Donor Engineering for NIR-II Molecular Fluorophores with Enhanced

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Donor Engineering for NIR-II Molecular Fluorophores with Enhanced Fluorescent Performance Qinglai Yang, Zhubin Hu, Shoujun Zhu, Rui Ma, Huilong Ma, Zhuoran Ma, Hao Wan, Tong Zhu, Zhengyan Jiang, Weiqiang Liu, Liying Jiao, Haitao Sun, Yongye Liang, and Hongjie Dai J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b10334 • Publication Date (Web): 16 Jan 2018 Downloaded from http://pubs.acs.org on January 16, 2018

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Donor Engineering for NIR-II Molecular Fluorophores with Enhanced Fluorescent Performance

Qinglai Yang†,||,§,‡, Zhubin Hu#,‡, Shoujun Zhuǂ,‡, Rui Ma†, Huilong Ma†, Zhuoran Maǂ, Hao Wan†,ǂ, Tong Zhuψ, Zhengyan Jiang†, Weiqiang Liu||, Liying Jiao§, Haitao Sun#,*, Yongye Liang†,*, and Hongjie Daiǂ,*

†

Department of Materials Science & Engineering, Shenzhen Key Laboratory of Printed Organic

Electronics, South University of Science & Technology of China, Shenzhen 518055, China #

State Key Laboratory of Precision Spectroscopy, School of Physics and Materials Science, East China

Normal University, Shanghai 200062, China ǂ

Department of Chemistry, Stanford University, Stanford, California 94305, USA

||

Research Center for Advanced Materials and Biotechnology, Research Institute of Tsinghua University

in Shenzhen, Shenzhen 518057, China §

Department of Chemistry, Tsinghua University, Beijing 100084, China.

ψ

School of Chemistry and Molecular Engineering, East China Normal University, Shanghai 200062,

China ‡

These authors contributed equally to this work.

*Correspondence should be addressed to Y.L. ([email protected]), H.S. ([email protected]) and H.D. ([email protected]).

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KEYWORDS: molecular fluorophores, second near-infrared window, biological imaging, donor engineering. ABSTRACT:

Organic fluorophores have been widely used for biological imaging in the visible and

the first near-infrared windows. However, their application in the second near-infrared window (NIR-II, 1000-1700 nm) is still limited mainly due to low fluorescence quantum yields (QYs). Here, we explore molecular engineering on the donor unit to develop high performance NIR-II fluorophores. The fluorophores are constructed by a shielding unit-donor(s)-acceptor-donor(s)-shielding unit structure. Thiophene is introduced as the second donor connected to the shielding unit, which can increase the conjugation length and red-shift the fluorescence emission. Alkyl thiophene is employed as the first donor connected to the acceptor unit. The bulky and hydrophobic alkyl thiophene donor affords larger distortion of the conjugated backbone and less interactions with water molecules compared to other donor units studied before. The molecular fluorophore IR-FTAP with octyl thiophene as the first donor and thiophene as the second donor exhibits fluorescence emission peaked at 1048 nm with a QY of 5.3% in aqueous solutions, one of the highest for molecular NIR-II fluorophore reported so far. Superior temporal and spatial resolutions have been demonstrated with IR-FTAP fluorophore for NIR-II imaging of the blood vessels of a mouse hindlimb. 1

■ INTRODUCTION

2

Organic fluorophores have been widely used in biological imaging in the visible (400-650 nm) and the

3

first near-infrared (NIR-I, 650-900 nm) windows,1-4 as well as photoacoustic imaging and photothermal

4

therapy applications.5-10 Structural engineering on organic molecules can afford powerful tunability on

5

the optical properties of the fluorophores and versatile conjugation methods with target

6

biomolecules.11-13 Further, organic fluorophores generally exhibit good biocompatibility. For example,


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Journal of the American Chemical Society Journal of the American Chemical Society

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CyX series14-17 and boron-dipyrromethene (BODIPY)18-19 dyes have been well studied for cellular and

2

tissue applications. Indocyanine green (ICG)20-22 and methylene blue (MB)23-25 have been approved by

3

the US Food and Drug Administration (FDA) for in vivo imaging. However, there are very few organic

4

fluorophores reported so far in the second near-infrared (NIR-II, 1000-1700 nm) window, which has

5

recently demonstrated superior imaging performance in tissue penetration and signal/noise ratio

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compared to the visible and the NIR-I windows.26-32 It should be pointed out that very recent studies

7

revealed that some NIR-I fluorophores, such as ICG and IR800, could exhibit fluorescence tail in

8

NIR-II window.33 However, it is important to pursue new fluorophores with fluorescence peaked at

9

NIR-II window for high fluorescence quantum yield (QY).

10

Through increase of conjugation length and heteroatom substitutions, a few polymethine molecules,

11

such as IR-26, IR-1048, IR-1051, IR-1061, can emit fluorescence peaked over 1000 nm.34-37 Another

12

type of NIR-II organic fluorophores is based on donor-acceptor-donor (D-A-D) structure.38-42 Strong

13

acceptors with stabilized quinoidal structure (such as benzo[1,2-c:4,5-c']bis[1,2,5]thiadiazole, BBTD)

14

are often employed to drive the fluorescence emission to the NIR-II window. Most of these NIR-II

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organic fluorophores are hydrophobic and cannot be used directly for biological imaging. 43-45 To make

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them water soluble, these molecules are either encapsulated in hydrophilic polymer matrixes or

17

functionalized with polyethylene glycol (PEG) or ionic groups in the side chains. 38-42,

18

problem of NIR-II organic fluorophores is their low fluorescence quantum yields (QYs), especially in

19

aqueous solutions. A D-A-D molecule, CH1055-PEG, with BBTD as the acceptor, triphenyl amine as

20

the donor and PEG as side chains, only exhibited QY of < 0.3% in aqueous solutions. 38 In another case,

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the QY of a quaternary ammonium salt molecule, Q-FITBBTTFI, is less than 0.1% in water.49 Such low

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QYs present a major bottleneck for NIR-II organic fluorophores to achieve ultrafast and high resolution

23

biological imaging.


46-49

A vital

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NIR-II organic fluorophores generally have large conjugated backbones in order to emit

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fluorescence in the NIR-II window. As a result, the molecules tend to have strong intermolecular

3

interactions, and the excited states of the fluorophore molecules could be easily attacked and then

4

quenched. Particularly, it seems the low QYs of NIR-II organic fluorophores in aqueous media is highly

5

related to the interactions with water molecules.41,50 To address these problems, we introduced shielding

6

unit (S) to construct S-D-A-D-S type fluorophores and 2,6-dialkoxy substituted benzene or 9,9’-dialkyl

7

substituted fluorene was used as the shielding units.40-42 The side chains on the shielding units extend

8

out of the conjugated backbone and are able to reduce intermolecular interactions. Further,

9

3,4-ethoxylene dioxythiophene (EDOT) or 3-(2-(2-(2-methoxyethoxy)ethoxy)ethoxy) (TEG) substituted

10

thiophene was employed as the donor to distort the conjugated backbones, which could also reduce

11

intermolecular interactions and energy transfer from the excited states of the molecules to the

12

surrounding water.41-42 The organic fluorophore IR-FE, which is constructed with BBTD as the

13

acceptor, EDOT as the donor and fluorene as the shielding units, exhibits fluorescence peaked at 1013

14

nm with a QY of 31% in toluene.41The analogue IR-FEP conjugated with PEG chains exhibits

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fluorescence QY of 2.0% in aqueous solution. In contrast, the counterpart with thiophene as donor,

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IR-FTP, exhibits a low QY of 0.02%.41

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Donor units play important roles on the fluorescence performance of D-A-D fluorophores. They are

18

not only coupled with the acceptor units to afford low bandgap (accurately speaking, the gap between

19

the highest occupied molecular orbital (HOMO) and the lowest unoccupied molecular orbital (LUMO))

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and fluorescence emission in the NIR-II window, but also able to significantly affect the fluorescence

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QYs as revealed by previous studies.40-42Here, we explore donor engineering to develop high

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performance NIR-II molecular fluorophores and reveal the relationship between the donor structures

23

and fluorescence properties. Since the photon scattering in tissue is reduced with increasing wavelength,

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fluorescence emission at longer wavelength is favored.51-54 As a result, a thiophene ring is added as the

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second donor next to the shielding unit to increase the conjugation length. Such thiophene insertion ACS Paragon Plus Environment

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results in redshift of the absorption and fluorescence emission, but decrease of QYs. The first donor

2

connected to the acceptor is further tuned. Besides oxygen containing EDOT and TEG substituted

3

thiophene, alkyl chain substituted thiophene is also introduced as the first donor. It is found that alkyl

4

thiophene can increase the dihedral angle between acceptor and donor and hydrophobicity for improved

5

QY, but results in blue-shift of the absorption and emission spectra. The fluorophore IR-FTAP with

6

octylthiophene as the first donor and thiophene as the second donor shows the optimized performance

7

with fluorescence peaked at 1048 nm and a high QY of 5.3% in aqueous solutions. The high fluorescent

8

performance of IR-FTAP allows for ultrafast (>25 frames s-1) biological imaging in the NIR-II window

9

with superior spatial resolution for molecular fluorophores.

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■ RESULTS AND DISCUSSION

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Donor engineering on the molecular fluorophores

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As illustrated in Scheme 1, the molecular fluorophores employ an S-D2-D1-A-D1-D2-S structure with

13

BBTD as the acceptor and dialkyl fluorene as the shielding unit. To increase the conjugation length for

14

redshift of fluorescence emission, a thiophene ring is inserted as the second donor (D2) connected to the

15

shielding unit. To investigate donor engineering, thiophene (T), EDOT (E), 3-TEG substituted

16

thiophene (G) and 3-alkyl (octyl) substituted thiophene (A) are used as the first donor (D1) conjugated

17

to the acceptor to afford the molecular fluorophores IR-FTXs: IR-FTT, IR-FTE, IR-FTG and IR-FTA,

18

respectively (Scheme 1). Compared with the oxygen containing EDOT and TEG thiophene, alkyl

19

thiophene is more hydrophobic and can afford larger dihedral distortion considering the bulkier CH2

20

linked on the thiophene ring. Molecular fluorophores without the second donor (IR-FXs: IR-FT, IR-FE,

21

IR-FG and IR-FA) are also included for comparison (Scheme 1).

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The molecular fluorophores were feasibly synthesized by coupling reactions. Generally, the donor

23

units were conjugated to the shielding unit first, and then coupled to the acceptor to yield the molecules. ACS Paragon Plus Environment

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The synthetic procedures and structural characterizations of the new fluorophores IR-FA, IR-FTT,

2

IR-FTE, IR-FTG, IR-FTA are detailed in Supporting Information (SI). To enable water solubility, the

3

molecular fluorophores were first functionalized with azide on the termini of the side chains and then

4

conjugated with 2 equivalences of PEG-alkyne (Mn = 600) to afford the PEGylated molecules IR-FTTP,

5

IR-FTEP, IR-FTGP, IR-FTAP (Scheme 1). The successful PEGylation was confirmed by comparing

6

the molecular weight difference before and after reaction from SEC measurements (Figure S1 and

7

Table S1). Most of the PEGylated fluorophores exhibit good water solubility. However, the PEG600

8

version of IR-FTAP is not water soluble due to the more hydrophobic nature of the alkyl chain on the

9

thiophene donor. As a result, the PEG5000 (Mn = 5000) version of IR-FTAP was prepared.

10

Scheme 1. Schematic illustration of the design of molecular fluorophores.

11 12

Frontier molecular orbitals and geometric structures

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Density functional theory (DFT) and time-dependent DFT (TDDFT) calculations were performed to

14

analyze the electronic properties and geometries of the molecular fluorophores at the optimally-tuned ACS Paragon Plus Environment

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ωB97XD*/6-31G(d) level55-60 using the Gaussian 09 software61 (see SI for computational details).

2

Figure 1 shows the HOMOs and LUMOs of the molecular fluorophores. All the HOMOs are

3

delocalized along the conjugated backbone in the molecular fluorophores. With the added thiophene

4

donor unit, IR-FTX series show higher HOMO energy levels than IR-FX series. The LUMOs in

5

IR-FTX series are mainly localized on the D1-A-D1 core, thus the LUMO energy levels are similar to

6

those of the IR-FX series. The LUMO distribution for each fragment in the molecule was quantified

7

using Hirshfeld method by Multiwfn software62 (see Table S2). The results confirm that the proportion

8

of LUMO localized on the BBTD acceptor is the largest in the conjugated backbone. IR-FTA with

9

alkyl thiophene as D1 has the highest value of 87.8%, while IR-FTT with thiophene as D1 has the

10

lowest value of 73.1% (Table S2), indicating that the LUMO distribution is more localized on the

11

BBTD moiety of IR-FTA than that of IR-FTT. It should be noticed that there is a significant portion in

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D2 in the IR-FTX fluorophores. The D2 portion decreases following the order: IR-FTT (1.4%) >

13

IR-FTE (0.9%) > IR-FTG (0.8%) > IR-FTA (0.4%). The extension of LUMOs can increase the

14

probability of intermolecular interactions and is not favorable for the energy maintenance of excited

15

states.

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The optimized ground-state (S0) and first singlet excited-state (S1) geometries of the molecular

17

fluorophores are shown in Figure 2. For the S0 geometries, the dihedral angle between A and D1

18

gradually enlarges with the increase of side-chain steric hindrance on D1: 2° for IR-FTT, 42° for

19

IR-FTE, 45°for IR-FTG, and 58°for IR-FTA. IR-FTE exhibits a small dihedral angle between D1

20

and D2 due to the oxygen (EDOT) – sulfur (D2 thiophene) interaction.63-65 The dihedral angles between

21

D2 and fluorene shielding unit (S) in the IR-FTX fluorophores are similar with values of 30-33°. By

22

adding up all the three dihedral angles (A-D1, D1-D2 and D2-S), it yields the twist between shielding

23

unit and acceptor, which follows the order: IR-FTA > IR-FTG > IR-FTE > IR-FTT. The larger the

24

value, the side chains on the shielding units stretch out the conjugated backbone to a larger extent,

25

which can reduce the intermolecular interactions. For the S1 geometries, all the dihedral angles decrease ACS Paragon Plus Environment

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compared to those in the S0 geometries, indicating a geometric reorganization in the excited-state to

2

allow for planarization of the π-conjugated backbone and more delocalized character of the S1 states.

3

Interestingly, IR-FTA (S0 vs S1 = 58°vs 40°), IR-FTG (45°vs 33°), and IR-FTE (42°vs 31°) exhibit

4

much larger decrease of D1-A dihedral angles than IR-FTT (2°vs 0.5°).

LUMO

HOMO

5 6

IR-FTT

-5.68 eV

-4.41 eV

IR-FTE

-5.69 eV

-4.24 eV

IR-FTG

-5.82 eV

-4.34 eV

IR-FTA

-6.20 eV

-4.50 eV

IR-FE

-5.85 eV

-4.29 eV

IR-FT

-5.86 eV

-4.46 eV

Figure 1. Calculated HOMOs and

LUMOs of the molecular fluorophores

at the

7

tuned-ωB97XD*/6-31G(d) level. The HOMO and LUMO energy levels are also presented in the figures.

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To reduce the computational requirements, side chains on the fluorene units are replaced by methyl

9

groups. Note that the LUMO levels are obtained by subtracting the optical gap from the HOMO levels.


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IR-FE

IR-FT

1 2

Figure 2. Optimized ground-state (S0) and first singlet excited state (S1) geometries of the molecular

3

fluorophores at the optimally-tuned ωB97XD*/6-31G(d) level. To reduce the computational

4

requirements, side chains on the fluorene units are replaced by methyl groups. The dihedral angles are

5

shown in the figures.

6 7


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Optical properties of the fluorophores

2

The absorption and emission spectra of the molecular fluorophores were measured in toluene (Figure 3

3

and Figure S3). The optical properties data are summarized in Table 1 and Table S4. Compared to

4

IR-FX fluorophores, IR-FTX fluorophores exhibit substantial redshift of the absorption, which could

5

be attributed to the raising of HOMO energy levels in IR-FTX. For example, the absorption peaks of

6

IR-FTE and IR-FTA locate at 810 nm and 721 nm, while the ones of IR-FE and IR-FA locate at 767

7

nm and 680 nm, respectively. In the IR-FTX series, the absorption peaks are gradually blue-shifted

8

following the order: IR-FTT (889 nm), IR-FTE (810 nm), IR-FTG (757 nm), IR-FTA (721 nm),

9

owing to the more torsion of conjugated backbones and more localized LUMOs from IR-FTT to

10

IR-FTA. The extinction coefficient of IR-FTX decreases after PEGylation (IF-FTXP) (Table 1), which

11

could be due to the interaction of the polar ethylene glycol units (-OCH2CH2O-) of PEG chains with the

12

conjugated backbones, reducing the probability of an electronic transition from ground state S 0 to

13

excited state S1 (absorption S0-S1). The fluorescence emission spectra (excited at 808 nm) of both the

14

IR-FTX and IR-FX fluorophores are located at 900-1400 nm (Figure 3b). The IR-FTX emission

15

spectra are slightly red-shifted compared to the ones of IR-FX. For example, the emission peak of

16

IR-FTE (1052 nm) is longer than the one of IR-FE (1013 nm). As IR-FA exhibits very weak

17

absorption at 808 nm, its emission spectrum was not measured. In the IR-FTX fluorophores, the

18

emission peaks are gradually blue-shifted in the order: IR-FTT (1070 nm), IR-FTE (1052 nm),

19

IR-FTG (1036 nm), IR-FTA (1005 nm). The emission peak difference is smaller than absorption peak

20

difference, mainly due to the increasing reorganization energies from IR-FTT to IR-FTA: 0.24 eV for

21

IR-FTT, 0.35 eV for IR-FTE, 0.44 eV for IR-FTG, 0.49 eV for IR-FTA (Figure S2 and Table S3).

22

Theoretical calculations confirm that all the S1 excitations arise from the dominated HOMO to LUMO

23

transitions (Table S3). Therefore, building a hydrophobic environment around the BBTD acceptor

24

where the LUMO mainly distributes could efficiently prevent the intermolecular interactions with water,

25

reduce non-radiative energy transfer and afford a high fluorescence QY in water. From the analysis of ACS Paragon Plus Environment

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electronic configurations (Table S3), the simulated results of E01/E10 (eV) and corresponding

2

absorption/emission peaks (nm) are 1.27/1.01, 1.45/1.08, 1.48/1.06, 1.70/1.21 and 977/1227, 855/1150,

3

839/1166, 730/1024 for IR-FTT, IR-FTE, IR-FTG, IR-FTA respectively.

1.4

Absorbance

1.2 1.0 0.8 0.6

IR-FE IR-FA IR-FTT IR-FTE IR-FTG IR-FTA

(b) Normalized Fluorescence

(a)

In Toluene

0.4 0.2 0.0

808 nm 600

1.2 1.0 0.8

800

900

1000

1100

Wavelength (nm)

(c) 1.4

700

IR-FEP IR-FTTP IR-FTEP IR-FTGP IR-FTAP

(d) Normalized Fluorescence

500

Absorbance

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

In Water

0.6 0.4 0.2 0.0 500

4

600

700

800

900

1000

1100

1.2

IR-FE QY=31% IR-FTT QY=12% IR-FTE QY=15% IR-FTG QY=17% IR-FTA QY=27%

1.0 0.8 0.6

In Toluene

0.4 0.2 0.0 900

1000

1100

1200

1300

1400

1500

Wavelength (nm) 1.2

IR-FEP QY=2.0% IR-FTTP QY=0.1% IR-FTEP QY=1.0% IR-FTGP QY=1.4% IR-FTAP QY=5.3%

1.0 0.8 0.6

In Water

0.4 0.2 0.0 900

Wavelength (nm)

1000

1100

1200

1300

1400

1500

Wavelength (nm)

5

Figure 3. (a) Absorption spectra and (b) Fluorescence emission spectra of the molecular fluorophores in

6

toluene solutions. The absorption spectra in (a) were measured with 50 mmol/L solutions. The emission

7

spectra in (b) were measured with optical density (OD) of 0.08 at 808 nm and the fluorescence was

8

normalized with IR-FE peak intensity. (c) Absorption spectra and (d) Fluorescence emission spectra of

9

the PEGylated fluorophores in aqueous solutions. The absorption spectra in (c) were measured with 150

10

mmol/L solutions. The emission spectra in (d) were measured with OD of 0.08 at 808 nm and the

11

fluorescence was normalized with IR-FTAP peak intensity. (An 808 nm laser was used for excitation

12

with exposure time of 50 ms.)


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The fluorescence QYs (determined with emission > 900 nm) were determined with the

2

single-walled carbon nanotube (HIPCO SWCNT) as the reference fluorophore (QY = 0.40%, see SI).38,

3

66

4

IR-FTT show lower QY values of 15% and 12% in toluene, respectively. It could be due to the increase

5

of intermolecular interactions with the conjugation length increase. The emission QY of IR-FTA is 27%

6

in toluene, substantially higher than those of IR-FTG (17%), IR-FTE (15%) and IR-FTT (12%). As

7

revealed by our previous work,41 the less delocalization of the LUMO in IR-FTA could weaken

8

interactions between excited state (S1) and external stimuli to reduce non-radiative energy transfer.

9

Table 1. Optical Data of the NIR-II fluorophores. Dye ε (103 L/mol·cm) λabs(nm)

Compared to IR-FE (31%) and IR-FT (19%), the thiophene inserted fluorophores IR-FTE and

λex (nm)

Φf%

IR-FE

19

767

1013

Stokes shift (nm/cm-1) 246/3170

IR-FTT

24

889

1070

181/1900

12±0.4

IR-FTE

23

810

1052

242/2840

15±0.4

IR-FTG

18

757

1036

279/3560

17±0.2

IR-FTA

17

721

1005

284/3920

27±0.4

IR-FEP

5.7

780

1047

267/3270

2.0±0.04

IR-FTTP

7.9

895

1112

217/2180

0.10

IR-FTEP

7.1

820

1100

280/3100

1.0±0.04

IR-FTGP

5.5

784

1068

284/3390

1.4±0.03

IR-FTAP

5.0

733

1048

315/4100

5.3±0.02

31±0.3

10 11

*The data were measured in toluene solutions for IR-FE and IR-FTX series and in aqueous solutions for IR-FEP and IR-FTXP series.

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In aqueous solution, the IR-FTXP fluorophores also exhibit significant redshifts in absorption and

13

fluorescence emission compared to the counterparts in the IR-FXP series, suggesting that the added

14

thiophene for extended conjugation can drive the absorption and emission to longer wavelength

15

effectively (Figure 3c-d). The emission peak of IR-FTTP, IR-FTEP, IR-FTGP and IR-FTAP locates

16

at 1112, 1100, 1068, 1048 nm, respectively. It follows the trend of the molecular fluorophores in toluene.

17

However, the measured fluorescence QYs of IR-FTEP (1.0%) and IR-FTGP (1.4%) are lower than

18

IR-FEP (2.0%) and IR-FGP (1.9%). IR-FTTP is similar to IR-FTP, showing little QYs in aqueous ACS Paragon Plus Environment

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1

solution. It confirms our previous finding that thiophene as a donor next to the acceptor is detrimental to

2

the fluorescence emission in aqueous solutions.41 Although IR-FTA exhibits a lower QY than IR-FE in

3

toluene, the water soluble analogue IR-FTAP exhibits a higher QY of 5.3% than the previous champion

4

IR-FEP (2.0%) in aqueous solution. We also prepared IR-FTEP and IR-FTGP with PEG5000 and find

5

that their fluorescence QYs is similar to the PEG600 ones (Figure S3e-f). It can exclude the PEG

6

molecular weight as a major contributor for the high QYs of IR-FTAP. The better maintenance of the

7

fluorescence QY from IR-FTA in toluene to IR-FTAP in water should be attributed to the hydrophobic

8

alkyl thiophene as D1. We also tested the photostability of IR-FTAP in water, phosphate buffer saline

9

(PBS), and fetal bovine serum (FBS). As shown in Figure S4, there is a small decay (< 10%) on the

10

fluorescence signal under continuous laser exposure (808 nm, 0.33 W/cm2) for over 150 minutes. In

11

contrast, the fluorescence signals of ICG decay very fast, and almost disappear after 30 minutes. It

12

indicates that IR-FTAP possesses good photostability for bio-imaging. Long pass filters with

13

wavelength >1100 nm are often applied to improve the imaging quality.38,

14

proportions of longer fluorescence emission (>1100 nm) in the molecular fluorophores are further

15

quantitatively analyzed, and the data are as followed: IR-FTEP (56%) vs IR-FEP (32%), IR-FTGP

16

(43%) vs IR-FGP (28%), IR-FTAP (33%). And the fluorescence QY (>1100 nm) of the molecular

17

fluorophores are 0.56% for IR-FTEP vs 0.64% for IR-FEP, 0.60% for IR-FTGP vs 0.54% for

18

IR-FGP, 1.7% for IR-FTAP, respectively. It is clearly that the thiophene insertion as the second donor

19

can increase the portion of fluorescence emission at longer wavelength. Such increase can compensate

20

or even outweigh the loss of fluorescence QY in the whole window. The combination of thiophene as

21

the second donor and alkyl thiophene as the first donor makes IR-FTAP to exhibit the highest >1100

22

nm QY.

66-67

As a result, the

23 24


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1

Decay effects for fluorescence quantum yield

2

In general, QY = kr / kr + knr, where kr is the radiative decay rate and knr is the nonradiative decay rate.

3

68,69

4

by Einstein spontaneous emission equation kr = ƒΔE2/1.499 for a two-level system. And ƒ is the

5

dimensionless oscillator strength and ΔE is the energy gap in units of cm−1.The values of kr for the four

6

fluorophores are then calculated to be in the range of 3~4.5 x 107 s-1 (Table S3). As shown in Figure S5,

7

since the kr values are close for the four fluorophores, the suppression of knr is more important to obtain

8

high QYs. There are generally three major pathways for nonradioactive decay: (i) internal conversion

9

(kIC), (ii) intersystem crossing (kISC) from singlet excited state to triplet one, and (iii) the charge/energy

10

transfer (kqh) during the production of excited state, often induced by solvents. So the fluorescence QYs

11

are determined by the competition of these decay rates.

The kr constant is mainly determined by the electronic structures of molecules and can be calculated

12 13

Figure 4. Torsional potential of the bond (in red) bridging the central BBTD and the first donor unit

14

based on the optimized excited-state geometries of the IR-FTT, IR-FTE, IR-FTG and IR-FTA

15

molecules.

16

The internal conversion (kIC) decay rate can be greatly suppressed with a more rigid structure since

17

the rotation of functional groups contributes largely to the internal conversion processes.68,70 The 14 ACS Paragon Plus Environment

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1

calculated torsional potential as a function of dihedral angle between BBTD acceptor and the first

2

thiophene donor is shown in Figure 4. The steep potential energy barrier and narrow distribution of

3

torsion angles indicate that the π-conjugated skeleton of IR-FTA is more rigid than other molecules. As

4

a result, the kIC decay rate should be in the order: IR-FTA < IR-FTG < IR-FTE < IR-FTT, that are

5

consistent with the trend of torsional potential.

6

For a singlet−triplet (S−T) conversion process, kISC can be typically increased through the

7

heavy-atom substitution or significantly small singlet-triplet gap.71,72 As shown in Table S5, the S−T

8

gaps (ΔEST) of the four molecules are calculated to be around 0.9~1.0 eV. Neither the small

9

singlet-triplet gap nor heavy-atom substitution exists in the molecular systems studied herein.

Thus, a

10

large kISC value should not be expected. However, it has been pointed out that the dissolved molecular

11

oxygen could be a mediator for this decay process. To study the effects of the molecular oxygen, we

12

measured the fluorescence behaviors of IR-FTAP in aqueous solutions after continuously feeding

13

nitrogen or oxygen, respectively. As shown in Figure S6, the feeding of nitrogen to remove oxygen in

14

solutions can cause an increase of the fluorescence intensity of 13%, while the continuous feeding of

15

oxygen greatly decreases the fluorescence intensity. The T1 energies of the four molecular fluorophores

16

are all below the energy gap between triplet ground-state oxygen and singlet oxygen and the intersystem

17

crossing between S1 and T1 of the fluorophores cannot be efficient due to the relatively large ΔEST.

18

Therefore, the direct energy transfer from the S1 state of molecular fluorophore to the ground-state 3O2

19

with the subsequent generation of singlet 1O2 is more possible.72 The relatively large ΔEST gaps of the

20

four molecular fluorophores can also allow for sufficiently long singlet state lifetimes to effectively

21

contact with oxygen molecules. In addition, the activated singlet oxygen is capable of reacting with the

22

molecular fluorophores to induce oxidative effects that deteriorate the fluorophores.

23

Considering the significantly different QYs between IR-FTXP in toluene (12 ~ 27 %) and in water

24

(0.1~ 5.3 %), the quenching process owing to the existence of water should be considered as an


15

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Page 16 of 26

1

important nonradiative decay path. In order to better understand the interactions between fluorophores

2

and water molecules, the molecular dynamics (MD) simulations on the designed NIR-II fluorophores

3

were performed. Both the PEG chains and the shielding units of substituted fluorene are included in the

4

models. The simulations were initiated by solvating the geometry-optimized molecules in a cubic water

5

box of 10 Å side lengths and equilibration runs of 60 ns durations were then conducted (see

6

computational details in SI). The MD results show that both the PEG chains (in blue) and the side

7

chains of substituted fluorene (in grey) can twine around the π-conjugated skeletons to some extent. The

8

number of water molecules around the fluorophore core region (i.e., BBTD acceptor) is revealed by the

9

radial distribution functions (RDFs) of oxygen atoms in water molecules around BBTD (Figure S7a). It

10

shows that the water molecules are very likely to exist around the BBTD acceptor center when the

11

sphere radii R > 3.5 Å, although most water molecules are excluded outside the π-conjugated skeletons

12

(Figure S7b). As shown in Figure 5 and Figure S7b, the water molecules are obviously less structured

13

around the BBTD acceptor of IR-FTAP compared to that of IR-FTTP. This can be mainly attributed to

14

the existence of two octyl chains (in yellow) of IR-FTAP, favoring to excluding water molecules due to

15

their steric hindrance and hydrophobicity.

16

In addition, a computational model based on DFT calculations was also introduced to evaluate the

17

probability of water molecules presenting in the fluorophore core region (see SI). The model including a

18

water molecule and a molecular fluorophore is constructed as shown in Figure 6 (top left). The

19

interaction energy (in kcal/mol) on each position is calculated based on the equation: ΔE =

20

E(FTT…H2O) – E(FTT) – E(H2O). The larger the ΔE values, the more unstable the water molecule will

21

exist therein. The red region corresponding to lower energy indicates where the water molecule can

22

easily exist. The almost red region in IR-FTTP indicates that its surrounding of the D1-A-D1 core has a

23

great chance to interact with water molecules. As a comparison, IR-FTEP, IR-FTGP, and IR-FTAP

24

whose D1 units are substituted thiophene possess significant non-red areas, indicating water molecules

25

are more difficult to enter into the D1-A-D1 core region of these molecules than that of IR-FTTP. ACS Paragon Plus Environment

16

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1

IR-FTAP possesses the minimum red area, suggesting that the D1-A-D1 core of IR-FTAP has the least

2

interactions with water molecules compared to other molecular fluorophores (Figure 6). This

3

computational model provides a direct schematic description of the interactions between molecular

4

fluorophore core region and a water molecule, which is in line with the MD simulations. Such

5

computations confirmed the role of the alkyl thiophene due to the obvious steric hindrance and

6

hydrophobic nature, which can account for the highest fluorescence QY of IR-FTAP in aqueous

7

solutions.

R

IR-FTTP

IR-FTAP

8 9

Figure 5. Representative schematic diagram of water molecules surrounding the BBTD acceptor of the

10

IR-FTAP and IR-FTTP. The π-skeleton of molecular fluorophore is shown in grey; the PEG chain in

11

blue; the alkyl chain in yellow; the water molecules around the BBTD center in the effective contact

12

distance (R = 6 ~ 7 Å) are shown as explicit water model. The water molecules outside the whole

13

backbone of fluorophore are shown as implicit water model (transparent water).


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Page 18 of 26

IR-FTT/IR-FTTP

1

IR-FTE/IR-FTEP

IR-FTG/IR-FTGP

IR-FTA/IR-FTAP

2

Figure 6. Schematic illustration of the molecular fluorophores’ interactions with a water molecule. The

3

model including a water molecule and a molecular fluorophore is constructed (top left). The water

4

molecule is placed 2 Å above the BBTD plane and it is further located on the circular plane as a

5

function of radius (2Å, 4Å, 6Å) and angle (0°~360°). All the energies are calculated at the

6

ωB97XD*/6-31G (d) level of theory.

7

Further, to confirm the influence of water molecules on the excited states of the molecular

8

fluorophores. We thus performed the time-dependent density functional theory (TDDFT) calculation at

9

the ωB97XD*/6-31G(d) level on the IR-FTT molecule which is surrounded by six water molecules.

10

Interestingly, as shown in Figure S9, the water molecules do have certain contribution to the LUMO,

11

which is closely related to the excited-state property.

12 13 ACS Paragon Plus Environment

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1

Ultrafast imaging of blood vessels

2

High fluorescence QY of the NIR-II fluorophores can benefit biological imaging with longer

3

wavelength emission to reduce light scattering and shorter exposure time to increase imaging rate. The

4

high brightness of IR-FTAP allows for ultrafast NIR-II imaging (>25 frames s-1) at >1200 nm with

5

small-molecule organic fluorophore for the first time. We performed an ultrafast video (Supplement

6

Video 1) rate imaging of the mouse hindlimb with tail vein injection of IR-FTAP PBS solution.

7

Immediately after injection, the superior imaging of blood vessels of hindlimb is clearly observed from

8

the surrounding background tissue, which allows us to identify the change in blood flow within a single

9

cardiac cycle (≈118-143 ms) (Figure 7a-d). The signal to surrounding tissue ratio (S/T) (obtained from

10

the red line) is 4.9±0.3, which is higher than that of IR-FEP (3.8±0.2) with the same injection dose

11

and imaging conditions. In addition, the NIR-II fluorescence images of hindlimb at sequential long pass

12

filters (4 ms exposure time for 1100 nm LP, 20 ms exposure time for 1200 nm LP, 100 ms exposure

13

time for 1300 nm LP, respectively) were performed and the results are shown in Figure 7a, e-g. The

14

line profile of the fluorescence intensity with 1100, 1200 and 1300 nm LP filter shows vessel to

15

surrounding tissue ratio of 3.6±0.3, 4.9±0.3 and 6.7±0.4, respectively. IR-FTAP demonstrates

16

significantly higher spatial and temporal resolution than the reported NIR-II molecular fluorophores

17

CQS1000 (1000 nm LP, 200 ms exposure time) and CH1055 (1200 nm LP, 200 ms exposure time and

18

1300 nm LP, 1s exposure time) applied in biological imaging.38,

19

principal component analysis (PCA)73 was applied to observe behavior of organs/vessels that are not

20

easily seen or resolved from nearby features in the raw images. The vessels in hindlimb position, mixed

21

with vein and artery were distinguished by PCA through the group images of ultrafast video (Figure 7h).

22

All the results indicate the promising potentials of IR-FTAP as an NIR-II fluorophore for in vivo

23

imaging.


67

Dynamic contrast imaging by

19

page 20 of 26

(a)

(b)

IR-FTAP, 1200 nm LP, 20 ms

Page 20 of 26

IR-FTEP, 1200 nm LP, 20 ms

5 mm

(d)150

180 150

S/T=4.9±0.3

PL intensity / a.u.

PL intensity / a.u.

(c) Vessel

120 90

Tissue

60 30 0 0

50

100

150

200

250

Position / pixel

(e)

90 60 30 0 0

300

(f)

(g)

50

100

150

200

250

300

Position / pixel


(h)

250 200

S/T=3.8±0.2

120


PL intensity / a.u.

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


vein artery

1100 nm LP 1200 nm LP 1300 nm LP

150 100 50 120

1

140

160

180

200

Position / pixel

2

Figure 7. The hindlimb vessel imaging of IR-FTAP with 1100, 1200 and 1300 nm long pass filters,

3

respectively. IR-FTAP suspended in PBS (200 uL, OD = 6 at 808 nm) was injected intravenously into

4

an six-weeks-old C57BL/6 mouse (n = 3). (a) An NIR-II fluorescence image of the left hindlimb of a

5

mouse by IR-FTAP, (b) by IR-FEP with an ultrafast frame rate of 25.6 frames per second. (c-d) The

6

line profile of the fluorescence intensity in (a) and (b). Image from IR-FTAP possesses an improved

7

S/T ration compared with IR-FEP. The S/T ratio was calculated according to the value of vessel signal

8

divided by surrounding normal tissue signal. (e-f) NIR-II fluorescence images of hindlimb at sequential

9

long pass filters (4 ms exposure time for 1100 nm LP, 100 ms exposure time for 1300 nm LP,

10

respectively). (g) The line profile of the fluorescence intensity in (a, e, f) with vessel to surrounding

11

tissue ratio 3.6±0.3, 4.9±0.3 and 6.7±0.4, respectively. (h) Time-dependent PCA images of hindlimb

12

vessels in a C57BL/6 mouse. ACS Paragon Plus Environment

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1

■ CONCLUSIONS

2

In summary, molecular engineering on the donor units is performed to develop high performance NIR-II

3

molecular fluorophores with BBTD as the acceptor and fluorene as the shielding unit. Thiophene is

4

introduced as the second donor connected to the shielding unit, which increases the conjugation length

5

of the molecular fluorophore and red-shifts the absorption and emission spectra, but results in the

6

decrease of the fluorescence QY. Alkyl thiophene is used as the first donor connected to the acceptor in

7

NIR-II molecular fluorophores. Theoretical calculations reveal that it affords larger distortion of the

8

conjugated backbone and less interactions with water molecules than EDOT or TEG thiophene donor

9

studied before. As a result, IR-FTAP with octylthiophene as the first donor and thiophene as the second

10

donor exhibits fluorescence peaked at 1048 nm with a QY of 5.3% in aqueous solutions, one of the

11

highest for molecular fluorophore reported so far. IR-FTAP can be superior to the inorganic

12

fluorophores like HIPCO SWCNT, quantum dots and rare earth nanoparticles with its straightforward

13

and well-controlled synthesis and conjugation with biomolecules.74, 26, 75-76 The QY of IR-FTAP is

14

about 13 times of HIPCO SWCNT, though it is still inferior to some bright quantum dot or rare earth

15

nanoparticle systems.77-78 Preliminary imaging studies reveal the great potential of IR-FTAP as an

16

NIR-II fluorophore with superior spatial and temporal performance. With the powerful tunability of

17

optical properties by molecular engineering, organic fluorophores can become promising probes for

18

biological imaging in the NIR-II window.

19

■ ASSOCIATED CONTENT

20

Supporting Information.

21 22 23

This material is available free of charge via the Internet at http://pubs.acs.org. General information about materials and methods, synthesis and characterizations, NMR spectra of the compounds, Figure S1-S9 and tables S1-S5, etc.

24

■ AUTHOR INFORMATION

25

Corresponding Author ACS Paragon Plus Environment

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1 2 3

* Y.L. ([email protected]), H.S. ([email protected]), H.D. ([email protected]).

4

Author Contributions

5 6

‡Q.Y., Z.H. and S.Z. contributed equally. All authors have given approval to the final version of the manuscript.

7 8

Notes The authors declare no competing financial interest

9

■ ACKNOWLEDGMENT

10 11 12 13 14 15 16 17 18 19 20 21

Page 22 of 26

Y.L. acknowledges financial supports from the National Science Foundation of China (21772084), "The Recruitment Program of Global Youth Experts of China", the Shenzhen Key Lab funding (ZDSYS201505291525382), the Shenzhen Technical Research funding (JSGG20160301095829250), and the Shenzhen peacock program (KQTD20140630160825828). Q.Y acknowledges financial supported by China Postdoctoral Science Foundation (2017M612752), Shenzhen Basic Research funding (JCYJ20170307151634428). H.S. thanks the National Science Foundation of China (21603074 and 11474096), “Chenguang Program” by Shanghai Education Development Foundation and Shanghai Municipal Education Commission (16CG25) and Shanghai-International Scientific Cooperation Fund (16520721200) for funding support. This study was partially supported by grants from the Calbrain Program to H.D. Animal experiments were approved by Stanford University's administrative panel on Laboratory Animal Care. All experiments were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

22 23

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20 ms exposure

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5 mm

5 mm S/B=4.9

0.2 0.0

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Table of Contents artwork

Normalized FE

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900

QY = 5.3% in water 1000

1100

1200

1300

1400

1500

Wavelenght /nm

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Donor Engineering for NIR-II Molecular Fluorophores with Enhanced

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