Supporting Information

An In Cellulo-Activated Multicolor Cell Labeling Approach Used to Image Dying Cell Clearance

Yilong Shi,a Rui Zhu,a Zhongwei Xue,a Jiahuai Han,b and Shoufa Hana,*

aState Key Laboratory for Physical Chemistry of Solid Surfaces, Department of Chemical Biology, College of Chemistry and Chemical Engineering, the Key Laboratory for Chemical Biology of Fujian Province, The MOE Key Laboratory of Spectrochemical Analysis & Instrumentation, and Innovation Center for Cell Signalling Network, Xiamen University;

bState key Laboratory of Cellular Stress Biology, Innovation Center for Cell Signalling Network, School of Life Sciences, Xiamen University, Xiamen, 361005, China; Tel: 86-0592-2181728; E-mail: shoufa@xmu.edu.cn.

Content

Scheme S1. Synthesis and chemical deacetylation of compound 1……………..…………….……S3

Fig. S1 1H-NMR of compound 2…..……………..…………….…..……………..……….……..…S3

Fig. S2 13C-NMR of compound 2…..……………..…………….…..……………..……………..…S4

Fig. S3 1H-NMR of compound 3…..……………..…………….…..……………..………….…..…S4

Fig. S4 13C-NMR of compound 3…..……………..…………….…..……………..……………..…S5

Fig. S5 HRMS of compound 2 and compound 3………..………………………………….………S5

Scheme S2. Synthetic routes for DHA-Red and AP-Red. ………..………………………..………S6

Scheme S3. Synthetic routes for DHA-Blue and DHA-Green. . ………..……………..…..………S6

Fig. S6 Esterase-triggered protein labeling with DHA-Red. ………..……………………..………S6

Fig. S7 Incapability of AP-Red to label cellular proteins. ………..………………………..………S7

S1

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

Fig. S8 Dose- and Incubation time-dependent cell labelling with DHA-Red. ………..……………S7

Fig. S9 Covalent cell labelling with DHA-Green. ………..…………………………………..……S8

Fig. S10 Covalent cell labelling with DHA-Blue. . ………..………………………………….……S8

Fig. S11 cytotoxicity of Dye-DHA diads. ………..…………………………………………….……S9

Fig. S12 Comparison of DHA-Dye diads to Cell-tracker CFSE. . ………..…………………..……S10

Fig. S13 Fluorescence retention of DHA-medaited labeling in HeLa cells undergoing cell death….S11

Fig. S14 1H-NMR of compound 1. ………..………………………………………………..….……S12

Fig. S15 13C-NMR of compound 1 ………..………………………………………………..….……S12

Fig. S16 1H-NMR of DHA-Red………..………………………………………….…………………S13

Fig. S17 13C-NMR of DHA-Red………..……………………………………………….….….……S13

Fig. S18 HRMS of DHA-Red………..………………………………………….……………..……S14

Fig. S19 1H-NMR of AP-Red………..………………………………………….….……………..…S14

Fig. S20 13C-NMR of AP-Red………..………………………………………….…………….……S15

Fig. S21 HRMS of AP-Red………..………………………………………….….……………….…S15

Fig. S22 1H-NMR of DHA-Blue………..…………………………………………………….….……S16

Fig. S23 13C-NMR of DHA-Blue………..…………………………………………………...….……S16

Fig. S24 HRMS of DHA-Blue………..………………………………………….……………….……S17

Fig. S25 1H-NMR of DHA-Green………..…………………………………………………..….……S17

Fig. S26 13C-NMR of DHA-Green………..………………………………………….………….……S18

Fig. S27 HRMS of DHA-Green………..………………………………………….………….….……S28

S2

HOO

NH

N3OCH3

CH3ONa

HO

OH

O

NH

N3

2 3

1

AcO

OAc

O

NH

N3

HO

OH

O

OH

acetyl chloride AcO

OAc

O

OH

H2N N3

EDC/TEA

+

1

reaction solution

DHA

Scheme S1. Synthesis and chemical deacetylation of compound 1.

Fig. S1 1H-NMR of compound 2

S3

Fig. S2 13C-NMR of compound 2

Fig. S3 1H-NMR of compound 3

S4

Fig. S4 13C-NMR of compound 3

287.11115 273.09536

Fig. S5 HRMS of compound 2 and compound 3

S5

EDC/TEA

HNN O

CO2H

N

DHA-Red

DHA

AcO

OAc

O

NN O

CO2H

N

AcOO

OH

AP-Red

AcOO

NN O

CO2H

N

A B

Scheme S2. Synthetic routes for DHA-Red and AP-Red (A). Fluorescence properties of DHA-Red (4 μM) in PBS (pH 7.2) (B)

AcO

OAc

O

NN

O

O NO

DHA-Blue

HNN

O

O NO

EDC/TEA

DHA

AcO

OAc

O

NH

NH N

H

SO

OAc

OAcO

ODHA-Green

1

AcO

OAc

O

NH

N31) Pd/C/H22) FITC/TEA;3) Acetic anhydride/TEA

Scheme S3. Synthetic routes for DHA-Blue and DHA-Green.

A B

Protein Marker BSA Esterase

0 2 4 0 2 4 0 2 4 0 2 4

Protein Marker BSA Esterase

DHA-Red (µ M)

Fig. S6 Esterase-triggered protein labeling with DHA-Red. Esterase or BSA (1.5 μg) were treated DHA-Red for 1 h and then subjected to SDS-PAGE. The gel was analyzed for fluorescence emission using λex = 520 nm (A) or stained with Coomassie Brilliant Blue (B).

S6

DHA-Red AP-Red DHA-Red AP-Red A B

Fig. S7 Incapability of AP-Red to label cellular proteins

DHA-Red Merge LysoTracker-Blue

10 min

30 min

60 min

120 min

DHA-Red Merge Bright View

1 µM

2 µM

4 µM

8 µM

A B

Fig. S8 Dose- and Incubation time-dependent cell labelling with DHA-Red. (A) HeLa cells were co-stained with LysoTracker Blue (1 μM) and DHA-Green (2 μM) for 10, 30, 60, 120 min in DMEM and then imaged by confocal microscopy for intracellular fluorescence. (B) HeLa cells labeled with DHA-Green (1, 2, 4, 8 μM) for 60 min and then imaged by confocal microscopy for intracellular fluorescence. Scale bar: 10 μm.

S7

Green signal Bright view LysoTracker-Red Merge +

DH

A-G

reen

+

DH

A-G

reen

+

FIxa

tion

Green signal Merge Bright view

2 µM

4 µM

8 µM

A B

Fig. S9 Covalent cell labelling with DHA-Green. (A) HeLa cells were co-stained with LysoTracker Red (1 μM) and DHA-Green (4 μM) for 1 h. Cells were fixed with 4% Paraformaldehyde Fix Solution, washed and then imaged for intracellular fluorescence. (B) HeLa cells labeled with DHA-Green (2, 4, 8 μM) for 1 h and then fixed before confocal microscopy analysis. Scale bar: 10 μm.

Coumarin signal Bright view LysoTracker-Red Merge

Coumarin signal Merge Bright view

2 µM

4 µM

8 µM

A B

+ D

HA

-Blu

e +

DH

A-B

lue

+ Fi

xatio

n

Fig. S10 Covalent cell labelling with DHA-Blue. (A) HeLa cells were co-stained with LysoTracker Red (1 μM) and DHA-Blue (4 μM) for 1 h. The cells were fixed with 4% paraformaldehyde Fix Solution, washed and then imaged for intracellular fluorescence. (B) HeLa cells labeled with DHA-Blue (2, 4, 8 μM) for 1 h and then fixed before confocal microscopy analysis. Scale bar: 10 μm

S8

Fig. S11 cytotoxicity of Dye-DHA diads.

S9

0 h 2 4 h 4 8 h0

2 0 0 0 0

4 0 0 0 0

6 0 0 0 0

A c -R B

t im e

Av

g S

ign

al

0 h 2 4 h 4 8 h0

1 0 0 0

2 0 0 0

3 0 0 0

4 0 0 0

C F S E

t im e

Av

g S

ign

al

DHA-Red Commercial CFSE

0 h 2 4 h 4 8 h0

1 0 0 0

2 0 0 0

3 0 0 0

4 0 0 0

A c -C M

t im e

Av

g S

ign

al

0 h 2 4 h 4 8 h0

1 0 0 0 0

2 0 0 0 0

3 0 0 0 0

A c -F IT C

t im e

Av

g S

ign

al

DHA-Green DHA-Blue

0 h 24 h 48 h

0 h 24 h 48 h 0 h 24 h 48 h

Com

mer

cial

CFS

E B

right

vie

w

Mer

ge

0 h 24 h 48 h

DH

A-G

reen

B

right

vie

w

Mer

ge

DH

A-R

ed

Brig

ht v

iew

Mer

ge

DH

A-B

lue

Brig

ht v

iew

Mer

ge

CFSE DHA-Red

DHA-Green DHA-Blue

A B

O

AcO

OAcO

OCFSE

Esterase

HN

O

HO

O

CO2H

Cell labeling

ON

O

O

O

O

Protein

C

Fig. S12 Comparison of DHA-Dye diads to Cell-tracker CFSE. HeLa cells were stained with CFSE (4 μM), DHA-Blue (4 μM), DHA-Green (4 μM), or DHA-Red (4 μM) for 60 min, respectively. The cells were further maintained in fresh DMEM for 0, 24, 48 h. A portion of the cells were harvested at indicated time points, rinsed with DMEM, and then analyzed by confocal microscopy (A) or flow cytometry (B) for intracellular fluorescence. (B) Chemistry of DHA-Green deacetylation in live cells. Scale bar: 10 μm.

S10

4 h 2 h 0 h 12 h

DH

A-R

ed

Brig

ht v

iew

Mer

ged

4 h 2 h 0 h 12 h

DH

A-R

ed

Brig

ht v

iew

Mer

ged

Apoptosis Necrosis

4 h 2 h 0 h 12 h 4 h 2 h 0 h 12 h

DH

A-B

lue

Brig

ht v

iew

Mer

ged

DH

A-B

lue

Brig

ht v

iew

Mer

ged

Apoptosis Necrosis

4 h 2 h 0 h 12 h 4 h 2 h 0 h 12 h

DH

A-G

reen

B

right

vie

w

Mer

ged

DH

A-G

reen

B

right

vie

w

Mer

ged

Apoptosis Necrosis

A B C

Fig. S13 Fluorescence retention of DHA-medaited labeling in HeLa cells undergoing cell death. RIP3+ HeLa cells were stained with DHA-Blue (4 μM), DHA-Red (4 μM), or DHA-Green (4 μM) for 1 h, and then rinsced with PBS, and then treated with Smac/TNF to triggered apoptosis or with Smac/TNF/Z-VAD to trigger necrosis. The dying cells were maintained in fresh medium and then monitored by confocal fluorescence microscopy for intracellualr fluorescence over time. Scale bars: 20 μm.

S11

Fig. S14 1H-NMR of compound 1

Fig. S15 13C-NMR of compound 1

S12

Fig. S16 1H-NMR of DHA-Red

Fig. S17 13C-NMR of DHA-Red

S13

DHA-Red

Fig. S18 HRMS of DHA-Red

Fig. S19 1H-NMR of AP-Red

S14

Fig. S20 13C-NMR of AP-Red

AP-Red

Fig. S21 HRMS of AP-Red

S15

Fig. S22 1H-NMR of DHA-Blue

Fig. S23 13C-NMR of DHA-Blue

S16

DHA-Blue

Fig. S24 HRMS of DHA-Blue

Fig. S25 1H-NMR of DHA-Green

S17

Fig. S26 13C-NMR of DHA-Green

Fig. S27 HRMS of DHA-Green

S18