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Design of NIR Chromenylium-Cyanine Fluorophore Library for “Switch-ON” and Ratiometric Detection of Bio-Active Species in Vivo Yanfen Wei, Dan Cheng, Tianbing Ren, Yinhui Li, Zebing Zeng, and Lin Yuan Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.5b04169 • Publication Date (Web): 05 Jan 2016 Downloaded from http://pubs.acs.org on January 6, 2016

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Analytical Chemistry

Design of NIR Chromenylium-Cyanine Fluorophore Library for “Switch-ON” and Ratiometric Detection of BioActive Species in Vivo Yanfen Wei, Dan Cheng, Tianbing Ren, Yinhui Li, Zebing Zeng, Lin Yuan* State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082 (PR China) ABSTRACT: The real-time monitoring of key bio-species in the living systems has received thrusting attention during the past decades. Specifically, fluorescent detection based on near-infrared (NIR) fluorescent probes is highly favorable for live cells, live tissues and even animal imaging, owing to the substantial merits of NIR window, such as minimal phototoxicity, deep penetration into tissues, and low auto-fluorescence background. Nevertheless, developing potent NIR fluorescent probes still poses serious challenges to the chemists because traditional NIR fluorophores are less tunable than visible-wavelength fluorophores. To address this issue, here we report a set of novel NIR hybrid fluorophores, namely the hybrid chromenylium-cyanine fluorophore (CC-Fluor), in which both the fluorescence intensity and the emission wavelength can be easily adjusted by the conformational changes and substitution groups. Compared to known NIR fluorophores, the new CC-Fluors are substantially advantageous for NIR probe development: (1) CC-Fluors display tunable and moderate Stokes shifts and quantum yields; (2) the fluorophores are stable at physiological conditions after long-term incubation; (3) the absorption maxima of CC-Fluors coincide with the common laser spectral lines in mainstream in vivo imaging systems; (4) most importantly, CC-Fluors can be easily modified to prepare NIR probes targeting various bio-species. To fully demonstrate the practical utility of CC-Fluors, we report two innovative NIR probes-a ratiometric pH probe and a turn-on Hg2+ probe-both are successfully employed in live animal imaging. Hence, the detailed studies allow us to confirm that CC-Fluors can work as an excellent platform for developing NIR probes for the detection of species in living systems.

The development of novel and potent fluorescent probes with enhanced photo-physical properties continues to attract attention from biologists and clinicians owing to their widespread application prospects in the area of cell biology and clinical 1-5 surgeries. Recent research has identified that fluorescent probes are featuring extraordinary analytical sensitivity, facile tenability and high 6-12 spatiotemporal resolution. However, when it comes to in vivo imaging, fluorescent probes in the visible optical window suffer from shallow penetration, high auto-fluorescence and terrible scattering issues from the biological samples. Hence, researchers have shifted their attention to near infrared (NIR) fluorescent probes long ago because they are particularly useful benefitted live animal imaging owing to the deep 13-15 penetration and minimum ambient interference. A number of NIR fluorescent probes with reasonable efficiency have been disclosed in the literatures and some of them were used to successfully tackle 13-15 biological and clinical issues. Even though there are classic NIR dye-based fluorescent probes, i.e. probes derived from cyanine moieties, majority of them still 13-15 suffer from various problems. In most cases, the tuning of NIR emission wavelengths and intensity is

less efficient than that of visible-wavelength fluorophores by traditional mechanisms such as fluorescence resonance energy transfer (FRET) or photo-induced electron transfer (PET). Moreover, to reach the NIR optical window, NIR fluorophores generally sacrifice quantum yields by adopting the loose π-conjugated structures; as a result, such loose scaffolds easily diffuse the excited state energy. Therefore, the development of novel NIR fluorophores with facilely tunable photo-physical properties has faced severe challenges. For efficient in vivo imaging, the general requirements for NIR probes are: 1) high quantum yields/fluorescence brightness under biological conditions; 2) moderate Stokes shifts to avoid auto fluorescence from tissues; and 3) drastic fluorescence intensity variations or wavelength shifts upon the interaction with targets of interest. In addition, due to the instrumental limitation, the common excitation wavelengths equipped by mainstream NIR microscopy are restricted. Therefore, the absorption/excitation maxima of the probes should closely match the common laser excitation wavelengths to allow highest excitation/emission efficiency.

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Towards this end, recently, others and our groups have constructed a series of novel NIR dyes, whose fluorescence can be easily controlled by the unique spirolactam ring-opening/-closing process or the 16-20 Various optically tunable hydroxyl group. fluorescent turn-on and ratiometric NIR probes based 21-31 on these scaffolds have been reported; however, they still suffer from multiple drawbacks, including the small Stokes shifts, limited spectral range and the unmatched absorption/excitation maxima with the commercial imaging system. In addition, most of the above fluorophores require complicated multiple-step synthesis, thereby restricting their substituent modification potential. These results prompted us to exploit new designs to obtain NIR dyes that are facilely synthesized and easily tuned. Herein, to obtain NIR probes with desired photophysical properties, a small library based on the hybrid chromenylium-cyanine fluorophore (CC-Fluor) core were successfully constructed. In total, 35 fluorescent compounds were prepared by facile one or two-step synthesis. The CC-Fluor has several key features: 1) its synthetic method is highly versatile and can be easily modulated to produce further structural shifts. 2) The absorption maxima of these dyes well coincide with the common laser spectral lines of the in vivo imaging system. 3) The optical properties of CC-Fluors are accommodated by structural variations such as tunable hydroxyl/amino groups or spirolactam ringopening and -closing mechanisms. Such features could significantly facilitate the development of NIR fluorescent turn-on or ratiometric probes, which can be applied to in vivo imaging. EXPERIMENTAL SECTION Materials and General experimental Methods. All chemical reagents for the dye and probe synthesis were obtained from commercial supplies (Sigma Aldrich, JK chemical, Aladdin, TCI, etc.), and used without further purification unless otherwise specified. Column chromatography was carried out on Silica Gel 60 (100−200 mesh). Synthetic reactions and analytical characterization were monitored by HPLC-MS (Agilient-1200 series) with a DAD detector and a single quadrupole mass spectrometer with an ESI probe. 1 13 NMR spectra ( H-400 MHz and C-100 MHz) were recorded on Bruker Avance 400 NMR spectrometers. Spectroscopic and quantum yield data were measured on spectroscopic measurements, performed on a HITACHI F4600 fluorometer and LabTech UV Power spectrometer. DFT calculations were performed using Gaussian 09 (Revision A.02) with B3LYP functional and 6-31 G(d) basis set. The in vivo imaging was carried out using an IVIS Lumina XR in vivo imaging system. Synthesis of CC35: To a stirred solution of CC1 (50.2 mg, 0.08 mM) in 5 mL DCM, 0.1 mL (0.21 mM) phosphorus oxychloride was added drop-wise under

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argon atmosphere. The mixture was stirred at room temperature for 30 min and then heated to reflux for another 5 h. The solvent was removed under reduced pressure and 5 mL acetonitrile was added into the mixture. Then thiosemicarbazide (30.0 mg, 0.33 mM) and triethylamine (40 μL) were added respectively and stirred at room temperature for overnight. The solvent was removed under reduced pressure and residue was purified by column chromatography on silica gel (CH2Cl2 : MeOH = 30 : 1) to afford the compound CC35 1 as a solid (yield: 32 mg, 68%). H NMR (400 MHz, CDCl3) δ 7.95 (d, J = 7.6 Hz, 1H), 7.90 (d, J = 7.8 Hz, 1H), 7.63 (t, J = 7.4 Hz, 1H), 7.57 (s, 1H), 7.54 (t, J = 7.4 Hz, 1H), 7.34 (t, J = 7.5 Hz, 1H), 7.31 – 7.27 (2H), 7.23 (d, J = 7.5 Hz, 1H), 7.19 (s, 1H), 7.12 (s, 1H), 6.47 (d, J = 2.3 Hz, 1H), 6.36 (d, J = 8.8 Hz, 1H), 6.31 (dd, J = 8.9, 2.3 Hz, 1H), 6.06 (s, 2H), 3.85 (s, 3H), 3.37 (q, J = 7.1 Hz, 4H), 2.87 – 2.65 (2H), 2.09 – 1.90 (2H), 1.73 – 1.69 (2H), 1.20 13 (t, J = 7.0 Hz, 6H). C NMR (100 MHz, CDCl3) δ 184.89, 167.14, 153.55, 149.41, 149.39, 149.30, 136.51, 134.08, 129.00, 128.78, 128.43, 128.41, 127.04, 125.41, 124.21, 124.01, 122.39, 119.78, 119.38, 116.28, 112.35, 109.28, 108.83, 103.03, 102.59, 98.52, 68.97, 44.35, 33.09, 29.70, 22.19, 28.33, + 12.62. MS (ESI): m/z = 590.0[M+H] . HRMS (EI): [MH] calcd for [C35H34N5O2S] 588.2439, found 588.2448. In Vivo image Studies. Kunming mice (20-25 g) were anesthetized by ip injection of xylazine (10 mg/kg) and ketamine (80 mg/kg). Then the mice was given an ip injection of CC35 (50 μL, 1 mM in a mixture of PBS buffer (pH 7.4, 10 mM) and DMSO (4/1, v/v)) 2+ and followed by ip injection with the Hg (50 μL, 1 mM in a mixture of water) after 5 min. The mice were then imaged by using an IVIS Lumina XR in vivo imaging system. RESULTS AND DISCUSSION Design and Synthesis the CC-Fluor Library. During the course of our studies, the reported attempts to develop novel NIR fluorophores CS-NIR 16, 17 and HD-NIR dye (Scheme 1) inspired our scaffold design. Structural analysis showed that these dyes are composed of a 2, 3-dihydro-1H-xanthene core connected to an indolium moiety, bridged by a doublebond. The fluorescence of CS-NIR and HD-NIR dyes was on/off controlled by the characteristic spirolactam ring-opening/-closing process or through the protection of the hydroxyl group on the xanthene. Such protected phenol derivatives or ring-closed derivatives would be further used as molecular probes for detection and imaging of species or cellular events with a turn-on model. We consider that 2, 3-dihydro1H-xanthene core and indolium moieties can be replaced by other electron-deficient chromenylium or electron-rich indoles or analogues, thereby creating a family of hybrid chromenylium-cyanine fluorophores (CC-Fluor). In addition, the chemical stability of fluorophores is of prime importance for their long time


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imaging in living system. We found that compound 1 was prone to bleach when exposed in aqueous solution (Figure S1). This instability is caused by the lack of chemical capping at C-4 of chromenylium, which is the most reactive position of the molecule. Herein, a benzoic acid was rationally introduced at C-4 site, thus not only improving the stability of the dye, but also taking use of a spirolactam ring opening/closing modulator to switch fluorescence on/off. As a result, the combination of eight 4-(2-carboxyphenyl)chromenylium cores with thirteen indole aldehydes or its analogues lead to 35 discrete hybrid chromenyliumcyanine fluorescent compounds, namely CC-Fluor (Scheme 1). Their names and photo-physical properties are listed in table 1-4 and table S1-2, with their purity characterized by HPLC-MS (Table S1).

Scheme 1. (A-C) Chemical structures of CS-NIR and HD-NIR dye (A), potential 4-(2-carboxyphenyl)-chromenylium moieties (B) and indole aldehydes or its analogues (C). (D) Preparation of CC-Fluor.

Effects of Indole Substituents on the Photophysical Properties of CC-Fluor. To elaborate the structure-properties relationship, the library was divided into several sub-groups, each sharing a common moiety. In group 1, a strong electrondonating diethyl amine moiety decorates the chromenylium core at 7-position, where only the indole or its analogue moiety is changed. Several general postulations can be deducted on the basis of integrated analysis data in Table 1: 1) It is clear that the dyes containing electron-rich nitrogen in the indole (e.g. indole derivatives CC1-CC2, CC5, CC10 and CC12) demonstrated higher fluorescence quantum yields than that of the compounds containing exocyclic electron-rich nitrogen within R1 functional group (e.g. CC7). 2) The quantum yields of CC-Fluor decrease with the introduction of extra electron donor groups to the indole ring (e.g. CC4, and CC6 vs CC2). This is possibly due to the excited state energy diffusion caused by the

extra groups overwhelming their electron ability. 3) The excitation/emission wavelengths showed bathochromic shift from 627/677 (CC1) to 698/729 (CC10) and finally to 718/749 nm (CC12) with enhanced conjugation of the indole ring, which also maintained their quantum yields moderate and stable. Table 1. Chemical structures and photo-physical properties of CC1-12 in MeOH.

Compd

R1

λabs

λem /nm

Stokes shift/nm

Φ (100 %)

/nm CC1

627

677

50

42

CC2

622

672

50

31

CC3

575

660

85

0.8

CC4

619

678

59

6

CC5

636

684

48

22

CC6

618

676

58

8

CC7

642

717

75

0. 7

CC8

570

654

84

12

CC9

618

668

50

4

CC10

698

729

31

35

CC11

626

667

41

16

CC12

718

749

31

27

An interesting compound worth our investigation here is CC2, a non-N-alkylated analogue of compound CC1. CC2 showed similar absorption and emission spectra to CC1 (Figure S2), while both absorption and emission wavelength displayed slightly hypochromic shift due to the difference of electron donating ability. However, compound CC2 displayed pH-insensitive absorption and emission spectra (Figure S3), in contrast to the pH dependency generally observed in 32-33 non-N-alkylated cyanine dyes. This result indicated that the positive charge of compound CC2 mainly distributed among the chromenylium moiety rather than the indole moiety. Thus, compound CC2 lost “cyanine limit” characteristic and is a typical intra34 molecular charge transfer (ICT) dye. This



Effect of Chromenylium Substituent on the Photo-physical Properties of CC-Fluor. Other than the effects of indole electron, the electron-donating capability of the substituents on the chromenylium should also affect the CC-Fluor. In this section, we have extracted the effects of R2 substituent on the photo-physical properties through a systematic analysis of a series of fluorescent compounds with same core skeletons: 2, 3-dihydro-1H-xanthene attached to either 1-methyl-3-vinyl-1H-indole or (E)-2allylidene-1,3,3-trimethylindoline (Figures 1, S6 and Table 2). The fluorescence intensity (quantum yield) increased when the R2 substituent changed from a mild electron donating group methoxyl (CC13, CC16) to a strong electron donating hydroxyl (CC14, CC17). On the other hand, the absorption and emission wavelengths displayed a bathochromic shift trend when the hydroxyl group changed from hydroxyl (CC14, CC17), to diethyl amino (CC1, CC10) and then to fused amino (CC15, CC18) group. Not surprisingly, the enhanced electron-donating ability of R2 group and hyper-conjugation between the electrons in the σ bonds of the substituents on the amino group with the adjacent p and π orbitals. Table 2. Photo-physical Properties of CC1, CC10 and CC13-18 (CC13-14 and CC16-17 were measured in PBS (pH = 10, 10%DMF), others recorded in MeOH).

Com pd

R2

λabs

Εmax

λem

/nm

/10 M −1 cm

4

/nm

−1

/nm

Φ (100 %)

Stoke s shift

CC13

OCH3

586

2.5

648

62

12

CC14

OH

577

2.8

645

68

42

CC1

N(Et)2

627

3.4

677

50

42

CC15

N(CH2C H2)2

634

3.6

686

52

33

CC16

OCH3

605/6 62

4.9

685

80

5

CC17

OH

685

9.4

715

30

27

CC10

N(Et)2

698

13.9

729

31

35

CC18

N(CH2C H2)2

708

12.5

743

35

30

a)

CC13 CC14 CC1 CC15

Normalized absorbance

experimental result was further supported by DFT calculation (Figure S4), which disclosed that the HOMO orbital mainly distributed in the whole πconjugated chromenylium indole framework (mainly in indole) and LUMO mainly distributed in the chromenylium-vinyl moiety. Significantly, these results are in accordance with the absorption spectrum of compound CC2 (Figure S3b), which bears a broad and intense absorption band in the NIR spectra, a characteristic of ICT dyes. This ICT characteristic can also explain the decreasing quantum yields of CC4, CC5, CC6, because introduction of extra electron donor groups to the indole ring causes too strong ICT 35-36 that results in the fluorescence quench. We also investigated the optical properties of compound CC3, an acetylated derivative of compound CC2. However, the optical profiles of CC3 are drastically distinct from those of CC2. CC3 displays roughly 40-fold lower quantum yield (Φ = 0.008) than CC2 (Φ = 0.31) (Figure S5), which is due to the acetyl group on indole nitrogen atom that significantly diminishes the electron donating ability of the indole nitrogen atom. As a result, both absorption and emission spectra exhibit hypochromic shifts due to the decrease of the strength of intra-molecular charge transfer (ICT).

400

500

600

700

Wavelength, nm b)

CC13 CC14 CC1 CC15

Normalized intensity

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

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600

650

700

750

Wavelength, nm

Figure 1. Normalized absorption (a) and emission (b) spectra of CC13-15 and CC1. The emission intensity of CC13-15 and CC1 were normalized while keeping the peak ratios between CC14 and CC13 unchanged.

Effect of Ring Size on the Photo-physical Properties of CC-Fluor. Other than the substituent effects, considering that minimal structural change 37 may significantly affect the photo-physical of dyes, we then investigated how different linkers at C-3, 1’ position of 2-vinylchromenylium could modulate the optical profiles. As shown in Figure 2 and Table 3, the


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excitation/emission wavelength displayed bathochromic shift when the number of saturated carbon linker decreased from four to three, and then to two. For example, both the absorption and excitation wavelengths red-shifted from CC19 (λab/λem = 709/735) to CC10 (λab/λem = 713/739) and CC20 (λab/λem = 719/744) in CH2Cl2 with fixed R2 group as diethylamino and R1 group as 1-methyl-3- vinyl-1H-indole. This because the molecular planarity between chromenylium moiety and indolium moiety gradually enhances when the ring size shrink, thus consequently extends the π-electron system and narrows the energy gap (Figure S7), which enables the CC-Fluor derivatives to emit in the long-wavelength region. However, the excitation/emission wavelength displayed notable blue shift when the saturated carbon number in ring decreased to zero. CC21 has an absorption/emission maximum at 688/714 in CH2Cl2, being blue-shifted by 31/30 nm in comparison with that of CC20. The blue-shifted absorption/emission maximum can be attributed to the lack of hyperconjugation effects of the electrons in the σ bonds of the substituents in the ring linker (Figure S7). Similar results were observed when the R2 group was changed, thus indicating that the saturated carbon number in the ring also significantly affects the optical properties (Table S2).

b)

Normalized absorbance

CC19 CC10 CC20 CC21

500

600

700

800

Wavelength, nm c)

CC19 CC10 CC20 CC21

Normalized intensity

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


680

720

760

800

Wavelength, nm

Figure 2. Structure (a), normalized absorption (b) and emission (c) spectra of CC19-21 and CC10 in CH2Cl2.

Table 3. Photophysical properties of CC19-21 and CC10 in CH2Cl2. Compd

λabs

λem

Stokes shift

Φ

CC19

709

735

27

0.12

CC10

713

739

26

0.31

CC20

719

744

25

0.27

CC21

688

714

26

0.12

Developing NIR CC-Fluor with Optically Tunable Hydroxyl Group and the Applications for Ratiometric Imaging in Living Mice. The fluorescent dyes with optically tunable hydroxyl groups have received extensive interest as they can be commonly employed in biochemical assays, 16, fluorescent sensing, and molecular optical imaging. 25-30, 38-39 However, there is scarce report of NIR fluorescent dyes with optically tunable hydroxyl groups that could display large fluorescence turn-on response or even well-resolved ratiometric response. The above structure-property studies provides solid evidence that the optical properties of CC-Fluor can be fine-modulated by changing the R1/R2 substituent groups or subtly tuning the size of saturated carbon ring in C-3, 1’ position of 2-vinylchromenylium. We then synthesized a series of CC-Fluors with various hydroxyl groups (R2) to evaluate whether CC-Fluor were suitable for fluorescent turn-on or ratiometric sensing. As shown in Table 4, Figures 3 and S8-15, both R1 substituent group and the ring size in C-3, 1’ position of 2-vinylchromenylium can notably affect the optical properties. For example, CC14 and CC30, which consisted of 3-vinyl-1H-indole unit at R1 site and three saturated carbons at C-3, 1’ linker, exhibited only fluorescence turn-on response (~6.4-fold) when pH increased from 1 to 10 with negligible fluorescence emission shift (Figures S8-9). When the ring size shrink, compounds CC31-32 still showed fluorescence turn-on response when pH increased from 1 to 4, with slight fluorescence emission wavelength shifts (11 nm) as the pH continues to increase from 4 to 10 (Figures S10-12). More significant fluorescence enhancement and wavelength shift were observed when R1 changed from 3-vinyl-1H-indole to (E)-2-allylidene-1, 3, 3trimethyl-indoline (Figures 3a, S13). For example, when pH increased from 4 to 8, CC17 displayed about 100fold fluorescence enhancement excited at 670 nm (Figure S13). More importantly, as shown in Figure 3a, two discernable emission bands were observed with excitation at the isosbestic point. These emission wavelength shifts and fluorescence intensity changes



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are attributed to the protonation/de-protonation of the hydroxyl groups, which changes the electron donating ability and the strength of ICT. Owing to such sharp fluorescence intensity changes and improved stability at physiological condition compared to 1 (Figure S1D-E), CC17 possesses remarkable advantages to be employed as NIR fluorescent “turnon” imaging reagents by easy modulations on the hydroxyl group. However, CC17 is unfavorable for the high-resolution ratiometric imaging in living animals because of the serious cross talk between two emission bands.

compared to CC17 (Figure 3c, Figure S15). CC33 displayed two well-resolved emission bands than other dyes with a superior stability under physiological condition. In addition, CC33 was even more stable in presence of intracellular nucleophilic reagent H2S (Figure S16), indicating that the new dye CC33 might be exploited as a candidate dye to develop ratiometric probes for high-resolution analyte-responsive imaging. As a showcase, CC33 was developed as NIR ratiometric fluorescent probe to visualize pH changes in an abdominal model. As shown in Figure S17, the mouse incubated with PBS (pH 5) and then CC33 displayed a strong fluorescence in Channel 1 (650 nm) and slight Nevertheless, the above studies imply that the fluorescence in Channel 2 (720 nm). However, the emission band shifts depend on the ring size and R1 fluorescence intensity in the mouse treated with PBS group: small ring size and (E)-2-allylidene-1,3,3(pH 8) and CC33 changed remarkably, a partial trimethylindoline as R1 substituent can benefit the decrease of fluorescence in Channel 1 along with an emission shift. Then an optimized dye CC33 and apparently increase of fluorescence in Channel 2 were controllable compound CC34 were synthesized. As observed. These results indicated that CC33 is capable expected, from Figure 3b and Figure S14 we observed of ratio monitoring pH changes in vivo, which was that CC33 displayed two well-resolved emission bands superior than many known NIR fluorescent pH while excited at the isosbestic point. In contrast, CC34 32,33,40 probes. showed a negative change of the emission bands Table 4. Chemical Structures and Spectroscopic Data of CC-Fluor with optically tunable hydroxyl group.

Compd

CC14

CC30

CC31

CC32

CC17

CC33

CC34

pH a

λabs

λem

Stokes shift

Φb

10.0

594

655

61

0.32

1.0

594

655

61

0.05

pKa c

Δλfl d

Ratio changes e

4.1

0

1.0

4.6

1

1.0

11

1.3

11

1.3

10.0

586

650

64

0.30

1.0

586

649

63

0.05

10.0

615

656

41

0.65

5.5

4.0

596

645

49

0.41

3.1

10.0

603

648

45

0.51

5.4

4.0

596

637

41

0.29

2.9

10.0

685

715

30

0.27

6.7

30

14.8

4.0

660/610

685

75

0.06

10.0

690

725

35

0.22

7.1

42

35.8

4.0

656/608

683

75

0.14

10.0

671

704

33

0.18

6.6

27

8.51

4.0

647/603

677

74

0.02

a

Condition: PBS/DMF (8: 2); b quantum yield were estimated using ICG (Φ = 0.13 in DMSO) as standard; c The pKa was calculated using the Henderson-Hasselbach-type mass action equation (log[(Fmax − F)/(F − Fmin)] = pKa – pH; d The shift of maximum emission wavelength (Δλfl) at pH 10 and 4. e The fluorescence intensity ratio changes at pH 10.0 over that at pH 4.0.


40 20 4 5 6 7 8 9 10

40

pH

20

0 700

750

Wavelength, nm

800

60

80

40 20 4

40

5

6

7

8

9

10

pH

20

60

750

40

800

40 20 0

4 5 6 7 8 9 10 Wavelength, nm

20

0 660

0 700

c)

60

Fl. intensity (a. u.)

60


b)

80


60

650

60

80

a)


80 Fl. intensity (a. u.)

680

700

720

740

760

Wavelength, nm

Wavelength, nm

Figure 3. Fluorescence emission spectra of CC17 (λex = 630 nm), CC33 (λex = 640 nm), CC34 (λex = 620 nm) in buffer solution at 5 µM with pH changing from 4.0 to 10.0. Inset: pH plot of normalized emission intensities at 715 nm for CC17 (a), at 725 nm for CC33 (b) and at 704 nm for CC34 (c). Measurements were made in 50 mM sodium phosphate buffer at various pH values in the presence of 5% DMF as a co-solvent.

concentration of CC35 used was 1 μΜ (Figure S22), which was superior than many known NIR fluorescent 2+ 19, 42-45 Hg probes (Table S3). The reaction-based NIR 2+ probe CC35 was highly selective to Hg over other 2+ + 3+ relevant metal ions represented by Cu , Cu , Fe , 2+ 2+ 2+ 2+ 3+ 2+ 2+ + + Cd , Zn , Mn , Pb , Al , Ca , Mg , Na , K (Figure 4c). In addition, the standard MTT assay indicates that the probe CC35 at the low micromolar concentrations exhibited no marked cytotoxicity to the cells (Figure S23), proving the promise of CC35 for biological applications.

750

b)


750


600 450 300

600 450 300 150 0 0 1 2 3 4 5 6 7 8

[Hg2+], µM

150 0 680

700

720

740

760

780

Wavelength, nm

750

c)

600 450 300 150


2+

g H

2+

2+

Zn

2+

+

3+

2+

M g M n N a Pb

3+

+

K Al

+

2+

2+

C u C u Fe

Ca Cd

2+

0 Fr ee

Development of a New NIR Fluorescent Turn-on Sensor for Mercury Ions and the Applications for Biological Imaging in Living Mice. Compared to previously reported NIR fluorescent dyes, CC-Fluor offers the following additional advantages: 1) Compatibility with common excitation wavelength (e.g. 605 nm, 640 nm, 675 nm, 710 nm) of living imaging system. 2) Bathochromic shifted fluorescence emission with modest Stokes shift and quantum yields. 3) Stability under biological conditions. 4) Moderate water solubility. 5) Easily synthesized by one-step reaction. 6) Importantly, facile control of the optical profiles of CC-Fluor through Spiro-lactam ringopening/-closing process or substituents. To further validate the possibility of using CC-Fluor for in vivo detection and imaging, we designed and synthesized 2+ CC35 as a NIR fluorescent turn-on probe for Hg 2+ (Figure 4a). The design was based on the Hg promoted 1,3,4-oxadiazoles formation from 41 thiospirolactone. Firstly, we measured the absorption and emission spectra of CC35 at pH 7.4 (Figures 4 and S18). CC35 intrinsically showed negligible absorption or fluorescence at the NIR region because it formed an intramolecular spirocyclic ring. In the presence of 2+ Hg , CC35 elicited peak absorption intensity at 663 nm and fluorescence intensity at 702 nm (Figures 4b 2+ and S18). Remarkably, one equivalent of Hg saturated the fluorescence of CC35 with a 105-fold enhancement (Figure 4b). High performance liquid chromatographymass spectrometry (HPLC-MS) analysis confirmed that 2+ the fluorescence turn-on was indeed due to the Hg mediated formation of a ring-opened fluorescent dye CC36 (Figures S19-20). Analysis of the titration data revealed that the emission intensity at 702 nm increased linearly with the increasing concentrations 2+ of Hg in a dynamic range of 0.1–4 μM (Figure S21) with CC35 5 uM and a detection limit of 50 nM based on calculation (3σ/s). It is worth noting that the detection limit can be further improved by lowering the concentration of CC35. For example, the detection 2+ limit of probe for Hg was lower than 10 nM when the


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Analytical Chemistry 2+

Figure 4. (a) Designing Hg probe. (b) Fluorescence spectra of NIR fluorescent probe CC35 (5 μM) in the presence of 2+ various concentrations of Hg (0−10 μM) in PBS buffer (50 mM, pH 7.4, containing 20% DMF) with excitation at 670 nm. Inset: Fluorescence intensity of CC35 (5 μM) at 702 nm 2+ vs Hg concentration (0−8 μM). (c) The fluorescence intensity of probe CC35 (5 μM) at 702 nm excited at 670 nm + + in the presence of various metal ions (50 mM for K , Na , 1 2+ 2+ mM for Ca , Mg and 10 μM for others).

After in vitro tests, we examined the ability of CC35 2+ to visualize Hg in living animal. Kunming mice were selected as our model and were given a skin-pop 2+ injection of CC35 and Hg in order. Live mice injected with CC35 for 0.5 hour showed very weak emission at NIR channel of 700−755 nm (Figure 5a). However, 2+ when the living mice were injected with Hg under the same conditions, a significant enhancement was observed with time (Figure 5b-f), indicating that probe CC35 can serve as a fluorescent agent for mercury ion imaging in vivo. 6 Normalized intensity

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sensor and a fluorescent turn-on Hg animal imaging.

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probe) in living

ASSOCIATED CONTENT Supporting Information Synthesis detail and characterization data, density functional theory calculations, HPLC−MS analysis, cytotoxicity of the probe, and data from the control sample (PDF). This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * Email: [email protected].

Author Contributions The manuscript was written through contributions of all authors and all authors have given approval to the final version of the manuscript.

Notes The authors declare no competing financial interest.

g)

ACKNOWLEDGMENT 4

2

0

a b c d e f

Figure 5. Representative fluorescence images (pseudocolor) of Kunming mouse given a skin-pop injection of CC35 (50 μL, 1 mM in a mixture of PBS buffer (pH 7.4, 10 mM) and DMSO 2+ (4/1, v/v)) and a subsequent skin-pop injection of Hg (50 μL, 1 mM in water). Images were taken after incubation for 0 (a), 1 (b), 5 (c), 15 (d), 20 (e) and 30 (f) min, respectively. (g) Quantification of imaging data.

CONCLUSION In this study, we have reported a novel NIR novel fluorophore with controllable optical properties. Our design was based on the hybrid chromenylium-cyanine scaffold (CC-Fluoros) that can be easily optically tuned. A series of chromenylium cores and indole substituents were incorporated into the framework of dye structure to generate a dye library (35 dyes) with the fluorescence emission lying in the NIR region (620760 nm). These dyes contain various advantages: moderate Stokes shift, substantial quantum yields and stability under physiological conditions. More importantly, the absorption maximum of these dyes covers most of common laser spectral lines in vivo imaging system, which is important for in vivo imaging with optimal excitation/emission efficiency. Finally, we demonstrated that CC-Fluoros can be exploited as NIR fluorescent probes for in vivo imaging applications with high brightness, which was verified by two innovative NIR probes (a ratiometric fluorescent pH

This work was financially supported by NSFC (21302050, 21502049, 51573040, J1210040), the Hunan Provincial Natural Science Foundation of China (14JJ2047), the Fundamental Research Funds for the Central Universities, and the Startup Fund of Hunan University (531109020043).

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