Electronic Supporting Information Anthracene-BODIPY dyads as

Electronic Supporting Information

Anthracene-BODIPY dyads as fluorescent sensors for biocatalytic Diels-Alder reactions

Alexander Nierth*, Andrei Yu. Kobitski‡, G. Ulrich Nienhaus‡# and Andres Jäschke*

*Institute of Pharmacy and Molecular Biotechnology, Heidelberg University, Im

Neuenheimer Feld 364, Heidelberg, 69120, Germany, ‡Institute of Applied Physics

and Center for Functional Nanostructures, Karlsruhe Institute of Technology,

Wolfgang-Gaede-Strasse 1, 76131 Karlsruhe, Germany, #Department of Physics,

University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA

E-mail: [email protected]

1. Supplementary Figures p2-10 2. Experimental Section p11 2.1. Materials and General Procedures p11-12 2.2. Synthetic Procedures p12-18 2.3. Chromatography p19-22 2.4. Optical Spectroscopy p23-28 2.5. References p29 2.6. Complete Reference (6) of Main Text p30 3. Spectra Appendix p31-45 List of Abbreviations:

BODIPY = 4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-indacene,

DAse = Diels-Alderase ribozyme, DCM = dichloromethane, DDQ = 2,3-dichloro-5,6-

dicyano-p-benzoquinone, DIPEA = N,N-diisopropylethylamine, DTBPPS = 3-(di-tert-

butylphosphonium)propane sulfonate, EtOAc = ethylacetate, EtOH = ethanol, MeCN

= acetonitrile, FCS = fluorescence correlation spectroscopy, MeOH = methanol, NPM

= N-pentylmaleimide, TEA = triethylamine, TEAA = triethylammonium acetate, TFA =

trifluoroacetic acid, THF = tetrahydrofurane.

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1. Supplementary Figures

Figure S1 (A) Image section of Figure 2B. Absorption (solid, ≈10 µM) and

normalized fluorescence emission spectra (dashed, 1.0 µM) of 1-AB (blue), 9-AB

(red) and 9-DAPB (black) at 488 nm excitation wavelength. (B) Comparison of

fluorescence intensities while exciting with 488 nm (solid) or 405/422 nm (dashed).

(C) Comparison of absorption (solid) and normalized fluorescence excitation

(dashed) spectra at 515 nm emixion wavelength. All spectra were recorded in

standard DAse buffer.

(A) (B)

(C)

S3

Figure S2 Fluorescence emission spectra (Ex. 488 nm) of 100 µM 1-AB (A, B),

9-AB (C, D) and 0.4 µM 9-DAPB (E, F). Spectra were recorded in unbuffered

aqueous solutions of different salts A, C and E (1.0 M each; dashed, no salt) and at

varying concentrations of NaCl B, D, F (100 mM-1.0 M; dashed, no salt). Spectro-

meter settings in panels E and F were different from those in the other panels.

(E) 9-DAPB (F) 9-DAPB

(A) 1-AB

(C) 9-AB

(B) 1-AB

(D) 9-AB

S4

Figure S3 Fluorescence emission of 1-AB (black circles, 90 µM), 9-AB (light gray

circles, 90 µM) and 9-DAPB (dark gray circles, 3.0 µM) as function of buffer pH

normalized to the maximum fluorescence (pH 2.1). Buffer solutions of pH 2.1-8.0

were prepared from appropriate mixtures of 0.1 M citric acid and 0.2 M Na2HPO4

(“Mc Ilvaine’s buffer”).1 Measurements of samples (100 µl) in 96 well-plates were

performed with a Tecan, Safire2 multiplate reader (Männedorf, Switzerland) using the

following settings: Ex./Em.: 488/515 nm, bandwidths: 5/5 nm, emission scan number:

31, number of reads: 10, step size: 1.0 nm, integration time: 40 µs, z-position:

11201 µm and gain: 150 (1-AB, 9-AB) or gain: 130 (9-DAPB). Error bars correspond

to the standard deviation of three to six separately prepared samples.

S5

Figure S4 Absorption (A) and emission spectra (B) (Ex. 488 nm) of 9-DAPB

(straight lines, 100 µM) in the presence of excess DAse ribozyme (dashed lines,

150 µM) in standard DAse buffer.

S6

Figure S5 Dependence of absorption (A) and fluorescence intensity (B) of 100 µM

1-AB (blue), 9-AB (red) and 9-DAPB (black) on the ribozyme concentration in

standard DAse buffer. Values were normalized with respect to the unbound probes.

For 9-DAPB half of the emission intensity of the free probe was substracted from all

other values to compensate for the fluorescence of the “wrong” enantiomer. The

fraction of bound RNA was calculated according the law of mass action, using Kd

values from the FCS measurements.

(A) (B)

S7

Figure S6 Fluorescence decay curves of free (black symbols) and bound (gray

symbols) fluorescent probes (A) 1-AB (40% bound), (B) 9-AB (50% bound) and (C) 9-DAPB (90% bound) in standard DAse buffer. The fraction of bound RNA was

calculated according the law of mass action, using Kd values from the FCS

measurements. The instrumental response function is shown as a dashed line; the

best-fit curves are depicted as solid lines.

(A) (B)

(C)

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Figure S7 Burst intensity averaged over busts containing 20 – 100 counts

(Figure 5C), as a function of the DAse ribozyme concentration for 1-AB (circles) and

9-DAPB (squares).

S9

Figure S8 Fluorescence kinetics (bulk) including calculated fits of Diels-Alderase

catalyzed reactions (dashed lines) of fluorescent dyads 1-AB (blue) or 9-AB (red) at

room temperature in standard DAse buffer. Panels A and B are identical to Figure 6

(main text) and shown here for comparison only. (C) Multiple-turnover conditions of

10 pmol DAse (0.67 µM) and 7.5 nmol NPM (500 µM), 75 pmol 1-AB (5.0 µM).

Excitation wavelength is 488 nm. For all experimental setups, background reactions

(light blue for 1-AB; orange for 9-AB) were recorded at the same conditions as in the

catalyzed reactions. Emission wavelength is in all cases 515 nm. (D) Numeric results

of the corresponding fits.

(A) (B)

(C) (D)

S10

Figure S9 Progress curves of the ribozyme-catalyzed Diels-Alder reaction at

multiple-turnover conditions measured on the confocal microscopy setup. The

reactions were carried out at RT in standard DAse buffer. 1-AB fluorescent probe

was excited at 488 nm and the emission in the 500 - 525 nm spectral range was

detected.

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2. Experimental Section 2.1. Materials and General Procedures

All reactions were carried out under argon atmosphere with dry solvents.

MeCN and THF (stored over molecular sieves) were purchased from Acros Organics.

Other dry solvents (DIPEA, DCM, toluene, DMSO) and chemicals were purchased

from Sigma-Aldrich and used without further purification, unless otherwise stated.

Yields refer to chromatographically and spectroscopically (1H NMR) homogeneous

materials. Tertiary amines, water and mixtures thereof were degassed by repetitive

freezing and thawing under reduced pressure and thorough purging with a stream of

argon for 10 min.

Microwave assisted reactions were performed with a Discover® LabMate

microwave reactor by CEM2, equipped with a focused single-mode reaction chamber

(2.45 GHz) in heavy-walled glass vials (10.0 ml capacity) that were sealed with

Teflon caps. Reaction temperatures were monitored with a built-in IR temperature

sensor and kept constant by automatic power control. Stirring and continuous active

cooling via compressed air was used to remove latent heat from all reactions.

Reactions were monitored by thin-layer chromatography (TLC) using silica gel

sheets Polygram® Sil G/UV254 (0.2 mm, 40 x 80 mm) and Polygram® ALOX N/UV254

(0.2 mm 40 x 80 mm) by Macherey-Nagel or RP-18 F245s by Merck. Visualisation was

performed using UV light or an ethanolic solution of phosphomolybdic acid and

cerium sulphate with heat as developing agent. Flash column chromatography was

carried out using silica gel 60 M (0.04-0.063 mm/230-240 mesh ASTM) and neutral

aluminiumoxide (activity grade I) by Macherey-Nagel or basic aluminiumoxide 90 (pH

8.5-10.4) by Carl Roth. Activity grade II was obtained by treating the alumina with 3%

water (relative to the mass of alumina) in THF and evaporation to dryness.

Preparative reversed phase (RP-18) chromatography was carried out using pre-

packed Lobar® glass columns (LiChroprep® RP-18, 310 x 25 mm) by Merck, in

combination with a peristaltic pump using a constant flow rate of 7.0 ml/min. Cation

exchange was performed using pre-packed columns Strata-X-C® with polymeric

strong cation resin (33 µm, 200 mg or 500 mg sorbent mass) by Phenomenex.

NMR spectra were recorded on a Varian 500 MHz NMR system (1H: 500 MHz, 13C: 126 MHz, 31P: 202 MHz) and calibrated using residual undeuterated solvent as

internal reference. 19F NMR spectra were recorded on a Varian 300 MHz Mercury

Plus spectrometer (19F: 282 MHz). CDCl3 was stored over dry K2CO3. The following

S12

abbreviations were used to explain the multiplicities in 1H and 19F NMR spectra: s =

singlet, d = doublet, t = triplet, q = quartet, m = multiplet, brs = broad singlet. The

following abbreviations were used to assign carbon saturations in APT NMR spectra:

q = quaternary, t = tertiary, s = secondary, p = primary. High-resolution mass spectra

(HRMS) were recorded on a Bruker ApexQe hybrid 9.4 T FT-ICR (ESI) or JEOL

JMS-700 sector field (EI, FAB). MALDI-TOF mass spectra were recorded on a Bruker

BIFLEX III spectrometer. Infrared (IR) spectra were recorded on a Jasco FT/IR-4100

spectrophotometer with ATR feature.

Purity of the target compounds was verified by reversed phase HPLC (RP-

HPLC) on an Agilent 1100 Series system, equipped with diode array and

fluorescence detectors. Separations on analytical scale were performed on a

Phenomenex Luna® C18 column (5 µm, 250 x 4.6 mm), eluting with appropriate

gradients of buffers A (0.1 M TEAA in H2O, pH 7.4) and B (0.1 M TEAA in

MeCN/H2O, 8:2, pH 7.4) at 1.0 ml/min. Semi-preparative separations were performed

on a Phenomenex Luna® C18 column (5 µm, 250 x 15 mm) at 6.0 ml/min. Prior to

injection samples were filtered through a 0.22 µm teflon syringe filter.

2.2. Synthetic Procedures Palladium ligand 3-(Di-tert-butylphosphonium)propane sulfonate (DTBPPS)3

3-(Di-tert-butylphosphonium)propane sulfonate (DTBPPS) was used as pre-

ligand for aqueous phase palladium-catalyzed cross-coupling reactions, according to

K. Shaugnessy and co-workers. Substance is reported to be indefinitely stable to air

and becomes activated through deprotonation of the phosphonium ion with bases.3

A reaction flask was charged with di-tert-butylphosphine (1.27 ml, 1.00 g,

6.84 mmol) and 1,3-propane sultone (562 mg, 4.56 mmol) in 6.0 ml dioxane. The

mixture was refluxed for 12 h, during which time a white precipitate formed. The

product was filtered off and washed with THF (3 x 10.0 ml) and Et2O (3 x 10.0 ml).

Drying in vacuo gave 987 mg (3.68 mmol, 81%) as white solid. 1H NMR (D2O):

1.49-1.52 (d, 3JP-H = 16.80 Hz, 18H), 2.21-2.29 (m, 2H), 2.52-2.57 (m, 2H), 3.06-3.09

S13

(t, J = 7.02 Hz, 2H), 3.76 (s, 1H) ppm; 31P-NMR (D2O): δ = 46.19-46.88 (t, J =

69.94 Hz); IR νMax (cm-1) = 2362 (br, PH), 1205, 1184 (s, νas(SO3)), 1029 (s,

νss(SO3)), 1022 (s, P-C); HRMS (ESI-): m/z calcd. for C11H24O3PS [M-H]-: 267.11893,

found: 267.11915.

Synthesis of (anthracen-1-ylethynyl)trimethylsilane A mixture of trimethylsilylacetylene (1.20 g, 12.20 mmol) in 20.0 ml THF was

cooled to 0°C, treated dropwise with ethylmagnesium bromide (1.0 M in THF,

11.30 ml, 1.50 g, 11.30 mmol), and stirred for 1 h at this temperature (gas evolution

was observed). The solution was transferred via a long needle into a mixture of

1-chloroanthracene (1.00 g, 4.70 mmol), Ni(acac)2 (4.8 mg, 18.80 µmol) and

triphenylphosphine (9.9 mg, 37.62 µmol) in 7.0 ml THF and heated to reflux for

4 days. The solvent was evaporated and the residue redissolved in 100.0 ml

petroleum ether. The solution was washed with water (3 x 100.0 ml) and the

combined aqueous phases were extracted with 100.0 ml petroleum ether/EtOAc

(1:1). The combined organic phases were dried over MgSO4 and the solvent was

evaporated. The crude product was purified by silica gel chromatography (petroleum

ether) to yield 1.15 g (4.19 mmol, 89%) as pale yellow oil. Rf = 0.29 (petroleum

ether); 1H NMR (CDCl3): δ = 0.41 (brs, 9H), 7.38-7.41 (dd, J = 6.88 Hz, J = 8.55 Hz,

1H), 7.53-7.48 (m, 2H), 7.71-7.72 (dd, J = 1.07 Hz, J = 6.88 Hz, 1H), 7.98-8.03 (m,

2H), 8.08-8.11 (m, 1H), 8.43 (s, 1H), 8.91 (s, 1H) ppm; APT NMR (CDCl3): δ = 0.16

(p), 99.96 (q), 103.33 (q), 120.79 (q), 124.50 (t), 125.12 (t), 125.76 (t), 125.78 (t),

126.85 (t), 127.99 (t), 128.63 (t), 129.38 (t), 130.66 (t), 131.01 (q), 131.09 (q), 131.88

(q), 132.06 (q) ppm; HRMS (EI+): m/z calcd. for C19H18Si [M]+: 274.11778, found:

274.1188.

Synthesis of 1-ethynylanthracene (general deprotection procedure) A reaction flask was charged with K2CO3 (322 mg, 2.33 mmol) in 3.0 ml MeOH

and (anthracen-1-ylethynyl)trimethylsilane (0.80 g, 2.92 mmol) in 7.0 ml DCM was

added dropwise. The suspension was stirred vigorously until TLC showed complete

conversion (~2-3 h). The reaction was poured into Et2O/water (1:1) and the organic

phase was separated. The combined organic phases were washed several times

with water, dried (Na2SO4) and subjected to flash column chromatography (silica gel,

petroleum ether, 4% toluene) to give 560 mg (2.77 mmol, 95%) as yellow solid. Rf =

S14

0.39 (petroleum ether); 1H NMR (CDCl3): δ = 3.60 (s, 1H), 7.40-7.43 (dd, J = 6.86 Hz,

J = 8.57 Hz, 1H), 7.49-7.54 (m, 2H), 7.76-7.77 (dd, J = 1.06 Hz, J = 6.86 Hz, 1H),

8.00-8.04 (m, 2H), 8.08-8.11 (m, 1H), 8.44 (s, 1H), 8.94 (s, 1H) ppm; APT NMR

(CDCl3): δ = 82.01 (q), 82.34 (q), 119.82 (q), 124.41 (t), 124.90 (t), 125.87 (t),

125.89 (t), 126.92 (t), 127.99 (t), 128.55 (t), 129.69 (t), 131.03 (q), 131.05 (q),

131.14 (t), 131.92 (q), 132.18 (q) ppm; HRMS (EI+): m/z calcd. for C16H10 [M]+:

202.07825, found: 202.0774.

Synthesis of (anthracen-9-ylethynyl)trimethylsilane4 In a reaction flask, 9-bromoanthracene (1.00 g, 3.88 mmol), Pd(Ph3)2Cl2

(68 mg, 97.00 µmol) and CuI (30 mg, 155.20 µmol) were combined in 20.0 ml THF.

Subsequently, trimethylsilylacetylene (603 µl, 419 mg, 4.26 mmol) and 20.0 ml TEA

were added and the mixture was stirred and heated to reflux for 12 h. Purification by

flash column chromatography (silica gel, hexane, 0.5% toluene) gave the protected

compound in yields of 60-85%, which then was additionally recrystallized from MeCN

before further usage. Rf = 0.41 (hexane, 0.5% toluene); 1H NMR (CDCl3): δ = 0.51

(brs, 9H), 7.51-7.53 (dd, J = 1.44 Hz, J = 8.19 Hz, 1H), 7.52-7.54 (dd, J = 1.27 Hz,

J = 8.36 Hz, 1H), 7.62-7.63 (dd, J = 1.27 Hz, J = 8.71 Hz, 1H), 7.63-7.65 (dd, J =

1.27 Hz, J = 8.71 Hz, 1H), 7.98-7.99 (m, 1H), 8.00-8.00 (m, 1H), 8.39 (s, 1H),

8.63-8.63 (m, 1H), 8.64-8.65 (m, 1H) ppm; APT (CDCl3): δ = 0.26 (t), 101.57 (q),

106.11 (q), 117.00 (q), 125.54 (t), 126.62 (t), 126.65 (t), 127.83 (t), 128.57 (t),

130.96 (q), 132.81 (q) ppm; HRMS (FAB+): m/z calcd. for C19H18Si [M]+: 274.11778,

found: 274.1184.

Synthesis of 9-ethinylanthracene Synthesis according to the general deprotection procedure. Purification was

performed by filtering through a short plug of silica (DCM) and the product was

obtained as red solid (97%). The compound is unstable over prolonged storage and

should be carried on directly to the next step. Rf = 0.31 (hexane, 0.5% toluene); 1H NMR (CDCl3): δ = 3.99 (s, 1H), 7.50-7.51 (dd, J = 1.16 Hz, J = 8.20 Hz, 1H),

7.51-7.53 (dd, J = 1.18 Hz, J = 8.34 Hz, 1H), 7.58-7.60 (dd, J = 1.29 Hz, J = 8.70 Hz,

1H), 7.59-7.61 (dd, J = 1.30 Hz, J = 8.69 Hz, 1H), 8.00-8.01 (m, 1H), 8.02-8.03 (m,

1H), 8.46 (s, 1H), 8.57-8.58 (m, 1H), 8.59-8.60 (m, 1H) ppm; APT NMR (CDCl3): δ =

88.18 (q), 116.01 (q), 125.67 (t), 126.55 (t), 126.82 (t), 126.82 (t), 128.22 (t), 128.66

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(t), 131.02 (q), 133.16 (q) ppm; HRMS (EI+): m/z calcd. for C16H10 [M]+: 202.07825,

found: 202.0788.

Synthesis of TMS-protected 9-ethynyl-Diels-Alder product A mixture of (anthracen-9-ylethynyl)trimethylsilane (250 mg, 0.91 mmol) and

N-pentylmaleimide5 (229 mg, 1.37 mmol) in 7.0 ml toluene was heated by microwave

irradiation for 12 h at 115°C (~120 W). The solvent was evaporated and the crude

purified by normal phase chromatography (EtOAc/petroleum ether, 1:10). The

product was obtained as colourless oil (269 mg, 0.61 mmol, 67%). Rf = 0.27

(EtOAc/petroleum ether, 1:10); 1H NMR ((CD3)2CO): δ = 0.41 (brs, 9H), 0.78-0.81 (m,

7H), 1.08-1.14 (m, 2H), 3.00-3.03 (m, 2H), 3.24-3.26 (d, J = 8.52 Hz, 1H), 3.30-3.32

(dd, J = 3.34 Hz, J = 8.52 Hz, 1H), 4.81-4.81 (d, J = 3.32 Hz, 1H), 7.18-7.30 (m, 5H),

7.50-7.52 (m, 1H), 7.62-7.63 (m, 1H), 7.78-7.80 (m, 1H) ppm; APT NMR ((CD3)2CO):

δ = 1.28 (p), 15.03 (p), 23.82 (s), 28.70 (s), 30.21 (s), 39.67 (s), 46.69 (t), 49.06 (q),

49.26 (t), 52.47 (t), 97.31 (q), 103.28 (q), 125.08 (t), 125.53 (t), 125.90 (t), 126.83 (t),

128.37 (t), 128.58 (t), 128.98 (t), 129.05 (t), 140.16 (q), 140.39 (q), 143.06 (q), 143.08

(q), 175.34 (q), 177.31 (q) ppm; HRMS (ESI+): m/z calcd. for C28H32NO2Si [M-H]+:

442.21968, found: 442.22008.

Synthesis of deprotected ethinyl-Diels-Alder product Synthesis according to the general deprotection procedure. After chromato-

graphy (silica gel, EtOAc/petroleum ether, 1:10) the product was obtained as gray

solid (79%). Rf = 0.13 (EtOAc/petroleum ether, 1:10); 1H NMR ((CD3)2CO): δ =

0.75-0.81 (m, 7H), 1.07-1.13 (m, 2H), 3.02-3.04 (m, 2H), 3.27-3.29 (d, J = 8.52 Hz,

1H), 3.32-3.34 (dd, J = 3.34 Hz, J = 8.52 Hz, 1H), 3.65 (s, 1H), 4.83-4.83 (d, J =

3.32 Hz, 1H), 7.19-7.31 (m, 5H), 7.51-7.53 (m, 1H), 7.62-7.64 (m, 1H), 7.79-7.81 (m,

1H) ppm; APT NMR ((CD3)2CO): δ = 15.01 (p), 23.81 (s), 28.68 (s), 30.18 (s), 39.70

(s), 46.67 (t), 48.39 (q), 49.22 (t), 52.37 (t), 80.93 (q), 81.92 (q), 124.97 (t), 125.42 (t),

125.94 (t), 126.88 (t), 128.39 (t), 128.61 (t), 129.03 (t), 129.11 (t), 140.16 (q), 140.26

(q), 142.98 (q), 143.03 (q), 175.52 (q), 177.30 (q) ppm; HRMS (ESI+): m/z calcd. for

C25H24NO2 [M-H]+: 370.18016, found: 370.18032.

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Synthesis of Di-triethylammonium 2,6-disulfonate-1,3,5,7-tetramethyl-8-(4'-bromophenyl)-4,4-difluoro-4-bora-3a,4a-diaza-s-indacence (bromo-BODIPY)6

1,3,5,7-Tetramethyl-8-(4'-bromophenyl)-4,4-difluoro-4-bora-3a,4a-diaza-s-

indacence) was synthesized starting from para-bromobenzaldehyde by a combi-

nation of literature procedures using freshly distilled 2,4-dimethylpyrrole.7-9 The non-

sulfonated BODIPY core (423 mg, 1.05 mmol) was dissolved in 18.0 ml DCM and

cooled to -40°C using a MeCN/CO2 bath. While stirring vigorously, a fresh solution of

chlorosulfonic acid (140 µl, 245 mg, 2.10 mmol) in 8.0 ml DCM was added dropwise

within 10 min. The temperature was slowly raised to -10°C and an orange precipitate

formed. A pre-cooled solution NaHCO3 (176 mg, 2.10 mmol) in 10.0 ml H2O was

added dropwise until neutral pH and the mixture was stirred at room temperature for

10 min. The organic phase was separated, extracted with water and the combined

aqueous phases were washed with DCM. The aqueous phase was lyophilized to

dryness, redissolved in 5.0 ml H2O and purified with liquid chromatography (RP,

buffer A/B, 7:3). The solvent and excess TEAA buffer were removed by repetitive

lyophilisation with water/MeCN (1:1) yielding 710 mg (88%) bromo-BODIPY (triethyl-

ammonium salt) as orange foam. Rf = 0.35 (buffer A/B, 7:3); 1H NMR (D2O/

(CD3)2CO, 5:2): δ = 1.36 (t, J = 7.3 Hz, 18H), 1.75 (s, 6H), 2.84 (s, 6H), 3.26-3.30

(dd, J = 7.3 Hz, 12H), 7.41-7.43 (m, 2H), 7.90-7.91 (m, 2H) ppm; APT NMR

(D2O/(CD3)2CO, 5:2): δ = 9.6 (p), 14.18 (p), 15.02 (p), 47.97 (s), 125.12 (q), 131.11

(t), 131.47 (q), 134.03 (q), 134.35 (t), 135.44 (q), 144.11 (q), 145.89 (q), 156.91

(q) ppm; 19F NMR (D2O): δ = -141-142 (dd, J = 32 Hz, J = 63 Hz, BF2) ppm. HRMS

(ESI-): m/z calcd. for C19H16BBrF2N2O6S2 [M]2-: 279.98526, found: 279.98534.

Synthesis of Di-triethylammonium 2,6-disulfonate-1,3,5,7-tetramethyl-8-(4'-iodo-

phenyl)-4,4-difluoro-4-bora-3a,4a-diaza-s-indacence (iodo-BODIPY)6 Analogous procedure to bromo-BODIPY using para-iodobenzaldehyde. Rf =

0.35 (buffer A/B, 7:3); 1H NMR (CD3CN): δ = 1.17 (t, J = 7.3 Hz, 18H), 1.59 (s, 6H),

2.67 (s, 6H), 3.04-3.08 (dd, J = 7.3 Hz, 12H), 7.13-7.15 (m, 2H), 7.89-7.92 (m,

2H) ppm; APT NMR (CD3CN): δ = 9.06 (p), 13.49 (p), 14.48 (p), 47.42 (s), 95.90 (q),

130.95 (t), 131.09 (q), 134.68 (q), 135.59 (q), 139.58 (t), 142.98 (q), 145.06 (q),

156.21 (q) ppm; 19F NMR (D2O): δ = -141-142 (dd, J = 32 Hz, J = 63 Hz, BF2); HRMS

(ESI-): m/z calcd. for C19H16BF2IN2O6S2 [M]2-: 303.97833, found: 303.97821.

S17

Synthesis of 1-AB The catalyst solution was prepared by equipping a schlenk flask with Pd(OAc)2

(8.8 mg, 39.19 µmol), DTBPPS (10.5 mg, 39.19 µmol) and CuI (2.5 mg, 13.06 µmol).

DIEA (273 µl, 203 mg, 0.16 mmol) in 5.0 ml degassed H2O. After addition of 15.0 ml

MeCN the yellow solution was ultrasonicated for 5 s. Subsequently, 2.00 ml of the

catalyst solution were transferred to another reaction flask, equipped with bromo-

BODIPY (di-triethylammonium salt, 100 mg, 0.13 mmol) and 1-ethynylanthracene

(29 mg, 0.144 mmol) and stirred at 70°C for 1 h in a microwave reactor. The mixture

was poured into water (10.0 ml) and extracted several times with hexanes. The

aqueous phase was lyophilized and the crude re-dissolved in 5.0 ml buffer A/B (6:4)

and purified via low pressure LC (RP, buffer A/B, 7:3). The product fraction was

lyophilized and co-evaporated three times with MeCN/H2O (1:1) to give the title

compound as red-orange foam (54 mg, 60.32 µmol, 46%). The sodium salt was

obtained according to the ion exchange procedure. Rf = 0.16 (buffer A/B, 1:1); 1H NMR (D2O, 60°C): δ = 1.65 (s, 6H), 2.96 (s, 6H), 6.92-6.93 (d, J = 7.4 Hz, 2H),

7.18-7.21 (t, J = 7.5, 1H), 7.25-7.29 (m, 2H), 7.53-7.54 (d, J = 7.7 Hz, 3H), 7.62-7.66

(dd, J = 8.4 Hz, J = 8.7 Hz, 2H), 7.71-7.72 (d, J = 8.5 Hz, 2H), 8.00 (s, 1H), 8.68 (s,

1H) ppm; APT NMR (D2O, 60°C): δ = 13.24 (p), 13.96 (p), 89.91 (q), 94.21 (q),

119.68 (q), 124.03 (t), 124.52 (q), 124.67 (t), 126.15 (t), 126.37 (t), 127.44 (t), 127.79

(t), 128.00 (t), 128.27 (t), 129.81 (t), 130.28 (q), 130.38 (q), 130.94 (q), 131.05 (t),

131.81, 131.88 (q), 132.54 (t), 133.81, 134.24 (q), 142.78, 144.63 (q), 155.91

(q) ppm; 19F NMR (D2O): δ = -142 (m, BF2) ppm; 1H NMR (D3COD/D2O, 5:2, RT): δ = 1.67 (s, 6H), 2.85 (s, 6H), 7.05-7.07 (d, J =

8.2 Hz, 2H), 7.18-7.21 (dd, J = 6.9 Hz, 1H), 7.38-7.42 (m, 2H), 7.64-7.65 (d, J =

7.8 Hz, 1H), 7.67-7.69 (d, J = 8.2 Hz, 2H), 7.85-7.92 (m, 3H), 8.29 (s, 1H), 8.72 (s,

1H) ppm; APT NMR (D3COD/D2O, 5:2, RT): δ = 11.59 (p), 13.87 (p), 90.27 (q), 94.63

(q), 120.94 (q), 125.19 (t), 125.81 (q), 125.83 (t), 127.14 (t), 127.38 (t), 128.30 (t),

129.01 (t), 129.14 (t), 129.47 (t), 130.87 (t), 131.33 (q), 131.62 (q), 131.89 (q), 132.24

(t), 133.12, 133.24 (q), 133.62 (t), 135.31, 135.77 (q), 143.49, 145.80 (q), 156.78

(q) ppm. IR νMax (cm-1) = 1650 (s, C=N), 1389, 1309 (s, B-F), 1187 (brs, B-N), 1058

(s, SO3Na); HRMS (ESI-): m/z calcd. for C35H25BF2N2O6S2 [M]2-: 341.06131, found:

341.06131;

S18

Synthesis of 9-AB Sonogashira coupling procedure analogous to 1-AB using iodo-BODIPY (di-

triethylammonium salt, 90 mg, 110.76 µmol) and 9-ethinylanthracene (27 mg,

132.91 µmol). The title compound was obtained as red-orange foam (59 mg,

66.07 µmol, 60%). Rf = 0.29 (buffer A/B, 1:1); 1H NMR (CD3OD/D2O, 5:2): δ = 1.74

(s, 6H), 2.81 (s, 6H), 7.32-7.34 (m, 2H), 7.52-7.55 (m, 2H), 7.61-7.65 (m, 2H),

7.88-7.90 (m, 2H), 8.03-8.05 (m, 2H), 8.49 (s, 1H), 8.51-8.53 (m, 2H) ppm; APT NMR

(CD3OD/D2O, 5:2): δ = 13.83 (p), 14.63 (p), 88.55 (q), 100.79 (q), 116.95 (q), 125.94

(q), 126.95 (t), 127.04 (t), 128.36 (t), 129.57 (q), 129.72 (q), 129.95 (q), 131.39 (t),

132.35, 133.85 (t), 133.63 (q), 135.51 (q), 135.60 (q), 143.70 (q), 145.993 (q), 156.84

(q) ppm; 19F NMR (CD3OD/D2O, 5:2): δ = -143.66-143.34 (dd, J = 28.66 Hz, J =

61.39 Hz); IR νMax (cm-1) = 1650 (s, C=N), 1389, 1309 (s, B-F), 1187 (brs, B-N), 1058

(s, SO3Na); HRMS (ESI-): m/z calcd. for C35H25BF2N2O6S2 [M]2-: 341.06131, found:

341.06133.

Synthesis of 9-DAPB Sonogashira coupling procedure analogous to 1-AB using bromo-BODIPY (di-

triethylammonium salt, 58 mg, 75.76 µmol) and the deprotected ethinyl-Diels-Alder

Product (44 mg, 119.19 µmol). The title compound was obtained as orange foam

(33 mg, 38.84 µmol, 51%). Rf = 0.25 (buffer A/B, 4:6); 1H NMR (D2O/CD6CN, 1:3): δ

= 0.54-0.59 (m, 2H), 0.67-0.70 (m, 5H), 1.61 (s, 6H), 1.93-1.95 (m, 2H), 2.69 (s, 6H),

3.00-3.03 (m, 2H), 3.23-3.32 (m, 2H), 4.77 (d, J = 3.04 Hz, 1H), 7.12-7.20 (m, 3H),

7.21-7.25 (m, 2H), 7.28-7.29 (d, J = 7.91 Hz, 2H), 7.40-7.41 (dd, J = 1.04 Hz, J =

7.09 Hz, 1H), 7.62-7.64 (dd, J = 1.04 Hz, J = 7.09 Hz, 1H), 7.79-7.80 (d, J = 7.36 Hz,

1H), 7.84-7.85 (d, J = 8.11 Hz, 2H) ppm; APT NMR (D2O/CD6CN, 1:3): δ = 13.55 (p),

13.85 (p), 14.47 (p), 22.58 (s), 27.58 (s), 28.77 (s), 39.24 (q), 45.15 (t), 47.75 (s),

48.08 (t), 51.45 (t), 86.99 (q), 91.05 (q), 123.88 (t), 124.92 (t), 124.58 (q), 125.22 (t),

125.92 (t), 127.69 (t), 128.03 (t), 128.40 (t), 129.28 (t), 131.07 (q), 133.74, 133.78 (t),

134.87 (q), 135.26 (q), 138.74 (q), 138.85 (q), 141.30 (q), 141.57 (q), 143.39 (q),

145.75 (q), 156.25 (q), 176.61 (q), 179.06 (q) ppm; 19F NMR (D2O/CD6CN, 1:3): δ =

-141-142 (dd, J = 27.76 Hz, J = 60.93 Hz); IR νMax (cm-1) = 3442 (br, NCO2), 1774 (w,

C=O), 1695 (s, C=N), 1400, 1309 (s, B-F), 1187 (brs, B-N), 1058, 1008 (s, SO3Na);

HRMS (ESI-): m/z calcd. for C44H38BF2N3O8S2 [M]2-: 424.60862, found: 424.60862.

S19

2.3. Chromatography Ion exchange procedure

The ion exchange column was equilibrated with 6.0 ml MeCN/H2O (1:1),

conditioned with 15% aqueous NaCl solution (8.0-10.0 ml) until neutral pH and

washed thoroughly with H2O. The sample was loaded (10-15 mg in 1.0 ml H2O) and

eluted with 1.0 ml H2O/MeCN (1:4). The fraction was collected in silanized tubes and

lyophilized to dryness. 10 to 15 mg were weighed and dissolved in degassed H2O to

give 2.0 mM stock solutions for spectroscopical measurements.

S20

HPLC analysis to confirm purity of spectroscopic probes

RP-18, 45% buffer B (isocratic)

1. 1-AB: injection of 2.0 nmol (10.0 µl)

2. 9-AB: injection of 2.0 nmol (10.0 µl)

3. 9-DAPB: injection of 1.0 nmol (10.0 µl)

S21

HPLC analysis to confirm Diels-Alder product identity:

RP-18, 45% buffer B (isocratic)

1. Reference (starting material, 1-AB):

2. Catalyzed reaction (with RNA, 15.5 h, room temperature):

1-AB

1-AB NPM(excess)

DAPB DAPB

S22

3. Uncatalyzed reaction (without RNA, 19.5 h, 50°C):

4. UV-spectra of 1-AB, NPM (39 min) and DAPB peaks (43 and 61 min):

1-ABNPM(excess)

DAPB DAPB

S23

2.4. Optical Spectroscopy Fluorescent samples were always kept in brown vials and on ice to suppress

unwanted side reactions. For spectroscopic measurements of fluorescent probes

appropriate amounts of concentrated stock solutions (2.0 mM) were diluted in water

or buffer to obtain final concentrations as required. Diels-Alderase ribozyme (DAse,

wt RNA sequence: 5’-GGA GCU CGC CCG GGC GAG GCC GUG CCA GCU CUU

CGG AGC AAU ACU CGG C-3') synthesized on solid phase, was obtained from

CSS - Chemical Synthesis Services, Scotland, UK. Standard Diels-Alderase (DAse)

buffer (300 mM NaCl, 30 mM Tris-HCl, pH 7.4 and 80 mM MgCl2) was prepared as

5x stock solution and diluted as required (final: 10% ethanol). RNA concentrations

were measured in 2.0 µl sample volumes on a NanoDrop ND-1000 spectro-

photometer (Peqlab Biotechnologie GmbH, Erlangen, Germany) using the theoretical

extinction coefficient of the DAse sequence, ε260 = 453600 M−1cm−1.10 A 100 µM

DAse stock solution in water was prepared, which was used throughout all

measurements involving RNA (except FCS measurements). Prior to measurements

the ribozyme was dissolved in diluted 5x DAse buffer and refolded by heating for

2.0 min at 75 C and leaving at room temperature for 20 min.

Time-resolved fluorescence, fluorescence correlation spectroscopy (FCS) and

time-traces on freely diffusing molecules in solution were measured on a home-built

confocal microscopy setup based on the Zeiss Axiovert 135 TV inverted microscope,

a detailed description of which can be found elsewhere.11,12 Briefly, excitation for the

lifetime measurements was performed with a pulsed diode laser (LDH-P-C-470B,

PicoQuant, Berlin, Germany) operated at 470 nm by multi-channel pulsed diode laser

system (PDL-808 “Sepia”, PicoQuant, Berlin, Germany). The average laser power

was adjusted to be 30 µW at the repetition rate of 20 MHz before entering the

microscope. For FCS and burst analysis experiments the excitation was provided by

an Ar+ laser (Stabilite 174, Spectra-Physics, Mountain View, CA) operated in cw

mode at 488 nm with a power of 30 and 250 µW, respectively. A gaussian-shaped

laser beam profile was obtained by coupling the excitation light with a single-mode

optical fiber (QSMJ, OZ Optics, Ottawa, Canada). The fluorescence emitted by the

sample was collected by a water immersion objective (UPlan-Apo 60x/1.2w,

Olympus, Hamburg, Germany) and passed through a 50 µm confocal pinhole. A set

of dichroic mirror (Chroma z470/532/637, AHF, Tübingen, Germany) and bandpass

filter (Semrock F512/25, AHF, Tübingen, Germany) were used to reject all unwanted

S24

light from detection. In the case of the lifetime measurements, detection was done

with a photomultiplier (PMC-100-4, Becker & Hickl, Berlin, Germany) and registered

using a TCSPC card (TimeHarp 200, PicoQuant, Berlin, Germany). Instrumental

response function with the full width at half maximum of 0.4 ns was measured using a

Rose Bengal dye with the lifetime of 90 ps.13 In FCS and burst analysis experiments,

detection was done with an avalanche photo diode (SPCM-CD 3017, Perkin Elmer,

Fremont, MA) and the signal was registered either by a digital correlator (ALV-

5000/E, ALV, Langen, Germany) or by a multifunctional counter card (NI PCI-6229,

National Instruments, München, Germany). Fluorescence quantum yields were

calculated with respect to Rhodamine 6G (Φ = 0.95)14 by integrating the emission

spectra and normalizing them on the absorption at 488 nm. Fluorescence emission

anisotropy values r were calculated from fluorescence intensities I according to the

following relation:

r = (Ivv − G.Ivh)/(Ivv + 2G.Ivh),

with subindexes v and h denoting vertically and horizontally polarized light, while the

first subindex relates to excitation and last to emission. G = Ihv/Ihh is the correction

coefficient for the polarization-dependent detection efficiency. Data analysis was

performed either by a home-made software using Matlab 6.5 (MathWorks, Natick,

MA) or in Origin 8.0 (OriginLab, Northampton, MA).

Fluorescence excitation and emission spectra Fluorescence excitation and emission spectra were measured on a FP-6500

fluorophotometer with temperature control unit (Jasco GmbH, Groß-Umstadt,

Germany) in 15 µl fluorescence cuvettes at 25°C. Device settings were always

(except for kinetic measurements) 0.5 s (response time), 0.2 nm (data pitch),

sensitivity (high) and 200 nm/min (scanning speed). All fluorescence measurements

were corrected for the wavelength dependence of the detector efficiency and

recorded with an excitation bandwidth of 3 nm. Excitation spectra were recorded with

3 nm bandwidth for emission. In other cases emission bandwidths were 3 nm (for

spectra with excitation at 488 nm: 1-AB, 9-AB and bromo/iodo-BODIPY; for spectra

with excitation at 365 nm: 9-HMA), 5 nm (for 9-DAPB) and 10 nm (for spectra with

excitation at 405/422 nm: 1-AB, 9-AB). In all fluorescence emission scans three

consecutive scans were accumulated.

S25

Emission spectra in the presence of salts (LiCl, NaCl, KCl, MgCl2) or RNA

were recorded by preparing samples in triplicate amounts (45 µl), which were

measured separately (15 µl each). The resulting files were averaged to give the final

spectrum. Samples incorporating RNA were equilibrated at 25°C for 5.0 min before

measurement.

Absorption spectra of Figure S1A were recorded on a Cary 1 spectro-

photometer, others on a Cary 50 UV-Vis spectrophotometer (Varian) with a resolution

of 0.2 nm. The corresponding fluorescence spectra were recorded with a SPEX

Fluorolog II spectrometer (HORIBA Jobin Yvon, Edison, NJ) with the excitation line

width set to 2 nm. The resolution was set for 1 nm and corrected for the wavelength

dependence of the detector efficiency.

Quantum yield of the RNA bound probes Samples were prepared by lyophilisation of appropriate amounts of RNA,

dissolved by adding 5x DAse buffer and water, refolding (1.0 minute at 70°C and RT

for 20 min) followed by adding fluorescent dye and ethanol. Absorption (200-600 nm)

and fluorescence spectra (495-595 nm) were measured in a 15 µl cuvette at constant

dye concentrations (100 µM) and variable RNA concentrations. Photometer settings

were maintained equal for 1-AB, 9-AB and 9-DAPB. Each point in the plots

corresponds to the average value of at least three separate measurements. The

fraction of bound RNA was calculated according the law of mass action:

[ ] [ ] [ ] [ ] [ ] [ ] [ ]DyeRNAKDyeRNAKDyeRNADyeRNA dd ⋅−⎟⎠⎞

⎜⎝⎛ ++

−++

=⋅2

22

using Kd values from the FCS measurements. Absorbance (at 488 nm) and

fluorescence intensities (peak maximum) were plotted against the total fraction of

bound RNA and extrapolated to obtain the theoretical absorbance and fluorescence

for a 100% bound probe (Figure S5). For 9-DAPB half of the intensity of the free

probe was substracted from all other values to compensate for the fluorescence of

the “wrong” enantiomer. Calculation of quantum yield QY relative to the free

fluorophores was done with the following equation:

unboundbound

unbound

unbound

boundbound QY

AA

FLFLQY ⋅⋅=

S26

Fluorescence correlation spectroscopy A perfect fit of the measured FCS curves was obtained with a 3D diffusion

model15-17 including a fast relaxation phase due to dye dynamics in the form of

stretched exponential function18,

⎥⎥

⎦

⎤

⎢⎢

⎣

⎡

⎟⎟

⎠

⎞

⎜⎜

⎝

⎛⎟⎟⎠

⎞⎜⎜⎝

⎛−+

⎥⎥⎦

⎤

⎢⎢⎣

⎡⎟⎟⎠

⎞⎜⎜⎝

⎛+⎥

⎦

⎤⎢⎣

⎡+=

−− β

ττ

ττ

τττ

RDD

Fzr

NG exp1111)(

2/12

0

01

, (1)

where N is the number of particles in the observation volume, τD is the characteristic

diffusion time, r0 and z0 are the axial and radial components of the observation

volume, F is the equilibrium constant between dark and bright state, τR is the

characteristic relaxation time, and β is a stretching exponent. Relaxation kinetics in

the form of the stretched exponential function represent processes governed by

distributions of characteristic times. In our case, these relaxation processes occur on

the microsecond time scale and are likely related to the blinking behavior observed in

the burst analysis experiments; which may arise from intramolecular rotations around

the main axis of the anthracene-BODIPY dyads. In order to account for the presence

of different enantiomers for the 9-DAPB probe, eq. (1) was modified according to 19:

2

2/12

0

01

2 exp111

)(

⎟⎟⎠

⎞⎜⎜⎝

⎛

⎥⎥

⎦

⎤

⎢⎢

⎣

⎡

⎟⎟

⎠

⎞

⎜⎜

⎝

⎛⎟⎟⎠

⎞⎜⎜⎝

⎛−+

⎥⎥⎦

⎤

⎢⎢⎣

⎡⎟⎟⎠

⎞⎜⎜⎝

⎛+⎥

⎦

⎤⎢⎣

⎡+

=

∑

∑−−

iii

RDDi

ii

N

Fzr

N

G

α

ττ

ττ

ττα

τ

β

, (2)

where α accounts for the relative brightness of the corresponding species, and i

ascribes the bound and unbound states.

In the case of 1-AB and 9-AB, the dissociation coefficients, Kd, were extracted

by fitting the measured diffusion times to a modified Hill equation:

( )nd

SESSDEK ][1

1)(+

−+= ττττ , (3)

where τS and τES are the diffusion times of the free and bound substrates,

respectively, n is the cooperativity parameter, and [E] is the concentration of free

DAse ribozyme molecules. For both substrates the cooperativity parameter was set

to unity. Figure 3 shows deviations from the monotonic increase of τD for 1-AB and

9-AB at a DAse concentration of ~1.0 mM. Such a behavior may be attributed to a

concentration-dependent oligomerization of RNA molecules only in their substrate-

free form and, consequently, to a reduction of available binding partners for the

S27

substrates. At the highest substrate concentrations, binding dominates and, thereby,

suppresses oligomerization; thus, the diffusion time increases again. We modeled

this deviation from a normal binding curve heuristically by introducing a peak function

with a Gaussian shape. Finally, we assumed that the τES should be equal for all

bound probes (≈ 400 µs) because of much larger mass of the ribozyme molecules

compared to free probes. A global fit, shown in Figure 3 as black dashed lines,

revealed the binding constants of (300±42) and (1210±190) µM for the 1-AB and

9-AB, respectively. Though clear saturation of the diffusion time was not achieved in

the DAse ribozyme titration range due to the limited RNA concentration, the

evaluated diffusion times at the highest concentration correspond well to the diffusion

time obtained by means of FCS with the fluorescently labeled ribozyme molecules

(data not shown). For 9-DAPB, a global fit of all FCS curves at different DAse

ribozyme concentrations was performed using eq. (2). Here, the brightness of the

unbound species was fixed to 0.3 according to the measured quantum yield

(Table 1), whereas the brightness of the bound species was allowed to vary in the fit,

which returned a value of ~60%. The bound fraction was directly extracted from the

change of Nbound in eq. (2) and displayed in Figure 3. The data points were fitted with

the Hill equation, yielding Kd and n values of (12±5) µM and 0.65, respectively.

Burst analysis Fluorescence time traces of 1-AB and 9-DAPB were recorded on the confocal

microscope with 100-µs time resolution at sub-nanomolar probe concentrations.

Individual fluorescence emission bursts were analyzed by summing up the intensity

of consecutive time bins above a threshold of 5 counts. The corresponding threshold

of 50 kHz was chosen to reject out-of-focus light from fluorescent impurities at high

RNA concentrations. For the analysis, only bursts with durations between 200 and

1500 µs were taken into account; thus, the “average” burst length was larger than the

diffusion time extracted from the FCS experiments. Therefore, an increase of the

diffusion time upon binding to RNA was noticeable but not clearly resolved in the

burst analysis experiments. Bursts with more than 20 counts were selected for the

frequency histograms of burst occurrence. For each DAse ribozyme concentration,

measurements were carried out with and without fluorescent substrates. For

background correction, the respective histograms of burst occurrence with

fluorescent probes were corrected for the number of bursts registered in samples

S28

without probes. Comparison of normalized burst histograms calculated for different

DAse ribozyme concentration did not reveal any statistically significant difference of

the shapes of the distributions, only the total number of bursts was changed (Figure

5D and S7).

Fluorescence kinetic measurements A 1.0 M stock solution of NPM5 in DMSO was used for dilutions in pure EtOH,

which then was used in reaction mixtures. All catalyzed reactions were conducted by

equilibrating RNA and the fluorescent probes in standard DAse buffer for 2.0 min at

25°C. Reactions were started by addition of 1.5 µl NPM in EtOH to give 15 µl reaction

volume (final: 10% EtOH). Subsequently, the samples were transferred into a 15 µl

fluorescence cuvette, mixed thoroughly with the pipette and placed in the

spectrofluorometer. Measurements were started exactly 40 seconds after addition of

the last component. Control reactions were conducted by omitting NPM and ribozyme

(photoreactions) or ribozyme alone (background reactions).

All kinetic curves were measured with the following photometer settings:

response time (2.0 s), data pitch (1.0 s), sensitivity (high), excitation bandwidth

(3 nm) and 515 nm emission wavelength. Kinetic curves at single-turnover reaction

conditions were recorded with excitation at 488 nm and emission bandwidth set to

20 nm. Kinetic curves at multiple-turnover reaction conditions were recorded with

excitation at 405 nm (for 1-AB) or 422 nm (for 9-AB) with emission bandwidth set to

20 nm or at 488 nm (for 1-AB and 9-AB) with emission bandwidth set to 3 nm.

Reaction kinetics were also recorded on the confocal microscope setup under

conditions similar to burst analysis measurements. In these experiments, reactants

were mixed in appropriate proportions in a reaction tube and introduced into the

sample chamber immediately thereafter, so that the dead time between mixing and

measurement was < 5 s. Typical reaction progress curves at varying DAse ribozyme

concentration are shown in Figure S9.

S29

2.5. References (1) McIlvaine, T. C. J. Biol. Chem. 1921, 49, 183-186.

(2) www.cem.com.

(3) Brown, W. S.; Boykin, D. D.; Sonnier, M. Q.; Clark, W. D.; Brown, F. V.;

Shaughnessy, K. H. Synthesis 2008, 1965-1970.

(4) Heuft, M. A.; Collins, S. K.; Yap, G. P.; Fallis, A. G. Org. Lett. 2001, 3, 2883-

2886.

(5) Heitz, J. R.; Anderson, C. D.; Anderson, B. M. Arch. Biochem. Biophys. 1968,

127, 627-636.

(6) Li, L. L.; Han, J. Y.; Nguyen, B.; Burgess, K. J. Org. Chem. 2008, 73, 1963-

1970.

(7) Baruah, M.; Qin, W. W.; Basaric, N.; De Borggraeve, W. M.; Boens, N. J. Org.

Chem. 2005, 70, 4152-4157.

(8) Burghart, A.; Kim, H. J.; Welch, M. B.; Thoresen, L. H.; Reibenspies, J.;

Burgess, K.; Bergström, F.; Johansson, L. B. A. J. Org. Chem. 1999, 64, 7813-

7819.

(9) Chen, J.; Burghart, A.; Derecskei-Kovacs, A.; Burgess, K. J. Org. Chem. 2000,

65, 2900-2906.

(10) http://www.ambion.com/techlib/misc/oligo_calculator.html.

(11) Amirgoulova, E. V.; Groll, J.; Heyes, C. D.; Ameringer, T.; Röcker, C.; Möller,

M.; Nienhaus, G. U. ChemPhysChem 2004, 5, 552-555.

(12) Kobitski, A. Y.; Nierth, A.; Hengesbach, M.; Jäschke, A.; Helm, M.; Nienhaus,

G. U. Biophysical Reviews and Letters 2008, 3, 439-457.

(13) Szabelski, M.; Luchowski, R.; Gryczynski, Z.; Kapusta, P.; Ortmann, U.;

Gryczynski, I. Chem. Phys. Lett. 2009, 471, 153-159.

(14) Magde, D.; Rojas, G. E.; Seybold, P. G. Photochem. Photobiol. 1999, 70, 737-

744.

(15) Haustein, E.; Jahnz, M.; Schwille, P. ChemPhysChem 2003, 4, 745-748.

(16) Lamb, D. C.; Schenk, A.; Rocker, C.; Scalfi-Happ, C.; Nienhaus, G. U.

Biophys. J. 2000, 79, 1129-1138.

(17) Magde, D.; Elson, E. L.; Webb, W. W. Biopolymers 1974, 13, 29-61.

(18) Klafter, J.; Shlesinger, M. F. Proc. Natl. Acad. Sci. U. S. A. 1986, 83, 848-851.

(19) Lakowicz, J. R. Topics in Fluorescence Spectroscopy, Plenum Press: New

York, 1991, 337-374.

S30

2.6. Complete Reference (6) of Main Text

(6) Eid, J.; Fehr, A.; Gray, J.; Luong, K.; Lyle, J.; Otto, G.; Peluso, P.; Rank, D.;

Baybayan, P.; Bettman, B.; Bibillo, A.; Bjornson, K.; Chaudhuri, B.; Christians,

F.; Cicero, R.; Clark, S.; Dalal, R.; Dewinter, A.; Dixon, J.; Foquet, M.;

Gaertner, A.; Hardenbol, P.; Heiner, C.; Hester, K.; Holden, D.; Kearns, G.;

Kong, X.; Kuse, R.; Lacroix, Y.; Lin, S.; Lundquist, P.; Ma, C.; Marks, P.;

Maxham, M.; Murphy, D.; Park, I.; Pham, T.; Phillips, M.; Roy, J.; Sebra, R.;

Shen, G.; Sorenson, J.; Tomaney, A.; Travers, K.; Trulson, M.; Vieceli, J.;

Wegener, J.; Wu, D.; Yang, A.; Zaccarin, D.; Zhao, P.; Zhong, F.; Korlach, J.;

Turner, S. Science 2009, 323, 133-138.

S31

3. Spectra Appendix

0.01.32.53.85.06.37.58.8

013253850637588100113125138

7.257.507.758.008.258.508.75

1H NMR spectrum(CDCl3)

120.0122.5125.0127.5130.0132.5

APT NMR spectrum(CDCl3)

TMS

S32

S33

0.01.32.53.85.06.37.58.8

1H NMR spectrum(CDCl3)

8090100110120130

APT NMR spectrum(CDCl3)

7.508.008.509.00H

S34

S35

1.02.03.04.05.06.07.08.0

1H NMR spectrum(CD3OD/D2O, (5:2), RT)

0255075100125150

APT NMR spectrum(CD3OD/D2O, (5:2), RT)

120.0125.0130.0135.0140.0145.0150.0155.0

7.007.508.008.50

N NB

F F

SO3--O3S

Et3NH+ Et3NH+

S36

1.02.03.04.05.06.07.08.09.0

1H NMR spectrum(D2O, 60 °C)

0255075100125150175

APT NMR spectrum(D2O, 60 °C)

7.007.508.008.50

N NBF F

SO3--O3S

Et3NH+ Et3NH+

S37

Analysis InfoAnalysis Name 2/6/2009 3:23:14 PMD:\Data\Jaeschke\icr3229_000001.d Acquisition Date

Instrument ICR Apex-QeESI neg Arginin 150-1300MethodLang/MitschSample Name Operator1-Anthryl-BODIPY-2SO3Na-1

Nierth, 1-Anthryl-BODIPY-2SO3Na-1 in H2O/CH3CN Comment

Acquisition Parameters Collision Gas Flow Rate 0.5 L/secCollision Energy -0.5 eVCollision Cell RF 900.0 VQ1 Resolution 5.0 Q1 Mass 200.000 m/z

Laser Power 0.0 %MALDI Plate -230.0 VCalibration Date Fri Jan 30 08:13:07

2009

Accumulations 8 Broadband Low Mass 144.4 m/zBroadband High Mass 1500.0 m/zData Acquisition Size 1048576

Spectrum Display Report 1 of 16/22/2009Bruker Compass DataAnalysis 4.0 Page printed: 12:47:24 PM

S38

1.02.03.04.05.06.07.08.0

1H NMR spectrum(CD3OD/D2O, (5:2))

255075100125150

APT NMR spectrum(CD3OD/D2O, (5:2))

7.257.507.758.008.258.50

N NBF F

SO3--O3S

Et3NH+ Et3NH+

S39


Instrument ICR Apex-QeESI neg Arginin 150-1300MethodLang/MitschSample Name Operator9-Anthryl-BODIPY-2SO3Na-1

Nierth, 9-Anthryl-BODIPY-2SO3Na-1 5 ul 2mM in 1000 ul H2O/CH3CN nochmal 1:20 verd.Comment


Laser Power 0.0 %MALDI Plate -230.0 VCalibration Date Fri Jan 30 08:13:07

2009



S40

0.01.02.03.04.05.06.07.08.0

1H NMR spectrum((CD3)2CO)

0255075100125150175

APT NMR spectrum((CD3)2CO)

7.207.307.407.507.607.707.80

125.0130.0135.0140.0

NO

O

TMS

S41

Meas. m/z Formula m/z err [mDa] err [ppm] mSigma rdb N-Rule e¯ Conf442.22008 C 21 H 28 N 7 O 4 442.21973 -0.4 -0.8 17.4 11.5 ok even

C 28 H 32 N O 2 Si 442.21968 -0.4 -0.9 50.9 14.5 ok even

Mass Spectrum Formula ReportAcquisition DateAnalysis Info 7/6/2009 10:37:20 AM

M:\Data\Jaeschke\ic4302_000001.dAnalysis Name

Comment Nierth, AK: Jaeschke, 9-DAP-TMS in MeOH

7/17/2009 5:20:29 PM 1 of 1Bruker Compass DataAnalysis 4.0 printed: Page

S42

1.02.03.04.05.06.07.08.0

1H NMR spectrum((CD3)2CO)

1733506783100117133150167

APT NMR spectrum((CD3)2CO)

7.207.307.407.507.607.707.80

122.5125.0127.5130.0132.5

NO

O

H

S43

Meas. m/z Formula m/z err [mDa] err [ppm] mSigma rdb N-Rule e¯ Conf370.18032 C 25 H 24 N O 2 370.18016 -0.2 -0.4 7.2 14.5 ok even

Mass Spectrum Formula ReportAcquisition DateAnalysis Info 7/6/2009 10:28:34 AM

M:\Data\Jaeschke\ic4301_000001.dAnalysis Name

Comment Nierth, AK: Jaeschke, 9-DAP-H in MeOH

7/17/2009 5:18:34 PM 1 of 1Bruker Compass DataAnalysis 4.0 printed: Page

S44

1.02.03.04.05.06.07.08.0

1H NMR spectrum(D2O/CD3CN, (3:9))

0255075100125150175200225

APT NMR spectrum(D2O/CD3CN, (3:9))

7.607.707.807.908.008.108.20

NNB

FF

NaO 3S

SO3Na

NO

O

S45


Instrument ICR Apex-QeESI neg Arginin 150-1300MethodLang/MitschSample Name Operator9-DAP-BODIPY

Nierth, AG Jaeschke: 9-DAP-BODIPY in CH3CN/H2O 1:1Comment


Laser Power 0.0 %MALDI Plate -230.0 VCalibration Date Mon Feb 23 09:50:56

2009



S46

Documents

Electronic Supporting Information Anthracene-BODIPY dyads as