Synthetic Pores with Reactive Signal Amplifiers as ... · Novabiochem or BioSystems, EYPC from...

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Synthetic Pores with Reactive Signal Amplifiers as Artificial Tongues

Svetlana Litvinchuk, Hiroyuki Tanaka, Tomohiro Miyatake, Dario Pasini, Takatsugu Tanaka,

Guillaume Bollot, Jiri Mareda and Stefan Matile

Department of Organic Chemistry, University of Geneva, Geneva, Switzerland,

[email protected]

Supplementary Information

Table of Content

1. Methods

1.1. Materials S2

1.2. Abbreviations S2

1.3. Synthesis S3

1.4. Vesicle preparation S8

1.5. Signal transduction S8

1.6. Signal amplification S9

1.7. Lactate sensing S10

1.8. Citrate sensing S15

1.9. Lactose sensing S18

1.10. Acetate sensing S21

1.11. Molecular modeling S22

1.12 Supporting references S23

2. Supporting figures and schemes S27

3. Supporting tables S44

© 2007 Nature Publishing Group

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1. Methods

1.1. Materials. As in (S1) or (S2), Supporting Information. Reagents, solvents CF, L-

lactate, citrate and polymer-bound p-toluenesulfonyl hydrazide were from Fluka/Aldrich,

Novabiochem or BioSystems, EYPC from Avanti Polar Lipids, CB-amplifier 2 from

Molecular Probes, all buffers, HEPES, MES, TES, Tris and inorganic salts were of the best

grade available from Sigma. Triton X-100, ATP, lactose, D-galactose, D-glucose, D-fructose

and enzymes were from Sigma. Dialysis was performed using 1 ml equilibrium dialysis cells

(Bel-Art Products), centrifugal filtration with a Centricon-3 device (Millipore). Large

unimellar vesicles (LUVs) were prepared by the Mini-Extruder with polycarbonate

membrane, pore size 100 nm, from Avanti Polar Lipids. Fluorescence spectra were recorded

on either a Fluoromax 2 or Fluoromax 3 from Jobin Yvon-Spex equipped with an injector

port, a magnetic stirrer and a temperature controller (25 oC).

1.2. Abbreviations. Boc: tert-Butoxycarbonyl; CF, 5(6)-carboxyfluorescein; DAN:

Dialkoxynaphthalene; DMF: N,N-Dimethylformamide; DMSO: Dimethylsulfoxide; EYPC-

LUVs: Egg yolk phosphatidylcholine large unilamellar vesicles; Gla: -OCH2CO- (H-Gla-

OH: glycolic acid); HBTU: N-[(1H-benzotriazol-1-yl)(dimethylamino)methylene]-N-

methylmethanaminium hexafluorophosphate N-oxide; HEPES: N-(2-

hydroxyethyl)piperazine-N’-(2-ethanesulfonic acid); His, H: L-Histidine; Leu, L: L-Leucine;

MES: 2-Morpholinoethanesulfonic acid monohydrate; NDI: Naphthalene diimide; Ndi, A:

artificial amino acid in pore 4; TES: N-[Tris(hydroxymethyl)methyl]-2-aminoethanesulfonic


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acid; TFA: Trifluoroacetic acid; Tris: Tris(hydroxymethyl)aminomethane; Z:

Benzyloxycarbonyl.

1.3. Syntheses

13,2

3,3

2,4

3,5

2,6

3,7

2,8

3-Octakis(Gla-Leu-Lys-Leu-His-Leu-NH2)-p-Octiphenyl 1

m.

Monomer 1m for self-assembly into pore 1 was prepared in 19 steps following previously

reported procedures (S1).

13,2

3,3

2,4

3,5

2,6

3,7

2,8

3-Octakis(Gla-Leu-Ndi-Leu-Lys-Leu-NH2)-p-Octiphenyl 4

m.

Monomer 4m for self-assembly into pore 4 was prepared in 24 steps as outlined in Scheme S1

following previously reported procedures (S2).

CB Hydrazide 2. Amplifier 2 was from Molecular Probes or prepared in 2 steps following

previously reported procedures (S3).

DAN Hydrazide 5. Amplifier 5 was prepared in 4 steps as follows (see Scheme S2 for

structures):

Compound 12. A solution of 1,5-dihydroxynaphthalene (10, 2.0 g, 12.5 mmol), potassium

carbonate (2.3 g, 16.3 mmol) and t-butyl bromoacetate (11, 2.1 ml, 13.7 mmol) in acetone (20

ml) was heated up to 70°C and stirred overnight. After cooling, the mixture was poured into

1N HCl (50 ml), extracted with EtOAc, washed with water (2 times) and brine, dried with

anhydrous Na2SO4, filtered and concentrated. Purification of the crude product by column


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chromatography (CH2Cl2) yielded 12 (830 mg, 24%) as a pale yellow powder. TLC

(CH2Cl2): Rf 0.29; mp: 132-133 °C; IR: 3369 (m), 2984 (w), 1720 (s), 1598 (m), 1521

(m), 1415 (s), 1366 (s), 1243 (s), 1228 (s), 1145 (s), 1095 (s), 910 (m), 838 (m), 759 (s),; 1H

NMR (400 MHz, CDCl3): 7.94 (d, 3J (H,H) = 8.6 Hz, 1H), 7.79 (d, 3J (H,H) = 8.4 Hz, 1H),

7.33 (t, 3J (H,H) = 8.1 Hz, 1H), 7.27 (t, 3J (H,H) = 8.5 Hz, 1H), 6.80 (d, 3J (H,H) = 7.6 Hz,

1H), 6.72 (d, 3J (H,H) = 7.6 Hz, 1H), 4.72 (s, 2H), 1.53 (s, 9H); 13C NMR (100 MHz, CDCl3):

168.6 (s), 153.8 (s), 151.5 (s), 127.1 (s), 125.7 (s), 125.7 (d), 124.9 (d), 115.2 (d), 114.9 (d),

109.8 (d), 105.7 (d), 82.9 (s), 66.3 (t), 28.3 (q); ESI MS (MeOH/CH2Cl2 1:1): m/z 293 (100,

[M + H2O + H]+), 275 (16, [M + H]+), 219 (73, [M - t-Bu + H]+), 173 (57, [M - t-Bu - CO2 +

H]+).

Compound 14. A solution of 12 (830 mg, 3.0 mmol), cesium carbonate (1.27 g, 3.9 mmol)

and methyl bromoacetate (13, 0.29 ml, 3.6 mmol) in acetone (10 ml) was heated up to 70°C

and stirred 30 min. After cooling, the mixture was filtrated and poured into 0.1N HCl,

extracted with EtOAc, washed with water (2 times) and brine, dried with anhydrous Na2SO4,

filtered and concentrated. Purification of the crude product by column chromatography (Pet.

ether/CH2Cl2 1:1) yielded 14 (963 mg, 93%) as colorless powder. TLC (CH2Cl2): Rf 0.61;

mp: 87-88 °C; IR: 2988 (w), 2955 (w), 1756 (s), 1597 (m), 1514 (m), 1415 (s), 1238 (m),

1208 (m), 1150 (s), 1106 (s), 1070 (m), 844 (m), 769 (s); 1H NMR (400 MHz, CDCl3): 8.01

(d, 3J (H,H) = 8.6 Hz, 1H), 7.98 (d, 3J (H,H) = 8.3 Hz, 1H), 7.40-7.35 (m, 2H), 6.76 (d, 3J

(H,H) = 7.6 Hz, 2H), 4.82 (s, 2H), 4.70 (s, 2H), 3.83 (s, 3H), 1.51 (s, 9H); 13C NMR (100

MHz, CDCl3): 169.6 (s), 168.1 (s), 153.8 (s), 153.6 (s), 127.0 (s), 126.9 (s), 125.4 (d),

125.3 (d), 116.0 (d), 115.6 (d), 106.2 (d), 106.1 (d), 82.6 (s), 66.4 (t), 65.9 (t), 52.5 (q), 28.3

(q); ESI MS (MeOH/CH2Cl2 1:1): m/z 365 (27, [M + H2O + H]+), 347 (15, [M + H]+), 291


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(100, [M - t-Bu + H]+).

Compound 5. To a solution of 14 (460 mg, 1.33 mmol) in CH2Cl2 (3 ml), TFA (3 ml) was

added at rt. After stirring 2 hrs at rt, the solvent was evaporated. The residue was triturated

with pet. ether/CH2Cl2 (1/1) and filtrated to yield 15 (322 mg). Crude 15 was dissolved in

hydrazine monohydrate (5 ml) at rt and stirred overnight at 60 °C. After removal of

hydrazine, the precipitate was triturated with MeOH, filtered and washed with MeOH to give

pure hydrazine 5 as a colorless powder (318 mg, 86%, 2 steps). mp: 251 °C (decomposed);

IR: 3305 (w), 2916 (w), 2649 (w), 1665 (m), 1594 (m), 1557 (m), 1507 (s), 1440 (m), 1408

(s), 1371 (m), 1334 (m), 1264 (s), 1211 (m), 1090 (s), 1071 (s), 1017 (m), 944 (m) , 909 (m) ,

778 (s) , 712 (m); 1H NMR (400 MHz, DMSO-d6/TFA-d 2/1): 7.91 (d, 3J (H,H) = 8.6 Hz,

1H), 7.86 (d, 3J (H,H) = 8.6 Hz, 1H), 7.33 (t, 3J (H,H) = 8.1 Hz, 2H), 6.89 (d, 3J (H,H) = 7.6

Hz, 1H), 6.83 (d, 3J (H,H) = 7.6 Hz, 1H), 4.87 (s, 2H), 4.78 (s, 2H); 13C NMR (100 MHz,

DMSO-d6/TFA-d 2/1): 169.8 (s), 167.3 (s), 153.0 (s), 152.7 (s), 126.0 (s), 125.8 (s), 125.1

(d), 124.9 (d), 115.0 (d), 114.5 (d), 106.1 (d), 105.7 (d), 65.9 (t), 64.5 (t); ESI MS

(DMSO/TFA 3:1): m/z 291 (100, [M + H]+), 273 (90, [M - NH2 + H]+).

NDI Hydrazide 9. Amplifier 9 was prepared in 5 steps as follows (see Scheme S3 for

structures):

Compound 20 (S4). HBTU (4.17 g, 11 mmol) and triethylamine (1.5 ml, 11 mmol) were

added to a solution of Z-Ala-OH (19, 2.23 g, 10 mmol) in DMF (30 ml) at 0 °C. After 5 min

stirring, t-butyl carbazate (1.45 g, 11 mmol) was added to the mixture. After stirring for 1

hour at room temperature, the reaction mixture was pored into 1 M aqueous KHSO4, extracted

with EtOAc, washed with sat. aqueous Na2CO3 and brine, dried over anhydrous Na2SO4, and


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concentrated in vacuo. The crude product was triturated with pet. ether/CH2Cl2 (1:1), filtered

and concentrated in vacuo to yield pure 20 (3.28 g, 97%) as colorless powder. TLC

(CH2Cl2/MeOH 6/1): Rf 0.50; mp: 146-147 °C; IR: 3316 (m), 3237 (w), 1679 (s), 1553

(m), 1530 (s), 1367 (s), 1339 (m), 1260 (m), 1243 (s), 1160 (s), 1015 (s), 693 (s); 1H NMR

(400 MHz, DMSO-d6): 9.59 (s, 1H), 8.72 (s, 1H), 7.42-7.20 (m, 6H), 5.01 (s, 2H), 3.21 (q,

3J (H,H) = 6.8 Hz, 2H), 2.28 (t, 3J (H,H) = 7.2 Hz, 2H), 1.39 (s, 9H); 13C NMR (100 MHz,

DMSO-d6): 169.9 (s), 156.0 (s), 155.3 (s), 137.1 (s), 128.4 (d), 127.8 (d), 127.8 (d), 79.1

(s), 65.2 (t), 36.7 (t), 33.5 (t), 28.1 (q); ESI MS (MeOH): m/z 355 (100, [M + H2O + H]+),

338 (75, [M + H]+), 282 (66, [M - t-Bu + H]+), 238 (50, [M - Boc + H]+).

Compound 21 (S4). Pd/C (10% on charcoal, 424 mg, 0.4 mmol) was added to a solution of

20 (1.38 g, 4.1 mmol) in MeOH (15 ml). The suspension was degassed and set under a H2

atmosphere. After stirring for 1 hr, the reaction mixture was filtered and the filtrate was

concentrated in vacuo to yield 21 (831 mg, quant.) as colorless gum. IR: 3261 (m), 2980

(m), 1669 (m), 1481 (m), 1367 (m), 1246 (s), 1158 (s), 1046 (w), 1016 (w), 845 (m), 733 (s),

701 (m); 1H NMR (400 MHz, DMSO-d6): 2.74 (t, 3J (H,H) = 6.6 Hz, 2H), 2.16 (t, 3J (H,H)

= 6.6 Hz, 2H), 1.39 (s, 9H); 13C NMR (100 MHz, DMSO-d6): 171.2 (s), 155.3 (s), 79.0 (s),

38.2 (t), 37.3 (t), 28.1 (q); ESI MS (MeOH): m/z 204 (100, [M + H]+).

Compound 22. To a suspension of 1,4,5,8-naphthalenetetracarboxylicacid dianhydride 16

(1.00 g, 3.7 mmol) in water (25 ml), aqueous potassium hydroxide (1.50 g, 22 mmol in 5 ml

water) was added at 80 °C. After stirring for 15 min at 80 °C, concentrated phosphoric acid

(ca. 1 ml) was added to it to adjust the pH around 5 to 7. To this, -alanine 17 (330 mg, 3.7

mmol) was added and the mixture was stirred at 110 °C overnight. After cooling down to


S7

room temperature, the mixture was acidified with 1 M HCl (20 ml) and the resulted

suspension was stirred for 0.5 hour at 80 °C. The precipitated materials were filtered and

dried in vacuo to obtain crude product 18 (1.27 g). The crude product 18 (1.27 g) was

dissolved in DMF, amine 21 (830 mg, 4.1 mmol) was added, and the mixture was stirred at

110 °C for 6 hours. After cooling down to rt, the reaction mixture was poured into 0.1 M HCl

(150 ml). The resulting precipitate was filtrated and purified by column chromatography

(CH2Cl2/MeOH/AcOH 20:1:0.1) and washing with MeOH/AcOH (1:1) yielded 22 (1.06 g,

54%, 2 steps) as yellow powder. TLC (CH2Cl2/MeOH 10:1): Rf 0.44; mp: > 300 °C; IR:

3239 (w), 2981 (w), 1704 (m), 1691 (m), 1652 (s), 1616 (m), 1581 (m), 1455 (m), 1339 (s),

1239 (s), 1160 (s), 1037 (m), 765 (s); 1H NMR (400 MHz, DMSO-d6): 12.30 (s, 1H), 9.72

(s, 1H), 8.76 (s, 1H), 8.51 (s, 4H), 4.31-4.12 (m, 4H), 2.71-2.43 (m, 4H); 13C NMR (100

MHz, DMSO-d6): 172.4 (s), 172.0 (s), 169.4 (s), 162.2 (s), 155.3 (s), 130.3 (d), 126.1 (s),

125.9 (s), 125.8 (s), 125.7 (s), 79.1 (s), 36.4 (t), 36.1 (t), 31.9 (t), 30.9 (t), 28.0 (q); ESI MS

(MeOH/DMSO 1:2): m/z 547 (15, [M + Na]+), 525 (13, [M + H]+), 469 (46, [M - tBu + H]+),

425 (33, [M - Boc + H]+), 393 (100, [M - Boc - NHNH2 + H]+).

Compound 9. A solution of 22 (440 mg, 0.84 mmol) in TFA (3 ml) was stirred for 3 hours at

rt. After removal of TFA by N2 air, the residue was triturated with MeOH, separated by

centrifuge, washed with MeOH and dried in vacuo to yield pure product 9 (240 mg, 67%) as

yellow powder. mp: > 300 °C; IR: 3311 (m), 3078 (w), 1698 (s), 1647 (s), 1581 (s), 1456

(m), 1338 (s), 1241 (s), 1173 (s), 1032 (s), 1001 (m), 892 (m), 768 (s); 1H NMR (400 MHz,

DMSO-d6/TFA-d 3/1): 8.65 (s, 4H), 4.40-4.22 (m, 4H), 2.73-2.57 (m, 4H); 13C NMR (100

MHz, DMSO-d6/TFA-d 3/1): 172.2 (s), 169.4 (s), 162.1 (s), 162.0 (s),130.2 (d), 125.9 (s),

125.8 (s), 125.7 (s), 36.0 (t), 31.7 (t), 30.9 (t); ESI MS (DMSO /TFA 3:1): m/z 425 (95, [M +


S8

H]+), 407 (33, [M - NH2 + H]+), 393 (100, [M - NHNH2 + H]+).

1.4 Vesicle Preparation (EYPC-LUVs CF). As in refs (S1) or (S2). The final stock

solutions had the following characteristics:

EYPC-LUVs CF (HEPES): ~2.5 mM EYPC; inside, 50 mM CF, 10 mM NaCl, 10

mM HEPES, pH 7.4; outside, 107 mM NaCl, 10 mM HEPES, pH 7.4.

EYPC-LUVs CF (Tris): ~2.5 mM EYPC; inside, 50 mM CF, 10 mM NaCl, 10 mM

Tris, pH 7.4; outside, 10 mM Tris, 107 mM NaCl, pH 7.4

In the following, EYPC-LUVs CF with matching buffer were used.

1.5. Blockage of Pore 1 by Pyruvate (General procedure for signal transduction).

Analogous to refs (S1) or (S2): 100 l of above stock solutions of EYPC-LUVs CF were

added to 1.90 ml gently stirred, thermostated buffer in a fluorescence cuvette (10 mM Tris,

107 mM NaCl, pH 7.5). Fluorescence emission intensity Ft ( ex 492 nm, em 517 nm) was

monitored as a function of time during addition of pyruvate (from concentrated stock

solutions in water, final concentrations 1-98 mM, compare Fig. S2), pore 1 (20 l of 37.5 M

DMSO stock solution, final concentration 375 nM monomer 1m; 94 nM tetramer 1) and 40

l 1.2% aq triton X-100 for final calibration. The fluorescence time courses were normalized

to fractional emission intensity I using

I = (Ft – F0) / (F – F0) [S1],

where F0 = Ft at pore addition, F = Ft at saturation after lysis (Fig. S2). The obtained I was

further converted into fractional pore activity


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Y = (IF – IFMIN) / (IF

MAX – IFMIN) [S2],

where IF is I before lysis, IFMAX is the maximal IF and IF

MIN the minimal IF of a dose response

experiment. Fractional pore activities Y were fitted to the Hill equation

Y = Y + (Y0 – Y ) / {1 + (cBLOCKER / IC50)n} [S3],

where Y0 is Y without ligand, Y is Y with excess ligand, IC50 the concentration for 50%

inhibition and n the Hill coefficient (Fig. S3, ).

1.6. Signal Amplification

1.6.1. Pyruvate-CB Hydrazide (3). Solutions of 2 (500 M) and pyruvate (10-500 M) in a

1:1 mixture of DMSO and 0.4 M NaOAc/HOAc (pH 4.5) were incubated for 20 min at 50 °C.

The relative emission intensity Ft ( em = 517 nm, ex = 492 nm) was then recorded as a

function of time t during addition of 20 µL aliquots of these reaction mixtures to EYPC-LUVs

CF (100 µL) in 1.90 ml gently stirred, thermostated buffer in a fluorescence cuvette (10

mM Tris, 107 mM NaCl, pH 7.5), followed by addition of pore 1 (375 nM 1m) and Triton-X

100 (40 l, 1.2%), normalized and analyzed. Assuming the quantitative reaction, the

concentration of pyruvate-amplifier conjugate 3 was calculated based on the concentration of

pyruvate. A formal IC50 = 2.6 ± 0.2 M was obtained following the analysis described in

above section 1.5 (Fig. S3, ).


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Controls included dose response curves for 2 (Fig. S3, ) and pyruvate (Fig. S3, ),

and the dependence of hydrazone formation on pH (Fig. S4) (S5). Pertinent IC50s determined

for amplifier 2 compared to analytes and analyte-amplifier conjugates are listed in Tab. S1.

1.6.2. DAN or NDI hydrazones. Solutions of 5 or 9 (50 mM) and pyruvate or -ketoglutarate

(500 mM) in DMSO (10 eq) were incubated for 2 h at 50 °C. The reaction mixture was

diluted with buffer (10 mM HEPES, 107 mM NaCl, pH 6.5) adjusted to pH 6.5 if necessary,

and further diluted to give 2-4 mM stock solutions.

The relative emission intensity Ft ( em = 517 nm, ex = 492 nm) was then recorded as a

function of time t during addition 20 µL aliquots of these stock solutions to EYPC-LUVs

CF in 1.90 ml gently stirred, thermostated buffer in a fluorescence cuvette (10 mM HEPES,

107 mM NaCl, pH 6.5), followed by pore 1 (375 nM 1m) or pore 4 (375 nM 4m, 20 l 37.5

M DMSO stock solution) and Triton-X 100 (40 l, 1.2%), normalized and analyzed to

obtain formal IC50s as described in above section 1.5. The concentration of analyte-amplifier

conjugates was estimated based on the concentration of 5 or 9, assuming the quantitative

reaction. Pertinent IC50s determined for amplifiers 5 and 9 compared to analytes and analyte-

amplifier conjugates are listed in Tab. S1.

1.7. Lactate Sensing

1.7.1. Concept. In order to detect a specific component from a complex mixture, we

envisioned enzymes as specific signal generators (1). In the case of lactate sensing, lactate

oxidase was expected to specifically oxidize lactose to give pyruvate (Fig. S5). As we have

reported previously, enzymatic reactions to generate or consume pore blockers could be easily


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detected by simply following the change in pore blockage ability of the enzymatic reaction

mixture (S6). Thus, by determining the difference in blockage ability of the test sample

before and after enzymatic reaction, the concentration of particular enzyme substrate could be

determined (S6). Although both lactate and pyruvate could serve as pore blockers, their IC50

values are too high to be useful for lactate sensing in the dairy products. This problem of

sensitivity was overcome by introduction of anionic CB hydrazide (2). Namely, as hydrazides

selectively react with ketones or aldehydes, among all the compounds involved in the

enzymatic reaction only pyruvate should form a conjugate with the CB core, giving a potent

blocker. The exceptional affinity between CB and rigid-rod barrel 1 was observed

previously (S1). As described in section 1.6.1, the IC50 of pyruvate - CB hydrazone (3) was

much lower than pyruvate, lactate, or also CB-hydrazide (2). The reaction catalyzed by

lactose oxidase should therefore lead to a decrease in pore activity (increase in blockage

ability of the test sample).

1.7.2. Enzyme Activity. Lactate (10 mM) was incubated with lactate oxidase (0, 2.5 and 10

units/ml, EC 1.1.3.2, Pediococcus species) and catalase (2200 units/ml, EC 1.11.1.6, bovine

liver) in buffer (50 mM KH2PO4, pH 6.5) at 37 oC (S7). In meaningful intervals, 5 l of the

reaction mixture were taken, added to 45 l of a solution of 2 (500 M) in a 1:1 mixture of

DMSO and 0.4 M NaOAc/HOAc (pH 4.5), and incubated for 20 min at 50 °C. From the

resulting reaction mixture, 20 l were added to 1980 l EYPC-LUVs CF (100 l LUV

stock (1.4) with 1880 l 10 mM Tris, 107 mM NaCl, pH 7.5), CF emission intensity It ( em =

510 nm, ex = 495 nm) was monitored during the subsequent addition of pore 1 (375 nM 1m

final concentration) and triton X-100 (40 µL, 1.2% aq.), and fractional pore activity Y at each


S12

reaction time was determined from fluorescence kinetics using eq [S1] and [S2] following the

procedure described in 1.5 and plotted as a function of reaction time t (Fig. S5).

1.7.3. Calibration Curve. Lactate (cAnalyte = 0.01 - 10 mM) was incubated with lactate

oxidase (10 units/ml, EC 1.1.3.2, Pediococcus species) and catalase (2200 units/ml, EC

1.11.1.6, bovine liver) in buffer (50 mM KH2PO4, pH 6.5) at 37 oC, total volume Vo = 100 l.

After 20 min (see Fig. S5), 5 l of the reaction mixture was taken, added to 45 l of a solution

of 2 (500 M) in a 1:1 mixture of DMSO and 0.4 M NaOAc/HOAc (pH 4.5), and incubated

for 20 min at 50 °C. From the resulting reaction mixture, 20 µL aliquots were then added to

EYPC-LUVs CF (100 l stock with 1880 l 10 mM Tris, 107 mM NaCl, pH 7.5). CF

emission intensity It ( em = 510 nm, ex = 495 nm) was monitored during addition of pore 1

(375 nM 1m) and triton X-100 (40 µL, 1.2%) and fractional pore activity Y was determined

from fluorescence kinetics using eq [S1] and [S2] following the procedure described in 1.5

and plotted as a function of lactate concentration in the enzymatic reaction (cAnalyte, Fig. S6A).

Fitting of the data to eq [S3’],

Y = Y + (Y0 – Y ) / {1 + (cAnalyte / c50)n} [S3’]

where Y0 is Y without blocker, Y is Y with excess blocker, c50 the concentration of lactate for

50% blockage of pore 1 after enzymatic oxidation and amplification and n the Hill coefficient,

revealed c50 = 750 ± 90 M. With 10x dilution during amplification and 100x dilution during

transduction, this calculated to an IC50 = 0.75 M that was in the range of the IC50 = 2.6 M

determined for pure, enzyme-free pyruvate under different, overall less complex conditions

(Fig. S3, , Tab. S1, entry 3).


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1.7.4. Rivella Red. Rivella Red (V = 1-40 µl) (S8) was diluted to 100 l with buffer (50 mM

KH2PO4, pH 6.5), incubated with lactate oxidase / catalase, amplified with 2 (10x dilution),

and transduced with EYPC-LUVs CF plus pore 1 (100x dilution) as described in section

1.7.3. Data were analyzed using eq [S3”],

Y = Y + (Y0 – Y ) / {1 + (V / V50)n} [S3”]

where Y0 is Y without ligand, Y is Y with excess ligand, V50 the volume of Rivella Red used

to achieve 50% blockage of pore 1 after enzymatic oxidation / amplification and n the Hill

coefficient, to reveal an effective initial volume V50 = 13.4 ± 2.8 l (Fig. S6B). In parallel,

control experiments were performed with the sample prepared in nearly identical manner, but

without exposure to the enzymes. Namely, 50 l of Rivella Red was amplified with 2 (total

volume 100 l, 2x dilution) and transduced with EYPC-LUVs CF plus pore 1 (100x

dilution). No blockage observed with this sample indicated V50B >> 250 l.

1.7.5. Determination of lactate concentration in Rivella Red. The obtained values c50 (1.7.3),

V50, and V50B (1.7.4) can be correlated to the concentration of lactate (c) and ketone /

aldehyde: e.g., pyruvate (cP) present in Rivella Red as in the eq [S4] and [S5].

cP V50B = c50 V0 [S4]

(c + cP) V50 = c50 V0 [S5]


S14

where V0 is the volume of sample used for calibration (here V0 = 100 l), c50 the effective

lactate concentration that causes 50% pore blockage after enzymatic reaction / amplification

(here c50 = 750 ± 90 M, Fig. S6A), V50 the sample volume that causes 50% pore blockage

after enzymatic reaction / amplification (here V50 = 13.4 ± 2.8 l, Fig. S6B), and V50B the

sample volume that causes 50% pore blockage after dilution (without enzymatic reaction) and

amplification (here V50B >> 250 l, V0 / V50

B = negligibly low). Rearrangement of eqs [S4]

and [S5] gives [S6].

c = (V0 c50 / V50) - (V0 c50 / V50B) [S6]

Using eq [S6], a lactate concentration in Rivella Red c = 5.6 ± 1.1 mM was found.

1.7.6. Sour Milk. Sour milk (S9) was centrifuged and the supernatant was filtered through a

cotton plug. The filtrate (source phase) was dialyzed against 1 ml of buffer (receiving phase,

50 mM KH2PO4, pH 6.5) for 5 h at 4 oC (cut off = 5 kD), followed by the replacement of the

source phase with the fresh filtrate, and further dialysis (5 h at 4 oC, 1.33x dilution). The pH

of the receiving phase was adjusted to pH 6.5 (1.37x dilution). Aliquots of the resulting sour

milk sample (vS = 0.1 - 20 µl) were incubated with lactate oxidase / catalase (total volume 100

l), amplified with 2 (10x dilution), and transduced with EYPC-LUVs CF plus pore 1

(100x dilution) as described in 1.7.3 (calibration). Data analysis using [S3”] (here, V is the

calculated amount of original sour milk in the enzymatic reaction mixture: V = 0.55 vS)

revealed an effective initial volume V50 = 1.26 ± 0.10 l under these conditions (Fig. S6C).

The control experiments using the sample prepared in nearly identical manner but without


S15

exposure to the enzymes revealed the presence of pyruvate equivalents in sour milk (V50B =

14.4 ± 0.6 l).

The lactate concentration cL = 54.3 ± 4.2 mM in sour milk was calculated by applying

V0 = 100 l, c50 = 750 ± 90 M, V50 = 1.26 ± 0.10 l and V50B = 14.4 ± 0.6 l to equation [S6]

(Tab. S2). A lactate concentration c = 53.6 ~ 86.1 mM was expected (S10).

1.8. Citrate Sensing

1.8.1. Concept. By citrate lyase, citrate can be converted into oxaloacetate which is prone to

spontaneous decarboxylation giving rise to pyruvate (Fig. S7). Both pyruvate and

oxaloacetate could react with CB amplifier 2 to generate potent blockers of pore 1. Thus, the

pyruvate detection method developed in section 1.7 could be applied for citrate sensing.

1.8.2. Enzyme Activity. Citrate (10 mM) was incubated with citrate lyase (0.4 unit/ml, EC

4.1.3.6, Enterobacter aerogenes [from 10 u/ml stock solution prepared by dissolving 4.3 mg

in 1 ml buffer (454 mM (NH4)2SO4, 0.3 mM ZnCl2, 10 mM triethanolamine (HCl), pH 7.6)])

and ZnCl2 (5 mM) (S11) in 100 mM triethanolamine (HCl), pH 7.6 at 30 oC. After 5 min, 5

l of the reaction mixture was taken, added to 45 l of a solution of 2 (500 M) in a 1:1

mixture of DMSO and 0.4 M NaOAc/HOAc (pH 4.5), and incubated for 20 min at 50 °C.

From the resulting reaction mixture, 20 l were added to 1980 l EYPC-LUVs CF (100 l

LUV stock (1.4) with 1880 l 10 mM Tris, 107 mM NaCl, pH 7.5), CF emission intensity It

( em = 510 nm, ex = 495 nm) was monitored during the subsequent addition of pore 1 (375

nM 1m final concentration) and triton X-100 (40 µL, 1.2 %), and fractional pore activity Y


S16

was determined from fluorescence kinetics using eq [S1] and [S2] following the procedure

described in 1.5.

This procedure was repeated with 5 l enzymatic reaction mixture, amplified after 10

and 26 min reaction time, and the fractional pore activity Y was plotted as a function of

reaction time t (Fig. S7, ). Control experiments included repetition of above set of

experiments with more (1.0 units/ml) and no citrate lyase (Fig. S7, and ).

1.8.3. Calibration Curve. Citrate (cAnalyte = 0.01 - 10 mM) was incubated with citrate lyase

(1.0 units/ml, EC 4.1.3.6, Enterobacter aerogenes) and ZnCl2 (5 mM) in buffer (100 mM

triethanolamine (HCl), pH 7.6) at 30 oC, total volume Vo = 50 l. After 20 min (see Fig. S7,

), 5 l of the reaction mixture were taken, added to 45 l of a solution of 2 (500 M) in a

1:1 mixture of DMSO and 0.4 M NaOAc/HOAc (pH 4.5), and incubated for 20 min at 50 °C.

From the resulting reaction mixture, 20 l were added to 1980 l EYPC-LUVs CF (100 l

LUV stock (1.4) with 1880 l 10 mM Tris, 107 mM NaCl, pH 7.5), CF emission intensity It

( em = 510 nm, ex = 495 nm) was monitored during the subsequent addition of pore 1 (375

nM 1m final concentration) and triton X-100 (40 µL, 1.2 %), and fractional pore activities Y

were determined from fluorescence kinetics using eq [S1] and [S2] following the procedure

described in 1.5 and plotted as a function of citrate concentration (cAnalyte, Fig. S8A). An

effective initial citrate concentration c50 = 650 ± 75 M exposed originally to enzymatic

signal generation was found using eq [S3’] to finally block pore 1 by 50%, i.e., to correspond

to the IC50 under these conditions. With 10x dilution during amplification and 100x dilution

during transduction, this calculated to an IC50 = 0.65 M that was in the range of the IC50 =

2.6 M determined for pure, enzyme-free pyruvate and the IC50 = 1.3 M determined for

pure, enzyme-free oxaloacetate under different, overall less complex conditions. (Fig. S3, ,


S17

Tab. S1, entries 3 and 7). The question whether or not oxaloacetate decarboxylated to

pyruvate during signal generation or amplification was not investigated because it is irrelevant

for citrate sensing.

1.8.4. Orange Juice. Orange juice (S12) was subjected to centrifugal filtration (8 x 103

rev/min, cut off 3 kD). The filtrate (500 l) was incubated with 15 mg of polymer-bound p-

toluenesulfonyl hydrazide (2.3 mmol/g) for 8.5 h at 37 oC (Fig. S9, 1.8.5). After removal of

the resin by filtration, the pH was adjusted to 7.6 (1.04x dilution). Aliquots of this sample (vo

= 0.05 - 10 µl) were incubated with citrate lyase / ZnCl2 (total volume 50 l), amplified with

2 (10x dilution), transduced with EYPC-LUVs CF plus pore 1 (100x dilution) as described

in 1.8.3 (calibration) and analyzed using eq [S3”] to reveal an effective initial volume V50 =

0.71 ± 0.08 l as effective final IC50 under these conditions (V = vo / 1.04, Fig. S8B). Nearly

identical experiments without exposure to enzyme were performed to reveal V50B >> 3 l

(compare 1.8.5). The citrate concentration c in orange juice was calculated by applying V0 =

50 l, c50 = 650 ± 75 M, V50 = 0.71 ± 0.08 l and V50B >> 3 l (V0 / V50

B = negligibly low) to

equation [S6]. In orange juice, a citrate concentration c = 43.1 ~ 44.7 mM was expected (S13,

S14), c = 45.8 ± 5.2 mM was found (Tab. S2). With routine NMR assays (filtration over

celite, lyophilization, integration of citrate resonance against internal standard, (S13)), a

citrate concentration c = 51.1 ± 0.7 mM was found despite clear differences in sample

preparation (different bottles).

1.8.5. Reactive Filtration (Fig. S9). Orange juice (3 l) was subjected to signal generation

and amplification with (a) and without (b) preceding incubation with 15 mg of polymer-bound

p-toluenesulfonyl hydrazide (2.3 mmol/g) for 8.5 h at 37 oC. Clear difference in blockage of

pore 1 by these samples (Fig. S9, b vs c) confirmed the extensive interferences from intrinsic


S18

ketones and aldehydes in orange juice, and the practical relevance of reactive filtration before

signal generation and amplification.

1.9. Lactose Sensing

1.9.1. Concept. Using -galactosidase (S15) and hexokinase (S16) instead of invertase and

hexokinase but otherwise analogous to sucrose sensing (S17), the concentration of lactose can

be determined by monitoring the consumption of ATP (Fig. S10). Namely, lactose is

hydrolyzed by -galactosidase to give galactose and glucose. Only the latter is

phosphorylated by hexokinase to give glucose-6-phosphate. As IC50 of ATP is lower than that

of the other molecules involved in the sequence of reactions, the potency of the reaction

mixture to block the pore relates to the concentration of ATP. By monitoring the enzyme

catalyzed lowering of blockage ability, the concentration of consumed ATP can be

determined, which is equal to the concentration of lactose. This definition is based on the

(experimentally verified (S18)) facts that -galactosidase converts lactose but not sucrose

(S15, S19), and hexokinase converts glucose and fructose but not galactose (S16, S19).

1.9.2. Enzyme Activity. Lactose (33 mM) was incubated with -galactosidase (10 units/ml,

EC 3.2.1.23, Aspergillus oryzae) in buffer (53 ml, 9.4 mM Na2HPO4, 5.3 mM citric acid, pH

4.5) at 30 oC (S15). After 20 min, 150 l of the reaction mixture were taken and added to 350

l of a solution of hexokinase (0.47 units/ml, EC 2.7.1.1, Baker’s yeast), ATP (10 mM) and

MgCl2 (10 mM) in buffer (100 mM Tris, pH 8.0, S16, S17) and incubated at 30 °C (500 l,

3.33x dilution). After 20 min, 20 l were taken from the resulting reaction mixture and added

to 1980 l EYPC-LUVs CF (100 l LUV stock (1.4) with 1880 l 10 mM HEPES, 107


S19

mM NaCl, pH 6.5), CF emission intensity It ( em = 510 nm, ex = 495 nm) was monitored

during the subsequent addition of pore 1 (375 nM 1m final concentration) and triton X-100 (40

µL, 1.2%), and fractional pore activity Y (at reaction time t = 20 min) was determined from

fluorescence kinetics using eq [S1] and [S2] following the procedure described in 1.5.

This procedure was repeated with 20 l reaction mixture taken after 3, 40, 60, 80 and

100 min hexokinase reaction time, and the fractional pore activity Y was plotted as a function

of reaction time t (Fig. S10, ). Control experiments included repetition of above set of

experiments with more (2.4 units/ml, 0.95 units/ml) and no hexokinase (Fig. S10, , and

).

1.9.3. Calibration Curve. 20 l ATP (1~10 mM) and MgCl2 (10 mM) (S16, S17) were added

to 1980 l EYPC-LUVs CF (100 l LUV stock (1.4) with 1880 l 10 mM HEPES, 107

mM NaCl, pH 6.5, 25 oC), CF emission intensity It ( em = 510 nm, ex = 495 nm) was

monitored during the subsequent addition of pore 1 (375 nM 1m final concentration) and triton

X-100 (40 µL, 1.2 %), and fractional pore activities Y were determined from fluorescence

kinetics using eq [S1] and [S2] following the procedure described in 1.5 and plotted as a

function of ATP concentration in the presence of 100 M MgCl2 (Fig. S11A). An IC50 = 40.1

± 3.3 M was found for ATP under these conditions (Mg2+ is known to reduce ATP

recognition by pore 1 (S17)).

1.9.4. Rivella Red. Rivella Red (S8) (400 or 600 l) was incubated first with -galactosidase

(a) 10 units/ml, b) 0 units/ml; total volume 1 ml). The resulting mixture (vRR = 3 - 40 µl) was

treated with hexokinase (2.4 units/ml, Fig. S10) and ATP (10 mM, V0 = 250 l), and then

transduced with EYPC-LUVs CF plus pore 1 (100x dilution) as described in 1.9.2 to reveal


S20

an effective initial volumes (a) V50 10.6 ± 0.4 l with -galactosidase incubation and (b) V50B

= 20.5 ± 0.9 l without -galactosidase incubation that cause 50% pore blockage ((here V =

0.4 (or 0.6) vRR, Fig. S11B). The lactose concentration c in Rivella Red was calculated by

applying V0 = 250 µl (hexokinase reaction), c50 = 4.0 ± 0.3 mM (IC50 (ATP) x 1000), V50 =

10.6 ± 0.4 l and V50B = 20.5 ± 0.9 l to equation [S6]. In Rivella Red, a lactose

concentration c = 44.0 ± 1.0 mM (15.2 ± 0.5 g/L) was found.

1.9.5. Milk Serum. Cow milk (S20) was kept for 1 day at 25 oC, centrifuged and the

supernatant was filtered through cotton. The obtained milk serum (1 ml) was dialyzed against

buffer (1 ml; a) 9.4 mM Na2HPO4, 5.3 mM citric acid, pH 4.5; b) 10 mM Tris, pH 5.0) at 23

oC for 6.5 h. Aliquots of dialyzed milk serum (receiving phase a, 50, 100, 150 µl) were

incubated first with -galactosidase (10 units/ml, total volume 500 l, 9.4 mM Na2HPO4, 5.3

mM citric acid, pH 4.5). Then aliquots (vm = 10 ~ 50 l) were incubated with hexokinase (2.4

units/ml, Fig. S10) and ATP (10 mM; 10 mM MgCl2, 100 mM Tris, pH 8.0; 100 l), and then

transduced with EYPC-LUVs CF plus pore 1 (100x dilution) as described in 1.9.2 to reveal

an effective initial volume V50 = 3.3 ± 0.2 l that causes 50% pore blockage (here, V = 0.1 (or

0.2, 0.3) vm, Fig. S11C). In parallel, an aliquot of dialyzed milk serum (receiving phase b, 37

l) was treated with hexokinase and transduced. Lack of blockage activity observed with the

resulting solution indicated V50B >> 37 l (V0 / V50

B negligible).

The lactose concentration c in milk serum was calculated by applying V0 = 100 µl

(hexokinase reaction), c50 = 4.0 ± 0.3 mM (IC50 (ATP) x 1000), V50 = 3.3 ± 0.2 l and V50B >>

37 l (V0 / V50B = negligibly low) to equation [S6]. In cow milk, a lactose concentration c =

114 ~ 140 mM was expected (S21 - S23), c = 121.5 ± 5.5 mM (= 41.6 ± 1.9 g/L) was found

(Tab. S2).


S21

1.10. Acetate Sensing

1.10.1. Concept. As in lactose sensing, the concentration of acetate can be determined by

following the consumption of ATP in the enzymatic phosphorylation of acetate (Fig. S12).

1.10.2. Enzyme Activity. Sodium acetate (300 mM) was incubated with acetate kinase (1.0

units/ml, EC 2.7.2.1, Escherichia coli), ATP (10 mM), MgCl2 (10 mM) and NH2OH (270

mM) in buffer (50 mM triethanolamine (HCl), pH 7.6) at 29 °C) (S24 - S26). After 10 min,

20 l were taken from the resulting reaction mixture and added to 1980 l EYPC-LUVs CF

(100 l LUV stock (1.4) in 1880 l 10 mM HEPES, 107 mM NaCl, pH 6.5, 25 ºC), CF

emission intensity It ( em = 510 nm, ex = 495 nm) was monitored during the subsequent

addition of pore 1 (375 nM 1m final concentration) and triton X-100 (40 µl, 1.2 %), and

fractional pore activity Y at reaction time t = 10 min was determined from fluorescence

kinetics using eq [S1] and [S2] following the procedure described in 1.5.

This procedure was repeated with 20 l reaction mixture taken after 30 and 65 min

reaction time, and the fractional pore activity Y was plotted as a function of reaction time t

(Fig. S12, ). Control experiments included repetition of above set of experiments with more

(3 units/ml) and no acetate kinase (Fig. S12, and ).

1.10.3. Calibration Curve. Sodium acetate (cAnalyte = 10 - 300 mM) was incubated with

acetate kinase (3.0 units/ml, EC 2.7.2.1, Escherichia coli) ATP (10 mM), MgCl2 (10 mM) and

NH2OH (270 mM) in buffer (50 mM triethanolamine (HCl), pH 7.6) at 29 °C, total volume V0

= 100 l. After 1 h (see Fig. S12), 20 l of the reaction mixtures were taken and added to


S22

1980 l EYPC-LUVs CF (100 l LUV stock (1.4) with 1880 l 10 mM HEPES, 107 mM

NaCl, pH 6.5, 25 ºC), CF emission intensity It ( em = 510 nm, ex = 495 nm) was monitored

during the subsequent addition of pore 1 (375 nM 1m final concentration) and triton X-100 (40

µL, 1.2 %), and fractional pore activities Y were determined from fluorescence kinetics using

eq [S1] and [S2] following the procedure described in 1.5 and plotted as a function of acetate

concentration (Fig. S13A). An effective initial acetate concentration c50 = 44.8 ± 2.9 mM

exposed to enzymatic signal generation was found using eq [S3’] to finally block pore 1 by

50%, i.e., to correspond to the apparent IC50 under these conditions.

1.10.4. Rice Vinegar. The pH of Japanese rice vinegar (S27) (2.5 ml) was adjusted to 7.6

(1.1x dilution). A rice vinegar sample (vv = 1.5 - 30 l) was incubated with acetate kinase

(3.0 units/ml, EC 2.7.2.1, Escherichia coli) and ATP (10 mM; 10 mM MgCl2, 270 mM

NH2OH, 50 mM triethanolamine (HCl), pH 7.6; total volume 100 l), and then transduced

with EYPC-LUVs CF plus pore 1 (100x dilution) as described in 1.10.3 to reveal an

effective initial volume V50 = 6.6 ± 0.7 l that causes 50% pore blockage (here, V = 0.9 vv,

Fig. S13B). The acetate concentration in rice vinegar was calculated by applying V0 = 100 µl

(acetate kinase reaction), c50 = 44.8 ± 2.9 mM, V50 = 6.6 ± 0.7 l and V50B = n.d. (V0 / V50

B =

negligibly low) to equation [S6]. In rice vinegar, an acetate concentration c = 658 ~ 770 mM

was expected (S27, S28), c = 757 ± 80 mM was found (Tab. S2).

1.11. Molecular Modeling

The pore 4 was assembled and pre-optimized (100 iterations) using Maestro 4.1 graphical

interface (S29) coupled with MacroModel version 7.0 (S30, S31). MMFF94s force field (S32,


S23

S33) and the Polak-Ribiere conjugate gradient (PRCG) algorithm were used to eliminate close

contacts. Geometry optimizations together with molecular dynamic (MD) simulations were

carried out with AMBER 9 (S34) package using parm99 and gaff force fields (S35, S36).

Using the xLEaP module (S37) one or two ligands 6 were placed into the pore 4, which was

beforehand processed by MD simulations for 1 ns. The ligand-barrel complexes obtained with

xLEaP were further optimized (rmsd<0.05[kcal/mol], cutoff=50.0Å) with Sander module.

Each complex was heated to 300°K during 50 ps using Cartesian restraints of 2.0 [kcal/mol]

followed by short relaxation (50ps) with 1.0 [kcal/mol] restraints and then equilibrated in a

simulation of 900 ps. For complexes with substrate 6, an additional period of Cartesian

restraints during 400 ps was included in the MD simulation. All structures have been

subjected to the equilibration of at least 500 ps in vacuo where all atoms of the complexes

were free to move. Geometry issued from MD simulations of open pore 4 is shown in Fig.

S14. MD simulated structures of pore 4 with one and two -ketoglutarate-amplifier

conjugates 6 are displayed in Figs. S15 and S16, respectively. The structures shown in Figs.

S14-S16 were obtained after optimization from the average geometry from the last 50 ps of

the MD simulation.

1.12. Supporting References

S1. Litvinchuk, S., Bollot, G., Mareda, J., Som, A., Ronan, D., Shah, M. R., Perrottet, P.,

Sakai, N. & Matile, S. J. Am. Chem. Soc. 126, 10067-10075 (2004).

S2. Tanaka, H., Litvinchuk, S., Tran, D.-H., Bollot, G., Mareda, J., Sakai, N. & Matile, S. J.

Am. Chem. Soc. 128, 16000-16001 (2006).


S24

S3. Whitaker, J. E., Haugland, R. P., Moore, P. L., Hewitt, P. C., Reese, M. & Haugland, R. P.

Anal. Biochem. 198, 119-130 (1991).

S4. King, H. D., Yurgaitis, D., Willner, D., Firestone, R. A., Yang, M. B., Lasch, S. J.,

Hellstrom, K. E. & Trail, P. A. Bioconjugate Chem. 10, 279-288 (1999).

S5. Shao, J. & Tam, J. P. J. Am. Chem. Soc. 117, 3893-3899 (1995).

S6. Das, G.; Talukdar, P. & Matile, S. Science 298, 1600-1602 (2002).

S7. Gavagan, J. E. Fager, S. K., Seip, J. E., Payne, M. S., Anton, D. L. & DiCosimo, R. J.

Org. Chem. 60, 3957-3963 (1995).

S8. www.rivella.ch.

S9. Fjord, www.danone.com/wps/portal/jump/CHD.Produit?ref=3033490178000

S10. F. Mizutani, F., Yabuki, S. & Hirata, Y. Anal Chim. Acta 314, 233-239 (1995).

S11. Singh, M. & Srere, P. A. J. Biol. Chem. 246, 3847-3850 (1971).

S12. www.granini.com

S13. Metzger, A. & Anslyn, E. V. Angew. Chem. Int. Ed. 37, 649-652 (1998).

S14. Saavedra, L., Ruperez, F. J. & Barbas, C. J. Agric. Food Chem. 49, 9-13 (2001).

S15. Bergmeyer, H. U. & Bernt, E. Methods of Enzyme Analysis (Bergmeyer, H. U., ed, 2nd

edition) 3, 1205-1212 (1974).

S16. Raaflaub, J. & Leupin, I. Helv. Chim. Acta 99, 832-837 (1956).

S17. Litvinchuk, S., Sordé, N. & Matile, S. J. Am. Chem. Soc. 127, 9316-9317 (2005).

S18. Litvinchuk, S. & Matile, S., unpublished.

S19. Xu, L. Z., Weber, I. T., Harrison, R. W., Gidh-Jain, M. & Pilkis, S. J. Biochemistry 34,

6083-6092 (1995).

S20. www.coop.ch

S21. Cataldi, T. R. I., Angelotti, M. & Bianco, G. Anal. Chim. Acta 485, 43-49 (2003).


S25

S22. Rajendran, V. & Irudayaraj, J. J. Dairy Sci. 85, 1357-1361 (2002).

S23. Mullin, W. J. Food Res. Int. 30, 147 (1997).

S24. Fox, D. K. & Roseman, S. J. Biol. Chem. 261, 13487-13497 (1986).

S25. Rose, I. A., Grunberg-Manago, M., Korey, S. R. & Ochoa, S. J. Biol. Chem. 211, 737-

756 (1954).

S26. Lipmann, F. & Tuttle, L. C. J. Biol. Chem. 159, 21-28 (1945).

S27. http://www.migros.ch

S28. Mizutani, F. Sawaguchi, T., Sato, Y., Yabuki, S. & Iijima, S. Anal. Chem. 73, 5738-

5742 (2001).

S29. Maestro 4.1, Schrödinger Inc., Portland OR (2001).

S30. MacroModel 7.0, Schrödinger, Inc., Portland OR (1999).

S31. Mohamadi, F., Richards, N. G. J., Guida, W. C., Liskamp, R., Lipton, M., Caufield,

C., Chang, G., Hendrickson, T. & Still, W. C. J. Comput. Chem. 11, 440-467 (1990).

S32. Halgren, T. A. J. Comput. Chem. 17, 490-641 (1996).

S33. Halgren, T. A. J. Comput. Chem. 20, 720-748 (1999).

S34. Case, D. A., Darden, T. A., Cheatham, T. E. III, Simmerling, C. L., Wang, J., Duke, R.

E., Luo, R., Merz, K. M., Pearlman, D. A., Crowley, M., Walker, r. C., Zhang, W.,

Wang, B., Hayik, S., Roitberg, A., Seabra, G., Wong, K. F., Paesani, F., Wu, X.,

Brozell, S., Tsui, V., Gohlke, H., Yang, L., Tan, C., Mongan, J., Hornak, V., Cui, G.,

Beroza, P., Mathews, D. H., Schafmeister, C., Ross, W. S. & Kollman, P. A. (2006),

AMBER 9, University of California, San Francisco.

S35. Wang, J., Cieplak, P. & Kollman, P. A. J. Comput. Chem. 21, 1049-1074 (2000).


S26

S36. Duan, Y., Wu, C., Chowdhury, S., Lee, M. C., Xiong, G., Zhang, W., Yang, R.,

Cieplak, P., Luo, R., Lee, T., Caldwell, J., Wang, J. & Kollman, P. A. J. Comput.

Chem. 24, 1999-2012 (2003).

S37. Schafmeister, C. E. A. F., Ross, W. S. & Romanovski, v. LeaP University of

California, San Francisco (1995).


S27

2. Supporting Figures and Schemes

4m: R =

O

O

O

O

O

OO

R

O

OO

R

O

R

O

R

O

R

O

R

O

R

O

R

O

O

NO

NO

HO

HN

O

NHO

HN

O

NH

HN

O

O

H2N

NH2

HN

O

NHO

HN

O

NH

HN

O

O

H2N

1m: R =

OSO3-

SO3- SO3

-

O

NH2HN

2

-O

O O

O O

HN NH2

O

O

N

O HN

ON

O

O-O

NH2

5

9

HN

N

NH2

Fig S1. Molecular structures of pores and amplifiers synthesized for this study.


S28

time (min)

0.8

Y

0.4

0.2

0

1

0.6

1.0

0 2 3

a)b)c)d)e)f)g)h)i)j)

Fig S2. Pore blockage (signal transduction). Fractional change in CF emission I ( ex 492 nm,

em 517 nm) as a function of time after addition of pyruvate (0 (a), 4.9 (b), 9.8 (c), 19.6 (d),

24.5 (e), 29.4 (f) 39.2 (g), 49.0 (h), 68.6 (i) and 98.0 mM (j)) and 1m (375 nM, arrow) to

EYPC-LUVs CF (~125 M EYPC, 10 mM HEPES, 107 mM NaCl, pH 6.5).


S29

blocker (M)

0.8

Y

0.4

0.2

0

0.6

1.0

10-3 10-2 10-1 110-410-510-610-7

Fig S3. The CB amplifier (signal amplification). Dependence of the fractional activity Y of

pore 1 (375 nM 1m) on the concentration c of CB pyruvate 3 ( ), CB amplifier 2 ( ) and

pyruvate ( ).

pH

0.6

Y

0.4

0.3

0.5

6.05.55.04.5

Fig S4. The CB amplifier (pH dependence). Dependence of the fractional activity Y of pores

1 (375 nM 1m), i.e., the formation of CB pyruvate 3, on the pH of the reaction of CB amplifier

2 and pyruvate (1.6.1, (S5)).


S30

OO

NH2HN

SO3-

-O3S

-O3SO

O

NHN

O-O

SO3-

-O3S

-O3SCB amplifier 2

CB pyruvate 3

O

O-O

pyruvate

OH

O-O

lactate

lactate oxidase

(38 mM)

(23 µM)

(2.6 µM)- H2O

time (min)

0.8

Y

0.4

0.2

010

0.6

1.0

0 20 30 40

Fig S5. The activity of lactate oxidase (signal generation). Fractional activity Y of pore 1 as a

function of incubation t of lactate with 0 ( ), 2.5 ( ) and 10 units/ml ( ) lactate oxidase;

reaction mixtures were amplified with 2 before blockage experiments, for conditions, see

1.7.2. Top: Lactate sensing reaction scheme. Numbers in parentheses are IC50 values.


S31

initial lactate (mM)

0.8

Y

0.4

0.2

0

0.6

1.0

1010.10.01 1

initial Rivella Red (µl)

0.1 10 100

A B

1

initial sour milk (µl)

0.1 10

C

0.01

Fig S6. The lactate sensor. Fractional activity Y of pore 1 as a function of the concentration

of citrate (A) and the volume of Rivella Red (B) and sour milk (C) after incubation with

lactate oxidase and amplification with 2; for conditions, see (A) 1.7.3, (B) 1.7.4 and (C) 1.7.6.

Lactate concentrations in B and C were determined by comparison with calibration curve A

using equation [S6]. See Fig. S5 for lactate sensing reaction scheme.


S32

OO

NHN

O-O

SO3-

-O3S

-O3SO

O-O

O-O

CB amplifier 2

CB pyruvate 3

O

O-O

pyruvate

(38 mM)

(23 µM)

(2.6 µM)

- H2O

oxaloacetate

OH

O-O

citrate

citratelyase

- CO2O

-O

O-O

a)

(0.56 mM)(2.3 mM)

time (min)

0.8

Y

0.4

0.2

010

0.6

1.0

0 20 30

Fig S7. The activity of citrate lyase. Fractional activity Y of pore 1 as a function of

incubation time t of citrate with 0 ( ), 0.4 ( ) and 1.0 units/ml ( ) citrate lyase; reaction

mixtures were amplified with 2 before blockage experiments, for conditions, see 1.8.2. Top:

Citrate sensing reaction scheme. Numbers in parentheses are IC50 values. a) Likely

spontaneous decarboxylation of oxaloacetate during sample preparation is irrelevant for

citrate sensing (see Tab. S1).


S33

initial citrate (mM)

0.8

Y

0.4

0.2

0

0.6

1.0

1010.10.01 1

initial orange juice (µl)

0.1 10

A B

Fig S8. The citrate sensor. Fractional activity Y of pore 1 as a function of the concentration

of citrate (A) and the volume of orange juice (B) after incubation with citrate lyase and

amplification with 2; for conditions, see (A) 1.8.3 and (B) 1.8.4. Citrate concentrations in B

were determined by comparison with calibration curve A using equation [S6]. See Fig. S7 for

citrate sensing reaction scheme.


S34

N

HN

R5

R6

general filter

OR5

R6

sample

OHR3

R4

OHR1

R2

NH2

HN

solid support

sample

OHR3

R4

OHR1

R2

enzyme

sample

OR3

R4

OHR1

R2

general hydrazide amplifier

A

time (min)

0.8

Y

0.4

0.2

0

1

0.6

1.0

0 2 3

a)b)

c)

B

Fig S9. The concept of reactive filtration before signal generation and amplification (A)

exemplified with citrate sensing in orange juice (B). B: Original traces (as in Fig. S2)

comparing fractional activity Y of pore 1 in the presence of 3 l orange juice after signal

generation and amplification with (b) and without (c) preceding incubation with solid support

hydrazide; a: background (blocker-free pore 1). Extensive interference from intrinsic ketones

and aldehydes in orange juice (c) confirms the practical relevance of reactive filtration.


S35

OHO

HOHO

OH

OH

OHOHO

OHHO

OH

OHO

HOHO

O

OH

OHO

OHHO

OH

lactose

-galactosidasegalactose

glucose

hexokinase

OHO

HOHO

OH

OH

OHOHO

OHHO

OPO32-

galactose

glucose-6-phosphate

ATP ADP(40 µM) (394 µM)

(10.4 mM)

time (min)

0.8

Y

0.4

0.2

030

0.6

1.0

0 60 90 120

Fig S10. The activity of -galactosidase and hexokinase. Fractional activity Y of pore 1 as a

function of incubation time t of lactose with first -galactosidase (constant) and then

hexokinase (0 ( ), 0.47 ( ), 0.95 ( ) and 2.4 units/ml ( ) and ATP; for conditions, see

1.9.2. Top: Lactose sensing reaction scheme. Numbers in parentheses are IC50 values.


S36

initial ATP (mM)

0.8

Y

0.4

0.2

0

0.6

1.0

1001010.1 10

initial Rivella Red (µl)

3 30

A B

10

initial milk serum (µl)

1

C

Fig S11. The lactose sensor. Fractional activity Y of pore 1 as a function of the concentration

of ATP (A) and the volume of Rivella Red (B) and cow milk serum (C) after incubation with

( ) and without ( ) -galactosidase and then with hexokinase plus ATP (B, C only); for

conditions, see (A) 1.9.3, (B) 1.9.4 and (C) 1.9.5. Lactose concentrations in B and C were

determined by comparison with calibration curve A using equation [S6]. See Fig. S10 for

lactose sensing reaction scheme.


S37

O-O acetate

kinase

ATP ADP

OPO32-

O

NHOH

O

NH2OH HPO42-

(40 µM) (394 µM)

time (min)

0.8

Y

0.4

0.2

015

0.6

1.0

0 30 45 60

Fig S12. The activity of acetate kinase. Fractional activity Y of pore 1 as a function of

incubation time t of acetate with 0 ( ), 1.0 ( ) and 3.0 units/ml ( ) acetate kinase and ATP;

for conditions, see 1.10.2. Top: Acetate sensing reaction scheme. Numbers in parentheses

are IC50 values.


S38

initial acetate (mM)

0.8

Y

0.4

0.2

0

0.6

1.0

1010.1 1

initial vinegar (µl)

0.1 10

A B

100

Fig S13. The acetate sensor. Fractional activity Y of pore 1 as a function of the concentration

of ATP (A) and the volume of rice vinegar (B) after incubation with acetate kinase and ATP

(B only); for conditions, see (A) 1.10.3 and (B) 1.10.4. Acetate concentrations in B were

determined by comparison with calibration curve A using equation [S6]. See Fig. S12 for

acetate sensing reaction scheme.


S39

Fig S14. Axial (top) and side view (bottom) of geometry issued from MD simulations of

open pore 4. p-Octiphenyl rods are shown as ball-and-stick structures (silver), -sheets as

ribbons (yellow), NDI ligands A and 100% protonated lysines as wire (orange).


S40

Fig S15. Axial (top) and side view (bottom) of geometry issued from MD simulations of pore

4 with one “umami signal” substrate 6 situated at the inner pore surface. The NDI-DAN-NDI

charge transfer complex is highlighted with the naphthyl moiety of -ketoglutarate-amplifier

conjugate as space-filling (lime), rest of the substrate 6 in thick tubes (lime) and the two NDIs

acceptors involved in - stacking as thick tubes (orange). p-Octiphenyl rods are drawn as

ball-and-stick structures (silver), -sheets as ribbons (yellow), NDI ligands A and 100%

protonated lysines as wires (orange).


S41

Fig S16. Axial (top) and side view (bottom) of geometry issued from MD simulations of pore

4 with two -ketoglutarate-amplifier conjugates 6 -clamped on the opposite inner walls of

the pore. The NDI-DAN-NDI charge transfer complexes are highlighted: the naphthyl moiety

of -ketoglutarate-amplifier conjugate as space-filling (lime), rest of the substrate 6 in thick

tube (lime) and the four NDIs acceptors involved in - interactions as thick tubes (orange).

p-Octiphenyl rods are drawn as ball-and-stick structures (silver), -sheets as ribbons (yellow),

NDI ligands A and 100% protonated lysines as wires (orange).


S42

4t: R = A, R1 = TBDPS, R2 = Boc

4m: R = A, R1 = R2 = Hn)

m)

N2Cl

O

O

ClN2

4q

O

O

O

O

O

OO

R

O

OO

R

O

R

O

R

O

R

O

R

O

R

O

R

O

O

NO

NO

OTBDPS

BrO

O

4r

RHNO

HN

O

NH O

HN N

H

O

O

NH2

NHBoc

O

O

NO

NO

OTBDPS

RHNO

NH O

HN N

H

O

O

NH2

NHBoc

4n

4k: R = Z

4l: R = Hi)

4o: R = Fmoc

4p: R = Hk)

H2NO

HN N

H

O

O

NH2

NHBoc

4j

FmocHNO

OH

O

O

NO

NO

OTBDPS

ZHNO

OH

4i

ON

O

ZHNO

OH

4d

f)

H2N

OTBDPS

O

O

OHOH

ZHNO

OH

4b

O

O

OO

OO

4c

NH2

ZHNO

OH

4a

NH2

O

4g

ZHN

OTBDPS

4f

ZHN

OH

d)

4e

H2N

OH

c)

O

O

NO

NO

OR1

NH O

HN

O

NH O

HN N

H

O

O

NH2

NHR2A =

4s: R = OH

4h

e)

l)

b)

h)

j)

g)

a)

Scheme S1. Synthesis of 4m as described in (S2).


S43

O

OO OH O

OO O

O

O

HO

OO O

O

HNNH2

5

d)HO

OO O

O

O

15

c)O

OO O

O

O

14

14

b)

12

a)

HO OH

10

BrO

OO

OBr

11 13

Scheme S2. a) K2CO3, 70 ºC, 18 h, 24%. b) Cs2CO3, 70 ºC, 93%. c) TFA. d) NH2NH2

monohydrate (neat), 86% (2 steps).

NH2

HN

O

O

N

O

O

N

O

O

HO

9

e)

NHBocHN

O

O

N

O

O

N

O

O

HO

22

d)

21

H2N

ONHBocN

H

20

ZHN

ONHBocN

H

19

ZHN

O

OH

O

N

O

O

HO

O

O

O

OO

O

H2N

O

OH

O OH

OOH

a)16 18

b) c)

18

17

21

Scheme S3. a) 1. NaOH, 2. H3PO4, (crude). b) NH2NHBoc, HBTU, 97%. c) Pd/C, MeOH,quant. d) DMF, 110ºC, 6 h, 54% (2 steps). e) TFA, 67%.


S44

3. Supporting Tables

Table S1 Blockage Data for Pores 1 and 4 with Amplifiers 2, 5, and 9.a

Analytea Amplifiera,b PorecIC50 ( M)a,d

A e

D f

- 2 1 23.4 ± 2.7

pyruvate - 1 37’800 ± 6’300

pyruvate 2 1 2.6 ± 0.2 14’538

-ketoglutarate - 1 1’300 ± 100

-ketoglutarate 2 1 4.0 ± 0.4 325 0.7

oxaloacetate - 1 560 ± 20

oxaloacetate 2 1 1.3 ± 0.1 430

pyruvate 5 1 291.3 ± 24.7 130

-ketoglutarate 5 1 91.4 ± 4.9 14 3.2

pyruvate 9 1 180.7 ± 11.3 209

-ketoglutarate 9 1 73.4 ± 5.8 17 2.5

-ketoglutarate - 4 8’290 ± 590

pyruvate 5 4 14.4 ± 0.9

-ketoglutarate 5 4 2.7 ± 0.2 3’070 5.3

pyruvate 9 4 32.0 ± 3.3

-ketoglutarate 9 4 11.0 ± 1.0 754 2.9

aInhibitory constants IC50 for 50% blockage of pores 1 or 4 by amplifiers 2 (1.6.1), 5 and 9(1.6.2), analytes and analyte-amplifier conjugates were determined from dose response curvesfor fluorogenic CF efflux from EYPC-LUVs CF, compare Figs. S2 and S3. bAmplifiers arepore blockers with reactive functional groups (e.g. hydrazides) for covalent capture of analytes(e.g. ketones and aldehydes); see Figs. 3 and 4 for structures and reactions. cSee Fig. 2.dPertinent data are the average value ± SE of at least three independent measurements.eAmplification factor A = IC50 (analyte) / IC50 (analyte-amplifier conjugate). fDiscriminationfactor D = IC50 (pyruvate) / IC50 ( -ketoglutarate).


S45

Table S2 Multicomponent Sensing in Complex Matrixes with Pore 1.a

Analyte Sample Enzyme(Amplifier)

Expected (mM) Found (mM) Ref

Lactose Milk -Galactosidase,Hexokinase

114 - 140 122 ± 6 (S21)-(S23)

Acetate RiceVinegar

Acetate Kinase 658 - 770 757 ± 80 (S27)(S28)

Lactate Sour milk Lactate Oxidase(2)

54 - 86 54.3 ± 4.2 (S10)

Citrate OrangeJuice

Citrate Lyase(2)

43.1 - 44.7 45.8 ± 5.2 b (S13),(S14)

aSummary of selected data determined as specified in section 1. b With routine NMR assays(filtration over celite, lyophilization, integration of citrate resonance against internal standard, acitrate concentration c = 51.1 ± 0.7 mM was found (S13).


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