DOI: 10.1038/NMAT3074 Maltodextrin based imaging … · and in vivo SUPPLEMENTARY INFORMATION DOI: 10.1038/NMAT3074 ©

Maltodextrin based imaging probes detect bacteria in vivo with high

sensitivity and specificity

Xinghai Ning1†, Seungjun Lee1†, Zhirui Wang2, Dongin Kim1, Bryan Subblefield3, Eric

Gilbert3, and Niren Murthy1 *

1The Wallace H. Coulter Department of Biomedical Engineering and the Parker H. Petit Institute

for Bioengineering and Bioscience, Georgia Institute of Technology, Atlanta, GA 30332

2Complex Carbohydrate Research Center, University of Georgia, Athens, GA 30602

3Department of Biology, Georgia State University, Atlanta, GA 30302

X. N. Phone: 1- 404-385-1329, Email: [email protected]

S. L. Phone: 1- 404-385-1329, Email: [email protected]

Z. W. Phone: 1- 706-542-4468, Email: [email protected]

D. K. Phone: 1- 404-385-1329, Email: [email protected]

B. S. Phone: 1- 404-413-5137, Email: [email protected]

E. G. Phone: 1- 404-413-5137, Email: [email protected]

N. M. Phone: 1- 404-385-5145, Email: [email protected]

*Correspondence should be addressed to: N. M.

SUPPLEMENTARY INFORMATIONDOI: 10.1038/NMAT3074

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SUPPLEMENTARY INFORMATION

ABBREVIATIONS

Å………………………………………………………………………………………….Angstrom

Ac……………………………………………………………………………………………Acetyl

Ac2O……………………………………………………………………………...Acetic anhydride

C…………………………………………………………………………………………….Carbon

CCl3CN……………………………………………………………………….Trichloroacetonitrile

CDCl3……………………………………………………………………....Deuterated chloroform

CD3OD……………………………………………………………………….Deuterated methanol

CO2………………………………………………………………….….…………..Carbon dioxide

COSY…………………………………………………………..………..Correlation spectroscopy

CuI…………………………………………………………………………………...Copper iodide

DCM/CH2Cl2………………………………….……………Dichloromethane/Methylene chloride

DIPEA…………………………………… …………………………..N,N-Diisopropylethylamine

DI water……………………………………………………………………….......Deionized water

DMF……………………………………………………………………..N, N-Dimethylformamide

D2O……………………………………………………………………………….Deuterium oxide

EDTA…………………………………………………………...Ethylenediamine tetra-acetic acid

EtOAc………………………………………………………………………................Ethyl acetate

H……………………………………………………………………………..........................Proton

H2…………………………………………………………………….. .........………...…Hydrogen

HCl………………………………………………………...................................Hydrogen chloride

HSQC………………………………………...…............Heteronuclear single quantum coherence

Hz……………………………………………………............……………………………..…Hertz

HRMS………………………………………………................High resolution mass spectrometry

KSAc…………………………………………………………………...……Potassium thioacetate

LiOH……………………………………………………………………………Lithium hydroxide

LTQ…………………………………………………………….Linear ion trap mass spectrometer

M…………………………………………………………………………………...………...Molar

MALDI-TOF-TOF……….Matrix assisted laser desorption ionization spectroscopy-time of flight

MeOH/CH3OH..……………………………………………………………………….…Methanol

MS………………………………………………………………………….……..Molecular sieves

NaBH4…………………………………..………………………….................Sodium borohydride

NaH…………………………………………………………………..……………Sodium hydride

NaHCO3……………………………………………………………………….Sodium bicarbonate

NaN3………………………………………………………………………………….Sodium azide

NaOH………………………………………………………………...……........Sodium hydroxide

NaSO4……………………………………………………………….……...............Sodium sulfate

NH2NH2…………………………………………………………………........................Hydrazine

NMR……………………………………………………………….…Nuclear magnetic resonance

N2……………………………………………………………………………………….....Nitrogen

PBS…………………………………...…………………………………Phosphate buffered saline

PEG……………………………………………………………………………Polyethylene glycol

Py…………………………………………………………………………………………..Pyridine

RIPA…………………………………………………................Radio-immunoprecipitation assay

TiCl4………………………………………………………………………...Titanium tetrachloride

2 NATURE MATERIALS | www.nature.com/naturematerials

SUPPLEMENTARY INFORMATION DOI: 10.1038/NMAT3074



SUPPLEMENTARY INFORMATION

ABBREVIATIONS

Å………………………………………………………………………………………….Angstrom

Ac……………………………………………………………………………………………Acetyl

Ac2O……………………………………………………………………………...Acetic anhydride

C…………………………………………………………………………………………….Carbon

CCl3CN……………………………………………………………………….Trichloroacetonitrile

CDCl3……………………………………………………………………....Deuterated chloroform

CD3OD……………………………………………………………………….Deuterated methanol

CO2………………………………………………………………….….…………..Carbon dioxide

COSY…………………………………………………………..………..Correlation spectroscopy

CuI…………………………………………………………………………………...Copper iodide

DCM/CH2Cl2………………………………….……………Dichloromethane/Methylene chloride

DIPEA…………………………………… …………………………..N,N-Diisopropylethylamine

DI water……………………………………………………………………….......Deionized water

DMF……………………………………………………………………..N, N-Dimethylformamide

D2O……………………………………………………………………………….Deuterium oxide

EDTA…………………………………………………………...Ethylenediamine tetra-acetic acid

EtOAc………………………………………………………………………................Ethyl acetate

H……………………………………………………………………………..........................Proton

H2…………………………………………………………………….. .........………...…Hydrogen

HCl………………………………………………………...................................Hydrogen chloride

HSQC………………………………………...…............Heteronuclear single quantum coherence

Hz……………………………………………………............……………………………..…Hertz

HRMS………………………………………………................High resolution mass spectrometry

KSAc…………………………………………………………………...……Potassium thioacetate

LiOH……………………………………………………………………………Lithium hydroxide

LTQ…………………………………………………………….Linear ion trap mass spectrometer

M…………………………………………………………………………………...………...Molar

MALDI-TOF-TOF……….Matrix assisted laser desorption ionization spectroscopy-time of flight

MeOH/CH3OH..……………………………………………………………………….…Methanol

MS………………………………………………………………………….……..Molecular sieves

NaBH4…………………………………..………………………….................Sodium borohydride

NaH…………………………………………………………………..……………Sodium hydride

NaHCO3……………………………………………………………………….Sodium bicarbonate

NaN3………………………………………………………………………………….Sodium azide

NaOH………………………………………………………………...……........Sodium hydroxide

NaSO4……………………………………………………………….……...............Sodium sulfate

NH2NH2…………………………………………………………………........................Hydrazine

NMR……………………………………………………………….…Nuclear magnetic resonance

N2……………………………………………………………………………………….....Nitrogen

PBS…………………………………...…………………………………Phosphate buffered saline

PEG……………………………………………………………………………Polyethylene glycol

Py…………………………………………………………………………………………..Pyridine

RIPA…………………………………………………................Radio-immunoprecipitation assay

TiCl4………………………………………………………………………...Titanium tetrachloride





TLC……………………………………………………………………Thin layer chromatography

THF………………………………………………………………………………..Tetrahydrofuran

TMS………………………………………………………………...............….Tertramethyl silane

TMSOTf…………………………………………………Trimethylsilyl trifluromethane sulfonate

UV………………………………………………………………………………………Ultraviolet

EXPERIMENTAL PROCEDURES

1H-NMR spectra were recorded in either CDCl3, CD3OD or D2O on a Varian 400 spectrometer

equipped with a Sun workstation at 300K. TMS (δ (ppm)H = 0.00) was used as the internal

reference. 13C-NMR spectra were recorded in either CDCl3, CD3OD and D2O at a 100MHz on a

Varian 400 spectrometer, using the central resonances of CDCl3 (δ (ppm)C = 77.0) and methanol

(δ (ppm)C = 50.4) as the internal references. COSY and HSQC experiments were used to assist

assignment of the products. Chemical shifts are reported in ppm and multiplicities are indicated

by s (singlet), d (doublet), t (triplet), q (quartet), dd (doublet of doublets), and m (multiplet).

Coupling constants, J, are reported in hertz (Hz). High-resolution mass spectra (HRMS) were

obtained on a AB SCIEX TOF/TOF 5800 system and are reported as m/z (relative intensity).

Accurate masses are reported for the molecular ion (M+) or a suitable fragment ion.

Fluorescence spectroscopic studies were performed using a Shimadzu (RF-5301-PC)

spectrofluorometer. Chemicals were purchased from Aldrich or VWR and used without further

purification. All solvents were purified using standard methods. Flash chromatography was

carried out using silica gel (230-400 mesh). All reactions were performed under anhydrous

conditions under N2 and monitored by TLC on Kieselgel 60 F254 plates (Merck). Detection was

accomplished by examination under UV light (254 nm) and by charring with 10 % sulfuric acid

in methanol.

Synthesis of azide functionalized maltohexaose 1 (Supplementary Figure S1)

Supplementary Figure S1. Synthesis of azide functionalized maltohexaoside (1)

Synthesis of α-D-Glucopyranose,2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl-(1→4)-O-2,3,6-tri-

O-acetyl-α-D-glucopyranosyl-(1→4)-O-2,3,6-tri-O-acetyl-α-D-glucopyranosyl-(1→4)-O-2,3,6-

tri-O-acetyl-α-D-glucopyranosyl-(1→4)-O-2,3,6-tri-O-acetyl-α-D-glucopyranosyl-(1→4)-

1,2,3,6-tetraacetate (5).

To a stirred solution of Maltohexaose 4 (0.5 g, 0.51 mmol) in pyridine (10 mL) was added Ac2O

(5 mL). The reaction mixture was stirred at room temperature for 18 hours under nitrogen and

then concentrated in vacuo. The residue was dissolved in EtOAc (100 mL) and washed with

aqueous Na2CO3 (1 M, 10 mL x 3), aqueous HCl (0.1 M, 10 mL), and brine (10 mL x 2). The

organic layer was dried over Na2SO4, filtered and evaporated to dryness in vacuo. The residue

was purified by flash column chromatography on silica gel (hexane/EtOAc, 2:3) to afford

5 (0.85g, 90.1%). 1H NMR (CDCl3, 400 MHz,): δ (ppm) 6.18 (d, 0.5H, J = 3.2 Hz, α1-H), 5.66

(d, 0.5H, J = 8.0 Hz, β1-H), 5.43 (t, 1H, J = 10.0 Hz, 3-H), 5.34-5.22 (m, 10 H, 3-H), 5.00 (t, 1 H,

J = 10.0 Hz, 4-H), 4.88 (dd, 0.5 H, J = 3.7 and 10.0 Hz, α 2-H), 4.77 (dd, 1 H, J = 3.9 and 10.5





TLC……………………………………………………………………Thin layer chromatography

THF………………………………………………………………………………..Tetrahydrofuran

TMS………………………………………………………………...............….Tertramethyl silane

TMSOTf…………………………………………………Trimethylsilyl trifluromethane sulfonate

UV………………………………………………………………………………………Ultraviolet

EXPERIMENTAL PROCEDURES

1H-NMR spectra were recorded in either CDCl3, CD3OD or D2O on a Varian 400 spectrometer

equipped with a Sun workstation at 300K. TMS (δ (ppm)H = 0.00) was used as the internal

reference. 13C-NMR spectra were recorded in either CDCl3, CD3OD and D2O at a 100MHz on a

Varian 400 spectrometer, using the central resonances of CDCl3 (δ (ppm)C = 77.0) and methanol

(δ (ppm)C = 50.4) as the internal references. COSY and HSQC experiments were used to assist

assignment of the products. Chemical shifts are reported in ppm and multiplicities are indicated

by s (singlet), d (doublet), t (triplet), q (quartet), dd (doublet of doublets), and m (multiplet).

Coupling constants, J, are reported in hertz (Hz). High-resolution mass spectra (HRMS) were

obtained on a AB SCIEX TOF/TOF 5800 system and are reported as m/z (relative intensity).

Accurate masses are reported for the molecular ion (M+) or a suitable fragment ion.

Fluorescence spectroscopic studies were performed using a Shimadzu (RF-5301-PC)

spectrofluorometer. Chemicals were purchased from Aldrich or VWR and used without further

purification. All solvents were purified using standard methods. Flash chromatography was

carried out using silica gel (230-400 mesh). All reactions were performed under anhydrous

conditions under N2 and monitored by TLC on Kieselgel 60 F254 plates (Merck). Detection was

accomplished by examination under UV light (254 nm) and by charring with 10 % sulfuric acid

in methanol.

Synthesis of azide functionalized maltohexaose 1 (Supplementary Figure S1)

Supplementary Figure S1. Synthesis of azide functionalized maltohexaoside (1)



tri-O-acetyl-α-D-glucopyranosyl-(1→4)-O-2,3,6-tri-O-acetyl-α-D-glucopyranosyl-(1→4)-

1,2,3,6-tetraacetate (5).

To a stirred solution of Maltohexaose 4 (0.5 g, 0.51 mmol) in pyridine (10 mL) was added Ac2O

(5 mL). The reaction mixture was stirred at room temperature for 18 hours under nitrogen and

then concentrated in vacuo. The residue was dissolved in EtOAc (100 mL) and washed with

aqueous Na2CO3 (1 M, 10 mL x 3), aqueous HCl (0.1 M, 10 mL), and brine (10 mL x 2). The

organic layer was dried over Na2SO4, filtered and evaporated to dryness in vacuo. The residue

was purified by flash column chromatography on silica gel (hexane/EtOAc, 2:3) to afford

5 (0.85g, 90.1%). 1H NMR (CDCl3, 400 MHz,): δ (ppm) 6.18 (d, 0.5H, J = 3.2 Hz, α1-H), 5.66

(d, 0.5H, J = 8.0 Hz, β1-H), 5.43 (t, 1H, J = 10.0 Hz, 3-H), 5.34-5.22 (m, 10 H, 3-H), 5.00 (t, 1 H,

J = 10.0 Hz, 4-H), 4.88 (dd, 0.5 H, J = 3.7 and 10.0 Hz, α 2-H), 4.77 (dd, 1 H, J = 3.9 and 10.5





Hz, 2-H), 4.67- 4.63 (m, 4 H, 2-H), 4.43-4.40 (m, 4H), 4.22-3.81 (m, 21H), 2.16, 2.15, 2.13, 2.11,

2.10, 2.09, 2.08, 2.06, 2.02, 1.99, 1.97, 1.95, 1.93, 1.91, 1.88 (60 H, 15 s, CH3). 13C NMR

(CDCl3, 100 MHz): δ (ppm) 170.9, 170.8, 170.7, 170.6, 170.5, 170.3, 170.2, 170.1, 170.0, 169.9,

169.8, 169.7, 169.6, 169.2, 168.9 (C=O), 96.1 (1-C), 96.0 (1-C), 95.9 (1-C), 95.8 (1-C), 95.7 (1-

C), 91.4, 89.0, 77.6, 77.5, 77.3, 75.3, 73.6, 73.5, 73.4, 73.1, 72.5, 72.4, 71.9, 71.8, 71.7, 71.6,

71.1, 70.7, 70.6, 70.3, 70.2, 69.9, 69.5, 69.2, 69.1, 68.6, 68.0, 62.8, 62.7, 62.6, 62.5, 62.4, 62.3,

61.5, 29.8, 21.2, 21.1, 21.0, 20.9, 20.8, 20.7, 20.6, 20.5, 20.3. HRMS (MALDI) m/z Found:

1853.5298, calculated: 1853.5280 for C76H102NaO51 [M+Na]+.



tri-O-acetyl-α-D-glucopyranosyl-(1→4)-O-2,3,6-tri-O-acetyl-α-D-glucopyranosyl-(1→4)-2,3,6-

triacetate (6).

To a stirred solution of 5 (0.73 g, 0.4 mmol) in DMF (10 mL) was added N2H4·HOAc (46.0 mg,

0.5 mmol). The reaction mixture was heated to 60 ºC for 12 hours under nitrogen, and the

mixture was concentrated in vacuo. The residue was dissolved in EtOAc (100 mL) and washed

with water (30 mL x 2) and brine (10 mL). The organic phase was dried over Na2SO4, filtered

and evaporated to dryness in vacuo. The residue was purified by flash column chromatography

on silica gel (hexane/EtOAc, 1:3) to afford 6 (0.66 g, 93.1%). 1H NMR (CDCl3, 400 MHz,): δ

(ppm) 5.56 (t, 1H, J = 9.6 Hz, 3-H ), 5.42−5.25 (m, 10H), 5.06 (t, 1 H, J = 9.2 Hz, 4-H), ), 4.83

(dd, 1 H, J = 4.0 and 9.6 Hz, α 2-H), 4.78-4.69 (m, 5H), 4.54-4.45 (m, 4H), 4.33-3.58 (m, 21 H),

2.18-1.96 (s, 57 H, CH3). 13CNMR (100 MHz, CDCl3): δ (ppm) 171.0, 170.9, 170.8, 170.7,

170.6, 170.6, 170.4, 170.1, 170.0, 169.9, 169.8, 169.7, 169.6 (C=O), 95.9 (1-C), 95.8 (1-C),

90.2 (1-C), 77.6, 73.9, 73.5, 72.6, 72.5, 71.9, 71.8, 70.6, 70.2, 69.7, 69.1, 68.6, 68.1, 67.9, 63.1,

62.6, 62.5, 62.3, 61.6, 60.6, 21.2, 21.1, 21.0, 21.0, 20.9, 20.8, 20.7. HRMS (MALDI) m/z Found:





triacetate 1-(2,2,2-trichloroethanimidate) (7).

To a stirred solution of 6 (0.53 g, 0.3 mmol) in dry THF (10 mL) was added trichloroacetonitrile

(60 µL, 0.6 mmol), and the solution was cooled to 0 ºC. NaH (9.0 mg, 0.4 mmol) was then

added and the suspension was stirred at 0 ºC for 6 hours under nitrogen. The reaction mixture

was concentrated in vacuo to afford crude 7 (0.58 g, quantitative). The crude compound was

used for the next step without purification. 1H NMR (400 MHz, CDCl3): δ (ppm) 8.58 (s, 1H,

NH), 6.50 (d, J = 3.6 Hz, 1H, H-1), 5.60 (t, 1H, J = 9.6 Hz, 3-H ), 5.43−5.28 (m, 10H), 5.11 (t, 1

H, J = 9.2 Hz, 4-H), ), 4.87-4.73 (m, 6H), 4.56-3.60 (m, 25 H), 2.18-1.96 (s, 57 H, CH3).

Synthesis of β-D-Glucopyranose,2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl-(1→4)-O-2,3,6-tri-



triacetate 1-(3’-azidopropyl) (1).

To a stirred solution of crude 7 (0.38 g, 0.2 mmol) and 3-azidopropanol (0.1 g, 1.0 mmol) in dry

CH2Cl2 (10 mL) was added 4Å M.S. The mixture was stirred under nitrogen at 0 ºC for 1 hour.

TMSOTf (45 µL, 0.25 mmol) was then added and the mixture was stirred at 0 ºC for 1 hour. The

mixture was allowed to warm to room temperature After 1 hour the reaction was quenched with

Et3N and concentrated in vacuo. The residue was dissolved in EtOAc (50 mL) and washed with

water (10 mL x 2) and brine (10 mL). The organic phase was dried over Na2SO4, filtered and





Hz, 2-H), 4.67- 4.63 (m, 4 H, 2-H), 4.43-4.40 (m, 4H), 4.22-3.81 (m, 21H), 2.16, 2.15, 2.13, 2.11,

2.10, 2.09, 2.08, 2.06, 2.02, 1.99, 1.97, 1.95, 1.93, 1.91, 1.88 (60 H, 15 s, CH3). 13C NMR

(CDCl3, 100 MHz): δ (ppm) 170.9, 170.8, 170.7, 170.6, 170.5, 170.3, 170.2, 170.1, 170.0, 169.9,

169.8, 169.7, 169.6, 169.2, 168.9 (C=O), 96.1 (1-C), 96.0 (1-C), 95.9 (1-C), 95.8 (1-C), 95.7 (1-

C), 91.4, 89.0, 77.6, 77.5, 77.3, 75.3, 73.6, 73.5, 73.4, 73.1, 72.5, 72.4, 71.9, 71.8, 71.7, 71.6,

71.1, 70.7, 70.6, 70.3, 70.2, 69.9, 69.5, 69.2, 69.1, 68.6, 68.0, 62.8, 62.7, 62.6, 62.5, 62.4, 62.3,

61.5, 29.8, 21.2, 21.1, 21.0, 20.9, 20.8, 20.7, 20.6, 20.5, 20.3. HRMS (MALDI) m/z Found:





triacetate (6).

To a stirred solution of 5 (0.73 g, 0.4 mmol) in DMF (10 mL) was added N2H4·HOAc (46.0 mg,

0.5 mmol). The reaction mixture was heated to 60 ºC for 12 hours under nitrogen, and the

mixture was concentrated in vacuo. The residue was dissolved in EtOAc (100 mL) and washed

with water (30 mL x 2) and brine (10 mL). The organic phase was dried over Na2SO4, filtered

and evaporated to dryness in vacuo. The residue was purified by flash column chromatography

on silica gel (hexane/EtOAc, 1:3) to afford 6 (0.66 g, 93.1%). 1H NMR (CDCl3, 400 MHz,): δ

(ppm) 5.56 (t, 1H, J = 9.6 Hz, 3-H ), 5.42−5.25 (m, 10H), 5.06 (t, 1 H, J = 9.2 Hz, 4-H), ), 4.83

(dd, 1 H, J = 4.0 and 9.6 Hz, α 2-H), 4.78-4.69 (m, 5H), 4.54-4.45 (m, 4H), 4.33-3.58 (m, 21 H),

2.18-1.96 (s, 57 H, CH3). 13CNMR (100 MHz, CDCl3): δ (ppm) 171.0, 170.9, 170.8, 170.7,

170.6, 170.6, 170.4, 170.1, 170.0, 169.9, 169.8, 169.7, 169.6 (C=O), 95.9 (1-C), 95.8 (1-C),

90.2 (1-C), 77.6, 73.9, 73.5, 72.6, 72.5, 71.9, 71.8, 70.6, 70.2, 69.7, 69.1, 68.6, 68.1, 67.9, 63.1,

62.6, 62.5, 62.3, 61.6, 60.6, 21.2, 21.1, 21.0, 21.0, 20.9, 20.8, 20.7. HRMS (MALDI) m/z Found:





triacetate 1-(2,2,2-trichloroethanimidate) (7).

To a stirred solution of 6 (0.53 g, 0.3 mmol) in dry THF (10 mL) was added trichloroacetonitrile

(60 µL, 0.6 mmol), and the solution was cooled to 0 ºC. NaH (9.0 mg, 0.4 mmol) was then

added and the suspension was stirred at 0 ºC for 6 hours under nitrogen. The reaction mixture

was concentrated in vacuo to afford crude 7 (0.58 g, quantitative). The crude compound was

used for the next step without purification. 1H NMR (400 MHz, CDCl3): δ (ppm) 8.58 (s, 1H,

NH), 6.50 (d, J = 3.6 Hz, 1H, H-1), 5.60 (t, 1H, J = 9.6 Hz, 3-H ), 5.43−5.28 (m, 10H), 5.11 (t, 1

H, J = 9.2 Hz, 4-H), ), 4.87-4.73 (m, 6H), 4.56-3.60 (m, 25 H), 2.18-1.96 (s, 57 H, CH3).

Synthesis of β-D-Glucopyranose,2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl-(1→4)-O-2,3,6-tri-



triacetate 1-(3’-azidopropyl) (1).

To a stirred solution of crude 7 (0.38 g, 0.2 mmol) and 3-azidopropanol (0.1 g, 1.0 mmol) in dry

CH2Cl2 (10 mL) was added 4Å M.S. The mixture was stirred under nitrogen at 0 ºC for 1 hour.

TMSOTf (45 µL, 0.25 mmol) was then added and the mixture was stirred at 0 ºC for 1 hour. The

mixture was allowed to warm to room temperature After 1 hour the reaction was quenched with

Et3N and concentrated in vacuo. The residue was dissolved in EtOAc (50 mL) and washed with

water (10 mL x 2) and brine (10 mL). The organic phase was dried over Na2SO4, filtered and





evaporated to dryness in vacuo. The residue was purified by flash column chromatography on

silica gel (hexane/EtOAc, 1:2) to afford 1 (0.15 g, 39.3%). 1H NMR (400 MHz, CDCl3): δ (ppm)

5.41−5.22 (m, 11H), 5.06 (t, 1 H, J = 10.0 Hz, 4-H), ), 4.82 (dd, 1 H, J = 4.0 and 10.0 Hz, α 2-

H), 4.77-4.70 (m, 3H), 4.52-4.48 (m, 6H), 4.37-3.88 (m, 20 H), 3.68-3.55 (m, 2H,

N3CH2CH2CH2O), 3.36 (t, 2H, J = 6.4 Hz, N3CH2CH2CH2O), 2.24-1.93 (s, 57 H, CH3), 1.83 (m,

2H, N3CH2CH2CH2O). 13CNMR (100 MHz, CDCl3): δ (ppm) 170.9, 170.9, 170.9, 170.8, 170.7,

170.6, 170.6, 170.5, 170.3, 170.0, 169.9, 169.9, 169.8, 169.7, 169.7, 169.6 (C=O), 100.5 (β 1-C),

95.9 (1-C), 95.8 (1-C), 90.2 (1-C), 77.6, 75.5, 73.9, 73.6, 73.4, 73.3, 72.5, 72.4, 72.3, 71.9, 71.8,

71.7, 70.7, 70.6, 70.2, 69.5, 69.1, 68.6, 68.1, 66.6, 63.0, 62.7, 62.6, 62.5, 62.3, 61.6, 48.2, 29.1,

21.1, 21.1, 21.0, 21.0, 20.9, 20.8, 20.8, 20.7. HRMS (MALDI) m/z Found: 1894.5679,

calculated: 1894.5658 for C77H105N3NaO50 [M+Na]+.

Synthesis of alkyne functionalized perylene (2) (Supplementary Figure S2)

Supplementary Figure S2. Synthesis of alkyne functionalized perylene (2)

Synthesis of formylperylene (9)

To a stirred solution of perylene 8 (1.0 g, 4.0 mmol) in 1,2-dichlorobenzene (25 mL) was added

1,1-dichloromethyl methyl ether (0.68 g, 6.0 mmol) and TiCl4 (1.1 g, 6.0 mmol). The reaction

mixture was stirred at 0 ºC for 1 hour under nitrogen, and then allowed to warm to room

temperature. The reaction mixture was diluted with CH2Cl2 (100 mL) and acidified with

aqueous HCl (0.1 M, 10 mL). The mixture was washed with water (50 mL x 3) and brine (20

mL). The organic phase was dried over Na2SO4, filtered and evaporated to dryness in vacuo.

The residue was purified by flash column chromatography on silica gel (hexane/ EtOAc, 15:1) to

afford 9 (0.81 g, 72.3%). 1H NMR (CDCl3, 400 MHz): δ (ppm) 10.31 (s, 1H, CHO); 9.11 (d,

1H, J = 8.8 Hz, aromatic); 8.26-8.15 (m, 4H, aromatic); 7.85 (d, 1H, J = 8.0 Hz, aromatic); 7.78

(d, 1H, J = 8.0 Hz, aromatic); 7.71 (d, 1H, J = 8.0 Hz, aromatic); 7.65 (m, 1H, aromatic); 7.48

(m , 2H, aromatic). 13C NMR (CDCl3, 100 MHz): δ (ppm) 193.3, 137.1, 136.6, 133.8, 131.4,

130.6, 130.0, 129.7, 129.4, 129.3, 129.0, 128.6, 127.9, 127.2, 127.1, 126.9, 123.8, 123.4, 122.0,

121.3, 119.8. HRMS (MALDI) m/z Found: 317.0960, calculated: 317.0937 for C22H14NaO

[M+Na]+.

Synthesis of (Perylenyl-3-methyl)propargyl ether (2).

To a stirred solution of 9 (0.56 g, 2.0 mmol) in ethanol (20 mL) was added NaBH4 (0.11 g,

3.0 mmol). The reaction mixture was stirred at room temperature for 30 minutes under nitrogen,

and then quenched with aq NH4Cl (0.1 M, 5 mL). The solution was diluted with EtOAc (50 mL)

and the organic phase was washed with water (10 mL x 2) and saturated NaHCO3 (10 mL). The

organic phase was dried over Na2SO4, filtered and evaporated to dryness in vacuo. The residue

was dissolved in THF (20 mL), to which was added NaH (16 mg, 4.0 mmol) under vigorous

stirring. The mixture was stirred at room temperature for 10 minutes under nitrogen, and 80%

propargyl bromide in toluene (0.63 g, 4.0 mmol) was added. The reaction was kept at room

temperature for 2 hours, and the solvent was removed in vacuo. The residue was dissolved in

EtOAc (30 mL) and washed with water (10 mL x 2) and brine (10 mL). The organic phase was

dried over Na2SO4, filtered and evaporated to dryness in vacuo. The residue was purified by

flash column chromatography on silica gel (hexane/EtOAc, 5:1) to afford 2 (0.55 g, 85.9 %). 1H

NMR (CDCl3, 400 MHz): δ (ppm) 8.41-8.27 (m, 4H, aromatic); 7.96 (d, 1H, J = 8.0 Hz,






silica gel (hexane/EtOAc, 1:2) to afford 1 (0.15 g, 39.3%). 1H NMR (400 MHz, CDCl3): δ (ppm)

5.41−5.22 (m, 11H), 5.06 (t, 1 H, J = 10.0 Hz, 4-H), ), 4.82 (dd, 1 H, J = 4.0 and 10.0 Hz, α 2-

H), 4.77-4.70 (m, 3H), 4.52-4.48 (m, 6H), 4.37-3.88 (m, 20 H), 3.68-3.55 (m, 2H,

N3CH2CH2CH2O), 3.36 (t, 2H, J = 6.4 Hz, N3CH2CH2CH2O), 2.24-1.93 (s, 57 H, CH3), 1.83 (m,

2H, N3CH2CH2CH2O). 13CNMR (100 MHz, CDCl3): δ (ppm) 170.9, 170.9, 170.9, 170.8, 170.7,

170.6, 170.6, 170.5, 170.3, 170.0, 169.9, 169.9, 169.8, 169.7, 169.7, 169.6 (C=O), 100.5 (β 1-C),

95.9 (1-C), 95.8 (1-C), 90.2 (1-C), 77.6, 75.5, 73.9, 73.6, 73.4, 73.3, 72.5, 72.4, 72.3, 71.9, 71.8,

71.7, 70.7, 70.6, 70.2, 69.5, 69.1, 68.6, 68.1, 66.6, 63.0, 62.7, 62.6, 62.5, 62.3, 61.6, 48.2, 29.1,

21.1, 21.1, 21.0, 21.0, 20.9, 20.8, 20.8, 20.7. HRMS (MALDI) m/z Found: 1894.5679,

calculated: 1894.5658 for C77H105N3NaO50 [M+Na]+.

Synthesis of alkyne functionalized perylene (2) (Supplementary Figure S2)

Supplementary Figure S2. Synthesis of alkyne functionalized perylene (2)

Synthesis of formylperylene (9)

To a stirred solution of perylene 8 (1.0 g, 4.0 mmol) in 1,2-dichlorobenzene (25 mL) was added

1,1-dichloromethyl methyl ether (0.68 g, 6.0 mmol) and TiCl4 (1.1 g, 6.0 mmol). The reaction

mixture was stirred at 0 ºC for 1 hour under nitrogen, and then allowed to warm to room

temperature. The reaction mixture was diluted with CH2Cl2 (100 mL) and acidified with

aqueous HCl (0.1 M, 10 mL). The mixture was washed with water (50 mL x 3) and brine (20

mL). The organic phase was dried over Na2SO4, filtered and evaporated to dryness in vacuo.

The residue was purified by flash column chromatography on silica gel (hexane/ EtOAc, 15:1) to

afford 9 (0.81 g, 72.3%). 1H NMR (CDCl3, 400 MHz): δ (ppm) 10.31 (s, 1H, CHO); 9.11 (d,

1H, J = 8.8 Hz, aromatic); 8.26-8.15 (m, 4H, aromatic); 7.85 (d, 1H, J = 8.0 Hz, aromatic); 7.78

(d, 1H, J = 8.0 Hz, aromatic); 7.71 (d, 1H, J = 8.0 Hz, aromatic); 7.65 (m, 1H, aromatic); 7.48

(m , 2H, aromatic). 13C NMR (CDCl3, 100 MHz): δ (ppm) 193.3, 137.1, 136.6, 133.8, 131.4,

130.6, 130.0, 129.7, 129.4, 129.3, 129.0, 128.6, 127.9, 127.2, 127.1, 126.9, 123.8, 123.4, 122.0,

121.3, 119.8. HRMS (MALDI) m/z Found: 317.0960, calculated: 317.0937 for C22H14NaO

[M+Na]+.

Synthesis of (Perylenyl-3-methyl)propargyl ether (2).

To a stirred solution of 9 (0.56 g, 2.0 mmol) in ethanol (20 mL) was added NaBH4 (0.11 g,

3.0 mmol). The reaction mixture was stirred at room temperature for 30 minutes under nitrogen,

and then quenched with aq NH4Cl (0.1 M, 5 mL). The solution was diluted with EtOAc (50 mL)

and the organic phase was washed with water (10 mL x 2) and saturated NaHCO3 (10 mL). The

organic phase was dried over Na2SO4, filtered and evaporated to dryness in vacuo. The residue

was dissolved in THF (20 mL), to which was added NaH (16 mg, 4.0 mmol) under vigorous

stirring. The mixture was stirred at room temperature for 10 minutes under nitrogen, and 80%

propargyl bromide in toluene (0.63 g, 4.0 mmol) was added. The reaction was kept at room

temperature for 2 hours, and the solvent was removed in vacuo. The residue was dissolved in

EtOAc (30 mL) and washed with water (10 mL x 2) and brine (10 mL). The organic phase was


flash column chromatography on silica gel (hexane/EtOAc, 5:1) to afford 2 (0.55 g, 85.9 %). 1H

NMR (CDCl3, 400 MHz): δ (ppm) 8.41-8.27 (m, 4H, aromatic); 7.96 (d, 1H, J = 8.0 Hz,





aromatics); 7.81 (d, 2H, J = 8.0 Hz, aromatic); 7.62 (m, 1H, aromatics); 7.56 (m, 3H, aromatics);

4.93 (s, 2H, ArCH2); 4.31 (d, 2H, J = 2.3 Hz, CH2C); 3.54 (t, 1H, J = 2.3 Hz, CH). 13C NMR

(CDCl3, 100 MHz): δ (ppm) 134.1, 133.2, 132.5, 130.7, 130.1, 130.2, 127.9, 127.8, 127.8, 127.5,

127.3, 127.1 126.8, 124.0, 120.8, 120.7, 120.5, 120.2, 80.0, 77.5, 69.1, 57.1. HRMS (MALDI)

m/z Found: 343.1113, calculated: 343.1093 for C24H16NaO [M+Na]+.

Synthesis of alkyne functionalized IR786 (3) (Supplementary Figure S3)

Supplementary Figure S3. Synthesis of alkyne functionalized IR786 (3)

Synthesis of 2-(2-(2-(Prop-2-ynyloxy)ethoxy)ethoxy)ethanol (11).

To a stirred solution of triethylene glycol 10 (2.2 mL, 16.7 mmol) in THF was added sodium

hydride (0.24 g, 6.0 mmol). The mixture was stirred at room temperature for 1 hour under

nitrogen after which propargyl bromide (0.6 mL, 6.0 mmol) was added. The mixture was stirred

at room temperature overnight, diluted with water (10 mL) and then neutralized with 0.1 M HCl

(15 mL). The resulting mixture was extracted with EtOAc (100 mL x 3) and the extract was

washed with brine (100 mL). The organic phase was dried over Na2SO4, filtered and evaporated

to dryness in vacuo. The residue was purified by flash column chromatography on silica gel

(hexanes/EtOAc, 1:1) to afford 11 as a colorless oil (0.46 g, 41.3%). 1H NMR (400 MHz,

CDCl3): δ (ppm) 4.25 (d, 2 H, J = 2.4 Hz, CHCCH2O), 3.68−3.63 (m, 11 H, OCH2 and OH) 3.59

(t, 2 H, J = 4.4 Hz, CH2OH), 2.41 (t, 1 H, J = 2.4 Hz, CHCCH2). 13C NMR (100 MHz, CDCl3): δ

(ppm) 79.3, 74.5, 72.1, 70.5, 70.3, 70.1, 69.0, 61.5, 58.2. HRMS (MALDI) m/z Found: 211.0959,

calculated: 211.0941 for C9H16NaO4 [M+Na]+.

Synthesis of 2-(2-(2-(Prop-2-ynyloxy)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate (12).

To a stirred solution of 11 (0.37 g, 2.0 mmol) in pyridine (10 mL) was added 4-toluenesufonyl

chloride (0.80 g, 4.0 mmol). The mixture was stirred vigorously at room temperature for 6 hours

under nitrogen. The mixture was then poured into ice water and extracted with CH2Cl2 (50 mL x

3). The combined organic phase was washed with brine, dried over Na2SO4, filtered and


silica gel (hexanes/EtOAc, 2:1) to afford 12 as white crystals (0.65 g, 93.7%). 1H NMR (400

MHz, CDCl3): δ (ppm) 7.83 (d, 2 H, J = 8.0 Hz, ArH), 7.36 (d, 2 H, J = 8.0 Hz, ArH), 4.21 (d, 2

H, J = 2.4 Hz, CHCCH2O), 4.17 (t, 2 H, J = 4.8 Hz, CH2OTs), 3.73−3.69 (m, 4 H, OCH2),

3.67−3.62 (m, 2 H, OCH2), 3.60 (s, 4 H, OCH2), 2.49 (s, 3 H, ArCH3), 2.45 (t, 1 H, J = 2.4 Hz,

CHCCH2). 13C NMR (100 MHz, CDCl3): δ (ppm) 145.0, 133.1, 130.0, 128.3, 79.8, 74.5, 70.8,

70.7, 70.6, 70.0, 69.3, 68.7, 58.6, 21.5. HRMS (MALDI) m/z Found: 365.1043, calculated:

365.1029 for C16H22NaO6S [M+Na]+.

Synthesis of 2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)ethyl ethanethioate (13).

To a stirred solution of 12 (340 mg, 1.0 mmol) in DMF (10 mL) was added KSAc (220 mg, 2.0

mmol). The mixture was then stirred at 60 ºC for 12 hours under nitrogen and the DMF was

removed under vacuum. The residue was dissolved in EtOAc (50 mL) and washed with water

(10 mL x 2) and brine (10 mL x 2). The organic phase was dried over Na2SO4, filtered and

evaporated to dryness in vacuo. The obtained residue was purified by flash column





aromatics); 7.81 (d, 2H, J = 8.0 Hz, aromatic); 7.62 (m, 1H, aromatics); 7.56 (m, 3H, aromatics);

4.93 (s, 2H, ArCH2); 4.31 (d, 2H, J = 2.3 Hz, CH2C); 3.54 (t, 1H, J = 2.3 Hz, CH). 13C NMR

(CDCl3, 100 MHz): δ (ppm) 134.1, 133.2, 132.5, 130.7, 130.1, 130.2, 127.9, 127.8, 127.8, 127.5,

127.3, 127.1 126.8, 124.0, 120.8, 120.7, 120.5, 120.2, 80.0, 77.5, 69.1, 57.1. HRMS (MALDI)

m/z Found: 343.1113, calculated: 343.1093 for C24H16NaO [M+Na]+.

Synthesis of alkyne functionalized IR786 (3) (Supplementary Figure S3)

Supplementary Figure S3. Synthesis of alkyne functionalized IR786 (3)

Synthesis of 2-(2-(2-(Prop-2-ynyloxy)ethoxy)ethoxy)ethanol (11).

To a stirred solution of triethylene glycol 10 (2.2 mL, 16.7 mmol) in THF was added sodium

hydride (0.24 g, 6.0 mmol). The mixture was stirred at room temperature for 1 hour under

nitrogen after which propargyl bromide (0.6 mL, 6.0 mmol) was added. The mixture was stirred

at room temperature overnight, diluted with water (10 mL) and then neutralized with 0.1 M HCl

(15 mL). The resulting mixture was extracted with EtOAc (100 mL x 3) and the extract was

washed with brine (100 mL). The organic phase was dried over Na2SO4, filtered and evaporated

to dryness in vacuo. The residue was purified by flash column chromatography on silica gel

(hexanes/EtOAc, 1:1) to afford 11 as a colorless oil (0.46 g, 41.3%). 1H NMR (400 MHz,

CDCl3): δ (ppm) 4.25 (d, 2 H, J = 2.4 Hz, CHCCH2O), 3.68−3.63 (m, 11 H, OCH2 and OH) 3.59

(t, 2 H, J = 4.4 Hz, CH2OH), 2.41 (t, 1 H, J = 2.4 Hz, CHCCH2). 13C NMR (100 MHz, CDCl3): δ

(ppm) 79.3, 74.5, 72.1, 70.5, 70.3, 70.1, 69.0, 61.5, 58.2. HRMS (MALDI) m/z Found: 211.0959,

calculated: 211.0941 for C9H16NaO4 [M+Na]+.

Synthesis of 2-(2-(2-(Prop-2-ynyloxy)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate (12).

To a stirred solution of 11 (0.37 g, 2.0 mmol) in pyridine (10 mL) was added 4-toluenesufonyl

chloride (0.80 g, 4.0 mmol). The mixture was stirred vigorously at room temperature for 6 hours

under nitrogen. The mixture was then poured into ice water and extracted with CH2Cl2 (50 mL x

3). The combined organic phase was washed with brine, dried over Na2SO4, filtered and


silica gel (hexanes/EtOAc, 2:1) to afford 12 as white crystals (0.65 g, 93.7%). 1H NMR (400

MHz, CDCl3): δ (ppm) 7.83 (d, 2 H, J = 8.0 Hz, ArH), 7.36 (d, 2 H, J = 8.0 Hz, ArH), 4.21 (d, 2

H, J = 2.4 Hz, CHCCH2O), 4.17 (t, 2 H, J = 4.8 Hz, CH2OTs), 3.73−3.69 (m, 4 H, OCH2),

3.67−3.62 (m, 2 H, OCH2), 3.60 (s, 4 H, OCH2), 2.49 (s, 3 H, ArCH3), 2.45 (t, 1 H, J = 2.4 Hz,

CHCCH2). 13C NMR (100 MHz, CDCl3): δ (ppm) 145.0, 133.1, 130.0, 128.3, 79.8, 74.5, 70.8,


365.1029 for C16H22NaO6S [M+Na]+.

Synthesis of 2-(2-(2-(prop-2-yn-1-yloxy)ethoxy)ethoxy)ethyl ethanethioate (13).

To a stirred solution of 12 (340 mg, 1.0 mmol) in DMF (10 mL) was added KSAc (220 mg, 2.0

mmol). The mixture was then stirred at 60 ºC for 12 hours under nitrogen and the DMF was



evaporated to dryness in vacuo. The obtained residue was purified by flash column





chromatography on silica gel (hexane/EtOAc, 3 : 1) to afford 13 (160 mg, 67.1 %). 1H NMR

(400 MHz, CDCl3) δ (ppm) 4.10 (d, 2H, J = 2.0 Hz, OCH2CCH), 3.59-3.51 (m, 8H, OCH2), 3.49

(t, 2H, J = 6.4 Hz, CH2O), 2.98 (t, 2H, J = 6.4 Hz, CH2S), 2.38 (t, 1H, J = 2.0 Hz, CCH), 2.23 (s,

3H, Ac). 13C NMR (100 MHz, CDCl3): δ (ppm) 195.3, 79.6, 74.5, 70.4, 70.3, 70.1, 69.6, 68.9,

58.2, 30.4, 28.7. HRMS (MALDI) m/z Found: 269.0833, calculated: 269.0818 for C11H18O4S

[M]+.

Synthesis of 1,3,3-trimethyl-2-(-2-(-2-((2-(2-(2-(prop-2-yn-1-oxy)ethoxy)ethoxy)ethyl)thio)-3-(-2-

(1,3,3-trimethylindolin-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-3H-indol-1-ium (3).

To a stirred solution of 13 (120 mg, 0.5 mmol) in CH3OH (5 mL) was added NaOH (40 mg 1.0

mmol). The mixture was stirred at room temperature for 2 hours under nitrogen and the solvent

was removed in vacuo. The residue thus obtained was dissolved in CH2Cl2 (10 mL) and mixed

with a 10 mL CH2Cl2 solution of IR786 perchlorate (290 mg, 0.5 mmol). The reaction mixture

was stirred at room temperature overnight under nitrogen, and diluted with CH2Cl2 (20 mL).

The mixture was washed with water (10 mL x 2) and brine (10 mL). The organic phase was


flash chromatography on silica gel (CH2Cl2/CH3OH, 20 : 1) to afford 3 as a solid ( 250 mg,

76.8%). 1H NMR (400 MHz, CDCl3): δ (ppm) 8.77 (d, 2 H, J = 14.0 Hz, ArH), 7.37-7.26 (m, 4

H, ArH), 7.21-7.14 (m, 4 H, ArH), 6.15 (d, 2 H, J = 14.0 Hz, ArH), 4.11 (d, 2H, J = 1.2 Hz,

OCH2-C), 3.68 (s, 6H, NCH3), 3.63-3.55 (m, 12H, OCH2CH2O), 2.93 (d, 2H, J = 6.8 Hz, SCH2),

2.62 (t, 4H, J = 6.0 Hz, C=CCH2), 2.36 (t, 1H, J = 1.2 Hz, Alkyne), 1.88 (m, 2H, CH2CH2CH2),

1.69 (s, 12H, CCH3). 13C NMR (100 MHz, CDCl3): δ (ppm) 172.6, 158.5, 145.8, 142.8, 140.7,

134.1, 128.7, 125.1, 122.1, 110.6, 101.4, 74.6, 70.5, 70.3, 70.2, 69.0, 58.3, 49.0, 32.4, 27.9, 26.5.

HRMS (MALDI) m/z Found: 651.3635, calculated: 651.3615 for C41H51N2 NaO3S [M+Na]+.

Synthesis of MDP-1 (Supplementary Figure S4)

Supplementary Figure S4. Synthesis of MDP-1

Synthesis of β-D-Glucopyranose,-2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl-(1→4)-O-2,3,6-tri-



triacetate 1-(3′-triazolepropyl perylene) (14).

To a stirred solution of 1 (38.0 mg, 0.02 mmol) and 2 (13.0 mg, 0.04 mmol) in DMF (5 mL) was

added CuI (0.2 mg, 1.0 µmol) and DIPEA (1.2 mg, 0.01 mmol). The mixture was stirred at room

temperature for 12 hours under nitrogen and the solvent was removed in vacuo. The residue was

dissolved in CH2Cl2 (20 mL) and washed with water (5 mL x 2) and brine (5 mL). The organic

phase was dried over Na2SO4, filtered and evaporated to dryness in vacuo. The residue was

purified by flash column chromatography on silica gel (CH2Cl2/CH3OH, 15/1) to afford 14 (35.0

mg, 79.5%). 1H NMR (400 MHz, CDCl3): δ (ppm) 8.21-8.14 (m, 4H, Aromatic), 8.09 (d, 1 H, J

= 8.8 Hz, Aromatic), 7.90 (d, 1 H, J = 8.8 Hz, Aromatic), 7.66(m, 2 H, Aromatic), 7.56-7.43 (m,

4H, aromatic), 5.43−5.26 (m, 11H), 5.03 (t, 1 H, J = 9.6 Hz, 4"-H), 4.97 (m, 2H, ArCH2O), 4.79





chromatography on silica gel (hexane/EtOAc, 3 : 1) to afford 13 (160 mg, 67.1 %). 1H NMR

(400 MHz, CDCl3) δ (ppm) 4.10 (d, 2H, J = 2.0 Hz, OCH2CCH), 3.59-3.51 (m, 8H, OCH2), 3.49

(t, 2H, J = 6.4 Hz, CH2O), 2.98 (t, 2H, J = 6.4 Hz, CH2S), 2.38 (t, 1H, J = 2.0 Hz, CCH), 2.23 (s,

3H, Ac). 13C NMR (100 MHz, CDCl3): δ (ppm) 195.3, 79.6, 74.5, 70.4, 70.3, 70.1, 69.6, 68.9,

58.2, 30.4, 28.7. HRMS (MALDI) m/z Found: 269.0833, calculated: 269.0818 for C11H18O4S

[M]+.

Synthesis of 1,3,3-trimethyl-2-(-2-(-2-((2-(2-(2-(prop-2-yn-1-oxy)ethoxy)ethoxy)ethyl)thio)-3-(-2-

(1,3,3-trimethylindolin-2-ylidene)ethylidene)cyclohex-1-en-1-yl)vinyl)-3H-indol-1-ium (3).

To a stirred solution of 13 (120 mg, 0.5 mmol) in CH3OH (5 mL) was added NaOH (40 mg 1.0

mmol). The mixture was stirred at room temperature for 2 hours under nitrogen and the solvent

was removed in vacuo. The residue thus obtained was dissolved in CH2Cl2 (10 mL) and mixed

with a 10 mL CH2Cl2 solution of IR786 perchlorate (290 mg, 0.5 mmol). The reaction mixture

was stirred at room temperature overnight under nitrogen, and diluted with CH2Cl2 (20 mL).

The mixture was washed with water (10 mL x 2) and brine (10 mL). The organic phase was


flash chromatography on silica gel (CH2Cl2/CH3OH, 20 : 1) to afford 3 as a solid ( 250 mg,

76.8%). 1H NMR (400 MHz, CDCl3): δ (ppm) 8.77 (d, 2 H, J = 14.0 Hz, ArH), 7.37-7.26 (m, 4

H, ArH), 7.21-7.14 (m, 4 H, ArH), 6.15 (d, 2 H, J = 14.0 Hz, ArH), 4.11 (d, 2H, J = 1.2 Hz,

OCH2-C), 3.68 (s, 6H, NCH3), 3.63-3.55 (m, 12H, OCH2CH2O), 2.93 (d, 2H, J = 6.8 Hz, SCH2),

2.62 (t, 4H, J = 6.0 Hz, C=CCH2), 2.36 (t, 1H, J = 1.2 Hz, Alkyne), 1.88 (m, 2H, CH2CH2CH2),


134.1, 128.7, 125.1, 122.1, 110.6, 101.4, 74.6, 70.5, 70.3, 70.2, 69.0, 58.3, 49.0, 32.4, 27.9, 26.5.

HRMS (MALDI) m/z Found: 651.3635, calculated: 651.3615 for C41H51N2 NaO3S [M+Na]+.



Synthesis of β-D-Glucopyranose,-2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl-(1→4)-O-2,3,6-tri-



triacetate 1-(3′-triazolepropyl perylene) (14).







mg, 79.5%). 1H NMR (400 MHz, CDCl3): δ (ppm) 8.21-8.14 (m, 4H, Aromatic), 8.09 (d, 1 H, J

= 8.8 Hz, Aromatic), 7.90 (d, 1 H, J = 8.8 Hz, Aromatic), 7.66(m, 2 H, Aromatic), 7.56-7.43 (m,

4H, aromatic), 5.43−5.26 (m, 11H), 5.03 (t, 1 H, J = 9.6 Hz, 4"-H), 4.97 (m, 2H, ArCH2O), 4.79





(dd, 1 H, J = 4.0 and 9.6 Hz, α 2'-H), 4.73 (s, 2H, CH2-C=C), 4.72-4.68 (m, 3H), 4.52-3.91 (m,

26 H), 3.75-3.52 (m, 2H, NCH2CH2CH2O), 3.38 (m, NCH2CH2CH2O), 2.22-1.96 (s, 57 H, CH3),

1.85 (m, 2H, NCH2CH2CH2O). 13C NMR (100 MHz, CDCl3): δ (ppm) 170.6, 170.5, 170.4,

170.3, 170.2, 170.0, 169.8, 169.7, 169.6, 169.5, 169.5, 169.4 (C=O), 145.2, 134.6, 133.1, 132.9,

131.5, 131.1, 130.9, 128.9, 128.3, 127.9, 127.8, 127.5, 126.8, 126.6, 126.5, 123.9, 123.8, 123.0,

122.8, 120.4, 120.3, 119.5, 100.2 (β 1-C), 95.9 (1-C), 95.7 (1-C), 95.6 (1-C), 76.5, 75.2, 73.8,

73.5, 73.3, 73.2, 72.6, 72.3, 72.1, 71.6, 71.6, 71.5, 71.2, 71.1, 70.5, 70.4, 70.0, 69.5, 69.3, 68.9,

68.4, 67.8, 67.7, 65.8, 64.7, 63.7, 62.9, 62.5, 62.3, 62.2, 62.1, 61.3, 53.7, 46.8, 31.7, 29.9, 29.2,

21.0, 20.8, 21.8, 21.6, 20.5. HRMS (MALDI) m/z Found: 2214.6877, calculated: 2214.6859 for

C101H121N3NaO51 [M+Na]+.

Synthesis of MDP-1

To a stirred solution of 14 (32.0 mg, 0.015 mmol) in CH3OH (2 mL) was added aqueous LiOH

(1.0 M, 2 mL) under nitrogen, and the reaction mixture was stirred at room temperature for 24

hours. The mixture was then neutralized with Dowex 50W resin, filtered and concentrated in

vacuo. The residue was purified by flash column chromatography on silica gel

(CH2Cl2/CH3OH/H2O, 5/5/2) to afford MDP-1 (20.8 mg, quantitative). 1H-NMR (400 MHz,

D2O): δ (ppm) 7.88-7.74, (m, 3H, Aromatic), 7.70 (d, 1H, J = 8.0 Hz, ArH), 7.50 (d, 1H, J = 8.0

Hz, ArH), 7.43 (s, 1H, triazole), 7.23 (d, 2H, J = 8.0 Hz, ArH),7.10-7.01 (m, 4 H, ArH), 5.42-

5.39 (m, 5H), 4.65 (m, 2H, ArCH2O), 4.51 (s, 2H, CH2-C=C), 4.46 (d, 1H, J = 8.4 Hz, 1-H'),

4.32 (t, J = 6.8 Hz, 2H), 4.19-4.10 (m, 3H), 4.05-3.45 (m, 45H), 3.41 (m, 1H), 3.35 (m, 1H),

2.23-1.85 (m, 4H). 13C NMR (100 MHz, D2O): δ (ppm) 143.2, 134.6, 133.0, 132.5, 131.7, 131.5,

130.9, 128.8, 128.3, 127.8, 127.6, 126.7, 126.5, 126.0, 123.9, 123.6, 120.3, 120.3, 120.2, 119.4,

103.5(β 1-C), 101.1(1-C), 100.7(1-C), 100.2(1-C), 77.9, 77.8, 75.5, 74.1, 73.7, 73.6, 72.7, 72.3,

72.3, 71.9, 70.0, 70.2, 68.3, 67.2, 62.7, 62.5, 62.2, 62.1, 61.8, 61.9, 58.6, 47.9, 32.4, 30.0, 28.7.

HRMS (MALDI) m/z Found: 1416.4868, calculated: 1416.4852 for C63H83N3NaO32 [M+Na]+.



Synthesis of β-D-Glucopyranose, 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl-(1→4)-O-2,3,6-tri-



triacetate 1-(3′- triazolepropyl IR786) (15).











(dd, 1 H, J = 4.0 and 9.6 Hz, α 2'-H), 4.73 (s, 2H, CH2-C=C), 4.72-4.68 (m, 3H), 4.52-3.91 (m,

26 H), 3.75-3.52 (m, 2H, NCH2CH2CH2O), 3.38 (m, NCH2CH2CH2O), 2.22-1.96 (s, 57 H, CH3),

1.85 (m, 2H, NCH2CH2CH2O). 13C NMR (100 MHz, CDCl3): δ (ppm) 170.6, 170.5, 170.4,

170.3, 170.2, 170.0, 169.8, 169.7, 169.6, 169.5, 169.5, 169.4 (C=O), 145.2, 134.6, 133.1, 132.9,

131.5, 131.1, 130.9, 128.9, 128.3, 127.9, 127.8, 127.5, 126.8, 126.6, 126.5, 123.9, 123.8, 123.0,

122.8, 120.4, 120.3, 119.5, 100.2 (β 1-C), 95.9 (1-C), 95.7 (1-C), 95.6 (1-C), 76.5, 75.2, 73.8,

73.5, 73.3, 73.2, 72.6, 72.3, 72.1, 71.6, 71.6, 71.5, 71.2, 71.1, 70.5, 70.4, 70.0, 69.5, 69.3, 68.9,

68.4, 67.8, 67.7, 65.8, 64.7, 63.7, 62.9, 62.5, 62.3, 62.2, 62.1, 61.3, 53.7, 46.8, 31.7, 29.9, 29.2,

21.0, 20.8, 21.8, 21.6, 20.5. HRMS (MALDI) m/z Found: 2214.6877, calculated: 2214.6859 for

C101H121N3NaO51 [M+Na]+.

Synthesis of MDP-1


(1.0 M, 2 mL) under nitrogen, and the reaction mixture was stirred at room temperature for 24

hours. The mixture was then neutralized with Dowex 50W resin, filtered and concentrated in

vacuo. The residue was purified by flash column chromatography on silica gel

(CH2Cl2/CH3OH/H2O, 5/5/2) to afford MDP-1 (20.8 mg, quantitative). 1H-NMR (400 MHz,

D2O): δ (ppm) 7.88-7.74, (m, 3H, Aromatic), 7.70 (d, 1H, J = 8.0 Hz, ArH), 7.50 (d, 1H, J = 8.0

Hz, ArH), 7.43 (s, 1H, triazole), 7.23 (d, 2H, J = 8.0 Hz, ArH),7.10-7.01 (m, 4 H, ArH), 5.42-

5.39 (m, 5H), 4.65 (m, 2H, ArCH2O), 4.51 (s, 2H, CH2-C=C), 4.46 (d, 1H, J = 8.4 Hz, 1-H'),

4.32 (t, J = 6.8 Hz, 2H), 4.19-4.10 (m, 3H), 4.05-3.45 (m, 45H), 3.41 (m, 1H), 3.35 (m, 1H),

2.23-1.85 (m, 4H). 13C NMR (100 MHz, D2O): δ (ppm) 143.2, 134.6, 133.0, 132.5, 131.7, 131.5,

130.9, 128.8, 128.3, 127.8, 127.6, 126.7, 126.5, 126.0, 123.9, 123.6, 120.3, 120.3, 120.2, 119.4,

103.5(β 1-C), 101.1(1-C), 100.7(1-C), 100.2(1-C), 77.9, 77.8, 75.5, 74.1, 73.7, 73.6, 72.7, 72.3,

72.3, 71.9, 70.0, 70.2, 68.3, 67.2, 62.7, 62.5, 62.2, 62.1, 61.8, 61.9, 58.6, 47.9, 32.4, 30.0, 28.7.

HRMS (MALDI) m/z Found: 1416.4868, calculated: 1416.4852 for C63H83N3NaO32 [M+Na]+.



Synthesis of β-D-Glucopyranose, 2,3,4,6-tetra-O-acetyl-α-D-glucopyranosyl-(1→4)-O-2,3,6-tri-



triacetate 1-(3′- triazolepropyl IR786) (15).











mg, 73.1%). 1H NMR (400 MHz, CDCl3): δ (ppm) 8.78 (d, 2 H, J = 14.0 Hz, ArH), 7.36-7.32

(m, 4 H, ArH), 7.21-6.97 (m, 5 H, ArH), 6.13 (d, 2 H, J = 14.0 Hz, ArH), 5.38−5.21 (m, 11H),

5.03 (t, 1 H, J = 10.0 Hz, 4"-H), 4.81 (dd, 1 H, J = 4.0 and 10.0 Hz, α 2'-H), 4.71-4.68 (m, 3H),

4.47-3.52 (m, 60 H), 2.93 (d, 2H, J = 6.8 Hz, SCH2), 2.59 (m, 4, C=CCH2), 2.16-1.86 (m, 61 H),


170.3, 170.0, 169.8, 169.7, 169.6, 169.5, 169.4, 169.4 (C=O), 157.3, 154.1, 145.8, 142.8, 142.4,

140.7, 134.1, 128.7, 127.7, 127.6, 125.0, 122.1, 114.7, 110.4, 101.3, 100.2, (β 1-C), 95.8 (1-C),

95.7 (1-C), 95.6 (1-C), 76.7, 75.2, 73.7, 73.2, 72.4, 72.3, 72.1, 71.7, 71.6, 71.3, 70.4, 70.3, 70.2,

70.1, 70.0, 69.6, 69.3, 68.9, 68.4, 67.9, 66.1, 62.7, 62.4, 62.3, 62.1, 61.7, 61.3, 49.0, 46.8, 41.4,

36.6, 31.8, 31.5, 31.0, 30.2, 29.6, 29.2, 27.9, 26.2, 24.3, 22.6, 20.9, 20.8, 20.7, 20.6, 20.5.

HRMS (MALDI) m/z Found: 2522.9401, calculated: 2522.9381 for C118H156N5O53S [M]+.

Synthesis of MDP-2


(1.0 M, 2 mL), and the reaction mixture was stirred at room temperature for 24 hours. The

mixture was then neutralized with Dowex 50W resin, filtered and concentrated in vacuo. The

residue was purified by flash column chromatography on silica gel (CH2Cl2/CH3OH/H2O, 5/5/3)

to afford MDP-2 (33.8 mg, quantitative). 1H NMR (400 MHz, D2O): δ (ppm) 8.72 (d, 2 H, J =

12.8 Hz, ArH), 7.58 (s, 1H, Triazole)7.33-7.29 (m, 4 H, ArH), 7.20-6.93 (m, 4 H, ArH), 6.11 (d,

2 H, J = 12.8 Hz, ArH), 5.43-5.40 (m, 5H), 4.48 (d, 1H, J = 8.4 Hz, 1-H'), 4.33 (t, J = 6.8 Hz,

2H), 4.17-4.09 (m, 3H), 4.05-3.45 (m, 60H), 3.41 (m, 1H), 3.35 (m, 1H), 2.88 (d, 2H, J = 6.0 Hz,

SCH2), 2.62 (m, 4H, C=CCH2), 2.23-1.88 (m, 4H), 1.71 (s, 12H, CCH3). 13C NMR (100 MHz,

D2O): δ (ppm) 171.8, 153.9, 145.7, 143.2, 142.1, 140.3, 133.9, 128.2, 127.3, 127.0, 124.8, 121.9,

114.0, 110.1, 103.2(β 1-C), 100.9, 100.7(1-C), 100.3(1-C), 100.2(1-C), 77.9, 77.8, 75.4, 74.0,

73.9, 73.4, 72.5, 72.4, 72.3, 71.9, 70.0, 70.2, 68.3, 67.2, 62.7, 62.5, 62.2, 62.1, 61.8, 61.9, 58.5,


1724.7373 for C80H118N5O34S+ [M]+.

Mass spectrometry analysis of MDP-1 and MDP-2 with NanoSpray ionization-linear ion

trap mass spectrometry (LTQ) (Supplementary Figure S6 and S7)

The chemical structure and synthesis of MDP-1 and MDP-2 were confirmed by LTQ. MDP-1

and MDP-2 were suspended in methanol/water (1:1, 0.5 mg/mL) and infused directly into the

LTQ instrument (LTQ, Thermo Finnigan) at a constant flow rate of 0.5 µL/min. The capillary

temperature was set at 210 ºC and MS analysis was performed in the positive ion mode. For

tandom mass spectrometry experiments, the collision energy was set to 35~45%, the m/z ranged

from 400 to 2000, and was scanned with 2.2 mass units per window.





mg, 73.1%). 1H NMR (400 MHz, CDCl3): δ (ppm) 8.78 (d, 2 H, J = 14.0 Hz, ArH), 7.36-7.32

(m, 4 H, ArH), 7.21-6.97 (m, 5 H, ArH), 6.13 (d, 2 H, J = 14.0 Hz, ArH), 5.38−5.21 (m, 11H),

5.03 (t, 1 H, J = 10.0 Hz, 4"-H), 4.81 (dd, 1 H, J = 4.0 and 10.0 Hz, α 2'-H), 4.71-4.68 (m, 3H),

4.47-3.52 (m, 60 H), 2.93 (d, 2H, J = 6.8 Hz, SCH2), 2.59 (m, 4, C=CCH2), 2.16-1.86 (m, 61 H),


170.3, 170.0, 169.8, 169.7, 169.6, 169.5, 169.4, 169.4 (C=O), 157.3, 154.1, 145.8, 142.8, 142.4,

140.7, 134.1, 128.7, 127.7, 127.6, 125.0, 122.1, 114.7, 110.4, 101.3, 100.2, (β 1-C), 95.8 (1-C),

95.7 (1-C), 95.6 (1-C), 76.7, 75.2, 73.7, 73.2, 72.4, 72.3, 72.1, 71.7, 71.6, 71.3, 70.4, 70.3, 70.2,

70.1, 70.0, 69.6, 69.3, 68.9, 68.4, 67.9, 66.1, 62.7, 62.4, 62.3, 62.1, 61.7, 61.3, 49.0, 46.8, 41.4,

36.6, 31.8, 31.5, 31.0, 30.2, 29.6, 29.2, 27.9, 26.2, 24.3, 22.6, 20.9, 20.8, 20.7, 20.6, 20.5.

HRMS (MALDI) m/z Found: 2522.9401, calculated: 2522.9381 for C118H156N5O53S [M]+.

Synthesis of MDP-2


(1.0 M, 2 mL), and the reaction mixture was stirred at room temperature for 24 hours. The

mixture was then neutralized with Dowex 50W resin, filtered and concentrated in vacuo. The

residue was purified by flash column chromatography on silica gel (CH2Cl2/CH3OH/H2O, 5/5/3)

to afford MDP-2 (33.8 mg, quantitative). 1H NMR (400 MHz, D2O): δ (ppm) 8.72 (d, 2 H, J =

12.8 Hz, ArH), 7.58 (s, 1H, Triazole)7.33-7.29 (m, 4 H, ArH), 7.20-6.93 (m, 4 H, ArH), 6.11 (d,

2 H, J = 12.8 Hz, ArH), 5.43-5.40 (m, 5H), 4.48 (d, 1H, J = 8.4 Hz, 1-H'), 4.33 (t, J = 6.8 Hz,

2H), 4.17-4.09 (m, 3H), 4.05-3.45 (m, 60H), 3.41 (m, 1H), 3.35 (m, 1H), 2.88 (d, 2H, J = 6.0 Hz,

SCH2), 2.62 (m, 4H, C=CCH2), 2.23-1.88 (m, 4H), 1.71 (s, 12H, CCH3). 13C NMR (100 MHz,

D2O): δ (ppm) 171.8, 153.9, 145.7, 143.2, 142.1, 140.3, 133.9, 128.2, 127.3, 127.0, 124.8, 121.9,

114.0, 110.1, 103.2(β 1-C), 100.9, 100.7(1-C), 100.3(1-C), 100.2(1-C), 77.9, 77.8, 75.4, 74.0,

73.9, 73.4, 72.5, 72.4, 72.3, 71.9, 70.0, 70.2, 68.3, 67.2, 62.7, 62.5, 62.2, 62.1, 61.8, 61.9, 58.5,


1724.7373 for C80H118N5O34S+ [M]+.

Mass spectrometry analysis of MDP-1 and MDP-2 with NanoSpray ionization-linear ion

trap mass spectrometry (LTQ) (Supplementary Figure S6 and S7)

The chemical structure and synthesis of MDP-1 and MDP-2 were confirmed by LTQ. MDP-1

and MDP-2 were suspended in methanol/water (1:1, 0.5 mg/mL) and infused directly into the

LTQ instrument (LTQ, Thermo Finnigan) at a constant flow rate of 0.5 µL/min. The capillary

temperature was set at 210 ºC and MS analysis was performed in the positive ion mode. For

tandom mass spectrometry experiments, the collision energy was set to 35~45%, the m/z ranged

from 400 to 2000, and was scanned with 2.2 mass units per window.





LTQ of MDP-1

Supplementary Figure S6. LTQ of MDP-1

The tandom mass spectrometry results of MDP-1 are shown in Figure S7 and confirm the

structure of MDP-1. The peaks labeled with asterisks in the full mass spectrum are fragments

from the MDP-1 molecule with an m/z of 1416 (single charge) and an m/z of 719 (double

charge). The MS/MS spectrum of m/z 1416 shows the peak ladder with differences of m/z of

162, which is caused by loss of glucose during the collisions. The MS/MS data of m/z 1416

show a glucose-loss ladder and the loss of other fragments such as N2 and perylene dye, which

matches with the predicted fragmentation pattern of MDP-1.





LTQ of MDP-1


The tandom mass spectrometry results of MDP-1 are shown in Figure S7 and confirm the

structure of MDP-1. The peaks labeled with asterisks in the full mass spectrum are fragments

from the MDP-1 molecule with an m/z of 1416 (single charge) and an m/z of 719 (double

charge). The MS/MS spectrum of m/z 1416 shows the peak ladder with differences of m/z of

162, which is caused by loss of glucose during the collisions. The MS/MS data of m/z 1416

show a glucose-loss ladder and the loss of other fragments such as N2 and perylene dye, which

matches with the predicted fragmentation pattern of MDP-1.





LTQ of MDP-2


The tandom mass spectrometry results of MDP-2 confirm the structure of MDP-2 (see Figure

S8). The major peaks in the full mass spectrum are m/z 874 (double charge) and m/z 1725

(single charge). The MS/MS data of m/z 1725 show a glucose-loss ladder and the loss of other

fragments such as N2 and IR786 dye, which matches with the predicted fragmentation pattern of

MDP-2.

Synthesis of PEG-IR (18) (Supplementary Figure S8)

Supplementary Figure S8. Synthesis of PEG-IR (18)

Synthesis of azidoPEG1000 (17).

To a stirred solution of PEG 1000 (16) (2 g, 2.0 mmol) in Py (10 mL) was added TsCl (0.4 g, 2.1

mmol). The mixture was stirred at room temperature for 12 hours under nitrogen and the Py was



evaporated to dryness in vacuo. The resulting residue was dissolved in DMF (15 mL), to which

NaN3 (16 mg, 4.0 mmol) was added under stirring. The reaction was kept at 80 ºC for 12 hours,

the solvent was removed in vacuo, and the resulting residue was dissolved in EtOAc (200 mL)

and was washed with water (10 mL x 2) and brine (10 mL). The organic phase was dried over





LTQ of MDP-2


The tandom mass spectrometry results of MDP-2 confirm the structure of MDP-2 (see Figure

S8). The major peaks in the full mass spectrum are m/z 874 (double charge) and m/z 1725

(single charge). The MS/MS data of m/z 1725 show a glucose-loss ladder and the loss of other

fragments such as N2 and IR786 dye, which matches with the predicted fragmentation pattern of

MDP-2.

Synthesis of PEG-IR (18) (Supplementary Figure S8)

Supplementary Figure S8. Synthesis of PEG-IR (18)

Synthesis of azidoPEG1000 (17).

To a stirred solution of PEG 1000 (16) (2 g, 2.0 mmol) in Py (10 mL) was added TsCl (0.4 g, 2.1

mmol). The mixture was stirred at room temperature for 12 hours under nitrogen and the Py was



evaporated to dryness in vacuo. The resulting residue was dissolved in DMF (15 mL), to which

NaN3 (16 mg, 4.0 mmol) was added under stirring. The reaction was kept at 80 ºC for 12 hours,

the solvent was removed in vacuo, and the resulting residue was dissolved in EtOAc (200 mL)

and was washed with water (10 mL x 2) and brine (10 mL). The organic phase was dried over





Na2SO4, filtered and evaporated to dryness in vacuo. The residue was purified by flash column

chromatography on silica gel (CH2Cl2/CH3OH, 15:1) to afford 17 (0.83 g, 41.7 %). 1H NMR

(400 MHz, CDCl3) δ (ppm) 3.65-3.53 (m, 88H, OCH2CH2O), 3.33 (t, 2H, J = 6.8 Hz, CH2N3),

2.85 (s, 1H, OH).

Synthesis of PEG-IR786 (18)

To a stirred solution of azidoPEG1000 17 (200 mg, 0.2 mmol) and 3 (130 mg, 0.2 mmol) in

DMF (10 mL) was added CuI (1.0 mg, 0.05 mmol) and DIPEA (12 mg, 0.1 mmol). The mixture

was stirred at room temperature for 12 hours under nitrogen and the solvent was removed in

vacuo. The residue was dissolved in CH2Cl2 (100 mL) and washed with water (10 mL x 2) and

brine (10 mL). The organic phase was dried over Na2SO4, filtered and evaporated to dryness in

vacuo. The residue was purified by flash column chromatography on silica gel (CH2Cl2/CH3OH,

13/1) to afford 18 (210 mg, 63.6%). 1H NMR (400 MHz, CDCl3): δ (ppm) 8.81 (d, 2 H, J =

14.0 Hz, ArH), 7.38-7.33 (m, 4 H, ArH), 7.25-7.01 (m, 5 H, ArH), 6.16 (d, 2 H, J = 14.0 Hz,

ArH), 3.66-3.52 (m, 88H, OCH2CH2O), 3.41 (t, 2H, J = 6.8 Hz, CH2N).

General method for measuring uptake of MDP-1 and MDP-2 in bacteria (Figure 3a, Figure

3c and Supplementary Figure S9)

The uptake of MDP-1 and MDP-2 was investigated in Escherichia coli (ATCC 33456),

Pseudomonas aeruginosa (ATCC 47085), Bacillus subtilis (ATCC 23059), Staphylococcus

aureus (ATCC 6538), metabolically inactive Escherichia coli (ATCC 33456), and two

Escherichia coli mutant strains, which contained either a LamB mutation (JW3992-1) or a MalE

mutation (TL212). Bacteria were cultured overnight in Luria-Bertani (LB) medium at 37 ºC

under 5% CO2 in an incubator shaker (InnovaTM 4230, New Brunswick Scientific, Edison, NJ),

set at 220 rpm. Bacteria (100 µL from the overnight culture) were re-suspended in 30 mL of

fresh LB medium and cultured in a 250mL flask, as described above, to an optical density of 0.5

at 600 nm. The bacterial culture solution was transferred into 6 well plates, with each well

containing 3 mL of bacteria, and 60 µL of either MDP-1 or MDP-2 stock solutions (1 mM in

PBS) were added, generating a 20 µM MDP concentration. The bacteria were incubated with the

MDPs for 1 hour at 37 ºC under 5% CO2 in an incubator shaker (InnovaTM 4230, New

Brunswick Scientific, Edison, NJ), set at 220 rpm. At this stage, a small aliquot of the bacterial

culture was plated to determine the CFUs/mL, following literature procedures1.

The bacteria were harvested by centrifuging the bacterial solutions at 10,000 rpm for 15

minutes in 15 mL centrifuge tubes, using a Microfuge® 18 centrifuge (Beckman Coulter, Brea,

CA). The resulting pellets were washed 3 times with 10 mL PBS by resuspending the pellets in

PBS and centrifuging. The washed bacterial pellets were transferred into a centrifuge tube (BD

Falcon Centrifuge Tube, BD Biosciences), placed in an ice bath and lysed by sonication for 20

seconds in 2 mL DI water. Sonication was performed with a Branson Sonifier S-250A (Branson

Ultrasonics Corporation, Danbury, CT), using a constant duty cycle at a 200 Watt output, 10

sonication cycles were performed. The bacterial supernatant (diluted in a 2 mL volume) was

isolated by centrifuging at 10,000 rpm for 10 minutes. The fluorescence intensity of the

supernatant was measured in a Shimadzu spectrofluorometer (RF 5301PC) and the fluorescence

intensity of control bacteria (no MDP treatment) was subtracted. The concentration of MDPs in

the supernatant was calculated based on a MDP calibration curve.

The intracellular MDP concentration in the bacteria was determined by estimating the total

bacterial volume, based on the published dimensions of the bacteria2-4. A representative

calculation is given below using Escherichia coli and MDP-1: After 1 hour of incubation with





Na2SO4, filtered and evaporated to dryness in vacuo. The residue was purified by flash column

chromatography on silica gel (CH2Cl2/CH3OH, 15:1) to afford 17 (0.83 g, 41.7 %). 1H NMR

(400 MHz, CDCl3) δ (ppm) 3.65-3.53 (m, 88H, OCH2CH2O), 3.33 (t, 2H, J = 6.8 Hz, CH2N3),

2.85 (s, 1H, OH).

Synthesis of PEG-IR786 (18)

To a stirred solution of azidoPEG1000 17 (200 mg, 0.2 mmol) and 3 (130 mg, 0.2 mmol) in

DMF (10 mL) was added CuI (1.0 mg, 0.05 mmol) and DIPEA (12 mg, 0.1 mmol). The mixture

was stirred at room temperature for 12 hours under nitrogen and the solvent was removed in

vacuo. The residue was dissolved in CH2Cl2 (100 mL) and washed with water (10 mL x 2) and

brine (10 mL). The organic phase was dried over Na2SO4, filtered and evaporated to dryness in

vacuo. The residue was purified by flash column chromatography on silica gel (CH2Cl2/CH3OH,

13/1) to afford 18 (210 mg, 63.6%). 1H NMR (400 MHz, CDCl3): δ (ppm) 8.81 (d, 2 H, J =

14.0 Hz, ArH), 7.38-7.33 (m, 4 H, ArH), 7.25-7.01 (m, 5 H, ArH), 6.16 (d, 2 H, J = 14.0 Hz,

ArH), 3.66-3.52 (m, 88H, OCH2CH2O), 3.41 (t, 2H, J = 6.8 Hz, CH2N).

General method for measuring uptake of MDP-1 and MDP-2 in bacteria (Figure 3a, Figure

3c and Supplementary Figure S9)

The uptake of MDP-1 and MDP-2 was investigated in Escherichia coli (ATCC 33456),

Pseudomonas aeruginosa (ATCC 47085), Bacillus subtilis (ATCC 23059), Staphylococcus

aureus (ATCC 6538), metabolically inactive Escherichia coli (ATCC 33456), and two

Escherichia coli mutant strains, which contained either a LamB mutation (JW3992-1) or a MalE

mutation (TL212). Bacteria were cultured overnight in Luria-Bertani (LB) medium at 37 ºC


set at 220 rpm. Bacteria (100 µL from the overnight culture) were re-suspended in 30 mL of

fresh LB medium and cultured in a 250mL flask, as described above, to an optical density of 0.5

at 600 nm. The bacterial culture solution was transferred into 6 well plates, with each well

containing 3 mL of bacteria, and 60 µL of either MDP-1 or MDP-2 stock solutions (1 mM in

PBS) were added, generating a 20 µM MDP concentration. The bacteria were incubated with the

MDPs for 1 hour at 37 ºC under 5% CO2 in an incubator shaker (InnovaTM 4230, New

Brunswick Scientific, Edison, NJ), set at 220 rpm. At this stage, a small aliquot of the bacterial

culture was plated to determine the CFUs/mL, following literature procedures1.

The bacteria were harvested by centrifuging the bacterial solutions at 10,000 rpm for 15

minutes in 15 mL centrifuge tubes, using a Microfuge® 18 centrifuge (Beckman Coulter, Brea,

CA). The resulting pellets were washed 3 times with 10 mL PBS by resuspending the pellets in

PBS and centrifuging. The washed bacterial pellets were transferred into a centrifuge tube (BD

Falcon Centrifuge Tube, BD Biosciences), placed in an ice bath and lysed by sonication for 20

seconds in 2 mL DI water. Sonication was performed with a Branson Sonifier S-250A (Branson

Ultrasonics Corporation, Danbury, CT), using a constant duty cycle at a 200 Watt output, 10

sonication cycles were performed. The bacterial supernatant (diluted in a 2 mL volume) was

isolated by centrifuging at 10,000 rpm for 10 minutes. The fluorescence intensity of the

supernatant was measured in a Shimadzu spectrofluorometer (RF 5301PC) and the fluorescence

intensity of control bacteria (no MDP treatment) was subtracted. The concentration of MDPs in

the supernatant was calculated based on a MDP calibration curve.

The intracellular MDP concentration in the bacteria was determined by estimating the total

bacterial volume, based on the published dimensions of the bacteria2-4. A representative

calculation is given below using Escherichia coli and MDP-1: After 1 hour of incubation with





MDP-1 in LB media, the CFU count of the Escherichia coli was 1.8 x108, and had an

approximate intracellular volume of 134 nanoliters, based on a cylindrical geometry (diameter of

0.7 µm and length of 2 µm). This 134 nanoliter bacterial intracellular volume was diluted into 2

mL DI water, and the MDP fluorescence at (Ex/Em = 410/450 nm) was 214 fluorescence units,

which gave an MDP-1 concentration of 352 nM, representing 704 picomoles of fluorescent dye

in the 2 mL volume. The intracellular concentration of MDP-1 in Escherichia coli was

calculated by dividing 704 picomoles into a 134 nanoliter volume, giving a 5.25 mM

intracellular MDP-1 concentration. The specificity of MDP-1 and MDP-2 for bacteria over

mammalian cells was determined in a similar manner as described above, except that the

fluorescence intensities were normalized to bacterial protein content, determined by the BCA

assay5, rather than the bacterial cell volume.

Preparation of metabolically inactive Escherichia coli

Metabolically inactive Escherichia coli were prepared by treating Escherichia coli (ATCC

33456) with sodium azide. Escherichia coli were cultured in 30 mL of LB to an OD600 = 0.5,

and 300 µL of sodium azide stock solution (1 M in PBS) was added, generating a 10 mM sodium

azide concentration. The Escherichia coli were incubated with the sodium azide for 1 hour at 37

ºC in an incubator shaker, and harvested by centrifuging. The resulting pellets were washed 3

times with 10 mL PBS by resuspending the pellets in PBS and centrifuging. The washed pellets

were re-suspended in 30 mL of fresh LB medium, and were used for in vitro and in vivo

experiments.

Supplementary Figure S9. MDP-2 has high specificity for bacteria over mammalian cells.

Bacteria (EC, PA, BS and SA) and mammalian cells (RASMs, MAs and FBs) were incubated

with 20 µM MDP-2 for 1 hour and the intracellular MDP-2 concentration was determined by

IR786 fluorescence, and normalized to protein content (as described in Figure 3c). Bacteria

transport MDP-2 at a rate three orders of magnitude faster than mammalian cells. The results are

expressed as mean micromoles per gram of protein ± s.e.m. for n = 6 per group. The p values

between each group of bacteria and each group of mammalian cells were determined by a one-

way analysis of variance (ANOVA) using Bonferroni’s post hoc test, and were found to be

statistically significant (p ≤ 0.001).

Km and Vmax of MDP-1 and MDP-2 (Figure 3b and Supplementary Figure S10)

The Km and Vmax of MDP-1 and MDP-2 were determined in Escherichia coli (ATCC 33456)

using the Lineweaver-Burk method. Escherichia coli were cultured overnight in LB medium at

37 ºC under 5% CO2 in an incubator shaker (InnovaTM 4230, New Brunswick Scientific, Edison,

NJ), set at 220 rpm. Escherichia coli (100 µL from the overnight culture) were re-suspended in

30 mL of fresh LB medium and cultured in a 250 mL flask to an OD600 = 0.5, as described above,





MDP-1 in LB media, the CFU count of the Escherichia coli was 1.8 x108, and had an

approximate intracellular volume of 134 nanoliters, based on a cylindrical geometry (diameter of

0.7 µm and length of 2 µm). This 134 nanoliter bacterial intracellular volume was diluted into 2

mL DI water, and the MDP fluorescence at (Ex/Em = 410/450 nm) was 214 fluorescence units,

which gave an MDP-1 concentration of 352 nM, representing 704 picomoles of fluorescent dye

in the 2 mL volume. The intracellular concentration of MDP-1 in Escherichia coli was

calculated by dividing 704 picomoles into a 134 nanoliter volume, giving a 5.25 mM

intracellular MDP-1 concentration. The specificity of MDP-1 and MDP-2 for bacteria over

mammalian cells was determined in a similar manner as described above, except that the

fluorescence intensities were normalized to bacterial protein content, determined by the BCA

assay5, rather than the bacterial cell volume.

Preparation of metabolically inactive Escherichia coli

Metabolically inactive Escherichia coli were prepared by treating Escherichia coli (ATCC

33456) with sodium azide. Escherichia coli were cultured in 30 mL of LB to an OD600 = 0.5,

and 300 µL of sodium azide stock solution (1 M in PBS) was added, generating a 10 mM sodium

azide concentration. The Escherichia coli were incubated with the sodium azide for 1 hour at 37

ºC in an incubator shaker, and harvested by centrifuging. The resulting pellets were washed 3

times with 10 mL PBS by resuspending the pellets in PBS and centrifuging. The washed pellets

were re-suspended in 30 mL of fresh LB medium, and were used for in vitro and in vivo

experiments.

Supplementary Figure S9. MDP-2 has high specificity for bacteria over mammalian cells.

Bacteria (EC, PA, BS and SA) and mammalian cells (RASMs, MAs and FBs) were incubated

with 20 µM MDP-2 for 1 hour and the intracellular MDP-2 concentration was determined by

IR786 fluorescence, and normalized to protein content (as described in Figure 3c). Bacteria

transport MDP-2 at a rate three orders of magnitude faster than mammalian cells. The results are

expressed as mean micromoles per gram of protein ± s.e.m. for n = 6 per group. The p values

between each group of bacteria and each group of mammalian cells were determined by a one-

way analysis of variance (ANOVA) using Bonferroni’s post hoc test, and were found to be

statistically significant (p ≤ 0.001).

Km and Vmax of MDP-1 and MDP-2 (Figure 3b and Supplementary Figure S10)

The Km and Vmax of MDP-1 and MDP-2 were determined in Escherichia coli (ATCC 33456)

using the Lineweaver-Burk method. Escherichia coli were cultured overnight in LB medium at

37 ºC under 5% CO2 in an incubator shaker (InnovaTM 4230, New Brunswick Scientific, Edison,

NJ), set at 220 rpm. Escherichia coli (100 µL from the overnight culture) were re-suspended in

30 mL of fresh LB medium and cultured in a 250 mL flask to an OD600 = 0.5, as described above,





and stored on ice until use. The bacterial culture solution (3 mL) was transferred into 6 well

plates and preincubated at 37 ºC for 5 minutes under shaking (as described above). 60 µL of

either MDP-1 or MDP-2 stock solutions (0.005, 0.025, 0.05, 0.25, 0.5, 1.0, 2.5, 5.0, 10, 20, 40,

and 80 mM in PBS) were added to the Escherichia coli suspension, generating MDP solutions at

twelve different concentrations (0.1, 0.5, 1, 5, 10, 20, 50, 100, 200, 400, 800 and 1600 µM). The

Escherichia coli were incubated at 37 ºC under 5% CO2, and at various time points (1, 2, 5, 10,

20 and 60 min) 500 µL aliquots of the bacterial solutions were rapidly filtered through 0.45-µm-

pore-size nitrocellulose filters (diameter, 25 mm; Schleicher & Schuell GmbH, Dassel,

Germany). The filtered bacteria were recovered and washed three times with 5 mL PBS buffer,

via centrifugation, and the intracellular MDP concentration was determined as described above.

Initial velocities Vo were determined by plotting the intracellular MDP concentration verse time

at a fixed extracellular concentration. Vo values were calculated at MDP concentrations of 0.1,

0.5, 1, 5, 10, 20, 50, 100, 200, 400, 800 and 1600 µM. Km and Vmax were determined by plotting

,

where [S] is the extracellular MDP concentration.

Supplementary Figure S10. The uptake of MDP-2 in Escherichia coli is saturable and follows

Michaelis-Menten kinetics, with a Vmax of 2.6 nmol/min/109 cell and a Km of 1.2 µM. The results

are expressed as mean nanomoles per min per 109 cells ± s.e.m. for n = 6 per group.

Uptake of MDP-1 in anaerobic growth conditions (Supplementary Figure S11)

The ability of Escherichia coli (ATCC 33456) to internalize MDP-1 was determined under

anaerobic conditions. Oxygen free fresh LB media was prepared by bubbling sterile high purity

nitrogen at approximately 50 mL/min into LB media for 20 minutes. Bacteria were cultured to

an OD600 = 0.5 as described above (in aerobic conditions). 3 mL of this bacterial solution was

centrifuged and resuspended in 3 mL of the nitrogen bubbled LB medium, and transferred to 15

mL BD Falcon centrifuge tubes. The Falcon tubes containing bacteria were infused with

nitrogen gas and 60 µL of a MDP-1 stock solution (1 mM in PBS) was added, generating a 20

µM MDP-1 concentration. The Falcon tubes were tightly sealed and the bacteria were incubated

with MDP-1 for 1 hour at 37 ºC under nitrogen in an incubator shaker (InnovaTM 4230, New

Brunswick Scientific, Edison, NJ). The bacteria were harvested and the intracellular MDP-1





and stored on ice until use. The bacterial culture solution (3 mL) was transferred into 6 well

plates and preincubated at 37 ºC for 5 minutes under shaking (as described above). 60 µL of

either MDP-1 or MDP-2 stock solutions (0.005, 0.025, 0.05, 0.25, 0.5, 1.0, 2.5, 5.0, 10, 20, 40,

and 80 mM in PBS) were added to the Escherichia coli suspension, generating MDP solutions at

twelve different concentrations (0.1, 0.5, 1, 5, 10, 20, 50, 100, 200, 400, 800 and 1600 µM). The

Escherichia coli were incubated at 37 ºC under 5% CO2, and at various time points (1, 2, 5, 10,

20 and 60 min) 500 µL aliquots of the bacterial solutions were rapidly filtered through 0.45-µm-

pore-size nitrocellulose filters (diameter, 25 mm; Schleicher & Schuell GmbH, Dassel,

Germany). The filtered bacteria were recovered and washed three times with 5 mL PBS buffer,

via centrifugation, and the intracellular MDP concentration was determined as described above.

Initial velocities Vo were determined by plotting the intracellular MDP concentration verse time

at a fixed extracellular concentration. Vo values were calculated at MDP concentrations of 0.1,

0.5, 1, 5, 10, 20, 50, 100, 200, 400, 800 and 1600 µM. Km and Vmax were determined by plotting

,

where [S] is the extracellular MDP concentration.

Supplementary Figure S10. The uptake of MDP-2 in Escherichia coli is saturable and follows

Michaelis-Menten kinetics, with a Vmax of 2.6 nmol/min/109 cell and a Km of 1.2 µM. The results

are expressed as mean nanomoles per min per 109 cells ± s.e.m. for n = 6 per group.

Uptake of MDP-1 in anaerobic growth conditions (Supplementary Figure S11)

The ability of Escherichia coli (ATCC 33456) to internalize MDP-1 was determined under

anaerobic conditions. Oxygen free fresh LB media was prepared by bubbling sterile high purity

nitrogen at approximately 50 mL/min into LB media for 20 minutes. Bacteria were cultured to

an OD600 = 0.5 as described above (in aerobic conditions). 3 mL of this bacterial solution was

centrifuged and resuspended in 3 mL of the nitrogen bubbled LB medium, and transferred to 15

mL BD Falcon centrifuge tubes. The Falcon tubes containing bacteria were infused with

nitrogen gas and 60 µL of a MDP-1 stock solution (1 mM in PBS) was added, generating a 20

µM MDP-1 concentration. The Falcon tubes were tightly sealed and the bacteria were incubated

with MDP-1 for 1 hour at 37 ºC under nitrogen in an incubator shaker (InnovaTM 4230, New

Brunswick Scientific, Edison, NJ). The bacteria were harvested and the intracellular MDP-1





concentration was determined as described above for aerobic growth conditions. The results are

presented in Figure S11 and demonstrate that bacteria internalize MDP-1 under anaerobic growth

conditions.

Supplementary Figure S11. The maltodextrin transport pathway is functional under anaerobic

fermentative growth conditions. Escherichia coli were incubated with 20 µM MDP-1 for 1 hour

under either aerobic or anaerobic fermentative growth conditions and the intracellular MDP-1

concentration was determined. Escherichia coli internalized MDP-1 under both aerobic and

anaerobic fermentative growth conditions. Results are expressed as mean millimolar

concentration per CFU ± standard error of the mean (s.e.m.), for n = 6 per group.

Competitive inhibition of MDP-1 transport in Escherichia coli by maltose and

maltohexaose (Supplementary Figure S12)

The ability of maltose and maltohexaose to inhibit the uptake of MDP-1 in Escherichia coli was

investigated. Escherichia coli (ATCC 33456) were grown in LB medium at 37◦C under 5% CO2

to an OD600 = 0.5, as described above. The bacterial suspensions (3 mL) were transferred to 6

well plates, preincubated at 37 ºC for 5 minutes under shaking (as described above), and 60 µL of

various concentrations of either maltose or maltohexaose stock solutions (10, 100 and 1000 mM

in PBS) were added, generating a 200 µM, 2 mM and 20 mM final concentration of maltose or

maltohexaose. The bacteria were incubated with maltose or maltohexaose for 5 minutes and 60

µL of MDP-1 stock solutions (1 mM in PBS) were added, generating a 20 µM MDP-1

concentration. The bacteria were incubated at 37 ºC under 5% CO2 for 1 hour and the bacterial

suspensions were rapidly filtered through 0.45-µm-pore-size nitrocellulose filters (diameter, 25

mm; Schleicher & Schuell GmbH, Dassel, Germany). The filtered bacteria were recovered and

washed three times with 5 mL PBS buffer, via centrifugation (as described above). The

intracellular fluorescence of the bacteria was determined as described in Figure 3a. The results

are presented in Supplementary Figure S12 and demonstrate that the uptake of MDP-1 in

Escherichia coli can be inhibited by an excess of maltose or maltohexaose, confirming that

MDP-1 is internalized by maltodextrin transporters.

Supplementary Figure S12. Competitive inhibition of MDP-1 transport by maltose and

maltohexaose.





concentration was determined as described above for aerobic growth conditions. The results are

presented in Figure S11 and demonstrate that bacteria internalize MDP-1 under anaerobic growth

conditions.

Supplementary Figure S11. The maltodextrin transport pathway is functional under anaerobic

fermentative growth conditions. Escherichia coli were incubated with 20 µM MDP-1 for 1 hour

under either aerobic or anaerobic fermentative growth conditions and the intracellular MDP-1

concentration was determined. Escherichia coli internalized MDP-1 under both aerobic and

anaerobic fermentative growth conditions. Results are expressed as mean millimolar

concentration per CFU ± standard error of the mean (s.e.m.), for n = 6 per group.

Competitive inhibition of MDP-1 transport in Escherichia coli by maltose and

maltohexaose (Supplementary Figure S12)

The ability of maltose and maltohexaose to inhibit the uptake of MDP-1 in Escherichia coli was

investigated. Escherichia coli (ATCC 33456) were grown in LB medium at 37◦C under 5% CO2

to an OD600 = 0.5, as described above. The bacterial suspensions (3 mL) were transferred to 6

well plates, preincubated at 37 ºC for 5 minutes under shaking (as described above), and 60 µL of

various concentrations of either maltose or maltohexaose stock solutions (10, 100 and 1000 mM

in PBS) were added, generating a 200 µM, 2 mM and 20 mM final concentration of maltose or

maltohexaose. The bacteria were incubated with maltose or maltohexaose for 5 minutes and 60

µL of MDP-1 stock solutions (1 mM in PBS) were added, generating a 20 µM MDP-1

concentration. The bacteria were incubated at 37 ºC under 5% CO2 for 1 hour and the bacterial

suspensions were rapidly filtered through 0.45-µm-pore-size nitrocellulose filters (diameter, 25

mm; Schleicher & Schuell GmbH, Dassel, Germany). The filtered bacteria were recovered and

washed three times with 5 mL PBS buffer, via centrifugation (as described above). The

intracellular fluorescence of the bacteria was determined as described in Figure 3a. The results

are presented in Supplementary Figure S12 and demonstrate that the uptake of MDP-1 in

Escherichia coli can be inhibited by an excess of maltose or maltohexaose, confirming that

MDP-1 is internalized by maltodextrin transporters.

Supplementary Figure S12. Competitive inhibition of MDP-1 transport by maltose and

maltohexaose.





The uptake of MDP-1 in the presence of antibiotics (Supplementary Figure S13)

The ability of antibiotics to inhibit the uptake of MDP-1 in Escherichia coli was investigated.

Escherichia coli (ATCC 33456) were grown in LB medium at 37 ºC under 5% CO2 to an OD600

= 0.5, as described above. 3 mL of this bacterial suspension was transferred to 6 well plates,

preincubated at 37 ºC for 5 minutes under shaking (as described above), and 30 µL of various

concentarions of ampicillin stock solutions (0.3, 0.6, 1.2, and 1.8 mg/mL in PBS) were added,

generating a 3 (IC50), 6, 12 and 18 µg/mL final concentration of ampicillin. Escherichia coli

were incubated with ampicillin for 1 hour at 37 ºC under 5% CO2, and 60 µL of MDP-1 stock

solutions (1 mM in PBS) were added, generating a 20 µM MDP concentration. Escherichia coli

were incubated with MDPs in the presence of ampicillin for 1 hour at 37 ºC under 5% CO2 in an

incubator shaker. At this stage, a small aliquot of the bacterial culture was plated to determine

the CFUs of bacteria in the MDP solution. Escherichia coli were harvested by centrifuging and

the resulting pellets were washed 3 times with 10 mL PBS. The intracellular fluorescence of the

bacteria was determined as described in Figure 3a. The results are presented in Supplementary

Figure S13 and demonstrate that the maltodextrin transporter in Escherichia coli is still active in

the presence of antibiotics.

Supplementary Figure S13. Escherichia coli internalize MDP-1 in the presence of ampicillin.

The uptake of MDP-1 in biofilms (Figure 3d)

Biofilm preparation

Biofilms of Escherichia coli (ATCC 33456), Pseudomonas aeruginosa (ATCC 47085), Bacillus

subtilis (ATCC 23059), and Staphylococcus aureus (ATCC 6538), were cultivated using

parallel-plate flow cells according to the procedure described in Gilbert et al. 20046. Bench scale

parallel-plate flow cells were constructed to the dimensions of a standard microscope slide, with

two channels for fluid to enter and exit. A 25.4-mm piece of stainless steel tubing with a 1.0 mm

outer diameter was inserted into the entrance and exit of the parallel-plate flow cell. To allow

bacteria to colonize on the surface of the slide, Escherichia coli, Pseudomonas aeruginosa,

Bacillus subtilis, or Staphylococcus aureus (OD = 0.08) in 50 mM potassium phosphate buffer

(pH = 7.2) were recirculated through the parallel-plate flow cell at 0.84 mL/min for 2 hours. The

circulation system was then switched to a continuous-flow of fresh LB medium for 20 ± 1 hours





The uptake of MDP-1 in the presence of antibiotics (Supplementary Figure S13)

The ability of antibiotics to inhibit the uptake of MDP-1 in Escherichia coli was investigated.

Escherichia coli (ATCC 33456) were grown in LB medium at 37 ºC under 5% CO2 to an OD600

= 0.5, as described above. 3 mL of this bacterial suspension was transferred to 6 well plates,

preincubated at 37 ºC for 5 minutes under shaking (as described above), and 30 µL of various

concentarions of ampicillin stock solutions (0.3, 0.6, 1.2, and 1.8 mg/mL in PBS) were added,

generating a 3 (IC50), 6, 12 and 18 µg/mL final concentration of ampicillin. Escherichia coli

were incubated with ampicillin for 1 hour at 37 ºC under 5% CO2, and 60 µL of MDP-1 stock

solutions (1 mM in PBS) were added, generating a 20 µM MDP concentration. Escherichia coli

were incubated with MDPs in the presence of ampicillin for 1 hour at 37 ºC under 5% CO2 in an

incubator shaker. At this stage, a small aliquot of the bacterial culture was plated to determine

the CFUs of bacteria in the MDP solution. Escherichia coli were harvested by centrifuging and

the resulting pellets were washed 3 times with 10 mL PBS. The intracellular fluorescence of the

bacteria was determined as described in Figure 3a. The results are presented in Supplementary

Figure S13 and demonstrate that the maltodextrin transporter in Escherichia coli is still active in

the presence of antibiotics.

Supplementary Figure S13. Escherichia coli internalize MDP-1 in the presence of ampicillin.

The uptake of MDP-1 in biofilms (Figure 3d)

Biofilm preparation

Biofilms of Escherichia coli (ATCC 33456), Pseudomonas aeruginosa (ATCC 47085), Bacillus

subtilis (ATCC 23059), and Staphylococcus aureus (ATCC 6538), were cultivated using

parallel-plate flow cells according to the procedure described in Gilbert et al. 20046. Bench scale

parallel-plate flow cells were constructed to the dimensions of a standard microscope slide, with

two channels for fluid to enter and exit. A 25.4-mm piece of stainless steel tubing with a 1.0 mm

outer diameter was inserted into the entrance and exit of the parallel-plate flow cell. To allow

bacteria to colonize on the surface of the slide, Escherichia coli, Pseudomonas aeruginosa,

Bacillus subtilis, or Staphylococcus aureus (OD = 0.08) in 50 mM potassium phosphate buffer

(pH = 7.2) were recirculated through the parallel-plate flow cell at 0.84 mL/min for 2 hours. The

circulation system was then switched to a continuous-flow of fresh LB medium for 20 ± 1 hours





(medium flow rate of 0.35 mL/min, corresponding to 1 flow cell volume per minute) to provide

nutrients for biofilm growth and remove unattached bacteria.

Confocal laser scanning microscopy of MDP-1 treated biofilms

The uptake of MDP-1 in biofilms was investigated. Biofilms (in flow cells) were rinsed with

sterile 50 mM potassium phosphate buffer for 10 min, and were exposed to MDP-1 via

continuous pumping of a 20 µM MDP-1 (PBS) solution through the biofilms for 5 minutes at a

flow rate of 0.35 mL/min. The pump was then turned off and the bacteria were incubated with

the 20 µM MDP-1 (PBS) solution for 10 minutes. The biofilms were subsequently rinsed with

sterile 50 mM potassium phosphate buffer for 10 minutes at a flow rate of 0.35 mL/min. The

biofilms were counterstained with 20 µM SYTO 59 (Invitrogen) for 10 minutes and rinsed with

10 flow-cell volumes of sterile 50 mM potassium phosphate buffer. The stained biofilms were

imaged nondestructively using a Zeiss LSM 510 confocal laser scanning microscope (CLSM)

equipped with a Fluor 40 x oil immersion lens. Samples were excited simultaneously at

wavelengths of 430 nm and 622 nm. Biofilms were imaged with a 1-µm z-step increment. The

thickness of the biofilms was determined from the 3D confocal biofilm images.

In vivo imaging of bacterial infections with MDP-2 (Figure 4, Figure 5 and Supplementary

Figure S14)

In vivo imaging of bacteria

Female Wistar rats (10 weeks, 200-250 g, Harlan Laboratories, Inc) were anaesthetized with

isofluorane and the hair on the thigh and back were removed. A suspension of Escherichia coli

(105-107 CFUs) was injected into the left rear thigh muscle (injection depth 5 mm in 250 µL

saline), and 250 µL of saline, LamB mutants (107 CFUs), metabolically inactive bacteria or LPS

(1mg/kg) were injected into the right rear thigh muscle as a control (injection depth 5 mm in 250

µL saline). After 1 hour the rats were injected with MDP-2 (280-350 µL of 1 mM MDP-2 in

PBS) via the jugular vein. Fluorescence images were captured using an IVIS® Lumina Imaging

System (Caliper Life Sciences Inc) 16 hours after the MDP-2 injection. The fluorescence

intensity from the area around the bacterial or saline injection site (region of interest) was

integrated. Six rats were used for each experimental group. See Figure S14 for imaging of 106

Escherichia coli CFUs.

Specificity of MDP-2 for bacteria



(107 CFUs) in 250 µL saline was injected into the left rear thigh muscle (injection depth 5 mm),

and either LPS (1 mg/kg) or a suspension of NaN3 treated Escherichia coli (107 CFUs) in 250 µL

saline was injected into the right rear thigh muscle (injection depth 5 mm). After 1 hour the rats

were injected with MDP-2 (280-350 µL of 1 mM MDP-2 in PBS) via the jugular vein.

Fluorescence images were captured using an IVIS® Lumina Imaging System (Caliper Life

Sciences Inc) 16 hours after the MDP-2 injection. The fluorescence intensity from the bacteria

or LPS injection area (region of interest) was integrated. Six rats were used for each

experimental group.

Histology of bacterial infected muscles

At the end of the imaging procedure rats were sacrificed by CO2 inhalation, and the bacterial

infected muscle and the saline treated muscle were harvested. Tissues were cut to 3 x 4 x 5 mm

pieces, fixed in PBS containing 4% formaldehyde, washed in PBS, embedded in OCT compound

(Tissue Tek, Miles, Elkhart, IN), rapidly frozen in liquid nitrogen, and kept at -80 ºC until





(medium flow rate of 0.35 mL/min, corresponding to 1 flow cell volume per minute) to provide

nutrients for biofilm growth and remove unattached bacteria.

Confocal laser scanning microscopy of MDP-1 treated biofilms

The uptake of MDP-1 in biofilms was investigated. Biofilms (in flow cells) were rinsed with

sterile 50 mM potassium phosphate buffer for 10 min, and were exposed to MDP-1 via

continuous pumping of a 20 µM MDP-1 (PBS) solution through the biofilms for 5 minutes at a

flow rate of 0.35 mL/min. The pump was then turned off and the bacteria were incubated with

the 20 µM MDP-1 (PBS) solution for 10 minutes. The biofilms were subsequently rinsed with

sterile 50 mM potassium phosphate buffer for 10 minutes at a flow rate of 0.35 mL/min. The

biofilms were counterstained with 20 µM SYTO 59 (Invitrogen) for 10 minutes and rinsed with

10 flow-cell volumes of sterile 50 mM potassium phosphate buffer. The stained biofilms were

imaged nondestructively using a Zeiss LSM 510 confocal laser scanning microscope (CLSM)

equipped with a Fluor 40 x oil immersion lens. Samples were excited simultaneously at

wavelengths of 430 nm and 622 nm. Biofilms were imaged with a 1-µm z-step increment. The

thickness of the biofilms was determined from the 3D confocal biofilm images.

In vivo imaging of bacterial infections with MDP-2 (Figure 4, Figure 5 and Supplementary

Figure S14)

In vivo imaging of bacteria



(105-107 CFUs) was injected into the left rear thigh muscle (injection depth 5 mm in 250 µL

saline), and 250 µL of saline, LamB mutants (107 CFUs), metabolically inactive bacteria or LPS

(1mg/kg) were injected into the right rear thigh muscle as a control (injection depth 5 mm in 250

µL saline). After 1 hour the rats were injected with MDP-2 (280-350 µL of 1 mM MDP-2 in

PBS) via the jugular vein. Fluorescence images were captured using an IVIS® Lumina Imaging

System (Caliper Life Sciences Inc) 16 hours after the MDP-2 injection. The fluorescence

intensity from the area around the bacterial or saline injection site (region of interest) was

integrated. Six rats were used for each experimental group. See Figure S14 for imaging of 106

Escherichia coli CFUs.

Specificity of MDP-2 for bacteria



(107 CFUs) in 250 µL saline was injected into the left rear thigh muscle (injection depth 5 mm),

and either LPS (1 mg/kg) or a suspension of NaN3 treated Escherichia coli (107 CFUs) in 250 µL

saline was injected into the right rear thigh muscle (injection depth 5 mm). After 1 hour the rats

were injected with MDP-2 (280-350 µL of 1 mM MDP-2 in PBS) via the jugular vein.

Fluorescence images were captured using an IVIS® Lumina Imaging System (Caliper Life

Sciences Inc) 16 hours after the MDP-2 injection. The fluorescence intensity from the bacteria

or LPS injection area (region of interest) was integrated. Six rats were used for each

experimental group.

Histology of bacterial infected muscles


infected muscle and the saline treated muscle were harvested. Tissues were cut to 3 x 4 x 5 mm







examined. Tissues were cut with a rotary microtome (AO 820 microtome; American Optical,

Buffalo, NY) at an 8 µm thickness and mounted on poly-L-lysine-coated glass slides. Randomly

selected sections were stained with BD™ Gram Stain Kits to detect the presence of bacteria7 or

hematoxylin and eosin (H&E) to detect neutrophils and macrophages8-9.

Histology of LPS treated muscles


infected muscle and the LPS treated muscle were harvested. Tissues were cut to 3 x 4 x 5 mm





selected sections were stained with hematoxylin and eosin (H&E) to detect neutrophils and

macrophages, following standard H&E staining procedures8-9.

Supplementary Figure S14. MDP-2 can image bacterial infections and can detect 106 CFUs in

vivo.

a1, Escherichia coli (106 CFUs) were injected into the left thigh muscle of rats, and the right

thigh muscle was injected with saline as a control. After 1 hour, MDP-2 (280-350 µL of 1 mM

MDP-2 in PBS) was injected into the rats via the jugular vein, and the rats were imaged after 16

hours in an IVIS Lumina imaging machine. The rat image is a representative result of six

experiments, and identifies the infection site.

a2, Escherichia coli (106 CFUs) infected muscles have a 9 fold increase in fluorescence over un-

infected control muscles. Regions of interest in the infected and control muscles were identified

and integrated using software from the Lumina machine.

The results in a2 are expressed as mean numbers of photons per second per cm2 in the designated

ROI ± s.e.m. for n = 6 per group. The statistical significances in a2 were determined using a

two-sample Student t-test (***p ≤ 0.001).

Biodistribution of MDP-2 in infected rats (Figure 4b and Supplementary Figure S15)

Female Wistar rats (10 weeks, 200-250 g, Harlan Laboratories, Inc) were given thigh infections

with Escherichia coli (107 CFUs) and IV injected with either MDP-2 or PBS, following the

procedure described in the Materials and Methods. 16 hours after the MDP-2 or PBS IV

injection the rats were anesthetized, their vena cava was ligated, and the heart was perfused with

50 mL saline until the liver turned from reddish pink to pale white. The heart, liver, lung, kidney,

small intestine and large intestine from the rats were then harvested. The organs were weighed,

mixed with 1mL of RIPA buffer, homogenized, and centrifuged for 25 minutes at 9000 rpm.

The fluorescence intensity of the supernatant was measured in a Shimadzu spectrofluorometer

(RF 5301PC) and normalized to the tissue weight and plotted after subtracting background auto-

fluorescence, obtained from saline treated rats. Six rats were used for each experimental group.







selected sections were stained with BD™ Gram Stain Kits to detect the presence of bacteria7 or

hematoxylin and eosin (H&E) to detect neutrophils and macrophages8-9.

Histology of LPS treated muscles


infected muscle and the LPS treated muscle were harvested. Tissues were cut to 3 x 4 x 5 mm





selected sections were stained with hematoxylin and eosin (H&E) to detect neutrophils and

macrophages, following standard H&E staining procedures8-9.

Supplementary Figure S14. MDP-2 can image bacterial infections and can detect 106 CFUs in

vivo.


thigh muscle was injected with saline as a control. After 1 hour, MDP-2 (280-350 µL of 1 mM

MDP-2 in PBS) was injected into the rats via the jugular vein, and the rats were imaged after 16

hours in an IVIS Lumina imaging machine. The rat image is a representative result of six

experiments, and identifies the infection site.

a2, Escherichia coli (106 CFUs) infected muscles have a 9 fold increase in fluorescence over un-

infected control muscles. Regions of interest in the infected and control muscles were identified

and integrated using software from the Lumina machine.

The results in a2 are expressed as mean numbers of photons per second per cm2 in the designated

ROI ± s.e.m. for n = 6 per group. The statistical significances in a2 were determined using a

two-sample Student t-test (***p ≤ 0.001).

Biodistribution of MDP-2 in infected rats (Figure 4b and Supplementary Figure S15)

Female Wistar rats (10 weeks, 200-250 g, Harlan Laboratories, Inc) were given thigh infections

with Escherichia coli (107 CFUs) and IV injected with either MDP-2 or PBS, following the

procedure described in the Materials and Methods. 16 hours after the MDP-2 or PBS IV

injection the rats were anesthetized, their vena cava was ligated, and the heart was perfused with

50 mL saline until the liver turned from reddish pink to pale white. The heart, liver, lung, kidney,

small intestine and large intestine from the rats were then harvested. The organs were weighed,

mixed with 1mL of RIPA buffer, homogenized, and centrifuged for 25 minutes at 9000 rpm.

The fluorescence intensity of the supernatant was measured in a Shimadzu spectrofluorometer

(RF 5301PC) and normalized to the tissue weight and plotted after subtracting background auto-

fluorescence, obtained from saline treated rats. Six rats were used for each experimental group.





Supplementary Figure S15. Biodistribution of MDP-2 in infected rats. MDP-2 is efficiently

cleared from all the major organs and selectively accumulates in infected muscle tissue 16 hours

after injection of MDP-2.

Competitive inhibition of MDP-2 transport in Escherichia coli by maltose in vivo

(Supplementary Figure S16)



(107 CFUs) was injected into the left rear thigh muscle (injection depth 5 mm in 250 µL saline),

and 250 µL of saline were injected into right rear thigh muscle as a control (injection depth 5

mm in 250 µL saline). After 1 hour the rats were given maltose (500 µL of 1 M maltose in PBS)

via intraperitoneal injection, followed by injection of MDP-2 (280-350 µL of 1 mM MDP-2 in

PBS) via the jugular vein. The rats then received hourly intraperitoneal injections of 500 µL of 1

M maltose for a total of 16 hours. Fluorescence images were captured using an IVIS® Lumina

Imaging System (Caliper Life Sciences Inc) 16 hours after the MDP-2 injection. The

fluorescence intensity from the area around the bacterial or saline injection site (region of interest)

was integrated. The results are presented in Figure S16 and demonstrate that the uptake of

MDP-2 in Escherichia coli is inhibited by high concentrations of maltose in vivo. Three rats

were used for each experimental group.

Supplementary Figure S16. MDP-2 transport in Escherichia coli was inhibited by an excess of

maltose in vivo. Bacterial infected muscles have a only 1.7 fold increase in fluorescence over

un-infected control muscles, versus a 26 fold increase in Figure 4a.


thigh muscle was injected with saline as a control. Rats were injected with maltose and MDP-2

and imaged. The rat image is a representative result of three experiments and demonstrates that

maltose can inhibit the uptake of MDP-2 in vivo.

a2, Escherichia coli (107 CFUs) infected muscles have a only 1.7 fold increase in fluorescence

over un-infected control muscles. Regions of interest in the infected and control muscles were

identified and integrated using software from the Lumina machine.





Supplementary Figure S15. Biodistribution of MDP-2 in infected rats. MDP-2 is efficiently

cleared from all the major organs and selectively accumulates in infected muscle tissue 16 hours

after injection of MDP-2.

Competitive inhibition of MDP-2 transport in Escherichia coli by maltose in vivo

(Supplementary Figure S16)



(107 CFUs) was injected into the left rear thigh muscle (injection depth 5 mm in 250 µL saline),

and 250 µL of saline were injected into right rear thigh muscle as a control (injection depth 5

mm in 250 µL saline). After 1 hour the rats were given maltose (500 µL of 1 M maltose in PBS)

via intraperitoneal injection, followed by injection of MDP-2 (280-350 µL of 1 mM MDP-2 in

PBS) via the jugular vein. The rats then received hourly intraperitoneal injections of 500 µL of 1

M maltose for a total of 16 hours. Fluorescence images were captured using an IVIS® Lumina

Imaging System (Caliper Life Sciences Inc) 16 hours after the MDP-2 injection. The

fluorescence intensity from the area around the bacterial or saline injection site (region of interest)

was integrated. The results are presented in Figure S16 and demonstrate that the uptake of

MDP-2 in Escherichia coli is inhibited by high concentrations of maltose in vivo. Three rats

were used for each experimental group.

Supplementary Figure S16. MDP-2 transport in Escherichia coli was inhibited by an excess of

maltose in vivo. Bacterial infected muscles have a only 1.7 fold increase in fluorescence over

un-infected control muscles, versus a 26 fold increase in Figure 4a.


thigh muscle was injected with saline as a control. Rats were injected with maltose and MDP-2

and imaged. The rat image is a representative result of three experiments and demonstrates that

maltose can inhibit the uptake of MDP-2 in vivo.

a2, Escherichia coli (107 CFUs) infected muscles have a only 1.7 fold increase in fluorescence

over un-infected control muscles. Regions of interest in the infected and control muscles were

identified and integrated using software from the Lumina machine.





The results are expressed as mean numbers of photons per second per cm2 in the designated ROI

± s.e.m. for n = 3 per group. The statistical significances in a2 were determined using a two-

sample Student t-test (*p ≤ 0.05).

Systemic toxicity of MDP-2 in healthy rats (Supplementary Figure S17)

The toxicity of MDP-2 was assessed in female Wistar rats (10 weeks, 200-250 g, Harlan

Laboratories, Inc). A toxic dose was defined as a dose that caused a 15 % loss of initial weight.

Rats were accustomed to laboratory conditions for at least 1 week before experimental use and

were randomly divided into two groups (6 rats per group), each group was assigned to either

MDP-2 or the control group (saline). A dose of 50 mg/kg of MDP-2 was given to the rats for

this study, which was 20 times of the dose used in the bacterial imaging studies shown in Figures

4 and 5. Rats were anesthetized with isofluorine and MDP-2 (36 mg/mL) in PBS was injected

intravenously via the jugular vein, using a 25G 5/8 needle in a 280-350 µL volume. A control

group of rats were injected with 280-350 µL of PBS, which was also given via the jugular vein

with a 25G 5/8 needle. The weights and general health of the rats were monitored for 6 days

after the MDP-2 or control injections. The body weight changes of the rats are plotted in Figure

S17 and demonstrate that MDP-2 has no toxicity at a dose of 50 mg/kg. Six rats were used for

each experimental group.

Supplementary Figure S17. MDP-2 shows no systemic toxicity to rats at a dose of 50 mg/kg.

▲, rats treated with MDP-2; O, rats treated with PBS (control).

Uptake of PEG-IR in bacteria (Supplementary Figure S18)

The uptake of PEG-IR and MDP-2 was investigated in Escherichia coli (ATCC 33456).

Bacteria were cultured overnight in LB medium at 37 ºC under 5% CO2 in an incubator shaker

(InnovaTM 4230, New Brunswick Scientific, Edison, NJ), which was set at 220 rpm. Bacteria

(100 µL from the overnight culture) were re-suspended in 30 mL of fresh LB medium and

cultured in a 250 mL flask to an OD600 = 0.5, as described above. The bacterial culture solution

was transferred into 6 well plates, with each well containing 3 mL of bacteria, and 60 µL of

either PEG-IR or MDP-2 stock solutions (1 mM in PBS) were added, generating a 20 µM

concentration. The bacteria were incubated with either PEG-IR or MDP-2 for 1 hour at 37 ºC


set at 220 rpm. The uptake of either PEG-IR or MDP-2 by Escherichia coli was determined by

fluorescence (Ex/Em = 750/775 nm) following the procedures used to measure the uptake of





The results are expressed as mean numbers of photons per second per cm2 in the designated ROI

± s.e.m. for n = 3 per group. The statistical significances in a2 were determined using a two-

sample Student t-test (*p ≤ 0.05).

Systemic toxicity of MDP-2 in healthy rats (Supplementary Figure S17)

The toxicity of MDP-2 was assessed in female Wistar rats (10 weeks, 200-250 g, Harlan

Laboratories, Inc). A toxic dose was defined as a dose that caused a 15 % loss of initial weight.

Rats were accustomed to laboratory conditions for at least 1 week before experimental use and

were randomly divided into two groups (6 rats per group), each group was assigned to either

MDP-2 or the control group (saline). A dose of 50 mg/kg of MDP-2 was given to the rats for

this study, which was 20 times of the dose used in the bacterial imaging studies shown in Figures

4 and 5. Rats were anesthetized with isofluorine and MDP-2 (36 mg/mL) in PBS was injected

intravenously via the jugular vein, using a 25G 5/8 needle in a 280-350 µL volume. A control

group of rats were injected with 280-350 µL of PBS, which was also given via the jugular vein

with a 25G 5/8 needle. The weights and general health of the rats were monitored for 6 days

after the MDP-2 or control injections. The body weight changes of the rats are plotted in Figure

S17 and demonstrate that MDP-2 has no toxicity at a dose of 50 mg/kg. Six rats were used for

each experimental group.

Supplementary Figure S17. MDP-2 shows no systemic toxicity to rats at a dose of 50 mg/kg.

▲, rats treated with MDP-2; O, rats treated with PBS (control).

Uptake of PEG-IR in bacteria (Supplementary Figure S18)

The uptake of PEG-IR and MDP-2 was investigated in Escherichia coli (ATCC 33456).

Bacteria were cultured overnight in LB medium at 37 ºC under 5% CO2 in an incubator shaker

(InnovaTM 4230, New Brunswick Scientific, Edison, NJ), which was set at 220 rpm. Bacteria

(100 µL from the overnight culture) were re-suspended in 30 mL of fresh LB medium and

cultured in a 250 mL flask to an OD600 = 0.5, as described above. The bacterial culture solution

was transferred into 6 well plates, with each well containing 3 mL of bacteria, and 60 µL of

either PEG-IR or MDP-2 stock solutions (1 mM in PBS) were added, generating a 20 µM

concentration. The bacteria were incubated with either PEG-IR or MDP-2 for 1 hour at 37 ºC


set at 220 rpm. The uptake of either PEG-IR or MDP-2 by Escherichia coli was determined by

fluorescence (Ex/Em = 750/775 nm) following the procedures used to measure the uptake of





MDP-1 (Figure 3a). The results are presented in Figure S18 and demonstrate that Escherichia

coli do not internalize PEG-IR.

Supplementary Figure S18. PEG-IR is not internalized by bacteria.

a, Escherichia coli do not internalize PEG-IR. Escherichia coli were incubated with either 20

µM PEG-IR or 20 µM MDP-2 (as a control) for 1 hour and the intracellular concentration of

PEG-IR and MDP-2 were determined by IR fluorescence. Results are expressed as mean

millimolar concentration per CFU ± standard error of the mean (s.e.m.), for n = 6 per group.

b, Chemical structure of PEG-IR.

References

1. McKinney, J.D., et al. Persistence of Mycobacterium tuberculosis in macrophages and

mice requires the glyoxylate shunt enzyme isocitrate lyase. Nature 406, 735-738 (2000).

2. Miao, J., et al. Imaging whole Escherichia coli bacteria by using single-particle x-ray

diffraction. Proc Natl Acad Sci U S A 100, 110-112 (2003).

3. Lederberg, J. Encyclopedia of microbiology, (Academic Press, San Diego :, 2000).

4. Ryan, K.J.R., C. George. Sherris Medical Microbiology: An Introduction to Infectious

Diseases, (McGraw Hill, New York, 2000).

5. Yang, G., et al. Activation-induced deaminase cloning, localization, and protein

extraction from young VH-mutant rabbit appendix. Proc Natl Acad Sci U S A 102,

17083-17088 (2005).

6. Gilbert, E. & Keasling, J. scale flow cell for nondestructive imaging of biofilms. in

Environmental Microbiology Methods and Protocols (eds. Spencer, J. & Ragout de

Spencer, A.) (Humana Press, Totowa, NJ, 2004).

7. Mangels, J.I., Cox, M.E. & Lindberg, L.H. Methanol fixation. An alternative to heat

fixation of smears before staining. Diagn Microbiol Infect Dis 2, 129-137 (1984).

8. Haraoka, M., et al. Neutrophil recruitment and resistance to urinary tract infection. J

Infect Dis 180, 1220-1229 (1999).

9. Hang, L., et al. Macrophage inflammatory protein-2 is required for neutrophil passage

across the epithelial barrier of the infected urinary tract. J Immunol 162, 3037-3044

(1999).





MDP-1 (Figure 3a). The results are presented in Figure S18 and demonstrate that Escherichia

coli do not internalize PEG-IR.

Supplementary Figure S18. PEG-IR is not internalized by bacteria.

a, Escherichia coli do not internalize PEG-IR. Escherichia coli were incubated with either 20

µM PEG-IR or 20 µM MDP-2 (as a control) for 1 hour and the intracellular concentration of

PEG-IR and MDP-2 were determined by IR fluorescence. Results are expressed as mean

millimolar concentration per CFU ± standard error of the mean (s.e.m.), for n = 6 per group.

b, Chemical structure of PEG-IR.

References

1. McKinney, J.D., et al. Persistence of Mycobacterium tuberculosis in macrophages and

mice requires the glyoxylate shunt enzyme isocitrate lyase. Nature 406, 735-738 (2000).

2. Miao, J., et al. Imaging whole Escherichia coli bacteria by using single-particle x-ray

diffraction. Proc Natl Acad Sci U S A 100, 110-112 (2003).

3. Lederberg, J. Encyclopedia of microbiology, (Academic Press, San Diego :, 2000).

4. Ryan, K.J.R., C. George. Sherris Medical Microbiology: An Introduction to Infectious

Diseases, (McGraw Hill, New York, 2000).

5. Yang, G., et al. Activation-induced deaminase cloning, localization, and protein

extraction from young VH-mutant rabbit appendix. Proc Natl Acad Sci U S A 102,

17083-17088 (2005).

6. Gilbert, E. & Keasling, J. scale flow cell for nondestructive imaging of biofilms. in

Environmental Microbiology Methods and Protocols (eds. Spencer, J. & Ragout de

Spencer, A.) (Humana Press, Totowa, NJ, 2004).

7. Mangels, J.I., Cox, M.E. & Lindberg, L.H. Methanol fixation. An alternative to heat

fixation of smears before staining. Diagn Microbiol Infect Dis 2, 129-137 (1984).

8. Haraoka, M., et al. Neutrophil recruitment and resistance to urinary tract infection. J

Infect Dis 180, 1220-1229 (1999).

9. Hang, L., et al. Macrophage inflammatory protein-2 is required for neutrophil passage

across the epithelial barrier of the infected urinary tract. J Immunol 162, 3037-3044

(1999).





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DOI: 10.1038/NMAT3074 Maltodextrin based imaging … · and in vivo SUPPLEMENTARY INFORMATION DOI: 10.1038/NMAT3074 ©