A NIR-Emitting Cyanine with Large Stokes Shifts for Live Cell Imaging:

Large Impact of Phenol Group on Emission

Dipendra Dahal, Lucas McDonald, Sabita Pokhrel, Sailaja Paruchuri, Michael Konopka, Yi Pang*

Corresponding Author: yp5@uakron.edu

General Considerations: All starting materials and the essential solvents were purchased from Sigma-Aldrich, Ark

Pharma, Fischer Scientific, Alfa-Asaer and Across Organics and directly used without further purifications. All

deuterated solvents were purchased from Cambridge Isotopes and used as received. All NMR data were recorded on

Varian 300 and 500 MHz instruments with all spectra referenced to deuterated solvents. Mass spectra were obtained

from HP1100LC/MSD mass spectrometry. HRMS data were performed on an ESI-TOF MS system (Waters, Milford,

MA).

Synthesis of 2-hydroxy-5-methylisophthalaldehyde:

OH

CH3

OH

O OCF3COOH

Reflux 4 hrs

N

N

NN

In a 250 mL round-bottomed (RB) flask,1 g (9.25 mmol) of p-cresol and 2.6 g (18.55 mmol) of

hexamethylene tetraamine were mixed in a 250 mL RB flask and 30 mL trifluoroacetic acid was added. The mixture

was then refluxed for 6 hrs. After completion of the reaction, the solution was cooled in an ice-water bath and was

neutralized with KOH solution to the pH about 6 to get a yellow colored precipitate. The precipitate was then filtered,

washed several times with water and dried. Pure yellow crystalline solid 2-hydroxy-5-methylisophthalaldehyde was

obtained by recrystallization in methanol with >90 % yield.

1H NMR ( 300 MHz, DMSO-d6): δ = 11.45 ppm (s, 1H), 10.21 ppm (s, 2H), 7.77ppm (s, 2H), 7.26 ppm (solvent),

2.38 ppm (s, 3H).

Electronic Supplementary Material (ESI) for Chemical Communications.This journal is © The Royal Society of Chemistry 2019

Synthesis of 2,2'-((2-hydroxy-5-methyl-1,3-phenylene)bis(ethene-2,1-diyl))bis(3-ethyl benzothiazole-3-ium) diiodide (compound 2):

OH

O O

OHS

NI I

N

S

I

MeOH / RefluxOvernight at 600C

N

S

2

In a round-bottomed flask, 297 mg (0.98 mmol) of 1-ethyl-2methylbenzothiazolium salt was dissolved in 20

mL methanol, and 0.5 mL pyridine was added to it. The mixture was stirred at room temperature for 30 minutes, and

75 mg (0.46 mmol) of 2-hydroxy-5-methylisophthalaldehyde (dialdehyde) was added to the flask, and the mixture

was heated to 60oC and stirred overnight at 60oC. After completion of the reaction, the solvent was evaporated using

rotary evaporator and the crude solid obtained, was washed with 50 mL ethyl acetate which was filtered and dried to

get dark-colored solid. The solid still contained excess unreacted benzothiazolium salt which was washed with water

several times filtered and dried to get pure dark-colored solid compound 2 with 75% isolated yield. NMR and mass

spectroscopy then characterized compound 2.

1H NMR ( 500 MHz, DMSO-d6): δ = 8.22 ppm (d, 2H), 8.08 ppm (m, 6H), 7.70ppm (t, 2H), 7.67 ppm (s, 2H), 7.60

ppm (t, 2H), 4.71 ppm (q, 4H), 2.45 ppm (solvent DMSO), 2.19 ppm (s, 3H) and 1.42 ppm (t, 6H).

13C NMR ( 125 MHz, DMSO-d6): δ = 171.78, 143.55, 141.13, 134.17, 130.03, 128.70, 124.86, 124.19, 117.07,

113.19, 44.79, 40.02 (solvent), 20.51, 14.68.

Synthesis of 2,2'-(1,3-phenylenebis(ethene-2,1-diyl))bis(3-ethylbenzo[d]thiazol-3-ium) iodide (compound 3):

In a 50 mL RB flask, 100 mg (0.33 mmol) of 1-ethyl-2methylbenzothiazolium iodide salt was dissolved in

30 mL methanol in a round-bottomed flask, and 0.5 mL pyridine was added. The mixture was stirred for about 30

minutes, and 22 mg (0.16 mmol) of isophthalaldehyde was added to it. After mixing, it was then stirred at 600C for

overnight. When the reaction was completed, the solvent was evaporated in a rotary evaporator to get a red-colored

solid, which was washed with ethyl acetate, filtered and dried. The solid obtained was again washed with water several

times to remove unreacted excess benzothiazolium salt, filtered and dried to get pure compound 3 with the yield of

85%. NMR and mass spectroscopy characterized compound 3.

O OS

NI I

N

S

I

MeOH / RefluxOvernight at 600C

N

S2 equiv.

1H NMR ( 500 MHz, DMSO-d6): δ = 8.68 ppm (s, 1H), 8.49 ppm (d,2H), 8.35 ppm (d, 2H), 8.30 ppm (m, 4H), 8.20

ppm (d, 2H), 7.92 ppm (t, 2H), 7.84 ppm (t, 2H), 7.78 ppm (t, 2H), 5.04 ppm (q, 4H), 2.48 ppm (solvent), 1.52 ppm

(t, 6H).

13C NMR ( 126MHz, DMSO-d6): δ = 171.79, 147.97, 141.48, 135.33, 132.95, 131.79, 130.46, 130.22, 129.16,

129.08, 125.10, 117.39, 45.42, 40.02 (solvent), 14.87.

Calculation of quantum yield:

Zinc Phthalocyanine (Фref.≈ 0.20) was used as the standard of reference for calculation of quantum yield of

compound 2, and quinine sulfate ((Фref.≈ 0.53) was used as a standard of reference for compound 3. Quantum yield

of probes was calculated using an equation shown below, where ‘A’ is absorbance at the excitation wavelength, ‘I’ is

an integrated area of emission, ‘Фref’ is a quantum yield of reference compound and ‘η’ is the refractive index of

solvent used.

Ф𝑠𝑎𝑚𝑝𝑙𝑒=Ф𝑟𝑒𝑓 ∗𝐼𝑠𝑎𝑚𝑝𝑙𝑒

𝐼𝑟𝑒𝑓∗𝐴𝑟𝑒𝑓

𝐴𝑠𝑎𝑚𝑝𝑙𝑒∗𝜂 2𝑠𝑎𝑚𝑝𝑙𝑒

𝜂 2𝑟𝑒𝑓

NMR Study:

OH

OO

CH3

-OH-CHO

a a

aCD

Cl3

-CH3

Figure S1. 1H NMR spectra of 2-hydroxy-5-methylisophthalaldehyde in CDCl3.

OHS

NH2C

CH3CH3

N

S

CH2H3C

DM

SO

CH

3-C

H2-

CH3-CH2-

-CH3

ab

c

de

f

g g

f

ed

c

ba

d

b

f

a

g

e

c

Figure S2. 1H NMR spectra of compound 2. The top inset is an expanded region from 7.6 ppm to 8.5 ppm for clarity.

DMSO

OH

N

SN

S

Figure S3. 13C NMR spectra of compound 2 in DMSO-d6.

OHN

S N

S

Figure S4. ESI-TOF mass spectra of compound 2.

N

S N

SCH2

H3C

H2C CH3

abc

de

f g

hi

ab

cd

ef

hCH3-CH2-

DM

SOCH3-CH2-

i

cbfe,h

da

g

Figure S5. 1H NMR spectra of compound 3.

N

SN

S

CH2

H3C

H2C CH3

DM

SO

Figure S6. 13C NMR spectra of compound 3 (125MHz).

N

S N

S

Exact Mass: 454.1526Mass error: -7.05 ppm

Theoretical

Actual

Figure S7. ESI-TOF mass spectra of compound 3.

Study of Photophysical properties:

350 400 450 500 550 600 650 700 750 800 8500.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

DCM DMF DMSOMeCN EtOH MeOH Water

600

640

680

720

Abso

rban

ce W

avele

ngth

Solvent Polarity

Abso

rban

ce (A

U)

Wavelength (nm)

DCM DMF DMSO EtOH MeCN MeOH Water

589

633

728

717

385

Figure S8. UV-absorbance spectra of compound 2 (10 µM) in different solvents.

700 720 740 760 780 800 820 840 860 880 900

01x105

2x105

3x105

4x105

5x105

6x105

7x105

8x105

Inten

sity (

CPS)

Wavelength (nm)

DCM DMF DMSO EtOH MeCN MeOH Water

795803784

Figure S9. Fluorescence emission spectra of compound 2 (10 µM) in various solvents.

400 600 800 1000 1200 1400

0.0

0.2

0.4

0.6

0.8

1.0

Inte

nsity

Wavelength (nm)

absorption emission

390

714780

Figure S10. The calculated absorption/emission spectra of keto tautomer of 2 at the B3LYP/6-31+G(d) level in (CH2Cl2). The spectra were generated by using TD-SCF method, after the molecular geometry was optimized at the

B3LYP/6-31+G(d) level.

HOMO

LUMO

Figure S11. Calculated molecular HOMO/LUMO orbitals for compound 2 in its keto- tautomer after deprotonation at the B3LYP/6-31+G(d) level.

300 350 400 450 500 550

0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

Abso

rban

ce (A

U)

Wavelength (nm)

DCM DMF DMSO EtOH MeCN MeOH Water

380

Figure S12. UV absorbance spectra of compound 3 in different solvents.

425 450 475 500 525 550 575

0

1x104

2x104

3x104

4x104

5x104

6x104

7x104

8x104

Inte

nsity

(CPS

)

Wavelength (nm)

DCM DMF DMSO EtOH MeCN MeOH THF Water

460

Figure S13. Fluorescence emission spectra of compound 3 in various solvents.

600 650 700 750 800 8500.0

2.0x106

4.0x106

6.0x106

8.0x106

1.0x107

Fl. I

nten

sity

(CPS

)

Wavelength (nm)

-188 -160 -140 -112 -95 -73 -45 -28 -17 -5 20

700

788

800 (RT)

-188o C

-95o C

-73o C

Figure S14. Low-temperature fluorescence spectra of compound 2 (λ ex = 450).

690 720 750 780 810 840 870 900

0.0

4.0x105

8.0x105

1.2x106

1.6x106

2.0x106

-188 -160 -145 -132 -123 -116 -109 -101 -95 -89 -80 -74 -60 -50 -40 -30 -20 0 20

-180-150-120 -90 -60 -30 0700

720

740

760

780

800

Emiss

ion W

avele

ngth

(nm)

Temperature (0C)

Fluo

resc

ence

Inte

nsity

(CPS

)

Wavelength (nm)

700

800

Figure S15. Low-temperature fluorescence spectra of compound 2 (λ ex = 650).

400 450 500 550 600 650 700

0.2

0.4

0.6

0.8

1.0No

rmali

zed I

ntens

ity (C

PS)

Wavelength (nm)

@ -188 @ -53 @ RT

389

465

618

453

612

Figure S16. Excitation spectra of comound 2 at different temperature.

300 350 400 450 500 550 600 650 700 750 8000.000.050.100.150.200.250.300.350.400.45

Abso

rban

ce (A

U)

Wavelength (nm)

pH 1 pH 2 pH 3 pH 4 pH 5 pH 6 pH 7 pH 8 pH 9 pH 10

385

593

Figure S17. UV-absorbance spectra of compound 2 (10 µM) in different pH buffer in water.

0 1 2 3 4 5 6 7 8 9 100.0

0.1

0.2

0.3

0.4

0.5

pKa = 4.01Ab

sorb

ance

pH value

Absorbance at 385 nm Absorbance at 593 nm

Figure S18. Boltzmann’s curve fitting of absorbance of compound 2 to determine the pKa value.

350 400 450 500 550 600 650 700 750 800

0.0

0.1

0.2

0.3

0.4

0.5

0.6

Abso

rban

ce (A

U)

Wavelength (nm)

C2 Acetate ADP AMP ATP BSA ClO4

-

Cysteine F-

GSH H2O2

H2PO4-

HSO3-

PPi S2O3

2-

SH-

622

387a)

400 500 600 700 8000.0

0.1

0.2

0.3

0.4

0.5

0.6

Abso

rban

ce (A

U)

Wavelength (nm)

20 uM C2 200 uM GSH 400 uM GSH 1mM GSH 5mM GSH 10mM GSH

385620

390

b)

Figure S19. UV absorbance spectra of compound 2 (C2): (a) in the presence of one equivalent (10 µM) of different anions, and (b) in presence of different concentration of

glutathione (GSH) in in EtOH/PBS (1:1 by v/v).

650 700 750 800 850 9000

1x105

2x105

3x105

4x105

5x105

6x105

C2 Acetate ADP AMP ATP BSA ClO4

-

Cysteine F-

GSH H2O2

H2PO4-

HSO3-

PPi S2O3

2-

SH-

Emiss

ion

Inte

nsity

(CPS

)

Wavelength (nm)

768a)

650 700 750 800 8500.0

2.0x105

4.0x105

6.0x105

8.0x105Fl

. Iny

ensit

y (C

PS)

Wavelength (nm)

20 uM C2 200 uM GSH 400 uM GSH 1mM GSH 5mM GSH 10mM GSH

768b)

Figure S20. Fluorescence emission spectra of compound 2 (C2): (a) in the presence of one equivalent (10 µM)of different anions in EtOH/PBS (1:1 by v/v) and b) in the

presence of different concentration of glutathione (GSH) in EtOH/PBS (1:1 by v/v).

Cell viability test for compound 2:

Biocompatible photophysical properties (NIR absorption and emission) of compound 2 encouraged us to find

the application of compound 2 in live cells imaging. Before using in live cells imaging, Cell cytotoxicity of compound

2 was acquired using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide) assay. Normal Human

Lung Fibroblasts (NHLF) cells 5×103 per well were seeded in a 96-well plate in DMEM low glucose media with 10%

FBS and incubated 24 hrs. at 37⁰ C with 5% CO2. After 24 hrs., the cells were starved overnight with 0.4% FBS

DMEM low glucose media. The cells were then treated with a series of dilution of compound 2 (0-1 mM; n=8). A

final concentration of 0.5 mg/mL of MTT per well was used followed by incubation at 37⁰C for 2 hours. The

absorbance was detected using Epoch Bio-Tek microplate reader at 570nm, and the IC50 value was calculated using

GraphPad Prism. The IC50 value for 2 was found to be 24.72 µM.

Figure S21. Cell viability test for compound 2 in NHLF cells (IC50) was found to be 24.72 µM.

Cell culture and staining: Normal human lungs fibroblast (NHLF) cells and oligodendrocytes cells were maintained

in Dulbecco’s modified eagle’s medium (DMEM) with 10% FBS at 370C in 5% CO2 humidified incubator. The

solution of probe 2 and MitoTracker Green FM was made in DMSO. The final concentration of 1 µM solution of

probe 2, 200 nM concentrations of MitoTracker Green were applied in cells for in vivo imaging. For cell studies, the

NHLF cells were plated in a separate imaging petri dish with DMEM media and they were treated with probe 2 and

Red MitoTracker green for 30 minutes at 37oC. Cells were then washed three times with 1x PBS.

The excitation wavelength of compound 2 was 640 nm laser with 700 – 735 nm filter for emission.

MitoTracker green was excited at 488 nm with a 525/50 nm bandpass emission filter. An Okolab Bold Cage Incubator

at 37oC was used for the incubation of imaging cells, and images were processed using NIS Elements. Fiji ImageJ

software was used to calculate the colocalization coefficient of compound 2 with commercial trackers.

Figure S22. Confocal fluorescence images for normal human lungs fibroblast (NHLF) cells stained with compounds 1b and 2. A) 2 under 100x magnification, B) 4 times digitally enhanced image of yellow rectangular box in A, C) bright-field to show cells for compound 2, D) 1b under 100x magnification, E) 4 times digitally enhanced image of yellow rectangular box in D and F) bright-field to show cells for compound 1b. Compound 2 was excited with 640 nm laser with the emission filter of 680-735 nm, and 1b was excited at 405 nm lesser with emission filters of 680 -

720 nm.

Figure S23. Images of oligodendrocyte cells stained with A) MitoTracker Green (200 nM), B) compound 2 (1 M), C) merged A and B under magnification of 100x and D) correlation plot between Compound 2 and

MitoTracker Green. Excitation for MitoTracker Green was 488 nm with the emission of 525 nm, for compound 2 excitation was 640 nm with 680-735 nm filters for emission,

Figure S24. Images of fixed NHLF cells stained with compound 2 (1 µM). A) cells were stained with compound 2 first for 30 minutes and fixed, B) cells were fixed first, permeabilized and stained for 30 minutes and C) cells were fixed and treated with dye for 30 minutes. The compound 2 was excited at 640 nm excitation laser with the emission filter of 680-735 nm.

It was assumed that the probe 2 could be a functional dye to stain mitochondria as it had double positive charges, which would have strong interaction with the negatively charged mitochondrial matrix. In order to shed some light on its staining interaction with mitochondria, we further tested the probe 2 in fixed cells. The probe 2 remained inside the cells in a non-uniform pattern when cells were treated with the dye before fixing them (Figure S24A). After cells being fixed, the cells could be stained with 2 along with permeabilizer (to drive the probe molecules into cells)(Figure S24B). However, compound 2 alone did not show the ability to stain the fixed cells (Figure S24C), as the cellular functions (e.g. mitochondrial potential gradient or endocytosis) would have been lost upon fixation of cells. Therefore, probe 2 could be considered as a “functional mitochondrial probe” which is accumulated into mitochondria due to the potential gradient (∆ψm) of the mitochondrial matrix.