soluble cyanine dyes are sought-after owing to their broad potential in biological and photomedical applications, such as biomolecule labels and cell imaging. Amino acids are representative biologically species related closely to human health and play crucial roles in regulating various physiological and pathological processes. Owing to high water solubility of 2a-g, their interactions with 20 kinds of natural amino acids, homocysteine (Hcy), glutathione (GSH) and L-cystine are investigated to explore their applications in biomolecular detection (Table S2, SI). 2b is found to be active towards biothiols cysteine (Cys), Hcy or GSH, and detailed study is performed in the following parts. Concentration-dependence and time-dependence via UV−vis. As displayed in Figure 5 and Figure S3 (SI) the concentration-dependent UV-vis spectra are recorded in aqueous PBS to investigate the performance of 2b in detecting biothiols. In the absence of Cys, Hcy

12


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or GSH, the spectrum of 2b shows a single absorption peak at 540 nm. Upon addition of increasing concentrations of Cys (0-30 μM), Hcy (0-50 μM), or GSH (0-60 μM), the absorption peak of 2b at 540 nm decreases gradually. In the same time, a new absorption band along with a shoulder emerges and increases at 480 nm. A well-defined isosbestic point appears at 507 nm, indicating the generation of new species through the interaction between 2b and Cys, Hcy or GSH. Meanwhile, solution color changes prominently from fuchsia to bright yellow as observed by the naked eye. After the concentration increases to 30 μM for Cys, 45 μM for Hcy, and 55 μM for GSH, the absorption at 480 nm reaches plateau. The sensitivity consequence of 2b to biothiols is Cys > Hcy > GSH under the same conditions. From the intensity of the absorbance band at 540 nm, 2b provides the linear range of 0-17 μM for Cys, 0-30 μM for Hcy, and 5-20 μM for GSH with the corresponding detection limits (3δ) of 2.40 μM, 2.51 μM, and 2.61 μM. These results demonstrate that 2b can detect biothiols Cys, Hcy or GSH quantitatively.

Figure 5. UV-vis spectra of 2b (10 μM) upon addition of Cys (0-30 μM) in aqueous PBS. Inset shows the absorption intensity (A540) of 2b versus the concentrations of Cys. 13



Page 14 of 33

Quick response to the target is an essential factor for an ideal probe, in addition to high sensitivity. Then, the response of 2b towards Cys, Hcy or GSH is evaluated as a function of time by monitoring the changes at 540 nm in the absorbance spectra. After addition of Cys, Hcy or GSH, the absorption intensity decreases and keeps constantly at its minimum value within 4.4, 5.2 and 12.2 min (Figure 6), respectively. The reaction rate of 2b with biothiols increases in the following sequence: Cys > Hcy > GSH, which is consistent with the sensitivity order of 2b toward Cys, GSH and Hcy.

Figure 6. Time-dependent absorption spectra changes of 2b (10 μM) at 540 nm after treatment with 30 μM Cys, Hcy or GSH in aqueous PBS.

Selectivity and competitive studies. In the selectivity study, the response of 2b towards Cys is investigated in aqueous PBS when employing Hcy, GSH and other 19 kinds of natural amino acids as competitive candidates. The samples are prepared by mixing 2b (10 μM) with 3 equiv. of Cys, Hcy, GSH or 100 equiv. of 19 types of amino acids, and their absorption spectra are displayed in Figure S4 (SI). Nearly no absorption spectra changes are observed when incubating the solution of 2b with 19 types of amino acids (Phe, Ala, Gly, 14


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Glu, Gln, Met, Val, Arg, Lys, Tyr, Leu, Pro, Trp, Ser, Thr, Asn, Asp, Ile, His or L-cystine) for 30 min. However, under the same conditions, the absorption is significantly enhanced at 480 nm when 2b is incubated with sulfydryl-containing amino acids (Cys, Hcy or GSH). The high selectivity of 2b towards Cys, Hcy or GSH can be observed via naked eyes, i.e., an obvious color change from fuchsia to bright yellow (Figure S5, SI). When 3 equiv. of Cys is added to above solutions, their corresponding spectra are similar to that of Cys alone. This demonstrates that 2b exhibits high sensitivity and selectivity towards Cys in the presence of high concentration of competitive amino acids (Figure 7).

Figure 7. Absorbance ratio (A480/A540) of 2b towards various amino acids. Black bars are A480/A540 of 2b in the presence of Cys, Hcy, GSH (30 μM), other amino acids (1.0 mM), respectively. Red bars represent A480/A540 of the mixture of 2b with competitive amino acids and Cys (30 μM). The analytes from left to right are: Phe, Ala, Gly, Glu, Gln, Met, Val, Arg, Lys, Tyr, Leu, Pro, Trp, Ser, Thr, Asn, Asp, Ile, His, L-cystine, Cys, Hcy, GSH.

15



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Sensing mechanism. The reaction mechanism between 2b and Cys is investigated by the HRMS of 2b and 2b incubated with 3 equiv. Cys for 30 min in aqueous PBS (Figure S6, SI). After incubation 2b with Cys (Figure S6B and S6D), the peaks at m/z 122.0402 and 383.0254 disappears, which correspond to [Cys+H]+ (calcd. 122.0276 for C3H8NO2S, Figure S6A, SI) and [2b-I]+ (calcd. 383.0212 for C19H16N2BrS, Figure S6C, SI), respectively. Then, two peaks emerge at m/z 160.1124 and 305.1152, corresponding to [Cys+O+Na]+ (calcd. 160.0044 for C3H7NO3SNa) and [2b-I-Br+H]+ (calcd. 305.1107 for C19H17N2S), respectively. This is attributed to the fact that 2b can selectively oxidize the sulfydryl of Cys into hydrosulfinyl in aqueous PBS (Scheme 2). Proposed the detailed mechanism of interaction between 2b and Cys is described in Scheme S1 (SI). Oxidation of sulfydryl group in biothiols by meso-halogenated 2-thiazole orange (2a-c) involves the departure of halogen anion and capture of proton thereafter. The departing ability of halogen anion to function as a leaving group is I¯ > Br¯ > Cl¯, and the order of removing proton is Cl¯ > Br¯ > I¯. Consequently, meso-brominated 2-thiazole orange (2b) exhibits good reactivity in oxidation of sulfydryl group of Cys, Hcy and GSH.

S I

+

N CH3 Br 2b

N CH3

COOH HS

NH2

S

H 2O HBr I

Cys

N CH3 H 1

N CH3

+

O

COOH

HS

NH2 [Cys+O]

Scheme 2. The Proposed Detection Mechanism of Cys Using 2b Cell toxicity and imaging. The cytotoxicity of 2b is evaluated by determining the cell viability of 4T1 cells incubated with different concentrations of 2b (1.25, 2.5, 5, 10, 20, 40 16


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μM) using CCK-8 assay. As shown in Figure S7 (SI), the cell viability value stays above 80% even at a high concentration of 2b (10 μM) after 12 h. These results indicate that 2b has low toxicity. Then, the fluorescent imaging for detecting biothiols in living cells using 2b is performed. As control background, 4T1 cells show no fluorescence (Figure 8A). However, incubating 4T1 cells with 2b for 30 min gives rise to green fluorescence from intracellular nucleus regions (Figure 8B). After the cells are pretreated with N-ethylmaleimide (NEM) for 30 min, followed by incubating with 2b for 30 min, the green fluorescence is not observed (Figure 8C). This is attributed to the fact that NEM traps intracellular thiols. Together with the overlay of fluorescent images and bright field images, 2b is able to penetrate through cell membrane and detect endogenous biothiols. When 500 μM Cys is added to NEM-pretreated cells, strong green fluorescence is observe as consequence (Figure 8D), due to the interaction between 2b and exogenous biothiols Cys. These results demonstrate that 2b displays good ability to penetrate cell membrane and detect biothiols in living cells.

17



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Figure 8. Fluorescent microscope imaging of 4T1 cells. (A) Cells as background control. (B) Cells incubated with 2b (10 μM) for 30 min. (C) Cells pretreated with NEM (300 μM) for 30 min, then incubated with 2b (10 μM) for 30 min. (D) Cells pretreated with NEM (300 μM) for 30 min and further incubated with 2b (10 μM) for 30 min, and then incubated with Cys (500 μM). (A–D) Top: bright-field images; Middle: fuorescence images from green channel (λex = 488 nm); Bottom: merged images.

CONCLUSION A strategy to preclude aggregation and enhance water solubility of cyanine dyes has been presented. One-step synthesis of 2a-g is achieved via simple operating procedures under mild conditions, where heteroatom-containing substituent is directly introduced to the methine chain of 1. In this case, simultaneous control of aggregation behavior and water 18


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solubility of targeted dyes is realized, i.e., the addition of heteroatom-containing substituent to the methine chain of 1 renders no aggregation of 2a-g in aqueous PBS, and up to 1300 times enhancement of water solubility for 2g is obtained. A hypsochromic shift of λmax is observed for 2g containing electron-withdrawing formyl group, whilst introduction of alkyl groups or substituents such as Cl, Br, I or SCH3 (2a-f) results in bathochromic shift. Fast-response and high selectivity of 2b in identifying biothiols is achieved in aqueous PBS solution. It can further penetrate cell membrane to detect biothiols in living cells when performing fluorescent imaging. EXPERIMENTAL SECTION Materials and methods. 2-Thiazole orange was synthesized by the reaction of 2,3-dimethylbenzothiazolium

iodide

with

1-methyl-2-methylthioquinolinium

iodide

according to the literature.18 All the solvents and reagents were obtained from commercial sources and used without further purification. The solvents of DMF and DMSO were dried before use. Flash chromatography columns were packed with thin layer chromatography silica gel G. The absorbance spectra were performed on a Purkinje General TU-1900 UV-vis spectrometer. The fluorescence spectra were measured by FL-2700 fluorescence spectrophotometer. Melting points were taken on an X-4 micromelting apparatus. 1H and 13C{1H}

NMR spectra were recorded on a Bruker AVANCE 400MHz spectrometer at 400

MHz for 1H and at 100 MHz for

13C{1H}

and chemical shifts were reported relative to

internal Me4Si. HRMS was recorded on a Bruker microTOFQ II ESI-Q-TOF LC/MS/spectrometer. Elemental analyses were performed on a Perkin-Elmer 2400C 19



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elemental analyzer. Single crystal X-ray diffraction data were collected on a Bruker SMART APEX II CCD X-ray crystallography. Cell imaging was performed using Leica DMI8 Microscope. Measurement of solubility in aqueous PBS. To prepare saturated solutions of 2a-g, excess amount of dye was added to a certain amount of aqueous PBS, stirred for 30 min, and placed in the dark for 12 h at 25 °C. After that, the solutions were filtered and the remaining solids were dried and weighed. All solubility determination assays were performed three times in duplicate. General spectroscopic methods. The stock solutions of 2a-g (1.0×10−3 M) were prepared using DMSO and diluted to required concentration (1.0×10−5 M) using DMSO, MeOH, PBS and 90% glycerol-water, respectively. The UV-vis absorption spectra and fluorescence spectra were measured at room temperature. General procedure for biomolecular detection. The stock solution of 2b (1.0×10−3 M) was prepared using aqueous PBS (10 mM, pH 7.4). Then, all kinds of natural amino acids, homocysteine (Hcy), glutathione (GSH) and L-cystine were dissolved in aqueous PBS to obtain 1.0×10−2 M stock solutions. All measurements were carried out in the following steps. Sample solutions were prepared by pipetting 100 μL stock solution of 2b and appropriate portions of each the other analyte stock solutions into 10 mL colorimetric tube, and diluted to 10 mL using aqueous PBS. The UV-vis absorption spectra of the resulting solutions were recorded after 30 min at room temperature.

20


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Cytotoxicity assay. Cytotoxicity of 2b toward 4T1 cells were evaluated by CCK-8 assays. The 4T1 cells were seeded in 96-well plates at a density of 1×105 cells/mL and incubated in an incubator for overnight culture at 37 °C under 5% CO2. Next, the cells were incubated with 2b at different concentrations of (0-40 μM) for 12 h. Then the culture medium was removed and the cells were washed with aqueous PBS for three times. After that, 10 μL of CCK-8 solution was added into each well, after incubated for 3 h in the incubator, the absorbance intensity at 450 nm was measured with a microplate reader. All experiments were measured three times in parallel and the cell viability was calculated as a percentage of the average carrier control. Cell image experiment. The 4T1 cells were seeded in 8-well confocal dish containing sterile coverslips and adhered for 12 h. Then all kinds of cell samples were prepared such as (A) cells as background control, (B) cells incubated with 2b (10 μM) for 30 min, (C) cells pretreated with NEM (300 μM) for 30 min, then incubated with 2b (10 μM) for 30 min, (D) cells pretreated with NEM (300 μM) for 30 min and further incubated with 2b (10 μM) for 30 min, and then incubated with Cys (500 μM). Before the fluorescent microscope imaging, the cells were washed three times with PBS. The fluorescence images were acquired in the wavelength ranges of 512-542 nm at excitation wavelength of 488 nm. Synthesis and characterisation data. Synthesis of 2a. A mixture of 1 (0.22 g 0.50 mmol), 0.50 mL of 30% H2O2 and anhydrous AlCl3 (0.20 g, 1.50 mmol) were reacted in dichloromethane at 25 °C for 4 h. After completion of the reaction, water was added to quench the reaction. The resulting reaction mixture was extracted with dichloromethane, 21



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and the organic layer was concentrated under reduced pressure to give the crude product, which was submitted to flash gradient elution chromatography (methanol/dichloromethane, 0/100, 1/100, 1/50, 1/20 v/v) and afforded 2a as a green solid (0.13 g, 53%). m.p. 204−205 °C. UV-vis (MeOH) λmax (ε [M-1 cm-1]): 536 nm (3.66×104). 1H NMR (400 MHz, DMSO-d6): δ (ppm) 8.71 (d, J = 8.0 Hz, 1H, Ar-H), 8.23−8.28 (m, 2H, Ar-H), 8.10 (t, J = 8.0 Hz, 1H, Ar-H), 8.01 (d, J = 8.0 Hz, 1H, Ar-H), 7.82−7.88 (m, 2H, Ar-H), 7.59 (d, J = 4.0 Hz, 2H, Ar-H), 7.40−7.44 (m, 1H, Ar-H), 4.22 (s, 3H, N+CH3), 3.36 (s, 3H, NCH3). 13C{1H}

NMR (100 MHz, DMSO-d6): δ (ppm) 162.5 (Ar-C), 154.1 (Ar-C), 144.5 (Ar-C),

141.8 (Ar-C), 140.7 (Ar-C), 134.7 (Ar-C), 130.0 (Ar-C), 128.4 (Ar-C), 128.3 (Ar-C), 127.0 (Ar-C), 125.8 (Ar-C), 125.5 (Ar-C), 125.0 (Ar-C), 123.1 (Ar-C), 119.3 (Ar-C), 114.2 (Ar-C), 83.3 (meso-C), 44.2 (NCH3). HRMS in CH3OH (ESI/Q-TOF) m/z [M−I]+ calcd for C19H16ClN2S+ 339.0717; found 339.0709. Anal. Calcd for C19H16ClIN2S: C, 48.89; H, 3.46; N, 6.00; found: C, 48.79; H, 3.33; N, 5.90. Synthesis of 2b. A mixture of 1 (0.22 g, 0.50 mmol), 0.50 mL of liquid Br2 and 0.25 mL of Et3N in dichloromethane was reacted at 25 °C for 3 h. After the reaction was completed, water was added to quench the reaction. The resulting reaction mixture was extracted with dichloromethane, and the organic layer was concentrated under reduced pressure to give the crude product, which was submitted to flash gradient elution chromatography (methanol/dichloromethane, 0/100, 1/100, 1/50, 1/20 v/v) and afforded 2b as a green solid (0.23 g, 87%). m.p. 208−209 °C. UV-vis (MeOH) λmax (ε [M-1 cm-1]): 545 nm (3.21×104). 1H

NMR (400 MHz, DMSO-d6): δ (ppm) 8.73 (d, J = 8.0 Hz, 1H, Ar-H), 8.25−8.31 (m, 2H, 22


Page 23 of 33 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Ar-H), 8.11 (t, J = 8.0 Hz, 1H, Ar-H), 7.97 (d, J = 8.0 Hz, 1H, Ar-H), 7.92 (d, J = 8.0 Hz, 1H, Ar-H), 7.85 (t, J = 8.0 Hz, 1H, Ar-H), 7.52−7.58 (m, 2H, Ar-H), 7.40 (t, J = 8.0 Hz, 1H, Ar-H), 4.21 (s, 3H, N+CH3), 3.27 (s, 3H, NCH3). 13C{1H} NMR (100 MHz, DMSO-d6): δ (ppm) 163.2 (Ar-C), 155.7 (Ar-C), 144.8 (Ar-C), 142.0 (Ar-C), 140.6 (Ar-C), 134.8 (Ar-C), 130.0 (Ar-C), 128.6 (Ar-C), 128.2 (Ar-C), 127.1 (Ar-C), 126.6 (Ar-C), 125.4 (Ar-C), 124.9 (Ar-C), 122.9 (Ar-C), 119.5 (Ar-C), 114.1 (Ar-C), 69.4 (meso-C), 55.4 (N+CH3), 44.1 (NCH3). HRMS in CH3OH (ESI/Q-TOF) m/z [M−I]+ calcd for C19H16BrN2S+ 383.0212; found 383.0227. Anal. Calcd for C19H16BrIN2S: C, 44.64; H, 3.15; N, 5.48; found: C, 44.57; H, 3.11; N, 5.30. Synthesis of 2c. A mixture of 1 (0.22 g, 0.50 mmol), 0.50 mL of ICl and 0.25 mL of Et3N in dichloromethane was reacted at 25 °C for 4 h. After the reaction was completed, water was added to quench the reaction. The resulting reaction mixture was extracted with dichloromethane, and the organic layer was concentrated under reduced pressure to give the crude product, which was submitted to flash gradient elution chromatography (methanol/dichloromethane, 0/100, 1/100, 1/50, 1/20 v/v) and afforded 2c as a green solid (0.20 g, 72%). m.p. 186−187 °C. UV-vis (MeOH) λmax (ε [M-1 cm-1]): 537 nm (4.50×104). 1H

NMR (400 MHz, DMSO-d6): δ (ppm) 8.69 (d, J = 8.0 Hz, 1H, Ar-H), 8.24 (t, J = 8.0 Hz,

2H, Ar-H), 8.10 (t, J = 8.0 Hz, 1H, Ar-H), 7.99 (d, J = 8.0 Hz, 1H, Ar-H), 7.82−7.87 (m, 2H, Ar-H), 7.59 (d, J = 4.0 Hz, 2H, Ar-H), 7.40−7.44 (m, 1H, Ar-H), 4.22 (s, 3H, N+CH3), 3.36 (s, 3H, NCH3). 13C{1H} NMR (100 MHz, DMSO-d6): δ (ppm) 162.6 (Ar-C), 154.1 (Ar-C), 144.5 (Ar-C), 141.7 (Ar-C), 140.7 (Ar-C), 134.8 (Ar-C), 130.0 (Ar-C), 128.4 (Ar-C), 128.3 23



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(Ar-C), 127.0 (Ar-C), 125.8 (Ar-C), 125.5 (Ar-C), 125.1 (Ar-C), 123.0 (Ar-C), 119.2 (Ar-C), 114.2 (Ar-C), 83.4 (meso-C), 44.2 (NCH3). HRMS in CH3OH (ESI/Q-TOF) m/z [M−I]+ calcd for C19H16IN2S+ 431.0073; found 431.0065. Anal. Calcd for C19H16IN2S: C, 40.88; H, 2.89; N, 5.02; found: C, 40.79; H, 2.81; N, 4.95. Synthesis of 2d. A mixture of 1 (0.22 g, 0.50 mmol), 2 mL of 2-thiophenemethanol and anhydrous AlCl3 (0.02 g, 0.15 mmol) was reacted at 110 °C for 2 h. After the reaction was completed, water was added to quench the reaction. The resulting reaction mixture was extracted with dichloromethane, and the organic layer was concentrated under reduced pressure to give the crude product, which was submitted to flash elution chromatography (methanol/dichloromethane, 1/100 v/v) and afforded 2d as a green solid (0.22 g, 81%). m.p. 147−148 °C. UV-vis (MeOH) λmax (ε [M-1 cm-1]): 520 nm (2.96×104). 1H NMR (400 MHz, DMSO-d6): δ (ppm) 8.50 (d, J = 8.0 Hz, 1H, Ar-H), 8.12 (t, J = 8.0 Hz, 2H, Ar-H), 8.00 (t, J = 8.0 Hz, 1H, Ar-H), 7.94 (d, J = 8.0 Hz, 1H, Ar-H), 7.83 (d, J = 9.2 Hz, 1H, Ar-H), 7.74 (t, J = 8.0 Hz, 1H, Ar-H), 7.55 (d, J = 4.0 Hz, 2H, Ar-H), 7.30−7.40 (m, 2H, Ar-H), 7.04 (b, 1H, Ar-H), 6.93−6.95 (m, 1H, Ar-H), 4.40 (s, 2H, CH2), 3.98 (s, 3H, N+CH3), 3.21 (s, 3H, NCH3). 13C{1H} NMR (100 MHz, DMSO-d6): δ (ppm) 164.4 (Ar-C), 158.3 (Ar-C), 143.4 (Ar-C), 142.8 (Ar-C), 140.4 (Ar-C), 140.0 (Ar-C), 133.9 (Ar-C), 129.7 (Ar-C), 128.1 (Ar-C), 127.6 (Ar-C), 127.6 (Ar-C), 126.2 (Ar-C), 126.0 (Ar-C), 125.6 (Ar-C), 125.5 (Ar-C), 125.1 (Ar-C), 125.0 (Ar-C), 122.9 (Ar-C), 119.3 (Ar-C), 114.2 (Ar-C), 93.6 (meso-C), 55.4 (CH2), 43.2 (N+CH3), 36.7 (NCH3). HRMS in CH3OH (ESI/Q-TOF) m/z

24


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[M−I]+ calcd for C24H21N2S2+ 401.1141; found 401.1155. Anal. Calcd for C24H21IN2S2: C, 54.55; H, 4.01; N, 5.30; found: C, 54.51; H, 4.03; N, 5.21. Synthesis of 2e. A mixture of 1 (0.22 g, 0.50 mmol), piperonyl alcohol (1.52 g, 10.0 mmol) and anhydrous AlCl3 (0.02 g, 0.15 mmol) was reacted at 110 °C for 5 h. After the reaction was completed, water was added to quench the reaction. The resulting reaction mixture was extracted with dichloromethane, and the organic layer was concentrated under reduced pressure to give the crude product, which was submitted to flash elution chromatography (methanol/dichloromethane, 1/100 v/v) and afforded 2e as a green solid (0.19 g, 67%). m.p. 152−153 °C. UV-vis (MeOH) λmax (ε [M-1 cm-1]): 523 nm (2.89×104). 1H

NMR (400 MHz, DMSO-d6): δ (ppm) 8.52 (d, J = 8.0 Hz, 1H, Ar-H), 8.13−8.16 (m, 2H,

Ar-H), 8.02 (t, J = 8.0 Hz, 1H, Ar-H), 7.93 (d, J = 8.0 Hz, 1H, Ar-H), 7.83 (d, J = 8.0 Hz, 1H, Ar-H), 7.76 (t, J = 8.0 Hz, 1H, Ar-H), 7.56 (d, J = 4.0 Hz, 2H, Ar-H), 7.35−7.39 (m, 1H, Ar-H), 6.90 (s, 1H, Ar-H), 6.80−6.86 (m, 2H, Ar-H), 5.98 (s, 2H, OCH2O), 4.15 (s, 2H, CH2), 3.99 (s, 3H, N+CH3), 3.24 (s, 3H, NCH3).

13C{1H}

NMR (100 MHz, DMSO-d6): δ

(ppm) 164.1 (Ar-C), 158.9 (Ar-C), 148.0 (Ar-C), 146.4 (Ar-C), 143.6 (Ar-C), 140.4 (Ar-C), 140.0 (Ar-C), 133.9 (Ar-C), 132.6 (Ar-C), 129.6 (Ar-C), 128.0 (Ar-C), 127.6 (Ar-C), 126.2 (Ar-C), 125.8 (Ar-C), 125.6 (Ar-C), 124.8 (Ar-C), 122.9 (Ar-C), 121.1 (Ar-C), 119.2 (Ar-C), 114.0 (Ar-C), 108.8 (Ar-C), 108.7 (Ar-C), 101.4 (OCH2O), 93.4 (meso-C), 55.4 (CH2), 43.2 (N+CH3), 41.3 (NCH3). HRMS in CH3OH (ESI/Q-TOF) m/z [M−I]+ calcd for C27H23N2O2S+ 439.1475; found 439.1486. Anal. Calcd for C27H23IN2O2S: C, 57.25; H, 4.09; N, 4.95; found: C, 57.14; H, 4.10; N, 4.89. 25



Page 26 of 33

Synthesis of 2f. A mixture of 1 (0.60 g, 1.39 mmol) and 1.40 g of Py/SO3 in 4 mL DMSO was heated at 110 °C for 10 h under stirring. After the reaction was completed, water was added to quench the reaction. The resulting reaction mixture was extracted with dichloromethane, and the organic layer was concentrated under reduced pressure to give the crude product, which was submitted to flash gradient elution chromatography (methanol/dichloromethane, 0/100, 1/100, 1/50 v/v) and afforded 2f as a green solid (0.41 g, 61%). m.p. 221−222 °C. UV-vis (MeOH) λmax (ε [M-1 cm-1]): 520 nm (3.09×104). 1H NMR (400 MHz, DMSO-d6): δ (ppm) 8.56 (d, J = 8.0 Hz, 1H, Ar-H), 8.19 (t, J = 8.0 Hz, 2H, Ar-H), 8.04 (t, J = 8.0 Hz, 1H, Ar-H), 7.97 (d, J = 8.0 Hz, 1H, Ar-H), 7.91 (d, J = 8.0 Hz, 1H, Ar-H), 7.78 (t, J = 8.0 Hz, 1H, Ar-H), 7.55 (d, J = 4.0 Hz, 2H, Ar-H), 7.39−7.43 (m, 1H, Ar-H), 4.07 (s, 3H, N+CH3), 3.22 (s, 3H, NCH3), 2.39 (s, 3H, SCH3). 13C{1H} NMR (100 MHz, DMSO-d6): δ (ppm) 169.8 (Ar-C), 158.3 (Ar-C), 144.5 (Ar-C), 140.6 (Ar-C), 140.4 (Ar-C), 134.1 (Ar-C), 129.8 (Ar-C), 127.9 (Ar-C), 127.9 (Ar-C), 126.8 (Ar-C), 126.6 (Ar-C), 126.5 (Ar-C), 125.2 (Ar-C), 123.0 (Ar-C), 119.2 (Ar-C), 114.2 (Ar-C), 84.5 (meso-C), 43.3 (N+CH3), 38.6 (NCH3), 19.0 (SCH3). HRMS in CH3OH (ESI/Q-TOF) m/z [M−I]+ calcd for C20H19N2S2+ 351.0984; found 351.0988. Anal. Calcd for C20H19IN2S2: C, 50.21; H, 4.00; N, 5.86; found: C, 50.13; H, 3.98; N, 5.71. Synthesis of 2g. A mixture of 1 (0.95 g, 2.20 mmol) and 3 mL of SOCl2 in 5 mL DMF was reacted at 80 °C for 5 h. After the reaction was completed, and cooled, an excess of diethyl ether was added into the reaction solution to give precipitates. Then the mother liquor was concentrated under reduced pressure, and obtained solid was combined with 26


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above precipitates to give the crude product, which was submitted to flash elution chromatography (petroleum ether/dichloromethane, 1/1 v/v or pure dichloromethane) and afforded 2g as an orange solid (0.77 g, 72%). m.p. 281−282 °C. UV-vis (MeOH) λmax (ε [M-1 cm-1]): 451 nm (1.25×104). 1H NMR (400 MHz, D2O): δ (ppm) 8.89 (s, 1H, CHO), 8.25 (d, J = 8.0 Hz, 1H, Ar-H), 8.20 (d, J = 8.0 Hz, 1H, Ar-H), 8.12 (t, J = 8.0 Hz, 1H, Ar-H), 8.04 (b, 1H, Ar-H), 7.88−7.93 (m, 2H, Ar-H), 7.84 (d, J = 9.6 Hz, 1H, Ar-H), 7.75 (d, J = 8.0 Hz, 1H, Ar-H), 7.67 (t, J = 8.0 Hz, 1H, Ar-H), 7.56 (t, J = 8.0 Hz, 1H, Ar-H), 4.31 (s, 3H, N+CH3), 3.45 (s, 3H, NCH3). 13C{1H} NMR (100 MHz, D2O): δ (ppm) 171.3 (Ar-C), 157.9 (Ar-C), 153.9 (Ar-C), 146.2 (CHO), 142.4 (Ar-C), 140.3 (Ar-C), 136.2 (Ar-C), 130.5 (Ar-C), 130.0 (Ar-C), 129.4 (Ar-C), 128.4 (meso-C), 127.7 (Ar-C), 126.9 (Ar-C), 126.2 (Ar-C), 123.0 (Ar-C), 119.0 (Ar-C), 115.8 (Ar-C), 47.7 (N+CH3), 37.6 (NCH3). HRMS in CH3OH (ESI/Q-TOF) m/z [M−I]+ calcd for C20H17N2OS+ 333.1056; found 333.1076. Anal. Calcd for C20H17IN2OS: C, 52.18; H, 3.72; N, 6.09; found: C, 52.07; H, 3.85; N, 6.22.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: xx.xxxx/acs.joc.xxxxxxx. Full copies of NMR and HRMS spectra (PDF), and crystallographic data for 2a, 2b, and 2f 27



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(CIF). This material is available free of charge on the ACS Publications website. AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]. * E-mail: [email protected] Author Contributions §These

authors contributed equally to this work

Notes There are no conflicts to declare. ACKNOWLEDGEMENTS This project was funded by the Scientific and Technological Research and Development Projects in Shaanxi Province under Grant No. 2018GY-128, and the National Natural Science Foundation of China under Grant No. 21575111 and 21807086. REFERENCES (1) (a) Thomas, A. P.; Palanikumar, L.; Jeena, M. T.; Kim, K.; Ryu, J. H. Cancer-Mitochondria-Targeted Photodynamic Therapy with Supramolecular Assembly of HA and A Water Soluble NIR Cyanine Dye. Chem. Sci. 2017, 8, 8351-8356. (b) Li, B. H.; Lu, L. F.; Zhao, M. Y.; Lei, Z. H.; Zhang, F. An efficient 1064 nm NIR-II Excitation Fluorescent Molecular Dye for Deep-Tissue High-Resolution Dynamic Bioimaging. Angew. Chem. Int. Ed. 2018, 57, 7483-7487. (2) (a) Fu, N.; Su, D.; Cort, J. R.; Chen, B. W.; Xiong, Y. J.; Qian, W. J.; Konopka, A. E.; Bigelow, D. J.; Squier, T. C. Synthesis and Application of an Environmentally Insensitive 28


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