1

Development of a deep-red fluorescent glucose-conjugated bioprobe

for tumor targeting in vivo

Yinwei Cheng,†a Ghulam Shabir,†a,b Xiang Li,a Laiping Fang,b Liyan Xu,*a Hefeng Zhang,*b and Enmin Li*a

a. Department of Biochemistry and Molecular Biology, Comprehensive Building,

Shantou University Medical College, 22 Xinling Road, Shantou, 515041, China

b. Department of Chemistry, Shantou University, 243 Daxue Road, Shantou, 515063,

China

† These authors contributed equally.

*Corresponding author: hfzhang@stu.edu.cn, lyxu@stu.edu.cn; nmli@stu.edu.cn

Contents

1. Experimental

1.1 Materials and Instruments

1.2 Synthesis of Glu-1-O-DCSN

1.3 Preparation of stock solutions of Glu-1-O-DCSN

1.4 Preparation of staining solution of Glu-1-O-DCSN

1.5 Cell culture and Glu-1-O-DCSN uptake assay

1.6 Glu-1-O-DCSN treatment and cell viability assay

1.7 Incubation and Staining of Living Cells

1.8 Animal experiments and in vivo image

1.9 Western blot analysis of GLUT1

2. Figures

S1. FTIR spectrum of compound 1 and isophorone

S2. 1H NMR spectrum of compound 1

S3. 13C NMR spectrum of compound 1

S4. FTIR spectrum of compound 2

S5. 1H NMR spectrum of compound 2

S6. 13C NMR spectrum of compound 2

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

2

S7. FTIR spectrum of compound 3

S8. 1H NMR spectrum of compound 3

S9. FTIR spectrum of the Glu-1-O-DCSN probe and compound 3

S10. 1H NMR spectrum of the Glu-1-O-DCSN probe

S11. Cell imaging of HeLa, KYSE150 and NE1 cells labelled by probe Glu-1-O-DCSN

at concentration of 0.10 μM.

S12. Colocalization analysis of Glu-1-O-DCSN with NucBlue live cell stain in live-

cell imaging of HeLa, KYSE150 and NE1 cells.

S13. Glu-1-O-DCSN probe (0.10 μM) uptake was competitively inhibited by D-

glucose

1. Experimental

1.1 Materials and Instruments

All chemicals and reagents were purchased from Aladdin Chemical Company.

Solvents were purified and dried by standard methods before use. FTIR spectra were

run on a single beam Nicolet IR 100 (Fourier transform). UV-vis spectra were recorded

on a Shimadzu UV-2600 UV-Visible spectrophotometer. Fluorescence spectra were

measured on an F-7000 FL spectrophotometer. The absolute fluorescence quantum

yield was determined by using a Hamamatsu quantum yield spectrometer C11347

Quantaurus-QY. Proton and carbon nuclear magnetic resonance spectra (1H and 13C

NMR) were recorded on an AVANCE-400 MHz and 100 MHz NMR spectrometer,

respectively, with TMS as an internal reference. Compounds were routinely checked

by thin layer chromatography (TLC) on silica gel plates using petroleum ether

(PE)/ethyl acetate (EA) and chloroform: methanol. The crude products were purified

by flash column chromatography and re-crystallization techniques.

1.2 Synthesis of Glu-1-O-DCSN

The synthesis for the Glu-1-O-DCSN probe was profiled in Scheme 1 in the main

text, which involved the following steps:

a) malononitrile (3.96 g, 60.00 mmol) was dissolved in stirring ethanol (70 mL) at

room temperature followed by addition of 7.49 mL of isophorone (50.00 mmol). After

adding piperidine catalyst (10.00 mg), the reaction mixture was heated to reflux for

12 h. After cooled to room temperature, water (100.00 mL) was added into the

3

reaction mixture leading to precipitation. The precipitates were dissolved in a water

(60 mL)/ethanol (90 mL) for re-crystallization. Crystals of 2-(3,5,5- trimethylcyclohex-

2-en-1-ylidene) malononitrile (1) were collected and dried in an oven at 60oC (3.10 g,

75% yield, m.p. 71oC). Characteristic spectroscopic data of compound 1 was as follows:

FTIR (KBr, ῡ) = 2954, 2220, 1614, 1549, 1383, 1325, 893 cm-1. 1H NMR (400 MHz,

chloroform-d) δ 6.55 (q, J = 1.5 Hz, 1H), 2.44 (s, 2H), 2.10 (t, J = 1.3 Hz, 2H), 1.96 (t, J =

1.1 Hz, 3H), 0.94 (s, 6H). 13C NMR (100 MHz, CDCl3): δ=170.33, 159.69, 120.59, 113.16,

112.38, 99.98, 45.69, 42.64, 32.36, 27.81, 25.29;

b) To a stirred solution of acetobromo-α-D-glucose (3.92 g 9.60 mmol) in DCM

(60.00 mL), 4-diethyaminosalicylaldehyde (1.44 g, 8.00 mmol) and

tetrabutylammonium bromide (2.60 g, 8.00 mmol) were added at room temperature,

followed by the addition of 5% NaOH aqueous solution (40.00 mL). 2 h later, the

organic layer was collected, and the aqueous layer was extracted three times with

chloroform (90.00 mL). The organic layer was washed with brine solution (80.00 mL)

and dried with Na2SO4. After concentrated on a rotary evaporator, the resultant dark

violet residue was purified by silica gel column with PE : EA (4:1) as eluent to give

yellow oily liquid product of compound 2 (0.70 g, 25%). Characteristic spectroscopic

data of compound 2 is as follows: FTIR (KBr, ῡ) = 2975, 1752, 1664, 1596 cm-1. 1H NMR

(400 MHz, chloroform-d): δ= 7.76 (dd, J = 8.9, 1.6 Hz, 1H), 6.47 (d, 3JH,H = 9.1 Hz, 1H),

6.38 (s, 1H), 5.42-5.29 (m, 2H), 5.30-5.15 (m, 2H), 4.31 (dd, 3JH,H = 12.4, 4.5 Hz, 1H),

4.22 (dd, 3JH,H= 12.4, 2.4 Hz, 1H), 3.93-3.85 (m, 1H), 3.45 (qd, 3JH,H = 7.3, 2.7 Hz, 4H),

2.07 (dd,3JH,H = 6.8, 1.7 Hz, 12H), 1.25 (t, 3JH,H= 7.0 Hz, 6H).13C NMR (100 MHz, CDCl3):

δ= 186.66, 170.51, 170.21, 169.29, 169.18, 161.34, 145.96, 131.77, 130.05, 108.82,

107.22, 99.51, 72.53, 72.18, 70.94, 68.19, 61.91, 45.20, 20.65, 20.58, 12.46.

c) 2-(3,5,5- trimethylcyclohex-2-en-1-ylidene) malononitrile 1 (0.12 g, 0.66 mmol)

was added to compound 2 (0.35 g, 0.66 mmol) in 10 mL dry ethanol at room

temperature. After heated to 80oC, ammonium acetate (10.00 mg) was added and the

reaction solution was refluxed overnight. The reaction mixture was concentrated on a

rotary evaporator and the residue was purified on by silica gel column

chromatography (PE : EA = 1:1) to give compound 3 (0.20 g, 43%). Characteristic

spectroscopic data of compound 3 is as follows: FTIR (KBr, ῡ) = 2960, 2210, 1750, 1550,

1600, 1500, 1250, 1210, 1040 cm-1. 1H NMR (400 MHz, chloroform-d) δ= 7.51 (d, 3JH,H

4

= 7.3 Hz, 1H), 7.33 (d, 3JH,H= 16.0 Hz, 1H), 6.76 (d, 3JH,H = 16.0 Hz, 2H), 6.45 (s, 1H), 6.3-

6.07 (m, 1H), 5.47-5.31 (m, 2H), 5.26-5.10 (m, 2H), 4.32 (q, 1H), 3.88-3.57 (m, 1H), 3.41

(q, 3JH,H = 7.1, 4H), 2.54 (m, 3H), 2.21-1.87 (m, 12H), 1.85 (m, 1H), 1.22 (t, 3JH,H = 7.1 Hz,

7H), 1.09 (m, 5H).

d) To compound 3 (100.00 mg, 0.144 mmol) solution in methanol (5.00 mL), KOH

(4.00 mg, 0.72 mmol) was added at room temperature and the resultant solution was

stirred for 2 h. After the reaction solution was neutralized with acetic acid, methanol

was removed by using rotary evaporator followed by addition of water (15.00 mL).

The mixture was filtered and dried to give the final compound Glu-1-O-DCSN (39 mg,

55%). Characteristic spectroscopic data of compound Glu-1-O-DCSN is as follows:

FTIR (KBr, ῡ) = 2960, 2210, 1750, 1550, 1600, 1500, 1250, 1210, 1040 cm-1. 1H NMR

(400 MHz, methanol-d4): δ=7.73 (d, 3JH,H = 16.0 Hz, 1H), 7.57 (d, 3JH,H = 9.0 Hz, 1H), 6.91

(d, 3JH,H = 16.0 Hz, 1H), 6.69 (s, 1H), 6.58 (d, 3JH,H = 2.5 Hz, 1H), 6.47 (dd, 3JH,H = 9.0, 2.5

Hz, 1H), 4.85 (d, 3JH,H = 7.7 Hz, 1H), 4.56 (s, 2H), 3.88 (dd, 3JH,H = 11.9, 1.8 Hz, 1H), 3.79-

3.66 (m, 2H), 3.55 (t, J = 8.2 Hz, 1H), 3.49-3.46 (m, 1H), 3.42 (m, 9H), 3.20 (q, 3JH,H = 7.3

Hz, 2H), 2.61-2.54 (m, 2H), 1.31 (t, 3JH,H = 7.3 Hz, 2H), 1.19 (t, 3JH,H= 7.0 Hz, 6H), 1.07 (d, 3JH,H = 4.5 Hz, 6H).

1.3 Preparation of stock solutions of Glu-1-O-DCSN

To prepare a stock solution, Glu-1-O-DCSN was dissolved in absolute ethanol to a

final concentration of 0.10 mg/ml (0.19 mM) and the stock solution was stored in a

vial at room temperature and protected from light.

1.4 Preparation of staining solution of Glu-1-O-DCSN

Glu-1-O-DCSN stock solution (0.10 mg/ml, 0.19 mM) was diluted to the final working

concentration in live cell image solution (Cat. No. A14291DJ, Life Tech) and using

working concentration of 0.05 - 0.10 μM.

1.5 Cell culture and Glu-1-O-DCSN uptake assay

Human HeLa cervical carcinoma cells were cultured in Dulbecco’s modified Eagle’s

medium (Thermo Fisher Scientific) supplemented with 10% fetal bovine serum,

human KYSE150 esophageal cancer squamous cells were cultured in RPMI 1640

medium (Thermo) containing 10% fetal bovine serum (GIBCO), and normal

immortalized NE1 esophageal epithelial cells were cultured in defined keratinocyte

serum-free medium (dKSFM, Invitrogen) at 37 oC, 5% CO2 (V/V).

5

1.6 Glu-1-O-DCSN treatment and cell viability assay

The CellTiter 96® AQueous One Solution Cell Proliferation Assay kit (Promega) was

used for determining cell proliferation after Glu-1-O-DCSN and vehicle treatment

(absolute ethanol). Briefly, cells were plated in triplicate in 96-well plates, at a density

of 10,000 cells per well (triplicate blank wells without cells were conducted for

normalization) and allowed to incubate overnight, and then serial dilutions of Glu-1-

O-DCSN (0.01 µM, 0.10 µM and 1.00 µM) were added, as well as vehicle control. After

treatment for 24 h, MTS reagent was added, and plates were read on a multi-well

scanning spectrophotometer at 492 nm, after incubating at 37 °C and 5% CO2 for 2 h,

and the cell viability ratio was calculated compared with vehicle treatment.

1.7 Incubation and staining of live cells and cell fluorescence intensity measurement

Cells were plated in fibronectin-coated 24-well NEST glass bottom cell culture plates

at 5,000 cells per well and incubated at 37 °C and 5% CO2 overnight to reach cells

confluency 60-70%. Remove cell culture medium from the well and cells were washed

twice with live cell imaging solution and add prewarmed (37 °C) staining solution

containing Glu-1-O-DCSN probe in the absence or presence of D-glucose at the

indicated concentrations. Live-cell imaging was immediately performed after Glu-1-

O-DCSN probe added using Zeiss confocal laser scanning microscope (LSM880) in

living image chamber (37 °C and 5% CO2) with time series mode (duration 240 cycles,

interval time 1 min), at excitation wavelength λex = 488 nm and emission wavelength

λem = 645 nm. In live cell imaging by using LSM880, both wavelength at 488 nm and

514 nm can excite Glu-1-O-DCSN efficiently with emission wavelength collected at

600-700nm. We choose 488 nm to excite Glu-1-O-DCSN as this excitation wavelength

is widely supplied in most fluorescent microscopes.

To investigate the subcellular localization of Glu-1-O-DSCN, a commercial

mitochondria probe Mito-Tracker Green (Cat. no. M7514, Invitrogen) (λex = 490 nm,

λem = 516 nm) and nuclear probe NucBlue Live Cell Stain (Cat. No. R37605, λex = 360

nm, λem = 460 nm) were used for colocalization analysis respectively. In cell imaging

using GLU-1-O-DCSN and MitoTracker Green, cells were incubated with 0.10 µM Glu-

1-O-DSCN and 0.10 µM Mito-Tracker green together for 30 minutes. Due to the

excitation wavelength of Mito-Tracker Green is 490 nm (λem = 516 nm) and at this

wavelength Glu-1-O-DCSN can also be excited, to avoid fluorescence crosstalk

6

between Glu-1-O-DCSN and Mito-Tracker Green, the excitation wavelength of Glu-1-

O-DCSN was chosen λex = 514 nm and collecting fluorescence in a range of 613 - 662

nm (at λex = 514 nm, λem, collected = 613 - 662 nm, only Glu-1-O-DSCN is visible), and Mito-

Tracker Green was chosen λex = 488 nm, collecting at 499-517 nm (at λex = 488 nm, λem,

collected = 499 - 517 nm, only Mito-Tracker Green is visible). In cell imaging using GLU-1-

O-DCSN and NucBlue Live Cell Stain, cells were incubated with 0.10 µM Glu-1-O-DCSN

and two drops of NucBlue Live Cell Stain together for 30 minutes according to the

manufacturer’s instructions. Glu-1-O-DCSN was imaged at excitation wavelength λex =

488 nm and emission wavelength λem = 645 nm, and NucBlue Live Cell Stain was

imaged at λex = 360 nm, λem = 460 nm. All image processing was done by ZEN (blue

edition) software, and co-localization analysis was done by Fuji software.

For measuring fluorescence intensity of each cell by ZEN software, we draw an area

as ROIs (regions of interest) which contains a cell in phase-contract image. ZEN

analyses images and let us know the digitalized mean of fluorescence intensity in the

ROIs that we determined. After subtracting the background intensity from the

fluorescence intensity, we get the fluorescence intensity value that a cell contains at

every time point. For each experiment, at least 3 images were performed and at least

10 cells are measured of each image.

1.8 Animal experiments and in vivo imaging

All experiments involving animals were performed in compliance with the policy on

animal use and ethics of Shantou University Medical College (SUMC), and

experimental procedures were performed in accordance with protocols approved by

SUMC Medical Animal Care & Welfare Committee. Five-week-old female nude mice

were purchased from Vital River Laboratories (Beijing, China) and maintained on a 12

h light/dark cycle under specific pathogen-free conditions, with free access to food

and water (Permit Number: 2017-0079). Mice (n=16) were randomized into two

groups, one is inoculated with KYSE150 cells (n=10) and the other is inoculated with

phosphate buffer saline (PBS) using as tumor-free control (n=6). Suspensions of 1×106

KYSE150 cells in 100 μL of serum-free 1640 medium were inoculated subcutaneously

into the right armpit of nude mice to establish the tumor model. Tumor growth was

monitored daily and tumor size were measured (length × width2). When tumor

reached about 100 mm3, mice were applied with Glu-1-O-DCSN probe as described

7

below.

For tumor labelling, the Glu-1-O-DCSN probe (20 nM, 100 μL) was intratumorally

injected into the tumor tissue of tumor-bearing mice (n=5). For control experiments

(n=3), the same dose of probe was injected subcutaneously into the right armpit of

tumor-free nude mice. All mice were anesthetized with 2% isoflurane throughout all

procedures. Real-time in vivo fluorescent images were performed using a Caliper IVIS

Kinetic small animal in vivo imaging system with an excitation filter of 400–650 nm at

different time intervals (0.5 h, 1.5 h, 18 h, 22 h) after injection. At 24 hours, mice were

sacrificed (operated on ice) and the main organs (including heart, liver, brain, lung,

spleen, stomach, kidney, colon and rectum) and tumor were removed, and

fluorescence imaging of the main organs was performed using the same above

imaging system.

For tumor targeting, the Glu-1-O-DCSN probe (20 nM, 100 μL) was intravenously

injected into tumor-bearing mice (n=5) and tumor-free control mice (n=3) via tail vein.

Mice and organ imaging were performed in the same way as the tumor labelling

experiments. Mice and organ imaging analyzed in a longitudinal series for each mouse

and were normalized using a look-up-table with common minimum and maximum

values. Relative fluorescence intensity was measured by LivingImage software by

background-corrected Regions of interest (ROI) measurement. ROI for both tumor and

background were derived from equivalent sized areas containing the same number of

pixels. Fluorescent intensity was calculated by mean ± SD, n=5.

1.9 Western blot analysis of GLUT1

NE1, KYSE150 and HeLa cells were lysed in 100 μL 1ⅹlaemmli sample buffer (bio-

rad,161-0747) for 10 minutes and sonicated for 10 seconds. Protein was loaded for

SDS-PAGE separation without additional boiling of lysate. Proteins were transferred

to PVDF membrane and blocking with 5% nonfat milk, then incubated with primary

antibody GLUT1 (1;1000, abcam, ab15309) and β-actin (1:1000, Santa Cruze, sc-

47778) at 4° overnight. Membrane is washed in TBST for 3 times and then incubated

with secondary antibody HRP-conjugated anti-rabbit (1;2000, CST, 7074) at room

temperature for 1 h. Chemiluminescence images were captured using bi0-rad chemi

imaging system after developing with ECL solution.

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2. Figures

4000 3500 3000 2500 2000 1500 1000 500

020406080

100120140160180200

CN

CN

O

Tr

ansm

ittan

ce

Wavenumber/cm

2960

2930 22201610 1550

1430 1320

1660

14301380 1250

1150901

8912960

Isophorone

Compound 1

Figure S1. FTIR spectra of compound 1 and isophorone

CN

CNH

a

b

cd

a

b

c

d

e

e

9

Figure S2. 1H NMR spectrum of compound 1

CN

CN

b

a

c de

fg

h ij

k

a

b

cde

ij,kfg

h

Figure S3. 13C NMR spectrum of compound 1

4000 3500 3000 2500 2000 1500 1000 500

0

20

40

60

80

100

O

HO

N

OAcO

AcO

OAc

OAc

Tran

smitt

ance

Wavenumber/cm

3480

2970

17501590

16601520

136012201040

752Compound 2

Figure S4. FTIR spectrum of compound 2(图有改动)

10

OH

O

OO

O

O

O

O

O

O

O

Na

b

c

c

c

c

e

fg

hij

k

l m

a

b

c

nnk

l m

e, g, f

hi, j

Figure S5. 1H NMR spectrum of compound 2

OH

O

OO

O

O

O

O

O

O

O

Na

f

e

d

c

b

e

gh

kij

o

p r

n

n

m

m

ab,c,d,f

f

l

q

s

t

u

vs t

u, v

g

qo,p

r k,h,i,j,e

l

Figure S6. 13C NMR spectrum of compound 2

11

4000 3500 3000 2500 2000 1500 1000 500

0

20

40

60

80

100

CCN

CNO

N

OAcO

AcO

OAc

OAc

Tran

smitt

ance

Wavenumber/cm

2210

2960

1600

1750

1550 1500 1210

12701040

Compound 3

Figure S7. FTIR spectrum of compound 3(图有改动)

O

NC CN

N

O

O

O

O

O

O

O

OO

a

b

c

d

e

f

g

h

i

j

k

l

m n

o p

q

rs

tf,g,h,i

c

a

b

e d

l, n, m

k,u

u

o

p,q

rst

j

Figure S8. 1H NMR spectrum of compound 3

12

4000 3500 3000 2500 2000 1500 1000 500

020406080

100120140160180200

CCN

CNO

N

OAcO

AcO

OAc

OAc

CCN

CNHO

N

OHO

HO

OH

OH

Tran

smitt

ance

Wavenumber/cm

3390 2970 2210 1610

1540 1510 10701280

1390

2970

22101750

1610

1550 1510 12101270 10401150

Glu-1-O-DCSN

Compound 3

Figure S9. FTIR spectra of the Glu-1-O-DCSN probe and compound 3(图有改动)

OCN

CN

O

OH

HO

HOOH

Na

b

c

d e

rf

g

hi

j

k

l m

n

op

q

bi,l,hn

m j k

gf

o

p qr

a

ceb

Figure S10. 1H NMR spectrum of the Glu-1-O-DCSN probe

13

Figure S11. Cell imaging of HeLa, KYSE150 and NE1 cells labelled by probe Glu-1-O-DCSN. Three types of cells were incubated with 0.10 μM Glu-1-O-DCSN for 0.5 h and were visualized by using a confocal laser scanning microscope with excitation wavelength λex = 488 nm, λem, collected = 600-700 nm). Scale bar=20 μM

Figure S12. Colocalization analysis of Glu-1-O-DCSN (λex = 488 nm, λem, collected = 600-700 nm) and nuclei by probing with NucBlue live cell stain (λex = 305 nm, λem, collected = 460-490 nm) using live-cell imaging of HeLa, KYSE150 and NE1 cells. Scale bar = 20 μm (three cell lines were incubated in 0.10 μM aqueous Glu-1-O-DCSN solution for 0.5 h followed by NucBlue live cell stain for 0.5 h for visualization on a confocal laser scanning microscope).

14

Figure S13. Glu-1-O-DSCN probe (0.10 μM) (λex = 488 nm, λem, collected = 600-700 nm) in Cell imaging of HeLa, KYSE150 and NE1 cells under Glucose treatment (0 mM, 5.6 mM).

Table S1. Quantum yields of Glu-1-O-DCSN in different solvents Solvent Quantum yielda

Water 1.5%Water +20% ethanol 1.8%Water+50% ethanol 16.1%

Water + 20% DMSO

2.4%

Water + 50% DMSO

12.6%

DCM 7.8%THF 7.6%

Chloroform 5.8%Ethanol 17.2%

Methanol 15.2%DMF 24.3%

DMSO 35.1%PBS 7.4 0.1%

PBS 7.4+5%DMSO 2.4%

15

a. Determined at a concentration of 0.5 μM by using spectrophotometer with an integrating sphere detector.