A General Method to Increase Stokes Shift by Introducing Alternating

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A General Method to Increase Stokes Shift by Introducing Alternating Vibronic Structures Tian-Bing Ren, Wang Xu, Wei Zhang, Xing-Xing Zhang, ZhiYao Wang, Zhen Xiang, Lin Yuan, and Xiaobing Zhang J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.8b04404 • Publication Date (Web): 24 May 2018 Downloaded from http://pubs.acs.org on May 24, 2018

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Journal of the American Chemical Society

A General Method to Increase Stokes Shift by Introducing Alternat‐ ing Vibronic Structures Tian-Bing Ren,‡ Wang Xu,‡ Wei Zhang, Xing-Xing Zhang, Zhi-Yao Wang, Zhen Xiang, Lin Yuan,* Xiao-Bing Zhang State Key Laboratory of Chemo/Biosensing and Chemometrics, College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082 (PR China). *E-mail: [email protected] Supporting Information Placeholder

ABSTRACT: Fluorescent dyes have enabled the progress of the broad biomedical fields. However, many commercially available dyes suffer from small Stokes shifts, resulting in poor signal-to-noise ratio and self-quenching on current microscope configuration. In this work, we have developed a general method to significantly increase the Stokes shifts of common fluorophores. By simply appending a 1,4diethyl-decahydro-quinoxaline (DQ) moiety onto the conjugated structure, we introduced a vibronic backbone that could facilely expand the Stokes shifts, emission wavelength and photo-stability of eleven different fluorophores by more than 3-fold. This generalizable method could significantly improve the imaging efficiency of commercial fluorophores. As a demonstration, we showed that the DQ derivative of hemicyanine generated 5-fold signal in mouse models over indocyanine green (ICG). Furthermore, the DQ modified fluorophores could pair with their parent molecules to conduct one-excitation, multiple emission imaging and study the cell behavior more robustly. This approach is prospective in generating dyes suitable for super-resolution microscopy and second window NIR imaging.

Introduction

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Fluorescent dyes oversee a broad spectrum of in vitro biological applications owing to their optical and structural tenability, non-invasive treatment, cell compatibility and real time response.1 However, most widely adopted fluorescent dyes, e.g. fluorescein, rhodamine, oxazineand cyanine display small Stokes shifts of less than 30 nm.2 The severe crosstalk between the excitation and emission spectra generally results in poor signal-to-noise ratio and self-quenching due to back scattering from biological samples (Figure 1a).3 Next generation applications, e.g. single-molecule analysis, demand fluorescent dyes with large and tunable Stokes shifts to achieve precise imaging and accurate sensing.4 Notably, much effort was devoted to redshift the emission in order to meet clinical requirements such as surgical guidance.5 Prominent examples cover 10’-heteroatom rhodamines,6 cyanine/polymethine derivatives5a,7 and nanomaterials.8 By narrowing the HOMO-LUMO gap, one indeed observes redshifted emission, but not predictable Stokes shift changes. Individual efforts to enlarge the Stokes shifts of known fluorophores were sparsely reported. Collectively, they can be classified as: 1) nitrogen insertion at meso-position of cyanine or pyronine;3a,4 2) incorporating a rotary substituent to 6(diethylamino)-1,2,3,4-tetrahydroxanthylium;9 3) pseudo Stokes shift through donor-acceptor energy transfer.10 Additional alternatives on achieving fluorophores with large stokes shifts include but are not limited to: 1) solvent cage relaxation;11 2) local excitation followed by an ICT emission;3a,12 3) TICT;13 4) exciplex;14 5) excited stated proton transfer.15 However, none of these designs can be translated to fluorescent scaffolds outside the reported regime, whereas some of them even come at the expense of other photo-physical properties. For instance, meso-insertion of nitrogen was applied in designing dyes with improved Stokes shift for super-resolution microscopy and fluorescence sensing, yet the heteroatoms frequently cause hypsochromic emission shift.3a,4 Hence a focused and generalizable strategy to broaden the Stokes shifts of fluorescent dyes is garnering tremendous interests. The electronic structures of fluorescein, rhodamine, oxazine, squaraine and cyanine dyes have indicated a peculiar symmetry throughout both HOMO and LUMO.6c,16 Although the symmetrical electronic structures endow these fluorophores with high quantum yields,2,6a,c,17 the vibration suborbitals are also largely weakened. As a result, electrons at excited states cannot interconvert to the lower vibrational states, fixing the emission at a wavelength close to the excitation.16b,18 Thus we reason that by introducing asymmetric electronic structures to these fluorophores, vibronic contributions to the HOMO and LUMO can induce strong internal conversion. It should enable the electrons at excited states to transit to lower energy vibrational state, reducing the emission energy and increasing the Stokes

2


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shifts of modified fluorophores (Figure 1b). More importantly, we anticipate this strategy to be generalized to a wide range of symmetric fluorescent scaffolds.

Figure 1. a) Fluorescence microscope light path shows dichroic mirror filters signals of small Stokes shift dyes but not large Stokes shift dyes. b) A general method to increase Stokes shifts by introducing asymmetric electronic structures.

Results and Discussion Rhodols and Rhodamine coupled with quinoxaline moieties have been reported to display redshifted emission and larger Stokes shifts.19 To elucidate the underlying mechanisms, we started with simpler scaffolds and calculated (density functional theory (DFT) calculations20) the electronic orbitals of Opyronine (OP), Si-pyronine (SiP) and oxazine. All three fluorophores show symmetrical HOMO and LUMO structures (Figure 2). Although such a feature endows the molecules with high quantum yields as revealed by their oscillation strengths, the Stokes shifts are inevitably small. OP displays 16 nm Stokes shift, whereas SiP and oxazine are 14 nm and 23 nm (Figure 2). The small Stokes shifts significantly limit their applications in molecular imaging (Figure 1a). However, when we expanded one N, N-dimethylamino to 1,4-dimethyl-decahydro-quinoxaline (DQ), the calculated Stokes shifts of OP, SiP and oxazine increased to 113 nm, 196 nm and 154 nm. A closer look at the electronic structures indicates although LUMOs still remain symmetrical, the HOMOs now display clear asymmetrical vibronic features, effectively increasing the HOMO energy level over the initially symmetrical structure. Thus the electrons at excited states can transit back to a higher energy vibration state, manifesting smaller emission energy and broader Stokes shift (Figure 2). 3



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Figure 2. Chemical structures and DFT optimized molecular orbital plots (LUMO and HOMO) of O-pyronine (OP), Si-pyronine (SiP) and oxazine (OX) and their 1,4-diethyl-decahydro-quinoxaline (DQ) modified counterparts. In the table are the calculated absorption and emission wavelengths and oscillator strengths of the above dyes (CPCM solvation model with EtOH as the solvent). Scheme 1. Synthesis of DQF-594, DQF-683 and DQF-707.

4


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To corroborate the calculations, we synthesized DQ fused derivatives of OP, SiP and oxazine (Scheme 1) and compared their optical performance with the original molecules. As shown in Figure 3 and Table S1, all three dyes originally exhibit small Stokes shifts of 19 nm (OP), 12 nm (SiP) and 14 nm (oxazine). Their DQ derivatives, on the other hand, exhibit much larger Stokes shifts of 58 nm (DQF594), 122 nm (DQF-707) and 81 nm (DQF-683). The emission of DQF-594 was pushed to the far red range (652 nm), whereas DQF-707 and DQF-683 extended to the near infrared range (829 nm and 764 nm). It is noteworthy that DQF-594 and DQF-683 display obviously solvatochromism. All of these dyes have large Stokes shifts, which increase significantly in polar environment (Figure S1, Table S2). The gradual redshift and drop of emission with increasing solvent polarity indicate that OP and oxazine transform from a symmetrical structure to a dye with intramolecular charge transfer (ICT) features.20c,21 The asymmetric electronic structure of ICT dyes endows them with larger Stokes shifts and redshifted emission. It is worth noting that even though DQF dyes exhibit solvatochromism, the intrinsic alternating vibronic backbone remains the key structural contributor to the extended Stokes shifts at less polar environment. Furthermore, the electronic structure transformation brings the pyronine/oxazine dyes higher molar extinction coefficient while maintaining their quantum yields (Table S1). Essentially, DQ fused pyronines and oxazine display comparable or even better fluorescence brightness (ε×φ) than the original molecules.

500 600 700 800

Wavelength/nm

b)

SiP DQF-707

600

700

800

Wavelength/nm

c)

Normalized intensity

OP DQF-594


a)


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


Oxazine DQF-683

900 500 600 700 800 900 Wavelength/nm

Figure 3. The normalized absorption (dotted)/emission (solid) spectra of DQF-594 (a), DQF-707 (b), DQF-683 (c) and their original molecules in EtOH (DQF-594, DQF-683) or CH2Cl2 (DQF-707) at 25 oC.

To further demonstrate the feasibility and efficacy of DQ-induced Stokes shift increment, we then synthesized more DQ derivatives of rhodamine. Compounds rhodamine 101 (Rh101), rhodamine B (RhB), rhodamine 6G (Rh6G) and rhodamine 110 (Rh110) were each modified with DQ on xanthene to produce DQF-593, DQF-584, DQF-570 and DQF-565. The photo-physical properties of these new dyes were measured in EtOH together with their original molecules (Table 1 & Figure S2). Remarkably, DQ modification brings all the rhodamine compounds more than 3-fold increase of Stokes shifts. It should 5



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be noted that Stokes shifts were calculated from both absorbance and emission maxima wavelengths. Introduction of DQ inevitably expanded the conjugation through ICT structure, thus redshifting the absorbance by 30-60 nm. However, the emission red-shift exhibits a much grander level of vibronic structural perturbation. Effectively, the emission maximum of all four rhodamine dyes redshift more than 60 nm with such small structure changes. Furthermore, owing to the ICT feature brought by the DQ moiety, the extinction coefficients of the modified molecules are much larger than their original compounds, thus compensating for the quantum yields drop (Table 1 and Table S3). The new DQ modified rhodamines exhibit comparable brightness as the original rhodamines. Notably, the solvatochromic behavior show that all the DQ modificated rhodamine compounds have an increased Stokes shift with enhanced solvent polarity (Figure S3 and Table S3), which is in good according with above investigation. Table 1. Chemical structures and photo-physical properties of DQF-593, DQF-584, DQF-570 and DQF-565 in EtOH at 25 oC.

dye

λAbs

ε/M−1cm-1

/nm Rh10122

φ

Stokes

/nm 118604

εφ/M−1cm-1

shift/nm

587

0.91

23

107929

a

60

76970

DQF-593

593

179000

653

0.43

RhB23

553

117000

572

0.53

19

62010

a

DQF-RB 24

Rh6G

DQF-570

584

176000

660

0.35

76

61600

530

116000

551

0.95

21

110200

a

76

44020

570

142000

646

0.31

Rh110

498

76000

520

0.88

22

66880

DQF-565

565

128000

648

0.24a

83

30720

25

[a]

564

λEm

The quantum yields were determined using cresol purple as reference (Φf = 0.58 in ethanol).

To explore the generalizability of the vibronic structure, we then tested the DQ modification in dyes other than (O, Si)-pyronine, oxazine or rhodamine. Our selection includes rhodols, squaraine and cyanine (Cy3/Cy5) dyes that not only cover ring expansion, but also modifications on four-membered ring and chain structures (Scheme 2, Table 2). Rhodol enjoyed a three-fold increase of Stokes shift after 6


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ring expansion, from 26 nm to 80 nm with the emission color redshifting from yellow (546 nm) to red (640 nm). Importantly, the DQ modification on rhodol did not Scheme 2. Synthesis of DQF-560, DQF-680, DQF-692 and DQF-780.

Table 2.Structures and optical properties of DQFs in EtOH at 25 oC. dye

λAbs

ε(M−1cm-1)

/nm

φ

Stokes

/nm

εφ(M−1cm-1)

shift/nm

25

Rhodol

518

60000

546

0.21

26

12600

DQF-560

560

71000

640

0.35a

80

24850

637

a

9

35520

Squaraine DQF-680 Cy3

628 680 547

222000 180000 173000

775/805 562

0.16

b

95/125

12240

c

15

20760

b

60

56810

19

27400

56

7371

0.068 0.12

DQF-692

692

247000

752

0.23

Cy5

641

137000

660

0.20a

DQF-780 [a]

λEm

780

273000

836

b

0.027

φ was determined using cresol purple as reference (Φf = 0.58, EtOH22). [b]φ was determined using ICG as reference

(Φf = 0.13, DMSO22). [c]φ was determined using Rh6G as reference (Φf = 0.95, EtOH24).

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affect its extinction coefficient and quantum yield. Rhodol exhibits extinction coefficient of 6.0 × 104 M−1 cm−1 and a modest φ = 0.21 in EtOH. The modified molecule DQF-560 shows comparable extinction coefficient ε = 7.1 × 104 M−1 cm−1 and quantum yield φ = 0.35, similar to the original dye. On the other hand, squaraine, Cy3 and Cy5 were modified through a different route. All three dyes contain two indoline moieties connected by either cyclic squaric acid or ethylene/diene chains, which appear synthetically challenging to modify. Hence in all the cases, the indoline was replaced with benzoquinoxaline. Squaraine enjoyed over 13-fold increase of Stokes shift, from 9 nm to 125 nm in ethanol. The emission maximum sharply redshifted from far red (637 nm) to near infrared (805 nm). The original small Stokes shift of squaraine made it practically incompatible with any filter set of microscopes, whereas after modification, it could serve as a new NIR dye for in vivo applications. Similarly, Cy3 and Cy5 originally exhibit small Stokes shifts of less than 20 nm. After DQ modifications, their Stokes shifts increased more than 3-fold to 60 nm. These phenomenons are in good accordance with the DFT calculation results (Figure S6), which implies that the HOMOs of DQ modified dyes exhibit clear asymmetrical vibronic features, manifesting the larger Stokes shift. Furthermore, the emission maximum of Cy3 increased from orange (562 nm) to near infrared (752 nm), while the emission maximum of Cy5 increases from far red (660 nm) to near infrared (836 nm). Hence such simple structural modification provided great potential to produce near infrared dyes with distinct Stokes shifts. From the observed solvatochromism (Figure S5 and Table S4), we once again confirmed solvent-dependent Stokes shift in the reconstituted dyes. These results are consistent with our above observations. Moreover, it should be noted that the clear distinction between absorption and emission spectra could compensate for the quantum yields loss. The emission of the dyes can be fully captured by the detector and the loss due to the dichroic mirror is minimized (Figure 1a). Before evaluating the performance of the DQ modified fluorophores for imaging, we first tested the photo-stability of these dyes. The DQ modification on the electron donor aimed at preserving the photostability of the original symmetrical dyes. Surprisingly, the cells incubated with the modified fluorophore exhibited more superior photo-stability than the original dyes over continuous excitation. Rh6G completely bleached after 10 minutes of excitation, whereas DQF-570, the DQ modified Rh6G, still preserved 90% of the fluorescence (Figures 4 and S7). Similarly, rhodol completely bleached in 10 minutes whereas its modified compound DQF-560 still exhibited bright signals (Figure S8). In addition, oxazine, squaraine and cyanine dyes all suffer from bleaching after harsh condition of photo-excitation, while their DQ modified compounds display superior photo-stability (Figures S9-S11). We reason that 8


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the DQ rings provide a rigid structural support for the fluorophores, protecting the electron donating group and increasing the stability.26 In general, dyes modified with electron donors tend to exhibit increased HOMO energy levels. The downside of this phenomenon is instability resulted from the labile electron rich moieties.26c,27 DQ not only offers the benefit of increasing HOMO energy level, but also blocks the electrophilic attack on the modified compound owing to its large steric hindrance.26 Hence the modification not only increases the Stokes shift of the dyes, but also improves their photostaibilty, which can prove to be a very powerful approach to generate near infrared dyes. b)

0% 20%

Signal loss

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


40%

DQF-570 Rh6G

60% 80%

100%

0

2

5

8 10 12 15 20 30 Time/min

Figure 4. a) Confocal fluorescence images of live HepG2 cells cultured with DQF-570 (5.0 μM) and Rh6G (5.0 μM) with continuous irradiation using confocal microscope with the same parameters. b) Quantification of the relative mean fluorescence levels of cells from the images of DQF-570 and Rh6G. Scale bar = 20 μm. For DQF-570, λex = 560 nm, λem = 663-738 nm; for Rh6G, λex = 560 nm, λem = 580-620 nm.

Commercial dye Rh6G and its derivative DQF-570 were selected for comparison. 5 μM Rh6G and DQF-570 were each incubated with live cells for 20 minutes and then imaged with a confocal microscope (Figure 5). The excitation wavelength was selected to be 560 nm, close to the overlapping point of their absorbance spectra (Figure S12). The emission was collected from channels 580-640 nm and 655-755 nm. Figure 5 and S13 shows that in 580-640 nm channel, DQF-570 emits 2-time stronger emission than Rh6G, even though the quantum yield of DQF-570 is smaller. This indicates that majority of the emission from Rh6G was filtered by the embedding dichroic mirror because of its small Stokes shift. Furthermore, in 655-755 nm channel, DQF-570 solely emits strong emission while Rh6G is negligibly visible. Clearly, the large Stokes shift of DQF-570 enables it a much broader emission range and more versatile utility.

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Figure 5. Pseudo-colored fluorescence images of 5.0 μM Rh6G (a, b, c) and DQF-570 (d, e, f) in HeLa cells with the same parameter. The excitation wavelength was 560 nm. a) & d) Green emission channel (580-640 nm); b) & e) Red emission channel (655-755 nm); c) Overlay of channel a) and b). f) Overlay of channel d) and e). Scale bar = 20 µm.

Figure 6. Intracellular multi-color image of Rh6G (2 μM) and LysoDQF-570 (2 μM) in HepG2 cells simulated with chloroquine (100 μM) at different time-points. Scale bar: 20 μm. LysoDQF-570, λex = 560 nm, λem = 663−738 nm; Rh6G, λex= 560 nm, λem = 580−620 nm. Scale bar: 20 μm.

Given the distinct emission between Rh6G and DQF-570 under the same excitation wavelength, we seek to apply them as co-staining dyes in cells. DQF-570 was modified with morpholine as a lysosome anchoring group to afford lysoDQF-570 (Figure S14a). Fluorescence measurements of lysoDQF-570 under different pH indicate that its emission maximizes at pH 5, an acidic condition similar to the lysosomes (Figure S14b-c). Cell co-localization studies show that Rh6G concentrate in mitochondria whereas the modified lysoDQF-570 accumulate in lysosomes (Figure S15-S16). We anticipate that by changing the pH of lysosomes, fluorescence emission of lysoDQF-570 should change accordingly. Hence we first incubated HeLa cells with both Rh6G and lysoDQF-570, followed by stimulation with 10


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chloroquine (100 μM), a known inhibitor for endosomal acidification. Indeed, after chloroquine treatment emission from lysosome substantially decreased with time (Figures 6 and S17). Continuous excitation studies in the cells proved remarkable photostability of DQF-570 (Figure S7), thus the emission decrease in lysosome reflected the actual pH increase. As a contrast, the emission of Rh6G from mitochondria remained constant. The overlay image of both cell compartments indicate that dyes with extended Stokes shifts can be used for multi-color probing of cell behaviors (Figure 6). As an additional showcase of the application, we induced apoptosis in HepG2 cells with dexamethasone (2 μM). It is known that the initial manifestation of apoptosis includes release of lysosomal acidic sustances into cytosol, leading to increase of lysosomal pH. We observed a similar fluorescence quenching of lysoDQF-570 with the progress of cell apoptosis (Figure S18-19). Overlay images clearly visualize that no obvious fluorescence change is observed from mitochondria, with lysosome seeing a 2fold quenching (Figure S19). It again proves that we can study cell health and activities with multi-color imaging technique. To further examine the advantages of large Stokes dyes in live animal imaging, we selected the Cy3 DQ derivative, DQF-692 and compared its skin penetration and quantum yield with a commercial dye, indocyanine green (ICG) (Figure S20). We subcutaneously injected the same dose (5.0 μM, 0.05 mL) of both near infrared dyes into different mice and excited them with 675 nm, 710 nm and 745 nm excitation. The emission was collected accordingly from ICG emission filter (810-875 nm). Figure S20 shows that the signals of ICG are much weaker than DQF-692 from all three excitation wavelengths. Apparently, the large Stokes shift of DQF-692 allows it more flexibility to adapt to different excitation/ emission channels. The absorption of ICG at the three excitation channels are much lower than DQF692, hence resulting in poorer brightness. This indicates that DQ modified dyes can provide near infrared emission with large Stokes shifts, suitable for live animal imaging.

Conclusion In conclusion, we have fulfilled a generalized method to increase the Stokes shifts, emission wavelength and photostaibilty of fluorophores by appending DQ moiety to their structures. By introducing an asymmetrical alternating vibronic feature, we perturbed the originally symmetric electronic structure and created internal charge transfer states out of the parent molecules. Eleven different fluorophores that cover xanthene, squaraine and cyanine scaffolds were each modified with 11



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DQ through facile chemistry, with a minimum Stokes shift increase of 3-fold. Such a robust and generalizable method resulted influorophores that could adapt to current dichroic mirror microscope configuration and achieve much brighter imaging effects than their parent molecules. Furthermore, the DQ modified fluorophores could pair with their parent molecules to conduct one-excitation, multiple emission imaging and study the cell behavior more robustly. Lastly, the stable large stokes shift dyes could easily extend into the NIR range while absorbing in short red wavelength, thus allowing them better animal imaging efficiency. This approach is prospective in generating dyes suitable for superresolution microscopy and second window NIR imaging.

ASSOCIATED CONTENT Supporting Information Synthesis, NMR spectra, some absorption and emission spectra, and additional experimental results (PDF). This materialis available free of charge via the Internet athttp: // pubs.acs.org.

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] Author Contributions ‡

These authors contributed equally.

Notes The authors declare no competing financial interests.

ACKNOWLEDGMENT This work was financially supported by NSFC (21622504, 21521063, 21735001), the Science and Technology Project of Hunan Province (2017RS3019), and the Hunan University Fund for Multidisciplinary Developing (2015JCA04).

REFERENCES

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TOC "General dyes"

"DQF dyes" Rational design

N

A

D N

Small stokes shift dye Abs Em

600

700 800 Wavelength/nm



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


900

Breaking symmetry Enhanced ICT (D A) Improved photo-stability Redshift emission Increased Stokes shifts

Large stokes shift dye Abs Em

600

700 800 Wavelength/nm

900

15 ACS Paragon Plus Environment

A General Method to Increase Stokes Shift by Introducing Alternating

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