Molecularly Imprinted Polymers Prepared Using … Electronic Supplementary Information Molecularly...

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Electronic Supplementary Information

Molecularly Imprinted Polymers Prepared Using Protein-Conjugated

Cleavable Monomers Followed by Site-Specific Post-Imprinting

Introduction of Fluorescent Reporter Molecules

Yusuke Suga, Hirobumi Sunayama

, Tooru Ooya and Toshifumi Takeuchi*

Graduate School of Engineering, Kobe University, 1-1 Rokkodai-cho, Nada-ku, Kobe 657-8501, Japan.

Fax: +81 78 803 6158; Tel: +81 78 803 6158; E-mail: [email protected]

Experimental Section

Materials

N,N'-Diisopropylethylamine (DIEA), triethylamine (TEA), N,N'-diethyldithiocarbamate, glutaric anhydride,

sodium chloride, citric acid, sodium carbonate, ribonuclease A (RNase A), cytochrome c (Cyt), human serum

albumin (HSA), hemoglobin (Hb), trichloromethane (CHCl3), and dimethyl sulfoxide (DMSO) were purchased

from Nacalai Tesque, Inc. (Kyoto, Japan). Acrylamide, N,N'-methylenebisacrylamide, methacryloyl chloride,

2-({2-[bis(carboxymethyl)amino]ethyl}(carboxymethyl)amino) acetic acid (EDTA), acetic acid, hexane, acetone,

acetonitrile (CH3CN), ethanol (EtOH), diethyl ether (Et2O), N,N-dimethylacetamide (DMA), methanol (MeOH),

ethyl acetate (AcOEt), and dichloromethane (CH2Cl2) were purchased from Wako Pure Chemical Industries, Ltd.

(Osaka, Japan). Cystamine dihydrochloride, 4-(N,N-dimethylaminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole

(DBD-F), -mercaptoethanol, N-(9-acridinyl)maleimide and tris(2-carboxyethyl)phosphine were purchased from

Tokyo Chemical Industry Co., Ltd. (Tokyo, Japan). Sodium chloroacetate and bis(acryloyl)cystamine were

purchased from Sigma-Aldrich Co. (St. Louis, USA). Di-tert-butyl dicarbonate and 4N hydrogen chloride in

1,4-dioxane (4N HCl/dioxane) were purchased from Watanabe Chemical Industries, Ltd. (Hiroshima, Japan).

N-Hydroxysuccinimidyl-15-(3-maleimidopropionyl)-amido-4,7,10,13-tetraoxapentadecanoate (MAL-dPEG4-NHS

ester) was purchased from Toyobo Co., Ltd. (Osaka, Japan). CyTM

5 Maleimide Mono-reactive Dye 5-Packs (Cy5

maleimide), Au-coated SPR sensor chips and a Vivaspin 20 instrument were purchased from GE Healthcare (Tokyo,

Japan). One vial of Cy5 maleimide was dissolved in of 50 L DMF prior to use. Immunoglobulin G (IgG) was

purchased from MP Biomedicals LLC (USA), and a Float-A-Lyzer G2 (MWCO: 8–10 kD) was purchased from

Spectrum® Laboratories, Inc. (USA).

Apparatus

1H NMR spectra were measured using a 300 Hz FT-NMR apparatus (JNM-LA300 FT NMR system, JEOL Ltd.,

Tokyo, Japan). MALDI-TOF mass spectra were measured using a MALDI-TOF MS apparatus

(Voyager-DETM

-1000, AB SCIEX, Tokyo, Japan). Circular dichroic spectra were measured using a circular

dichroism spectrometer (J-725, JASCO Corporation, Tokyo, Japan). Fluorescence spectra were recorded on a

Electronic Supplementary Material (ESI) for Chemical CommunicationsThis journal is © The Royal Society of Chemistry 2013

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fluorescence spectrophotometer (F-2500, Hitachi High-Technologies Corporation, Tokyo, Japan). Surface plasmon

resonance (SPR) measurements were performed on a Biacore 3000 (GE Healthcare Japan, Tokyo, Japan).

Syntheses of maleimide-type cleavable monomer and template molecule 6 (Scheme 1)

Scheme 1. Syntheses of maleimide-type cleavable monomer 5 and template molecule 6: a) di-tert-butyl

dicarbonate/triethylamine, MeOH, 0°C, 6 h, 41.7%; b) methacryloyl chloride/N,N-diisopropylethylamine, CH2Cl2,

0°C, 24 h, 90.7%; c) HCl/dioxane, CH2Cl2, 0°C, 24 h, 100%; d) MAL-dPEG4-NHS

ester/N,N-diisopropylethylamine, DMA, r.t., 12 h, 39.3%; e) Cyt, 10 mM phosphate buffer (pH 7.3), 4°C, 2days.

N-tert-Butoxycarbonyl cystamine (2)

Cystamine dihydrochloride (2.252 g, 10.0 mmol) and triethylamine (4.18 mL, 30.0 mmol) were dissolved in

MeOH (30 mL) and added to a methanolic solution (20 mL) of di-tert-butyl dicarbonate (1.091 g, 5.00 mmol). The

reaction mixture was stirred for 6 h at 0°C, after which the solution was evaporated, and 1 M NaH2PO4 was added

to the residue. The aqueous solution was washed with diethyl ether to remove di-t-Boc-cystamine. The aqueous

solution was adjusted to pH 9 by addition of 1 M NaOH, and the solution was extracted with AcOEt. The organic

phase was dried over MgSO4 and then evaporated. The resulting yellow oil was dried in vacuo to give 2. Yield:

0.526 g (41.7%). TLC: Rf 0.35 (MeOH). 1H NMR (300 MHz, CDCl3): δ 4.97 (b, 1H, NH-Boc), 3.47 (q, 2H,

C(=O)-NHCH2), 3.03 (t, 2H, CH2NH2), 2.80 (q, 4H, CH2SSCH2), 1.46 (s, 9H, CH3).

N-tert-Butoxycarbonyl-N'-methacryloylcystamine (3)

Compound 2 (0.576 g, 2.28 mmol) and N,N-diisopropylethylamine (0.79 mL, 4.56 mmol) were dissolved in

CH2Cl2 (20 mL) and added to methacryloyl chloride (0.29 mL, 2.96 mmol) dissolved in CH2Cl2 (20 mL). The

reaction mixture was stirred for 24 h at 0°C, after which the reaction solution was washed with brine. The organic

phase was evaporated and purified by silica gel column chromatography (eluent: hexane:AcOEt = 1:1). Compound

3 was obtained as a yellow solid. Yield: 0.634 g (90.7%). TLC: Rf 0.40 (hexane:AcOEt = 1:1). 1H NMR (300 MHz,

CDCl3): δδ 6.52 (b, 1H, C(=O)-NH), 5.73 5.33 (s, 2H, H2C=C(CH3)C(=O)-), 5.03 (b, 1H, NH-Boc), 3.62 (q, 2H,

SSH2N

NH2 SSH2N

NH

O

O

HClHCl

SS

HN

NH

O

O

O1 2

SS

HN

NH2

O HCl

3

54

a)

d)

b)

c)

HN O

O

SS

NH

O

4NH

O

N

O

O S

HN O

O

SS

NH

O

4NH

O

N

O

O

e)

6


S3

C(=O)-NHCH2), 3.43 (q, 2H, CH2NH-Boc), 2.86 (t, 2H, C(=O)-NHCH2CH2S), 2.79 (t, 2H, SCH2CH2NH-Boc),

1.96 (s, 3H, H2C=C(CH3)C(=O)-), 1.46 (s, 9H, CH3).

N-Methacryloylcystamine (4)

Compound 3 (0.304 g, 0.99 mmol), dissolved in CH2Cl2 (40 mL), was added to 4 N HCl/dioxane (0.745 mL, 2.98

mmol) in CH2Cl2 (20 mL), and stirred for 24 h at 0°C. The solution was evaporated to give the crude product as a

white solid, which was washed with diethyl ether and dried in vacuo. Yield: 0.257 g (100%). 1H NMR (300 MHz,

(CD3)2SO): δ 8.19 (b, 1H, C(=O)-NH), 8.10 (b, 2H, NH2), 5.76 5.68 (s, 2H, H2C=C(CH3)C(=O)-), 3.43 (q, 2H,

C(=O)-NHCH2), 3.08 (quint., 2H, CH2NH2), 2.96 (t, 2H, C(=O)-NHCH2CH2S), 2.86 (t, 2H, SCH2CH2NH2), 1.86 (s,

3H, H2C=C(CH3)C(=O)-).

Maleimide-type cleavable monomer for conjugated template (5)

Compound 4 (0.028 g, 0.107 mmol) and N,N-diisopropylethylamine (0.019 mL) were dissolved in DMA (10 mL),

after which MAL-dPEG4-NHS ester (0.050 g, 0.097 mmol) was added to the solution. The reaction mixture was

stirred for 12 h at room temperature and evaporated in vacuo. The residue was dissolved in CHCl3 and washed with

brine, and the organic phase was dried over MgSO4 and evaporated. The resulting yellow oil was dried in vacuo to

give 5. Yield: 0.026 g (39.3%). TLC: Rf 0.24 (CHCl3). 1H NMR (300 MHz, CDCl3): δ 7.07 (b, 1H,

CH2C(=O)-NH(CH2CH2O)4), 6.69 (s, 2H, CH=CH of maleimide), 6.63 (b, 1H, CH2NHC(=O)-(CH2CH2O)4), 6.57

(b, 1H, H2C=C(CH3)C(=O)-NHCH2), 5.75, 5.35 (s, 2H, H2C=C(CH3)C(=O)-), 3.84 (t, 2H, CH2N(CO)2), 3.73 (t,

2H, CH2C(=O)-NH(CH2CH2O)4), 3.62 (t, 16H, NHC(=O)-CH2(CH2OCH2)4), 3.54 (m, 2H,

CH2NHC(=O)-(CH2CH2O)4), 3.41 (q, 2H, H2C=C(CH3)C(=O)-NHCH2), 2.86 (m, 4H, CH2SSCH2), 2.50 (m, 4H,

C(=O)-CH2(CH2OCH2)4CH2C(=O)-), 1.97 (s, 3H, H2C=C(CH3)C(=O)-). MS: 642.19 [M+Na].

Synthesis of template molecule; Cyt-conjugated cleavable monomer (6)

Native Cyt (11.7 mg, 0.95 µmol) and 5 (12 mg, 19.4 µmol) were dissolved in 10 mM phosphate buffer at pH 7.3

and stirred for 2 days at 4°C. The reaction mixture was dialyzed for 2 days in 10 mM phosphate buffer at pH 7.3

using a Float-A-Lyzer G2 device (MWCO: 8–10 kD) to remove unreacted monomers. The dialyzed solution was

ultra-filtered using a Vivaspin 20 (4000 rpm, 10 min) and lyophilized to give 6.

Carboxylic acid-type cleavable monomer (7)

Compound 4 (0.128 g, 0.500 mmol) and N,N-diisopropylethylamine (0.174 mL, 1.00 mmol) were dissolved in

CH2Cl2 (20 mL), and the solution was added to a CH2Cl2 solution (20 mL) of glutaric anhydride (0.074 g, 0.65

SS

HN

NH2

O

SS

HN

NH

O

O

OH

O

4 7

O OO


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mmol) at 0°C. The reaction mixture was stirred for 1.5 h and then evaporated, and the residue was dissolved in

AcOEt and washed with aqueous citric acid and brine. The organic phase was evaporated to precipitate a white

solid, which was washed with AcOEt and dried in vacuo to give 7. Yield: 0.042 g (25.2%). 1H NMR (300 MHz,

(CD3)2SO): δ 12.06 (s, 1H, COOH), 8.13 (s, 1H, H2C=C(CH3)C(=O)-NH), 8.03 (s, 1H, NHC(=O)-CH2CH2) 5.67,

5.35 (s, 2H, H2C=C(CH3)C(=O)-), 3.40 (m, 4H, CH2CH2SSCH2CH2), 2.80 (m, 4H, CH2SSCH2), 2.21 (t, 2H,

CH2COOH), 2.10 (t, 2H, CH2CH2CH2COOH), 1.85 (s, 3H, H2C=C(CH3)C(=O)-), 1.70 (t, 2H, CH2CH2COOH).

Preparation of water-soluble initiator

A water-soluble initiator was prepared by modification of a method reported by Tsuji and Kawaguchi.1 Sodium

chloroacetate (466 mg, 4 mmol) and sodium N,N-diethyldithiocarbamate (900.6 mg, 4 mmol) were dissolved in 10

mL of water, and the reaction mixture was stirred for 2 days at room temperature with light shielding. HCl was then

added to the solution to precipitate the product, which was filtered and dried in vacuo. TLC: Rf 0.75 (MeOH). 1H

NMR (300 MHz, CDCl3): δ 4.18 (s, 2H, CH2COOH), 4.03 (q, 2H, NCH2CH3), 3.78 (q, 2H, NCH2CH3), 1.31

(quint., 6H, CH3).

1(a) T. Sato, S. Tsuji and H. Kawagauchi, Ind. Eng. Chem. Res., 2008, 47, 6358-6361. (b) S. Tsuji and H.

Kawagauchi, Macromolecules, 2006, 39, 4338-4344.

Preparation of 7-(2-hydroxyethylsulfanyl)-benzo[1,2,5]oxadiazole-4-sulfonic acid dimethylamide

(coupling reaction between DBD-F and -mercaptoethanol)

DBD-F (24.5 mg, 0.1 mmol) and sodium carbonate (31.8 mg, 0.3 mmol) were dissolved in CH3CN (10 mL) and

added to -mercaptoethanol (42 µL, 0.6 mmol), and the reaction mixture was stirred for 2 h at 50°C. The solvent

was removed by evaporation and the residue was dissolved in AcOEt. The organic phase was washed with brine

and then evaporated and purified by silica gel column chromatography (eluent: hexane:AcOEt = 1:2) to give the

product as a white solid. Yield: 27.0 mg (89.0%). TLC: Rf 0.53 (AcOEt). 1H NMR (300 MHz, (CD3)2SO): δ 7.92 (d,

1H, C5H of benzofurazane), 7.51 (d, 1H, C

6H of benzofurazane), 5.17 (t, 1H, OH), 3.78 (q, 2H, CH2OH), 3.40 (t,

2H, CH2S), 2.78 (s, 6H, NCH3). MS: 327.94 [M+Na].

HO

O

S N

SO

HOCl

NS

S

Na

NO

N

F

SO2N

NO

N

S

SO2N

HO

HOSH


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Preparation of glass substrate for introduction of polymerizable groups

A glass substrate (9.7 11.7 mm, Matsunami Glass Co., Ltd., Japan) was washed with methanol and pure water,

and placed under UV-O3 light for 30 min. The cleaned glass substrate was immersed in 1%

3-methacryloxypropyltrimethoxysilane aqueous solution (containing 1% acetic acid). After 24 h, the glass substrate

was washed with ethanol and pure water and placed on a hot plate at 110°C for 1 min.

Preparation of Cyt-imprinted polymer using 6 (MIP), Cyt-imprinted polymer using 7 (comp-MIP) and

non-imprinted polymer (NIP)

Pre-polymerization solutions for MIP, comp-MIP and NIP were prepared, as shown in Table 1, by dissolving each

compound in 2.5 mL of 10 mM phosphate buffer (pH 7.3). The 3-methacryloxypropyltrimethoxysilane-modified

glass substrate was covered by a silicone rubber sheet (9.7 11.7 mm) with a shallow well (6.0 9.0 mm), and 55

L of the pre-polymerization solution was placed in the well, which was then sealed by a cover glass (24 24 mm)

that had been treated with trimethylchlorosilane. To initiate polymerization, the reaction mixture was irradiated

with LED light (365 nm, 250 mW, ThermoStage-AS, YMC Co. Ltd., Kyoto, Japan) from the bottom of the

substrate for 1 h at 4°C. After polymerization, the resulting polymeric film was washed with ethanol and pure

water.

Table 1. Recipe for pre-polymerization solution.a

Cleavable monomer

(0.83 µmol)

Functional

monomer

(27 µmol)

Cross-linker

(114.6 µmol)

MIP Cyt-conjugated

monomer 6

Acrylamide N,N'-methylenebis acrylamide Comp-MIP Monomer 7 + Cyt

b

NIP Monomer 4

a All compounds were dissolved in 2.5 mL of 10 mM phosphate buffer (pH 7.3).

b Cyt (0.83 µmol) was added as a template to the pre-polymerization solution of comp-MIP.

Introduction of a fluorescent reporter molecule into the polymeric thin film by post-imprinting

treatment

For disulfide linkage reduction accompanied by removal of Cyt, each polymeric thin film was immersed in 10

mM tris(2-carboxyethyl)phosphine solution (5 mL) for 4 h, followed by washing with pure water. The exposed

thiol group in the polymeric film was allowed to react with thiol-reactive fluorescent dye (DBD-F). The polymeric

thin film was immersed in 5 mM DBD-F solution (acetonitrile:borate buffer containing 1% EDTA = 1:1) and

allowed to stand for 1 h at 50°C. After the reaction, the film was washed with methanol and pure water.

N-(9-acridinyl)maleimide and Cy5 maleimide were also applied to the post-imprinting treatment. The polymeric

thin film, in which thiol group was exposed by the same procedure for the DBD-F introduction, was immersed in

40 mM N-(9-acridinyl)maleimide solution (acetone : 100 mM borate buffer (pH 9.3) containing 1% EDTA = 1:1)


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for 1h at room temperature. Similarly, the thiol-exposed film was immersed in 400 L of 10 mM phosphate

buffer (pH 7.3) dissolving Cy5 maleimide that was prepared by the addition of Cy5 maleimide dissolved in DMF

(50 L), and allowed to stand for 12 h at room temperature. After the reactions, each film was washed with

methanol and pure water.

Fluorescence titration of proteins with prepared polymer films

MIP, comp-MIP and NIP, in which DBD-F was introduced by post-imprinting treatment, were placed in a cuvette

(AS ONE Corporation, Osaka, Japan, 12.5 12.5 45 mm) containing 10 mM phosphate buffer (pH 7.3, 3 mL).

The fluorescence intensity at 537 nm (Ex: 410 nm) was measured after addition of 20 µL of 30 µM Cyt solution.

Hb, RNase A, HSA, and IgG were used as controls. The final concentration of each protein was varied from 0 to

1.0 µM. In the same way, MIPs, in which either N-(9-acridinyl)maleimide or Cy5 maleimide was introduced,

were subjected to fluorescence titration of Cyt. Fluorescence intensities at 439 nm (Ex: 360 nm) and 668 nm (Ex:

650 nm) were measured for N-(9-acridinyl)maleimide and Cy5 maleimide, respectively.

Fluorescence titration of proteins with DBD-F conjugated with -mercaptoethanol (DBD-F derivative)

The DBD-F derivative (1.74 µmol, 0.53 mg) was dissolved in DMSO (58 µL) to give a 30 µM DBD-F solution,

of which 20 µL was added to a cuvette filled with 10 mM phosphate buffer (pH 7.3, 3 mL). The fluorescence

intensity was measured after addition of 20 µL of a 30 µM solution of each protein (Cyt, Hb, RNase A, HSA, and

IgG). The final concentration of each protein was varied from 0 to 1.0 µM.

Preparation of polymeric thin film on Au substrate for SPR measurements

A gold sensor chip was washed with ethanol and water and placed under UV-O3 light for 30 min. The sensor chip

was immersed in a 5 mM ethanolic solution of bis(acryloyl)cystamine. After 24 h, the chip was washed with

ethanol and pure water. Polymeric thin films of MIP and NIP were prepared on the Au sensor chip using the same

manner of preparation as for the glass substrate. These films were then immersed in 10 mM

tris(2-carboxyethyl)phosphine solution (5 mL) for disulfide linkage reduction. After 4 h, the films were allowed to

react with DBD-F (in a 1:1 solution of acetonitrile and 100 mM borate buffer (pH 9.3) containing 1% EDTA) for 1

h at 50°C, followed by washing with methanol and pure water.

SPR measurements

Binding experiments for MIP and NIP prepared on the Au chip were performed at 25°C. Cyt, Hb, RNase A, HSA,

and IgG, dissolved in 10 mM phosphate buffer (pH 7.3), were used for the experiments. The measurement

conditions were as follows. Running buffer: 10 mM phosphate buffer (pH 7.3); flow rate: 20 µL/min; injection

volume: 20 µL; regeneration solution: 10 mM phosphate buffer containing 1 M NaCl and 0.3% SDS aq. with 200

mM NaCl (flow rate: 30 µL/min, 30 s). The amounts of the bound proteins were calculated from the signal intensity

(measured in resonance units, RU) 120 s after the end of each sample injection period. The concentration of each


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protein was varied from 0 to 1.0 µM. Binding isotherms were drawn using ΔRU values for each protein

concentration.

NMR charts

N-tert- Butoxycarbonyl cystamine, 2

PPM

7 6 5 4 3 2 1 0

DFILE No.005.alsCOMNT autoDATIM Wed May 11 19:13:42 2011OBNUC 1HEXMOD nonOBFRQ 300.40 MHzOBFIN 131150.00 HzPOINT 8192FREQU 6009.62 HzSCANS 32ACQTM 1.3631 secPD 5.6369 secPW1 5.50 usecIRNUC 1HCTEMP 22.6 cSLVNT CDCL3EXREF 0.00 ppmBF 0.12 HzRGAIN 17

auto

0.7

8

2.0

0

1.8

6

3.8

4

9.2

1

7.2

889

4.9

797

3.4

858

3.4

638

3.0

561

3.0

366

3.0

146

2.8

242

2.8

096

2.8

047

2.7

876

2.7

681

2.0

627

1.5

769

1.4

646

0.0

146

SSH2N

NH

O

O


S8

N-tert-Butoxycarbonyl-N’-methacryloylcystamine, 3

N-Methacryloylcystamine, 4

PPM

7 6 5 4 3 2 1

DFILE No.023.alsCOMNT autoDATIM Wed Jun 22 12:39:31 2011OBNUC 1HEXMOD nonOBFRQ 300.40 MHzOBFIN 131150.00 HzPOINT 8192FREQU 6009.62 HzSCANS 32ACQTM 1.3631 secPD 5.6369 secPW1 5.50 usecIRNUC 1HCTEMP 23.3 cSLVNT CDCL3EXREF 4.79 ppmBF 0.12 HzRGAIN 15

auto

0.8

2 1.0

0

1.0

2

0.8

3

2.0

9

2.0

9

4.2

3

3.4

8

9.7

6

7.4

165

6.6

915

5.8

836

5.4

906

5.1

976

3.8

087

3.7

892

3.7

672

3.7

477

3.6

183

3.5

963

3.5

768

3.5

548

3.0

349

3.0

154

2.9

959

2.9

617

2.9

397

2.9

177

2.1

830

2.1

122

1.7

851

1.5

776

1.4

190

1.3

946

1.3

653

0.2

058

0.1

350

PPM

8 6 4 2 0

DFILE No.024.alsCOMNT autoDATIM Thu Jun 23 18:14:01 2011OBNUC 1HEXMOD nonOBFRQ 300.40 MHzOBFIN 131150.00 HzPOINT 8192FREQU 6009.62 HzSCANS 32ACQTM 1.3631 secPD 5.6369 secPW1 5.50 usecIRNUC 1HCTEMP 24.2 cSLVNT DMSOEXREF 0.00 ppmBF 0.12 HzRGAIN 22

auto

3.5

3

0.9

8

1.0

0

1.9

9

1.8

21.7

91.8

5

3.0

1

8.1

896

8.0

993

5.7

608

5.6

827

5.3

580

5.3

531

4.6

599

3.5

688

3.4

467

3.4

272

3.4

028

3.3

832

3.1

196

3.1

001

3.0

781

3.0

586

2.9

829

2.9

585

2.9

365

2.8

804

2.8

560

2.8

340

2.7

364

2.5

167

2.5

094

2.5

045

2.4

972

2.4

923

1.8

552

1.1

155

1.0

911

1.0

692

0.0

122

0.0

000

-0.

0098

-0.

0391

SS

HN

NH

O

O

O

SS

HN

NH2

O HCl


S9

Maleimide-type cleavable monomer, 5

Carboxylic acid-type cleavable monomer, 7

PPM

7 6 5 4 3 2 1 0

DFILE 12-08-07 No042.alsCOMNT autoDATIM Tue Aug 07 19:55:27 2012OBNUC 1HEXMOD nonOBFRQ 300.40 MHzOBFIN 131150.00 HzPOINT 8192FREQU 6009.62 HzSCANS 32ACQTM 1.3631 secPD 5.6369 secPW1 5.50 usecIRNUC 1HCTEMP 24.2 cSLVNT CDCL3EXREF 0.00 ppmBF 0.12 HzRGAIN 25

auto

0.7

6 1.7

20.8

0

0.9

1

1.0

0

0.9

7 1.9

42.1

3

17.

39

2.0

9

4.1

1

3.6

2

3.0

7

7.2

791

7.0

667

6.7

177

6.6

347

6.4

516

5.7

705

5.3

702

3.8

861

3.8

617

3.8

373

3.7

689

3.7

494

3.7

299

3.6

713

3.6

566

3.6

420

3.6

200

3.5

980

3.5

785

3.5

541

3.5

370

3.4

589

3.4

418

3.4

247

3.4

052

2.9

194

2.8

975

2.8

828

2.8

609

2.8

389

2.5

655

2.5

411

2.5

191

2.5

045

2.4

849

1.9

919

1.6

184

0.0

854

0.0

269

0.0

171

PPM

12 10 8 6 4 2 0

DFILE No.029-2.alsCOMNT autoDATIM Fri Dec 09 22:31:11 2011OBNUC 1HEXMOD nonOBFRQ 300.40 MHzOBFIN 131150.00 HzPOINT 8192FREQU 6009.62 HzSCANS 32ACQTM 1.3631 secPD 5.6369 secPW1 5.50 usecIRNUC 1HCTEMP 20.1 cSLVNT DMSOEXREF 0.00 ppmBF 0.12 HzRGAIN 23

auto

1.1

5

2.1

5

1.0

0

1.0

1

21.

87

4.6

2

2.2

32.2

7 2.9

42.2

9

12.

0586

8.1

286

8.0

334

5.6

705

5.3

483

5.3

434

3.4

272

3.4

076

3.3

832

3.3

491

3.3

149

3.2

954

2.8

438

2.8

194

2.7

950

2.7

730

2.7

510

2.5

216

2.5

094

2.5

045

2.4

972

2.2

311

2.2

067

2.1

823

2.1

286

2.1

042

2.0

797

1.8

527

1.7

282

1.7

038

1.6

794

0.0

171

0.0

073

-0.

0049

-0.

0342

HN O

O

O

OO

HN

O

N

O

O

SS

NH

O

SS

HN

NH

O

O

OH

O


S10

7-(2-Hydroxy-ethylsulfanyl)-benzo[1,2,5]oxadiazole-4-sulfonic acid dimethylamide

Fig. SI-1. Mass spectra of (a) native Cyt and (b) Cyt-conjugated cleavable monomer 6.

PPM

8 7 6 5 4 3 2 1 0

DFILE 12-5-10 DBD.alsCOMNT autoDATIM Thu May 10 20:21:06 2012OBNUC 1HEXMOD nonOBFRQ 300.40 MHzOBFIN 131150.00 HzPOINT 8192FREQU 6009.62 HzSCANS 32ACQTM 1.3631 secPD 5.6369 secPW1 5.50 usecIRNUC 1HCTEMP 114.0 cSLVNT DMSOEXREF 4.65 ppmBF 0.12 HzRGAIN 23

auto

1.0

0

0.9

9

2.0

3

2.0

8

6.2

9

7.9

380

7.9

112

7.5

255

7.5

011

5.1

895

5.1

724

5.1

528

4.8

550

4.0

422

4.0

178

3.7

981

3.7

786

3.7

590

3.7

395

3.6

272

3.6

077

3.4

173

3.3

978

3.3

758

3.3

196

3.0

145

2.8

290

2.8

095

2.7

826

2.7

338

2.5

483

2.5

141

2.5

092

2.5

019

2.4

970

2.4

897

2.2

725

1.9

893

1.1

984

1.1

764

1.1

520

0.0

121

-0.

0001

-0.

0099

11000 12000 13000 14000 15000Mass (m/z)

0

10

20

30

40

50

60

70

80

90

100

% In

ten

sit

y

13649.58

14268.6513006.25

14870.74

11000 12000 13000 14000 15000Mass (m/z)

0

10

20

30

40

50

60

70

80

90

100

% In

ten

sit

y

12378.83

12587.77

Cyt

Cyt-conjugated cleavable monomer 6

NO

N

S

SO2N

HO


S11

(a)

(b) (c)

NO O

N

NN

O3SSO3K

HN

O

N

O

O

Fig. SI-2. (a) Relative fluorescence change at 537 nm (Ex: 410 nm) of MIPs modified modified by DBD-F (closed

circle), N-(9-acridinyl)maleimide (open triangle) and Cy5 maleimide (open square) after the addition of Cyt in

10mM phosphate buffer (pH 7.3). (b) Chemical structire of N-(9-acridinyl)maleimide and (c) Cy5 maleimide.

-0.05

0.05

0.15

0.25

0.35

0.45

0 0.2 0.4 0.6 0.8 1

(I0-I

)/I 0

protein conc. (μM)

DBD-F

N-(9-Acridinyl)maleimide

Cy5 maleimide


S12

Fig. SI-3. Binding isotherms of Cyt with MIP (closed circle) and NIP (open square), measured by SPR in 10 mM

phosphate buffer (pH 7.3).

Fig. SI-4. Relative fluorescence change at 537 nm (Ex: 410 nm) of NIP after the addition of each protein [Cyt

(closed circle), Hb (closed diamond), HSA (open triangle), IgG (open diamond), and RNase A (open square)] in a

10mM phosphate buffer (pH 7.3).

0

200

400

600

800

1000

1200

1400

1600

1800

0 0.2 0.4 0.6 0.8 1

⊿R

U

Cyt conc. (M)

MIP

NIP

-0.05

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0 0.2 0.4 0.6 0.8 1

(I0-I

)/I 0

protein conc. (μM)

Cyt Hb HSA IgG RNase A


S13

Fig. SI-5. Relative fluorescence change at 537 nm (Ex: 410 nm) of a DBD-F derivative conjugated with

2-mercaptoethanol (7-(2-hydroxy-ethylsulfanyl)-benzo[1,2,5]oxadiazole-4-sulfonic acid dimethylamide) after the

addition of each protein [Cyt (closed circle), Hb (closed diamond), HSA(open triangle), IgG (open diamond), and

RNase A (open square)] in a 10mM phosphate buffer (pH 7.3).

-0.1

-0.05

0

0.05

0.1

0.15

0.2

0 0.2 0.4 0.6 0.8 1

(I0-I

)/I 0

protein conc. (μM)

Cyt Hb HSA IgG RNase A


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