Chemical CommunicationsAll photophysical experiments were performed in quartz glass cuvettes (d = 1...

Chemical Communications

SUPPORTING INFORMATION FOR:NORBORNADIENE-BRIDGED DIARYLETHENES

AND THEIR CONVERSION INTO TURN-OFF FLUORESCENTPHOTOSWITCHES

S. M. BÜLLMANN AND A. JÄSCHKE

May 3, 2020

INSTITUTE OF PHARMACY

AND MOLECULAR BIOTECHNOLOGY

HEIDELBERG UNIVERSITY

Electronic Supplementary Material (ESI) for ChemComm.This journal is © The Royal Society of Chemistry 2020

Contents

1 General Information 11.1 Reaction control and purification . . . . . . . . . . . . . . . . . . . . . . . . . 1

1.2 Nuclear Magnetic Resonance (NMR) spectroscopy . . . . . . . . . . . . . . . 1

1.3 Mass spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

2 Photophysical experiments 22.1 Investigation of the cyclization reaction of the diarylethenes . . . . . . . . . . . 2

2.2 Determination of the fatigue resistance . . . . . . . . . . . . . . . . . . . . . . 2

2.3 Determination of the thermostability . . . . . . . . . . . . . . . . . . . . . . . 2

2.4 Determination of the switching efficiency . . . . . . . . . . . . . . . . . . . . 3

2.5 Determination of the cyclization and cycloreversion quantum yields . . . . . . 4

2.6 Determination of the second-order rate constant of the inverse electron-demandDiels-Alder reaction (iEDDA) . . . . . . . . . . . . . . . . . . . . . . . . . . 5

2.7 Determination of the fluorescence quenching of the turn-off fluorescent photo-switch (15) during cyclization of the DAE moiety . . . . . . . . . . . . . . . . 5

3 Supplementary Figures 6

4 Synthetic procedures and analytical data 22

5 Appendix 425.1 1H-and 13C-NMR-spectra of the final compounds . . . . . . . . . . . . . . . . 42

5.2 Shutter files generated by the QYDS . . . . . . . . . . . . . . . . . . . . . . . 49

1 GENERAL INFORMATION

1 General Information

The synthesis of oxygen- or moisture-sensitive compounds was performed under an argon at-mosphere using the Schlenk-technique. Directly before setting up the reaction, a Schlenk vessel(flask or tube) was evacuated and heated with a heat gun and afterwards flushed with argon threeconsecutive times. If not stated otherwise, anhydrous solvents were degassed using freeze-pump-thaw cycles. Chemicals were purchased from Sigma Aldrich, ABCR or TCI and usedwithout further purification.

1.1 Reaction control and puri�cation

Standard column chromatography was performed on silica gel (60 M, 0.04-0.063 mm) pur-chased from Sigma Aldrich. Automated column chromatography was performed with an Pu-riFlash system (model 420) from Interchim using commercially columns from Telos. Ana-lytical thin-layer chromatography (TLC) was done on Marchery Nagel’s precoated silica gelPolygramm Sil G/UV264 plates and visualized by irradiating the TLC-plate with a hand-lampfrom Krüss Optronic at 254 nm and 366 nm. Furthermore, two HPLC systems (1100 se-ries and 1200 series) from Agilent Technologies (USA) were used for analytical as well aspreparative purposes. HPLC was performed using buffered mixtures of water (buffer A: 100mM triethylammonium-acetate solution in water, pH 7.0) and acetonitrile (buffer B: 100 mMtriethylammonium-acetate solution in 4:1 acetonitrile:water, pH 7.0). Furthermore, water waspurified with a MilliQ purification system (model A10 Synthesis) from Merck. If not statedotherwise, HPLC-column from Phenomenex (Luna 5u C18(2) 100A, 4.6 mm) was used foranalytical purposes. For the purification of crude products, a semi-preperative HPLC columnfrom Phenomenex (Luna 5u C18(2) 100A, 15 mm) was used.

1.2 Nuclear Magnetic Resonance (NMR) spectroscopy

Substance samples were dissolved in deuterated solvents prior to the measurement. NMR sys-tems from Varian Inc. with 300 MHz (Mercury plus 300 MHz) and a resolution of 500 MHz(Mercury plus 500 MHz) were used. Chemical shifts were indicated in parts per million (ppm)in reference to trimethylsilane. Splitted signals were described with following abbreviations: s= singulet, d = doublet, t = triplet, q = quartett, qi = quintett and m = multiplet.

1.3 Mass spectrometry

A mass spectrometer from Bruker (micro TOFQ-II) was available. This system was operatedin an ESI- and APCI-Mode. The molecular ion was indicated as [M ]+ in the positive mode and

1

2 PHOTOPHYSICAL EXPERIMENTS

as [M ]− in the negative mode. Solvents for detection in mass spectrometry were acetonitrile ormethanol depending on the solubility of the measured compound.

2 Photophysical experiments

All photophysical experiments were performed in quartz glass cuvettes (d = 1 cm, volume =3 mL) from Hellma (Model 110-10-40, Macro) at 25◦C. Stock solutions of the photoswitcheswith a concentration of 10 mM in DMSO were prepared and stored at -21◦C. Prior to thephotophysical experiments, the stock solutions were diluted with HPLC grade acetonitrile to afinal concentration of 100µM . During irradiation, the sample solution was stirred with magneticstirring bars to guarantee homogenety of the mixture.

2.1 Investigation of the cyclization reaction of the diarylethenes

For the determination of the photoswitching process, 2 mL of a 100 µM solution of the samplein acetonitrile were prepared and added to a quartz cuvette from Hellma (Model: 110-10-40,Macro). After the irradiation of the sample with a 300 nm UV-light LED from Nikkiso (model:VPC1A1) at defined time points, an absorbance spectrum was recorded with a Cary 100 BioUV/VIS-spectrometer from Varian and the data were analyzed with the software CaryWinUV.The irradiation times are listed for each photoswitch in it’s respective shutter file, which areshown in section 5.2.

2.2 Determination of the fatigue resistance

For the determination of the fatigue resistance, 2 mL of a 60 µM solution of the sample in ace-tonitrile were prepared and added to a quartz cuvette from Hellma (Model: 110-10-40, Macro).Then the solution was irradiated with a 310 nm LED (Thorlabs, Mounted High Power LED, op-erated at 350 mA) until the photostationary state was reached and an absorption spectrum wasrecorded with a Cary 100 Bio UV/VIS-spectrometer from Varian. Afterwards the sample wasirradiated with visible light (Intralux 600-1) until the absorption band in the visible wavelengthrange was erased, and a spectrum was recorded. This process was repeated for ten cycles andthe absorption maximum in the visible wavelength range of the closed ring isomer was plottedin a graph.

2.3 Determination of the thermostability

For the determination of the thermostability, 2 mL of a 60 µM solution of the sample in ace-tonitrile were prepared and added to a quartz glass cuvette from Hellma (Model: 110-10-40,

2


Macro). Irradiation of the sample was performed with a 310 nm UV-LED (Thorlabs, MountedHigh Power LED, operated at 350 mA) until the photostationary state was reached. Then theabsorption maximum at 480-500 nm of the closed ring isomer was recorded over 60 min at 20◦C and 50 ◦C with a Cary 100 Bio UV/VIS-spectrometer from Varian. The recorded data wasevaluated with the software Cary Win UV and OriginPro 9.1. Data processing included base-line correction and plotting the absorbance at 480-500 nm of the closed isomer at a time t (A),divided by the absorption measured at the beginning of the measurement (Ao) versus time.

2.4 Determination of the switching e�ciency

For the determination of the switching efficiency, 50 µL of a 100 µM solution of the openedring isomer were prepared and injected into the HPLC. Furthermore, 50 µL of a 100 µM so-lution of the sample after irradiation with a 310 nm UV-LED (Thorlabs, Mounted High PowerLED, operated at 350 mA) until the photostationary state was reached were injected into theHPLC. Separation of the two isomeres was performed on an analytical HPLC column fromPhenomenex (250 x 4.6 mm, 5 micron, Luna 5u C18, 100A). The methode shown in table 1was used in order to separate the two isomers upon irradiation with UV-light.

Table 1: HPLC method for the determination of the switching e�ciency of the photoswitches

Time [min] Bu�er B [%] Flow rate[mL/min]

0 65 1.5

30 100 1.5

40 100 1.5

42 65 1.5

50 65 1.5

The composition of the photostationary state was calculated by integration of the peaks at anisobestic wavelength. The recorded data was evaluated with the software OriginPro 9.1. Dataprocessing included baseline correction as well as peak integration. To ensure the consistencyof the isobestic points in buffered mixtures of acetonitrile and water, an additional absorptionspectrum of the sample was measured in the mixture at which it eluted from the HPLC column.

3


2.5 Determination of the cyclization and cycloreversion quantum

yields

Quantum yields were measured on an updated Quantum yield determination setup (QYDS)by König and Riedle [6]. Irradiation of the photoswitches was performed by Nikkiso SMD-mounted 300 nm LED (model: VPC173) and Osram Oslon SSL80 470 nm LED (model:LBCP7P-GYHY). The camara lens was replaced by two best fit lenses. The LED output ra-diant power was calibrated against the output voltage of the solar cell by using a power meterfrom Coherent (model: PowerMax-USB PS19Q). The raw data measured with the QYDS wasfurther processed with a Mathcad script provided by the Riedle group. The calculation of thequantum yields is based on the "initial slope method". Therefore, the power of the LED wasturned down sufficiently enough that the back reaction is negligible and the formation of theproduct is linear. Data processing with the Mathcad script includes baseline correction, spectraldecomposition of each spectrum into the substrate and product basis spectrum, and calculationof the quantum yields by the number of incoming photons per second and wavelength. Conver-sion to the cyclization product was calculated using the extinction coefficients of both isomers.Hereby, the concentration changes of the two isomers are numerically simulated with accountto the spectral dependencies of molar absorptivities and LED ligth. The simulated curve is fit-ted to the concentration values. The spectral composition of the PSS is a linear combination ofthe open- and closed-isomer, which makes it possible to calculate the spectrum of pure closedisomer, if the composition of the PSS is known. The composition of the PSS was determined asdescribed earlier, by separation of both isomers on an analytical HPLC column and subsequentintegration of the peaks at an isobestic point. The calculation of the absorbance as well as themolar extinction coefficient for each measured wavelength is described in the following:

EPSS = ECF ·X + EOF · Y

ECF =EPSS − EOF · Y

X

εCF =ECF

c0

The script uses the molar extinction coefficients of the two isomers to calculate the concen-tration of generated product (closed isomer) at each irradiation step which is then fitted withthe absorbed radiant power from the solar cell in order to calculate the quantum yield. Duringthe measurement a "shutter file" is generated, which displays the exact irradiation time pointsas well as the absorbed radiant power of each step. The shutter files of the photoswitches,described here, are displayed in section 5.2.

4


2.6 Determination of the second-order rate constant of the in-

verse electron-demand Diels-Alder reaction (iEDDA)

Reaction rates of the iEDDA reaction were determined according to a procedure by A. C. Knallet al.[7]. Reaction rates of iEDDA reactions can be easily determined by monitoring the decayof tetrazine concentration in the presence of excess dienophile (pseudo first-order conditions).The UV-Vis absorption spectrum of 1,4-di-(2-pyridyl)-tetrazine (pyTz) shows a destinctive ab-sorption maximum at 550 nm [ε = 400Lmol−1cm−1], which is responsible for the pink colorof the compound. Solutions of pyTz and the respective alkene (both in a 1:1 mixture of DMSOand MeOH) were mixed in a 384-well-plate (Corning, black and white, working volume 20 µL-80 µL, model: 3820), so that a final concentration of 5 mM pyTz and 10, 20, 30 and 35 mMof alkene substrate was achieved and the iEDDA was initiated. Directly after mixing, the decayof the tetrazine concentration was monitored by measuring the absorbance of pyTz at 545 nmat 25◦C with a TECAN safire2 (Tecan Trading AG) plate reader. The pseudo-first order reac-tion rate constants were determined by linear fits of ln([pyTz]/[pyTz0]) plotted versus reactiontime. Linear regression of the pseudo-first order reaction rate constants with the alkene con-centrations yielded the second-order rate constant for the respective alkene. All measurementswere done in triplicates and and a negative control with no addition of the alkene was run inparallel, which showed no change of the pyTz concentration during the first 1000 s.

2.7 Determination of the �uorescence quenching of the turn-o�

�uorescent photoswitch (15) during cyclization of the DAE

moiety

For the determination of the fluorescence quenching, 2 mL of a 40 µM solution of the sample inmethanol were prepared and added to a quartz cuvette from Hellma (Model: 110-10-40, Macro).Then the solution was irradiated with a 310 nm LED (Thorlabs, Mounted High Power LED,operated at 350 mA) until the photostationary state was reached and an absorption spectrumwas recorded with a Cary 100 Bio UV/VIS-spectrometer from Varian. After each irradiationstep 10 µL of the 40 µM solution were extracted and dilluted to a 4 µM solution in methanol. 60µL of this diluted solution were then filled to a fluorescence cuvette from Hellma (Model: 101-QS, 50 µL) and an emission fluorescence spectrum was recorded with a MOS 250 fluorometer(excitation wavelength: 460 nm, emission wavelength: 510 nm , bandwith: 10 nm, HV: 750 V).

5

3 SUPPLEMENTARY FIGURES

3 Supplementary Figures

300 400 500 600 700 800

0.0

0.5

1.0

1.5

2.0

2.5

Abso

rbance

Wavelength (nm)

A

300 400 500 600 700 800

0.0

0.5

1.0

1.5

2.0

Ab

so

rba

nce

Wavelength (nm)

B

Figure S1 (A) Absorption spectrum of a 100µM solution of (1a) in acetonitrile, irradiated with

300 nm UV-light at various time points (see shutter �le in section 5.2). (B) Absorption spectrum

of a 100µM solution of (1b) in acetonitrile, irradiated with 300 nm UV-light at various time

points (see shutter �le in section 5.2).

300 400 500 600 700 800

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Abso

rbance

Wavelength (nm)

A

300 400 500 600 700 800

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Absorb

ance

Wavelength (nm)

B

Figure S2 (A) Absorption spectrum of a 100µM solution of (1c) in acetonitrile, irradiated with

300 nm UV-light at various time points (see shutter �le in section 5.2). (B) Absorption spectrum

of a 100µM solution of (11) in acetonitrile, irradiated with 300 nm UV-light at various time

points (see shutter �le in section 5.2).

6


300 400 500 600 700 800

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Ab

so

rba

cn

e

Wavelength (nm)

0 min20 min40 min90 min150 min200 min

A

300 400 500 600 700

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

Wavelength (nm)

Absorb

ance

B

Figure S3 (A) Absorption spectrum of a 50µM solution of (13) in methanol, irradiated with

310 nm UV-light at the indicated time points. (B) Absorption spectrum of a 50µM solution of

(14) in acetronitrile, irradiated with 300 nm UV-light at various time points (see shutter �le in

section 5.2).

300 400 500 600 700 800

0.0

0.5

1.0

1.5

2.0

Ab

so

rba

nce

Wavelength (nm)

0 min10 min20min30 min40 min60 min80 min100 min120 min140 min

A

Figure S4 (A) Absorption spectrum of a 40µM solution of (15) in methanol, irradiated with

310 nm UV-light at the indicated time points.

7


3.63.84.04.24.44.64.85.05.25.45.65.86.06.26.46.66.87.07.27.47.67.8(ppm)

0

100

200

300

400

500

600

700

800

900

1000

2.0

1

2.0

12

.01

2.0

2

4.0

5

4.0

2

3.8

43

.85

3.8

5

7.0

27

.02

7.0

27

.04

7.2

27

.24

7.2

57

.26

CD

Cl3

7.3

37

.34

7.3

67

.52

7.5

3

3.63.84.04.24.44.64.85.05.25.45.65.86.06.26.46.66.87.07.27.47.6(ppm)

0

200

400

600

800

1000

1200

1.0

91

.10

1.0

5

1.0

2

2.0

0

2.0

44

.05

4.1

5

3.6

23

.66

6.2

16

.22

6.2

36

.33

6.3

46

.35

6.5

1

7.2

6 C

DC

l37

.29

7.3

17

.34

7.3

67

.37

7.5

37

.54

SS

H (7.02)

H (3.85)

H (7.04)

S S

H (6.34 / 6.22)

H (3.66 / 3.64)

H (6.51)

A

B

Figure S5 (A) 1H-NMR-spectrum of (1a) in the open form. (B) 1H-NMR-spectrum of (1a)

in the photostationary state. Chemical shifts that occur during cyclization are indicated at

the displayed structure. Alkene signals of the NBD-structure are conserved, which indicates

the formation of closed isomer instead of quadricyclane isomer. Additionally, two diastereomers

are observed after the cyclization reaction. The two diastereomeres show di�erent chemical

shifts, especially for the thiophene signals at 6.34 ppm and 6.22 ppm. The formation of the two

diastereomeres also indicates the formation of the displayed closed isomer. New signals in the

aliphatic region, that would appear if the quadricyclane valence isomer is formed, can not be

detected.

8


0 10 20 300

50

100

150

0

50

100

150

PSS

Abso

rban

ce (m

Au)

Time (min)

OF

CF

OF

Figure S6 Baseline-corrected HPLC spectrum of compound (1a) before (OF, front layer) and

after (PSS, back layer) irradiation with 300 nm UV-light. Absorption was measured at 277 nm

which matches the isobestic point of (1a). Open form (OF) and closed form (CF) are labeled

in the graph. Integration of the separated peaks results in a switching yield of 97%.

0 10 20 30 400

20

40

60

0

20

40

60

Abso

rban

ce (m

Au)

Time (min)

OF

PSS

CF

OF

Figure S7 Baseline-corrected HPLC spectrum of compound (1b) before (OF, front layer) and


which matches the isobestic point of (1b). Open form (OF) and closed form (CF) are labeled


9


0 10 20 30 40 500

20

40

60

0

20

40

60

Abso

rban

ce (m

Au)

Time (min)

OF

PSS

CF

OF

Figure S8 Baseline-corrected HPLC spectrum of compound (1c) before (OF, front layer) and


which matches the isobestic point of (1c). Open form (OF) and closed form (CF) are labeled in

the graph. Integration of the separated peaks results in a switching yield of 92%.

0 5 10 15 20 25 30 350

50

100

150

200

0

50

100

150

200

PSS

Abso

rban

ce (m

Au)

Time (min)

OF

CF

OF

Figure S9 Baseline-corrected HPLC spectrum of compound (11) before (OF, front layer) and


which matches the isobestic point of (11). Open form (OF) and closed form (CF) are labeled


10


0 5 10 15 20 25 30 35 40 450

25

50

75

100

0

25

50

75

100

OF

OF

Abso

rban

ce (m

Au)

Time (min)

PSS

CF





0 5 10 15 20 25 30 35 40 450

10

20

30

40

0

10

20

30

40

Abso

rban

ce (m

Au)

Time (min)

CFOF

OF

PSS





11


0.0

0 1 2 3 4 5 6 7 8 9 10

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

Cycles

Absorb

ance

A

0 1 2 3 4 5 6 7 8 9 10

0.0

0.2

0.4

0.6

0.8

1.0

Cycles

Ab

so

rba

nce

B

Figure S12 (A) Reversibility measurement of (1a). (B) Reversibility measurement of (1b)

Cycles

0 1 2 3 4 5 6 7 8 9 10

0.0

0.2

0.4

0.6

0.8

Ab

so

rba

nce

Figure S13 Reversibility measurement of (1c)

12


Cycles

0 1 2 3 4 5 6 7 8 9 10

0.0

0.2

0.4

0.6

0.8

Abso

rba

nce

A

Cycles

Abso

rba

nce

0 1 2 3 4 5 6 7 8 9 100.0

0.2

0.4

0.6

0.8

1.0

1.2

Figure S14 (A) Reversibility measurement of (11). (B) Reversibility measurement of (14)

A/A

0

Time (min)

0.0

0.2

0.4

0.6

0.8

1.0

10 20 30 40 50 60

20°C

50°C

A

Time (min)10 20 30 40 50 60

A/A

0

0.0

0.2

0.4

0.6

0.8

1.0

20°C

50°C

B

Figure S15 (A) Thermostability measurement of (1a). (B) Thermostability measurement of

(1b)

13


Time (min)10 20 30 40 50 60

A/A

0

0.0

0.2

0.4

0.6

0.8

1.0

20°C

50°C

A

Time (min)10 20 30 40 50 60

A/A

0

0.0

0.2

0.4

0.6

0.8

1.0

20°C

50°C

B

Figure S16 (A) Thermostability measurement of (1c). (B) Thermostability measurement of

(11)

Time (min)10 20 30 40 50 60

A/A

0

0.0

0.2

0.4

0.6

0.8

1.0

20°C

50°C

A

Time (min)10 20 30 40 50 60

A/A

0

0.0

0.2

0.4

0.6

0.8

1.0

20°C

50°C

B

Figure S17 (A) Thermostability measurement of (14). (B) Thermostability measurement of

(15)

14


0 5 10 15 20 25 30 35 40

m/z: 411.1 (CPD)m/z: 437.1 (NBD)m/z: 617.2 (CPDC)

cNBD

: 0.01 Mm/z: 237.2 (pyTz)

cNBD

: 0.02 M

cNBD

: 0.027 M

Time (min)

cNBD

: 0.035 M

S SPh PhS SPh Ph S SPh Ph

Exact Mass: 436,1319 Exact Mass: 410,1163

NNN

N

Exact Mass: 616,1755

N

N N

NN

N

Exact Mass: 236,0810

(NBD) (CPD) (CPDC) (PyTz)

A

B

Figure S18 (A) Analytical HPLC runs after the iEDDA cascade reaction of 1,4-(2-dipyridyl)-

tetrazine with di�erent concentrations of compound (1a). Peaks were collected and analyzed by

mass spectrometry (MS). (B) Chemical structures of the molecules detected in MS. Analysis of

the MS data revealed that the dihydropyridazine (9) can not be isolated. The similarity of the

two determined iEDDA 2nd-order reaction rates prompted us to investigate if compound (1a)

could react with pyTz twice and thereby distort the kinetic study. Therefore, analytical HPLC

runs of the samples with di�erent alkene concentrations in the plate reader were performed sequal

to the kinetic experiment. According to the HPLC study, it can be concluded that (1a) does not

react twice with 1,4-(2-dipyridyl)-tetrazine at the concentrations used in the kinetic experiments.

15


20 25 30

5

10

15

205

10

15

20

Abso

rban

ce m/z: 619.2 (CPDCred)

m/z: 617.2 (CPDCox)

m/z: 411.1 (CPD)

120 min

80 min

40 min

Time (min)

0 min

m/z: 437.1 (NBD)

Figure S19 Analytical HPLC study of the iEDDA reaction cascade of pyTz with compound

(1a) at lower (0,05 mM) and equimolar concentrations. Peaks were collected and analyzed by MS

as indicated in the �gure. It can be concluded that the dihydropyridazine does not accumulate

during the iEDDA reaction cascade. Furthermore we determined that a dual iEDDA reaction

to the CPDC (see structures in Fig. S18) does not take place signi�cantly and thereby does not

distrot the kinetic measurements.

16


0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.511.0(ppm)

-10

0

10

20

30

40

50

60

70

80

90

100

110

120

130

140

150

3.0

7

3.0

7

1.0

01.0

21.0

72.0

31.0

97.1

91.2

72.0

11.0

51.0

21.0

5

1.0

31.0

21.0

4

1.8

7

2.3

2

6.6

37.0

77.0

87.0

97.0

97.1

07.1

87.1

97.2

17.2

37.2

47.2

6 C

DC

l37.2

87.2

97.3

07.3

07.3

17.3

57.3

57.3

67.3

77.3

87.3

87.4

57.4

67.4

87.4

87.4

97.5

07.6

17.9

67.9

67.9

77.9

87.9

97.9

98.4

78.4

88.6

08.6

18.8

68.8

7

7.07.58.08.5(ppm)

1.0

21.0

72.0

31.0

97.1

91.2

72.0

11.0

51.0

2

1.0

5

1.0

3

1.0

2

1.0

4

7.0

77.1

97.2

17.2

37.2

47.2

6 C

DC

l37.2

87.2

97.3

07.3

07.3

17.3

67.3

77.4

57.4

67.4

97.6

17.9

77.9

88.4

78.4

88.6

08.6

18.8

68.8

7

SS

NN

N

N

Figure S20 1H-NMR-spectrum of the main product isolated after the iEDDA reaction cascade

of (1a) and 1,4-(2-dipyridyl)-tetrazine. The NMR spectra shown here as well as the MS-Data

shown in Fig. S18-S19 clearly demonstrate the existence of the dual iEDDA reaction product.

0102030405060708090100110120130140150160170180190(ppm)

-80

-70

-60

-50

-40

-30

-20

-10

0

10

20

30

40

50

60

70

80

90

100

110

120

130

140

14.0

114.7

3

77.1

6 C

DC

l3

109.1

6115.9

9120.4

4123.6

9124.0

2124.8

1125.4

1125.5

2126.3

6126.8

2126.9

5127.5

7128.7

6128.8

8134.7

7134.8

2134.9

2135.2

2135.4

3136.2

0136.5

2137.3

6138.5

8139.2

5143.6

8149.1

6149.8

1

SS

NN

N

N

Figure S21 13C-NMR-spectrum of the main product isolated after the iEDDA reaction cascade

of (1a) and 1,4-(2-dipyridyl)-tetrazine.

17


0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.510.010.511.011.5(ppm)

0

50

100

150

200

250

300

350

400

450

1.9

8

2.0

3

2.0

04

.00

1.3

2 N

HE

t3+

3.1

4 N

HE

t3+

7.2

6 C

DC

l37.4

17.4

17.4

27.4

37.4

47.4

47.9

07.9

17.9

27.9

27.9

37.9

48.6

98.7

48.7

48.7

58.7

6

7.58.08.5(ppm)

1.9

8

2.0

3

2.0

04

.00

7.4

17

.41

7.4

27

.43

7.4

47

.44

7.9

07

.91

7.9

27

.92

7.9

37

.94

8.6

98

.74

8.7

48

.75

8.7

6

NNN

N

Figure S22 1H-NMR-spectrum of the 1,4-dipyridylpyrazine side product that is formed after the

rearangement of the dihydropyridazine (9). The NMR spectra shown here as well as the MS-Data

shown in the synthetic section clearly demonstrates the existence of the iEDDA-rearangement

reaction cascade.

0102030405060708090100110120130140150160170180190200210(ppm)

-11

-10

-9

-8

-7

-6

-5

-4

-3

-2

-1

0

1

2

3

4

5

6

7

8

9

10

11

12

13

77.1

6 C

DC

l3

121.9

4125.0

0125.4

0

137.5

2

149.5

1153.4

4158.2

2

NNN

N

Figure S23 13C-NMR-spectrum of the 1,4-(2-dipyridyl)-pyrazine side product that is formed

after the rearangement of the dihydropyridazine (9).

18


-2-101234567891011121314(ppm)

0

50

100

150

200

250

300

350

400

450

3.0

23

.01

1.8

3

0.5

92

.00

0.6

31

.01

1.0

00

.71

2.0

12

.04

1.5

04

.09

1.3

44

.01

2.0

5A

2.1

4A

2.2

8 B

3.2

9 B

3.5

1A

6.4

9 B

6.5

4A

6.5

5A

6.7

2A

6.7

3A

6.8

8 B

7.0

9 a

7.1

0 a

7.2

0A

7.2

2A

7.2

2A

7.2

4A

7.2

6 C

DC

l37

.28 B

7.3

0 B

7.3

1 B

7.3

3A

7.3

4A

7.3

6A

7.4

3 B

7.4

4 B

7.5

1A

7.5

2A

6.46.56.66.76.86.97.07.17.27.37.47.57.6

(ppm)

0.6

31

.01

1.0

0

0.7

1

2.0

1

2.0

41

.50

4.0

91

.34

4.0

1

6.4

9 B

6.5

4A

6.5

5A

6.7

2A

6.7

3A

6.8

8 B

7.0

9 a

7.1

0 a

7.2

0A

7.2

2A

7.2

2A

7.2

4A

7.2

6 C

DC

l37

.28

B7

.30

B7

.31

B7

.33

A7

.34

A7

.36

A7

.43

B7

.44

B7

.51

A7

.52

A

SS SS

A B

4:1

Figure S24 1H-NMR-spectrum of (11) that is formed as main product in the iEDDA reaction

of (1a) with equimolar concentrations of 1,4-(2-dipyridyl)-tetrazine.

-100102030405060708090100110120130140150160170180190200210220230

(ppm)

-50

-40

-30

-20

-10

0

10

20

30

40

501

4.3

91

4.5

01

4.7

3

40

.97

46

.01

77

.16

CD

Cl3

12

4.5

31

24

.81

12

5.3

71

25

.47

12

5.5

21

25

.60

12

7.0

51

27

.18

12

8.8

81

28

.96

13

1.8

01

32

.07

13

4.4

31

34

.60

13

4.7

21

34

.93

13

5.2

71

35

.64

13

5.8

21

36

.33

13

7.2

81

37

.41

13

9.3

51

39

.82

14

0.1

71

42

.45

SS SS

A B

4:1

Figure S25 13C-NMR-spectrum of (11) that is formed as main product in the iEDDA reaction

of (1a) with equimolar concentrations of 1,4-(2-dipyridyl)-tetrazine.

19


0 200 400 600 800

-0.6

-0.5

-0.4

-0.3

-0.2

-0.1

0.0

ln([

pyT

z]/[p

yT

z] 0

)

Time (s)

0,01 MNBD

0,02 MNBD

0,027 MNBD

0,035 MNBD

Equation y = a + b*x

Weight No Weighting

Residual Sum

of Squares

1,14364E-4 8,6865E-5 2,08914E-4 9,74251E-4

Pearson's r -0,99934 -0,99979 -0,99967 -0,99903

Adj. R-Square 0,99864 0,99956 0,99933 0,99801

Value Standard Error

ln(tz)/ln(tz0)

Intercept -0,00233 5,37958E-4

Slope -1,98689E-4 1,17463E-6

Intercept 0,00183 4,68841E-4

Slope -3,06063E-4 1,02371E-6

Intercept -0,00234 7,27087E-4

Slope -3,81729E-4 1,58759E-6

Intercept -0,01026 0,00157

Slope -4,79442E-4 3,42838E-6

0.010 0.015 0.020 0.025 0.030 0.035

0.0002

0.0003

0.0004

0.0005

ka

pp

(s-1)

cNBD

(M)


Weight Instrumental

Residual Sumof Squares

8,8375

Pearson's r 0,99965

Adj. R-Square 0,99895


kappIntercept 8,86308E-5 4,03836E-6

Slope 0,01091 2,03889E-4

A B

Figure S26 (A) Determination of the pseudo-�rst-order reaction rate by plotting

ln([pyTz]/[pyTz0]) versus reaction time with excess of compound (1a). (B) Linear regression of

pseudo-�rst-order constant versus alkene concentration.

0 200 400 600 800 1000

-0.5

-0.4

-0.3

-0.2

-0.1

0.0

ln([

pyT

z]/[p

yT

z0])

Time (s)

0,01 MCPD

0,02 MCPD

0,03 MCPD

0,035 MCPD


Weight No Weighting

Residual Sum

of Squares

1,88479E-4 3,40105E-4 5,81694E-4 5,23069E-4

Pearson's r -0,99883 -0,99926 -0,99921 -0,99948

Adj. R-Square 0,99761 0,99848 0,99839 0,99894


ln(pyTz/pyTz0)

Intercept -0,0011 5,41189E-4

Slope -1,32674E-4 9,17405E-7

Intercept -0,00294 7,26982E-4

Slope -2,23706E-4 1,23236E-6

Intercept -0,00504 9,50745E-4

Slope -2,84236E-4 1,61167E-6

Intercept -0,00905 9,01564E-4

Slope -3,31937E-4 1,5283E-6

0.010 0.015 0.020 0.025 0.030 0.035

0.00010

0.00015

0.00020

0.00025

0.00030

0.00035

0.00040

ka

pp

(s-1)

cCPD

(M)


Weight Instrumental

Residual Sum

of Squares

97,04409

Pearson's r 0,99666

Adj. R-Square 0,98998


kIntercept 5,72859E-5 1,01593E-5

Slope 0,00784 4,54719E-4

A B

Figure S27 (A) Determination of the pseudo-�rst-order reaction rate by plotting

ln([pyTz]/[pyTz0]) versus reaction time with excess of compound (11). (B) Linear regression of

pseudo-�rst-order constant versus alkene concentration.

20


0 10 20 30 40 50

0

200

400

600

800

1000

0 10 20 30 40 50

0

2

4

6

8

10

Time (min)

490 nm

0 10 20 30 40 50

0

20

40

60

80

100

120

140

Time (min)

Emission at 510 nm

Absorb

ance (

mA

u)

Absorb

ance (

mA

u)

Flu

ore

scence E

mis

sio

n310 nm

Time (min)

m/z = 940.2519 (M+H )+

NNMe

S SPh Ph

HN

O

O

O

OH

CO

2 H

Exact Mass: 939 2437.

B

C

0 10 20 30 40 50

0

20

40

60

80

100

Absorb

ance (

mA

u)

Time (min)

310 nm

m/z = 437.1308 (M+H )+

SSPh Ph

Exact Mass: 436 1319.

A

Figure S28 A: Analytical HPLC directly after mixing of the tetrazine modi�ed �uorescein with

compound (1a). An excess of the dye-tetrazine conjugate of 3 eq. in relation to compound

(1a) was used. The peak at 46 min was collected and identi�ed as compound (1a) by MS. B:

Analytical HPLC study of the iEDDA reaction cascade. Complete conversion of the dienophile

was observed after 10 h at room temperature. No signi�cant formation of side products was

detected as can be seen in the HPLC diagram. The peaks close the the injection peak were

identi�ed as excess dye-tetrazine conjugate as well as the pyridazine, that is formed during the

iEDDA reaction cascade. The product peak was collected and analyzed via MS. C: HPLC

diagrams of compound (15) after puri�cation.

21

4 SYNTHETIC PROCEDURES AND ANALYTICAL DATA

4 Synthetic procedures and analytical data

2,3-dibromobicyclo[2.2.1]hepta-2,5-diene (2)

Br

Br

Procedure A:Norbornadiene (NB-1) (18.4 g, 200 mmol) was added to a flame-dried 3-neck flask containingpotassium tert-butoxide (11.56 g, 103 mmol) and 180 mL anhydrous THF, which was cooledto -80 ◦C. n-Butyllithium (40 mL, 102 mmol, 2.5 M in hexane) was added dropwise over 1 hto the reaction mixture, while the temperature was maintained below -70 ◦C. The mixture wasstirred at -65 ◦C for 30 min, then at -40 ◦C for 30 min. Afterwards, the mixture was cooledto -78 ◦C, 1,2-dibromoethane (9.7 g, 51.5 mmol) was added and the mixture was stirred at -40 ◦C for 1.5 h. Excess of 1,2-dibromoethane (29.1 g, 155 mmol) was then added at -70 ◦C.The brown mixture was stirred at -40 ◦C for 2 h, then at room temperature over night. Afterquenching the mixture with a saturated solution of ammonium chloride (100 mL) and water(150 mL), the aqueous layer was extracted three times with diethyl ether and the combinedorganic layers were washed sequentially with water (200 mL) and brine (200 mL) and driedover anhydrous magnesium sulfate. The solvent was removed by rotary evaporation and thecrude product was purified by vacuum distillation to give three fractions. The first fraction(26-40 mbar at 40-60 ◦C) contained mainly the excess 1,2-dibromoethane and norbornadiene.The second fraction (13-20 mbar at 50-60 ◦C) contained mainly 2-bromonorbornadiene and alittle 2,3-dibromonorbornadiene. The third fraction (7-12 mbar at 80-100 ◦C) contained mainly2,3-dibromonorbornadiene (NB-2) (8.9 g, 35.6 mmol, 68%) as a colorless oil.

The above described precedure was adapted from Yoo et al. [1].

Procedure B:Potassium tert-butoxide (3.7 g, 33.3 mmol) was dissolved in THF (70 mL) and the solutionwas cooled to –85 ◦C. Norbornadiene (NB-1) (3.7 mL, 40 mmol) was added, followed by n-butyllithium (2.5 M in hexanes, 13 mL, 33.3 mmol) during 60 min. The yellow solution wasstirred for 5 min at –85 ◦C and 60 min at –40 ◦C. The solution was cooled to –85 ◦C andp-toluenesulfonyl bromide (TsBr)(4 g, 16.8 mmol) was added. The mixture was stirred for 15min at –85 ◦C and 60 min at –40 ◦C. The solution was cooled to –85 ◦C and p-toluenesulfonylbromide (4 g, 16.9 mmol) was added. The mixture was stirred for 15 min and was then heated to

22


ambient temperature on a room-tempered water bath. The reaction mixture was quenched withwater (50 mL), the layers were separated and the aqueous layer was extracted three times withdiethyl ether (20 mL). The solvents from the combined organic layers were slowly removedon a rotary evaporator (40 ◦C ). The residue was dissolved in pentane (30 mL), washed withwater (10 mL) and brine (20 mL) and dried over magnesium sulfate. The solvent was removedunder reduced pressure and the crude product distilled (2 mbar), using a short Vigreux column,collecting the main fraction at 40-50 ◦C. This afforded NB-2 as a colorless liquid. Analyticaldata were consistent with previous reports [2].

The above described procedure was adapted from Lennertson et al. [3].

1H-NMR (300 MHz, CDCl3): δ[ppm] = 6.90 – 6.87 (m, 2H), 3.62 (p, J = 1.8 Hz, 2H), 2.45(dt, J = 6.3, 1.6 Hz, 1H), 2.18 (dt, J = 6.3, 1.9 Hz, 1H).

4-methylbenzenesulfonyl bromide (Ts-Br)

SBr

O O

In a 250 mL one-neck flask, p-Toluenesulfonyl hydrazide (15 g, 80.5 mmol) was dissolved inchloroform (200 mL) at 0 ◦C. Bromine (8.2 mL, 161 mmol) was added in small portions allow-ing the orange color to disappear between each addition. If, when the last portion of brominewas added, the color was still white, a small amount of extra bromine was added until a lightorange colour persisted. The layers were separated and the organic layer was washed with asaturated sodium hydrogencarbonate solution (100 mL) and 20 mL of a 1% sodium thiosul-fate solution. The organic layer was dried over anhydrous magnesium sulfate, filtered and thesolvent was removed on a rotary evaporator, to give 4-methylbenzenesulfonyl bromide (Ts-Br)(12.5 g, 67%) as a colorless powder.

1H-NMR (300 MHz, CDCl3): δ[ppm] = 7.88 (d, J = 8.5 Hz, 2H), 7.39 (d, J = 8.1 Hz, 2H),2.49 (s, 3H).

23


2-bromo-3-chlorobicyclo[2.2.1]hepta-2,5-diene (3)

Br

Cl

tert-Butyllithium (18.8 ml, 32 mmol, 1.7 M in pentane) was added to a flame-dried Schlenkflask containing 2,3-dibromonorbornadiene (NB-2) (4g, 16 mmol) in 80 mL anhydrous THF at-78 ◦C. After the yellow mixture was stirred for 30 min, p-touenesulfonyl chloride (6,5 g, 34mmol) was added. The mixture was stirred at -78 ◦C for 1 h, then at room temperatur for oneadditional hour. After quenching the mixture with water (20 mL), the aqueous layer was ex-tracted three times with diethyl ether and the combined organic layers were washed sequentiallywith water and brine and dried over anhydrous magnesium sulfate. The solvent was removedby rotary evaporation and the crude product was purified by silica gel column chromatogra-phy (100% cyclohexane) to give 2-bromo-3-chlorobicyclo[2.2.1]hepta-2,5-diene (NB-3) (2.93g, 71%) as a yellow oil.

1H-NMR (300 MHz, CDCl3): δ[ppm] = 6.89 (tt, J = 5.1, 2.5 Hz, 2H), 3.62 – 3.57 (m, 1H),3.55 – 3.50 (m, 1H), 2.43 (dt, J = 6.3, 1.6 Hz, 1H), 2.19 (dt, J = 6.3, 1.9 Hz, 1H).

24


3,5-dibromo-2-methylthiophene (5)

S Br

Br

2-methylthiophene (1) (30 g, 306 mmol) was dissolved in 500 mL acetic acid and cooled to 0◦C. A solution of bromine (121.6 g, 765 mmol) in 50 mL acetic acid was slowly added to thereaction mixture via a dropping funnel. After the addition, the mixture was slowly heated toroom temperature and stirred over night. The reaction was quenched by addition of water (150mL) and then carefully neutralized by adding small portions of sodium carbonate. The precipi-tate was dissolved in 800 mL water and the resulting mixutre extracted three times with diethylether. The combined organic layers were dried over anhydrous magnesium sulfate, filtered andthe solvent was removed under reduced pressure. The obtained dark brown crude product waspurified via vacuum distillation and the main fraction (130 ◦C at 12-20 mbar) was collected.3,5-Dibromo-2-methylthiophene (2) (46.8 g, 62%) was obtained as a yellow oil.

1H-NMR (300 MHz, CDCl3): δ[ppm] = 6.83 (s, 1H), 2.33 (s, 3H).

(4-bromo-5-methylthiophen-2-yl)boronic acid (5)

S B(OH)2

Br

In a flame-dried Schlenk flask under argon atmosphere, 3,5-dibromo-2-methylthiophene (2)(12.8 g, 50 mmol) was dissolved in 250 ml anhydrous diethyl ether and cooled to -78 ◦C. n-Butyllithium (2,5 M in hexane, 3.52 g, 55 mmol) was slowly added to the reaction mixture andstirred at -78 ◦C for 1 h. Tributyl borate (12.6 g, 55 mmol) was added to the mixture and theresulting solution was slowly heated to room temperature and stirred overnight. The reactionmixture was quenched by addition of 1 M HCl solution (150 mL) and extracted three timeswith diethyl ether. The combined organic layers were extracted three times with 100 mL of a1 M NaOH solution. The combined aqueous layers were cooled to 0 ◦C and conz. HCl wasadded carefully. The precipitate was filtered and dried under reduced pressure. (4-bromo-5-methylthiophen-2-yl)boronic acid (3) (9.2 g, 84%) was obtained as a yellow solid.

1H-NMR (300 MHz, d6DMSO): δ[ppm] = 8.30 (s, 2H), 7.50 (s, 1H), 2.37 (s, 3H).

25


3-bromo-2-methyl-5-phenylthiophene (7a)

S

Br

In a flame-dried Schlenk flask under argon atmosphere, (4-bromo-5-methylthiophen-2-yl)boronicacid (3) (4 g, 18 mmol) was dissolved in 120 mL anhydrous THF. Iodobenzene (4.7 g, 23 mmol),Pd(dppf)Cl2 (658 mg, 5 mol%) and 10 ml of a sodium carbonate solution (20% in water) wereadded and the reaction mixture was heated under reflux (80 ◦C) overnight. Afterwards the blacksolution was extracted three times with ethyl acetate. The combined organic layers were sequi-entially washed with 100 mL water and 100 ml brine, dried over anhydrous magnesium sulfate,filtered and the solvent was removed under reduced pressure. The dark brown crude productwas purified via silica gel column chromatography (100% cyclohexane) to give 3-Bromo-2-methyl-5-phenylthiophene (4a) (3.8 g, 83%) as a colorless solid.

1H-NMR (300 MHz, CDCl3): δ[ppm] = 7.51 (dt, J = 8.3, 1.8 Hz, 2H), 7.41 – 7.33 (m, 2H),7.32 – 7.27 (m, 1H), 7.11 (s, 1H), 2.42 (s, 3H).

3-bromo-5-(4-methoxyphenyl)-2-methylthiophene (7b)

S

Br

O

In a flame-dried Schlenk flask under argon atmosphere, (4-bromo-5-methylthiophen-2-yl)boronicacid (3) (1.5 g, 6.8 mmol) was dissolved in 30 mL anhydrous THF. 4-bromoanisole (1.53 g, 8.16mmol), Pd(dppf)Cl2 (250 mg, 5 mol%) and 10 ml of a sodium carbonate solution (20% in wa-ter) were added and the reaction mixture was stirred at (80 ◦C) for 18 h. Afterwards, the blacksolution was extracted three times with ethyl acetate. The combined organic layers were sequi-entially washed with 20 mL water and 20 ml brine, dried over anhydrous magnesium sulfate,filtered and the solvent was removed under reduced pressure. The dark brown crude productwas purified via silica gel column chromatography (100% cyclohexane) to give 3-bromo-5-(4-methoxyphenyl)-2-methylthiophene (4b) (1.2 g, 63%) as a yellow solid.

1H-NMR (300 MHz, CDCl3): δ[ppm] = 7.43 (d, J = 8.8 Hz, 2H), 6.98 (s, 1H), 6.90 (d, J = 8.8Hz, 2H), 3.83 (s, 3H), 2.40 (s, 3H).

26


4,4,5,5-tetramethyl-2-(2-methyl-5-phenylthiophen-3-yl)-1,3,2-dioxaborolane

S

BO

O

A flame-dried 100 mL Schlenk flask under argon atmosphere was charged with 3-Bromo-2-methyl-5-phenylthiophene (4a) (500 mg, 2 mmol) and anhydrous diethyl ether (70 mL) at -78 ◦C. n-Butyllithium (2.5 M solution in hexane, 166 mg, 2.6 mmol) was added dropwise tothe reaction mixture, which was then stirred for 1 h at -78 ◦C. Then, isopropoxyboronic acidpinacol ester (484 mg, 2.6 mmol) was added dropwise at -78 ◦C and the mixture was stirredfor 1h at room tempetature. The reaction was quenched by addition of 1 M HCl solution un-til pH was neutral and extracted with ethyl acetate for three times. combined organic layerswere washed with brine and dried over anhydrous magnesium sulfate. Then the solvent wasevaporated and the crude product was purified with silica-gel column chromatography by usinga mixture of cyclohexane and ethyl acetate (50:1) as eluent to afford 4,4,5,5-tetramethyl-2-(2-methyl-5-phenylthiophen-3-yl)-1,3,2-dioxaborolane (5’a) (488 mg, 81%) as a colorless oil.

1H-NMR (300 MHz, CDCl3): δ[ppm] = 7.31 – 7.24 (m, 2H), 7.16 (s, 1H), 7.06 – 6.99 (m, 2H),6.94 – 6.87 (m, 1H), 2.41 (s, 3H), 1.03 (s, 12H).

27


(2-methyl-5-phenylthiophen-3-yl)boronic acid (8a)

S

(HO)2B

In a flame-dried Schlenk flask under argon atmosphere 3-bromo-2-methyl-5-phenylthiophene(4a) (2.4 g, 9.5 mmol) was dissolved in 50 mL anhydrous diethyl ether and cooled to -78 ◦C.Then a solution of n-Butyllithium (2.5 M in hexane, 0.8 g, 12.3 mmol) was slowly added tothe reaction mixture and stirred at -78 ◦C for 1 h before tributyl borate (2.8 g, 12.3 mmol) wasadded. The solution was slowly heated to room temperature and stirred overnight. The reactionmixture was quenched by addition of 20 mL of a 1 M HCl solution and extracted three timeswith diethyl ether. The combined organic layers were extracted three times with 20 mL of a 1M NaOH solution. The combined aqueous layers were cooled to 0 ◦C and conz. HCl was addedcarefully. The generated precipitate was filtered and dried under reduced pressure. (2-methyl-5-phenylthiophen-3-yl)boronic acid (5a) (1.6 g, 80%) was obtained as a colorles solid.

1H-NMR (300 MHz, d6DMSO): δ[ppm] = 7.93 (bs, 2H), 7.59 (s, 1H), 7.56 – 7.49 (m, 2H),7.38 (t, J = 7.6 Hz, 2H), 7.25 (t, J = 7.3 Hz, 1H), 2.60 (s, 3H).

28


(5-(4-methoxyphenyl)-2-methylthiophen-3-yl)boronic acid (8b)

S

(HO)2B

O

In a flame-dried Schlenk flask under argon atmosphere 3-bromo-5-(4-methoxyphenyl)-2-methyl-thiophene (4b) (0.7 g, 2.47 mmol) was dissolved in 30 mL anhydrous diethyl ether and cooledto -78 ◦C. Then a solution of n-butyllithium (2.5 M in hexane, 1.3 mL, 3.21 mmol) was slowlyadded to the reaction mixture and stirred at -78 ◦C for 1 h before tributyl borate (740 mg,3.21 mmol) was added. The resulting solution was slowly heated to room temperature andstirred overnight. The reaction mixture was quenched by addition of 1 M HCl solution (10mL) and extracted three times with diethyl ether. The combined organic layers were extractedthree times with 10 mL of a 1 M NaOH solution. The combined aqueous layers were cooledto 0 ◦C and conz. HCl was added carefully. The generated precipitate was filtered and driedunder reduced pressure. (5-(4-methoxyphenyl)-2-methylthiophen-3-yl)boronic acid (5b) (350mg, 58%) was obtained as a colorles solid.

1H-NMR (500 MHz, d6DMSO): δ[ppm] = 7.89 (s, 2H), 7.45 (s, 1H), 7.44 (d, J = 3.2 Hz, 2H),6.95 (d, J = 8.8 Hz, 2H), 3.76 (s, 3H), 2.58 (s, 3H).

3-(3-chlorobicyclo[2.2.1]hepta-2,5-dien-2-yl)-2-methyl-5-phenylthiophene (4a)

S

Cl

Procedure A:In a flame-dried Schlenk flask under argon atmosphere Pd2(dba)3 (16 mg, 2.5 mol%) and CsF(380 mg, 2.5 mmol) were dissolved in 10 mL anhydrous THF. Then the reaction mixture wascharged with a solution of 5’a (220 mg, 0.73 mmol) in 1 mL THF along with NB-3 (136 mg,0.66 mmol). The solution was stirred for a few min, before P(t-Bu)3 was added in order to startthe reaction. The mixture was stirred at room temperature for 24 h until TLC indicated com-plete conversion of the starting material. Afterwards, the reaction mixture was filtered over apad of silica using ethyl acetate as eluent. The solvent was removed under reduced pressure and

29


the resulting crude product was purified via silica gel column chromatography (100% cyclo-hexane). 3-(3-chlorobicyclo[2.2.1]hepta-2,5-dien-2-yl)-2-methyl-5-phenylthiophene (6a) (110mg, 62 %) was obtained as a yellow oil.

Procedure B:In a flame-dried Schlenk flask under argon atmosphere Pd2(dba)3 (105 mg, 2.5 mol%), CsF(2.3 g, 15.8 mmol) and 5a (1.1 g, 5 mmol) were dissolved in 10 mL anhydrous THF. Then thereaction mixture was charged with NB-3 (920 mg, 4.5 mmol) and stirred for a few min, beforeP(t-Bu)3 (70 mg, 7.5 mol%) was added in order to start the reaction. The mixture was stirred atroom temperature overnight until TLC indicated complete conversion of the starting material.Afterwards the reaction mixture was filtered over a pad of silica using ethyl acetate as an eluent.The solvent was removed under reduced pressure and the resulting crude product was purifiedvia silica gel column chromatography (100% cyclohexane). 3-(3-chlorobicyclo[2.2.1]hepta-2,5-dien-2-yl)-2-methyl-5-phenylthiophene (6a) (990 mg, 76 %) was obtained as a yellow oil.

1H-NMR (500 MHz, CDCl3): δ[ppm] = 7.54 (d, J = 7.2 Hz, 2H), 7.35 (t, J = 7.7 Hz, 2H), 7.28– 7.23 (m, 1H), 7.14 (s, 1H), 7.02 – 7.00 (m, 1H), 6.98 – 6.94 (m, 1H), 3.82 (s, 1H), 3.53 (s,1H), 2.44 (s, 4H), 2.17 (dt, J = 6.2, 1.7 Hz, 1H).

13C-NMR (126 MHz, CDCl3): δ[ppm] = 142.39, 142.18, 142.16, 140.16, 134.85, 134.32,132.84, 128.76, 127.14, 125.49, 122.75, 71.32, 57.43, 55.45, 30.32, 15.02.

ESI-HRMS (pos. MeOH) m/z: [M + H]+ calculated for [C18H16ClS]+: 299,0656; found:

299,1935.

30


3-(3-chlorobicyclo[2.2.1]hepta-2,5-dien-2-yl)-5-(4-methoxyphenyl)-2-methylthio-

phene (4b)

S

O

Cl

In a flame-dried Schlenk flask under argon atmosphere Pd2(dba)3 (21 mg, 2.5 mol%), CsF(560 mg, 3.7 mmol) and 5b (250 mg, 1 mmol) were dissolved in 10 mL anhydrous THF.Then the reaction mixture was charged with NB-3 (189 mg, 0.92 mmol) and stirred for afew min, before P(t-Bu)3 (14.4 mg, 7.5 mol%) was added in order to start the reaction. Themixture was stirred at room temperature for 24 h until TLC indicated complete conversion ofthe starting material. Afterwards the reaction mixture was filtered over a pad of silica usingethyl acetate as eluent. The solvent was removed under reduced pressure and the resultingcrude product was purified via silica gel column chromatography (100% cyclohexane). 3-(3-chlorobicyclo[2.2.1]hepta-2,5-dien-2-yl)-5-(4-methoxyphenyl)-2-methylthiophene (6b) (230 mg,76 %) was obtained as a yellow oil.

1H-NMR (300 MHz, CDCl3): δ[ppm] = 7.50 – 7.43 (m, 2H), 7.02 (s, 1H), 7.01 – 6.98 (m, 1H),6.98 – 6.93 (m, 1H), 6.93 – 6.85 (m, 2H), 3.83 (s, 4H), 3.57 – 3.48 (m, 1H), 2.42 (s, 4H), 2.16(dt, J = 6.2, 1.7 Hz, 1H).

13C-NMR (300 MHz, CDCl3): δ[ppm] = 174.93, 158.70, 142.14, 141.98, 141.91, 139.82,133.58, 132.43, 126.97, 126.52, 121.43, 71.04, 57.14, 55.19, 55.07, 50.25, 14.71.

ESI-HRMS (pos. MeOH) m/z: [M + Na]+ calculated for [C19H17ClOSNa]+: 351,0581;

found: 351,0575.

31


2,3-bis(2-methyl-5-phenylthiophen-3-yl)bicyclo[2.2.1]hepta-2,5-diene (1a)

SS

In a flame-dried Schlenk flask under argon atmosphere Pd2(dba)3 (25 mg, 2.5 mol%), CsF(520 mg, 3.4 mmol) and 5a (500 mg, 2.3 mmol) were dissolved in 30 mL anhydrous THF. Thenthe reaction mixture was charged with NB-1 (260 mg, 1 mmol) and stirred for a few min, beforeP(t-Bu)3 (16 mg, 7.5 mol%) was added in order to start the reaction. The mixture was stirredat room temperature for 24 h until TLC indicated product formation. Afterwards the reactionmixture was filtered over a pad of silica using ethyl acetate as eluent. The solvent was removedunder reduced pressure and the resulting crude product was purified via silica gel column chro-matography (cyclohexane:ethyl acetate, 100:1). 9a (185 mg, 54%) was obtained as a red solid.

1H-NMR (500 MHz, CDCl3): δ[ppm] = 7.53 (d, J = 7.5 Hz, 4H), 7.35 (t, J = 7.5 Hz, 4H), 7.25(d, J = 6.3 Hz, 2H), 7.05 (s, 2H), 7.04 – 7.00 (m, 2H), 3.85 (s, 2H), 2.43 (d, J = 7.7 Hz, 1H),2.16 (d, J = 6.1 Hz, 1H), 1.99 (s, 6H).

13C-NMR (126 MHz, CDCl3): δ[ppm] = 146.47, 143.65, 140.54, 137.02, 135.01, 134.39,129.36, 127.59, 125.90, 123.69, 72.19, 56.99, 14.96, 1.60.

ESI-HRMS (pos. MeOH) m/z: [M +Na]+ calculated for [C29H24S2Na]+: 459,1212; found:

459,1202.

32


2,3-bis(5-(4-methoxyphenyl)-2-methylthiophen-3-yl)bicyclo[2.2.1]hepta-2,5-diene

(1b)

SSO O

In a flame-dried Schlenk flask under argon atmosphere Pd2(dba)3 (4.4 mg, 5 mol%), CsF(58 mg, 0.38 mmol) and 5b (50 mg, 0,21 mmol) were dissolved in 5 mL anhydrous THF.Then the reaction mixture was charged with NB-1 (24 mg, 0.1 mmol) and stirred for a few min,before P(t-Bu)3 (2.4 mg, 15 mol%) was added in order to start the reaction. The mixture wasstirred at room temperature for 24 h until TLC indicated complete conversion of the startingmaterial. Afterwards the reaction mixture was filtered over a pad of silica using ethyl acetate aseluent. The solvent was removed under reduced pressure and the resulting crude product waspurified via silica gel column chromatography (100:1, 50:1, cyclohexane:ethyl acetate). 9b (35mg, 73%) was obtained as a red solid.

1H-NMR (500 MHz, CDCl3): δ[ppm] = 7.44 (d, J = 8.8 Hz, 4H), 7.02 – 6.98 (m, 2H), 6.91 (s,2H), 6.88 (d, J = 8.8 Hz, 4H), 3.84 – 3.83 (m, 2H), 3.82 (s, 6H), 2.41 (d, J = 6.0 Hz, 1H), 2.13(d, J = 6.1 Hz, 1H), 1.96 (s, 6H).

13C-NMR (126 MHz, CDCl3): δ[ppm] = 158.85, 145.81, 143.06, 139.78, 136.32, 132.74,127.39, 126.59, 122.10, 114.18, 71.54, 56.37, 55.34, 14.30.

ESI-HRMS (pos. MeOH) m/z: [M + Na]+ calculated for [C31H28O2S2Na]+: 519,1423;

found: 519,1414.

33


5-(4-methoxyphenyl)-2-methyl-3-(3-(2-methyl-5-phenylthiophen-3-yl)bicyclo[2.2.1]

hepta-2,5-dien-2-yl)thiophene (1c)

SSO

In a flame-dried Schlenk flask under argon atmosphere Pd2(dba)3 (4 mg, 2.5 mol%), CsF(101 mg, 0.67 mmol) and 5b (46 mg, 0.18 mmol) were dissolved in 4 mL anhydrous THF.Then the reaction mixture was charged with a solution of 6a (50 mg, 0.17 mmol) in 1 mL an-hydrous THF and stirred for a few min, before P(t-Bu)3 (2.5 mg, 7.5 mol%) was added in orderto start the reaction. The mixture was stirred at room temperature for 24 h until TLC indicatedcomplete conversion of the starting material. Afterwards the reaction mixture was filtered overa pad of silica using ethyl acetate as eluent. The solvent was removed under reduced pres-sure and the resulting crude product was purified via silica gel column chromatography (100:1,cyclohexane:ethyl acetate). In order to completly remove the deborylated and dehalogenatedstarting material the product was additionally purified via preparative HPLC (70% acetonitrileto 100% acetonitrile over 30 min) 10 (20 mg, 28%) was obtained as a red solid.

1H-NMR (500 MHz, CDCl3): δ[ppm] = 7.52 (d, J = 7.6 Hz, 2H), 7.44 (d, J = 8.4 Hz, 2H),7.34 (t, J = 7.6 Hz, 2H), 7.23 (t, J = 7.2 Hz, 1H), 7.03 (s, 1H), 7.01 (s, 2H), 6.91 (s, 1H), 6.88(d, J = 8.5 Hz, 2H), 3.84 (s, 2H), 3.82 (s, 3H), 2.41 (d, J = 5.9 Hz, 1H), 2.14 (d, J = 5.6 Hz,1H), 1.97 (d, J = 7.9 Hz, 6H).

13C-NMR (126 MHz, CDCl3): δ[ppm] = 159.04, 146.17, 145.87, 143.22, 140.02, 136.65,136.45, 134.62, 133.96, 132.93, 128.93, 127.54, 127.15, 126.77, 125.48, 123.31, 122.23, 114.35,71.74, 56.56, 55.51, 14.46.

ESI-HRMS (pos. MeOH) m/z: [M+Na]+ calculated for [C30H26OS2Na]+: 489,1317; found:

489,1316.

34


3,3'-(cyclopenta-1,3-diene-1,2-diyl)bis(2-methyl-5-phenylthiophene) (11)

SS SS

A B

4:1

In microwave tube, 9a (10 mg, 0.023 mmol) was dissolved in 1 ml DMF. Then a solution ofdimethyltetrazine (5,1 mg, 0.046 mmol) in 0.2 ml methanol was added and the reaction mix-ture was stirred at 50 ◦C for 48 h until TLC indicated complete conversion of the startingmaterial. The solvent was removed under reduced pressure and the crude product was purifiedvia silica gel column chromatography (100% cyclohexane) to give 3,3’-(cyclopenta-1,3-diene-1,2-diyl)bis(2-methyl-5-phenylthiophene) 11 (8 mg, 67%) as a violet solid. Analytical data isconsistent with published data from Lemieux et al. [4].

Isomer A:1H-NMR (500 MHz, CDCl3): δ[ppm] = 7.50 (d, J = 7.7 Hz, 4H), 7.33 (t, J = 7.6 Hz, 6H), 7.22(s, 1H), 7.09 (s, 1H), 7.08 (s, 1H), 6.71 (dt, J = 5.5, 1.5 Hz, 1H), 6.53 (dt, J = 5.5, 1.5 Hz, 1H),3.50 (t, J = 1.5 Hz, 2H), 2.12 (s, 3H), 2.03 (s, 3H).

Isomer B:1H-NMR (500 MHz, CDCl3): δ[ppm] = 7.42 (d, J = 7.7 Hz, 4H), 7.29 (t, J = 7.7 Hz, 4H), 7.21(t, J = 7.7 Hz, 2H), 6.87 (s, 2H), 6.48 (t, J = 1.5 Hz, 2H), 3.28 (t, J = 1.5 Hz, 2H), 2.27 (s, 6H).

13C-NMR (126 MHz, CDCl3): δ[ppm] = 140.77, 140.30, 138.61, 138.09, 136.99, 136.74,136.62, 136.10, 135.18, 133.47, 133.10, 129.98, 128.29, 126.10, 126.02, 125.76, 46.55, 14.65,14.46.

APCI-HRMS (pos. MeOH) m/z: [M +H]+ calculated for [C27H22S2H]+: 411,1236; found:411,1256.

35


1,4-dimethyl-6,7-bis(2-methyl-5-phenylthiophen-3-yl)-5,8-dihydro-5,8-methan-

ophthalazine (14)

SS

NN

In a microwave tube 9a (10 mg, 0.023 mmol) and (diacetoxyiodo)benzene (14.8 mg, 0.046 mmol)were dissolved 0.5 mL anhydrous DCM. Then a solution of 3,6-dimethyl-1,2,4,5-tetrazine(5,1 mg, 0.046 mmol) in 0.5 mL anhydrous DCM was added and the reaction mixture wasstirred at room temperature overnight until TLC indicated complete conversion of the startingmaterial. The solvent was removed under reduced pressure and the crude product was purifiedvia preparative HPLC (see method below) to give 14 (2.1 mg, 0,005 mmol, 21%) as a red solid.

Table 2: HPLC method for the puri�cation of 12.


0 80 6

30 95 6

50 95 6

1H-NMR (500 MHz, CDCl3): δ[ppm] = 7.51 (d, J = 7.3 Hz, 4H), 7.36 (t, J = 7.7 Hz, 4H), 7.28(s, 2H), 6.99 (s, 2H), 4.26 (s, 2H), 2.79 (d, J = 1.0 Hz, 1H), 2.77 (s, 6H), 2.42 (s, 1H), 1.80 (s,6H).

13C-NMR (126 MHz, CDCl3): δ[ppm] = 151.93, 144.91, 141.09, 135.40, 134.49, 134.08,129.09, 127.57, 125.43, 122.16, 77.16, 68.29, 54.51, 19.57, 14.55

APCI-HRMS (pos. MeOH) m/z: [M + H]+ calculated for [C33H28N2S2H]+: 517,1767;found: 517,1771.

ESI-HRMS (pos. MeOH) m/z: [M +H]+ calculated for [C33H28N2S2H]+: 517,1767; found:517,1784.

36


6,7-bis(2-methyl-5-phenylthiophen-3-yl)-1,4-di(pyridin-2-yl)-5H-cyclopenta[d]pyridazine

(13)

SS

NN

N

N

In a microwave tube 1a (10 mg, 0.023 mmol) was dissolved in 0,5 mL MeOH and 0,5 mLDCM. Then 3,6-dipyridyl-1,2,4,5-tetrazine (21.6 mg, 0.092 mmol) was added and the reactionmixture was stirred at 40◦C overnight until TLC indicated complete conversion of the startingmaterial. The solvent was removed under reduced pressure and the crude product was purifiedvia semi-preparative HPLC (see method below) to give 16 (4.5 mg, 0.010 mmol, 45%) as a redsolid.


Time [min] Acetonitrile[%]

Flow rate[mL/min]

0 80 6

30 95 6

50 95 6

1H-NMR (500 MHz, CDCl3): δ[ppm] = 8.86 (d, J = 4.5 Hz, 1H), 8.61 (d, J = 4.7 Hz, 1H),8.47 (d, J = 7.9 Hz, 1H), 7.97 (td, J = 7.7 Hz, 1.5 Hz, 1H), 7.61 (s, 1H), 7.49 (dd, J = 6.9 Hz,4.8 Hz, 1H), 7.45 (d, J = 7.4 Hz, 2H), 7.36 (td, J = 7.7 Hz, 1.6 Hz, 1H), 7.34-7.25 (m, 7H), 7.23(d, J = 7.8 Hz, 1H), 7.20 (t, J = 6.6 Hz, 1H), 7.09 (dd, J = 7.5 Hz, 4.9 Hz, 1H), 7.07 (s, 1H),6.63 (s, 1H), 2.32 (s, 3H), 1.87 (s, 3H)

13C-NMR (126 MHz, CDCl3): δ[ppm] = 149.81, 149.16, 143.68, 139.25, 138.58, 137.36,136.52, 136.20, 135.43, 135.22, 134.92, 134.82, 134.77, 128.88, 128.76, 127.57, 126.95, 126.82,126.36, 125.52, 125.41, 124.81, 124.02, 123.69, 120.44, 119.61, 115.99, 109.16, 77.16, 14.73,14.01


37


1,4-dimethyl-6,7-bis(2-methyl-5-phenylthiophen-3-yl)-5H-cyclopenta[d]pyridazine

(16)

NN

S S

In a microwave tube 1a (10 mg, 0.023 mmol) was dissolved in 0,5 mL MeOH and 0,5 mLDCM. Then 3,6-dipyridyl-1,2,4,5-tetrazine (10.1 mg, 0.092 mmol) was added and the reactionmixture was stirred at 40◦C overnight until TLC indicated complete conversion of the startingmaterial. The solvent was removed under reduced pressure and the crude product was purifiedvia semi-preparative HPLC (see method below) to give 13 as a red solid.


Time [min] Acetonitrile[%]

Flow rate[mL/min]

0 80 6

30 95 6

50 95 6

1H-NMR (500 MHz, CDCl3): δ[ppm] = 7.58 (d, J = 7.3 Hz, 2H), 7.41 (d, J = 7.2 Hz, 2H),7.36 (t, J = 7.8 Hz, 2H), 7.28 (d, J = 7.6 Hz, 2H), 7.25 (d, J = 2.7 Hz, 1H), 7.21 (s, 1H), 7.18(t, J = 7.4 Hz, 1H), 6.97 (s, 1H), 6.93 (s, 1H), 2.76 (s, 3H), 2.51 (s, 3H), 2.42 (s, 3H), 2.03 (s, 3H)

13C-NMR (126 MHz, CDCl3): δ[ppm] = 139.21, 138.95, 138.93, 136.48, 136.23, 136.09,134.94, 134.91, 134.59, 128.99, 128.84, 127.69, 127.09, 126.84, 126.18, 125.49, 121.15, 118.62,116.37, 107.15, 77.16, 18.16, 17.90, 14.80, 14.18


38


6-Methyl-Tetrazine-5-FAM-CPD conjugate (15)

S S

NN

NHO

OO OH

COOH

In a microwave tube 9a (10 mg, 0.023 mmol) was dissolved 0.5 mL anhydrous DCM. Then asolution of 6-Methyl-Tetrazine-5-FAM (86.5 mg, 0.092 mmol) in 0.5 mL MeOH was added andthe reaction mixture was stirred at 50◦C overnight until TLC indicated complete conversion ofthe starting material. A saturated solution of NaNO2 in water was added and the mixture wasstirred at room temperature for 1h. Afterwards, the solvent was removed under reduced pressureand the crude product was purified via HPLC (see method below) to give 13 as a yellow solid.

Table 5: HPLC method for the puri�cation and analysis of the click product 13. The product

eluted at 32 min. The identity and purity of the compound was veri�ed by analytical HPLC (see

ref.) and HR-MS.


0 50 1.0

40 90 1.0

50 95 1.0

55 50 1.0

ESI-HRMS (pos. MeOH) m/z: [M + Na]+ calculated for [C58H41N3O6S2Na]+: 962,2334;

found: 962.2327.

39


6-Methyl-Tetrazine-5-FAM

OO OH

COOH

ON

HNN

NN

In a microwave tube under argon atmosphere 5-carboxyfluorescein-NHS-ester (124 mg, 0.33 mmol)was dissolved in dry DMF. Then (4-(6-methyl-1,2,4,5-tetrazin-3-yl)phenyl)methanamine as hy-drochloric salt (50 mg, 0.21 mmol) and DIPEA (0.17 mL, 0.95 mmol) were added and thereaction mixture was stirred at room temperature overnight. The solvent was removed underreduced pressure and the title product was purified by reverse phase collumn chromatography(Collumn: IR-50SI-F0025) using an automated puriflash instrument (PuriFlash PF420) fromInterchim with the method displayed in table 6. The title product was obtained as an orangesolid (110 mg, 0.20 mmol, 47%).

Table 6: Method for the puri�cation of 6-Methyl-Tetrazine-5-FAM.


0 30 15

50 70 15

1H-NMR (300 MHz, CD3OD: δ[ppm] = 8.58 – 8.47 (m, 3H), 8.27 (dd, J = 8.1, 1.5 Hz, 1H),7.64 (d, J = 8.4 Hz, 2H), 7.32 (d, J = 8.1 Hz, 1H), 6.68 (d, J = 2.2 Hz, 2H), 6.63 – 6.50 (m, 4H),4.75 (s, 2H), 3.03 (s, 3H).

ESI-HRMS (pos. MeOH) m/z: [M +H]+ calculated for [C31H21N5O6H]+: 560.1565; found:560.1541.

The above described procedure was adapted from Asare-Okai et al. [5]

40

REFERENCES

References

[1] W.-J. Yoo, G. C. Tsui, and W. Tam. Eur. J. Org. Chem., pages 1044–1051, 2005.

[2] G. K. Tranmer, C. Yip, S. Handerson, R. W. Jordan, and W. Tam. Can. J. Chem., 78:527–535, 2000.

[3] A. Lennartson, M. Quant, and K. M. Poulsen. Synlett., (26):1501–1504, 2015.

[4] V. Lemieux, S. Gauthier, and N. R. Branda. Angew.Chem.Int.Ed., 45:6820–6824, 2006.

[5] D. Fabrisa M. Royzen P. N. Asare-Okai, E. Agustin. Site-specific fluorescence labellingof RNA using bio-orthogonal reaction of trans-cyclooctene and tetrazine. Chem.Commun.,50:7844, 2014.

[6] U. Megerle, R. Lechner, B. Köning, and E. Riedle. Photochem. Photobiol. Sci., (9):1400–1406, 2010.

[7] A. C. Knall, M. Hollauf, and C. Slugovc. Tetrahedron Lett., 55:4763–4766, 2014.

41

5 APPENDIX

5 Appendix

5.1 1H-and 13C-NMR-spectra of the �nal compounds

Figure 1: Top: 1H-NMR-spectrum of (1a). Bottom: 13C-NMR-spectrum of (1a).

42

5 APPENDIX

Figure 2: Top: 1H-NMR-spectrum of (1b). Bottom: 13C-NMR-spectrum of (1b).

43

5 APPENDIX

Figure 3: Top: 1H-NMR-spectrum of (1c). Bottom: 13C-NMR-spectrum of (1c).

44

5 APPENDIX

Figure 4: Top: 1H-NMR-spectrum of (11). Bottom: 13C-NMR-spectrum of (11).

45

5 APPENDIX


46

5 APPENDIX


47

5 APPENDIX


48

5 APPENDIX

5.2 Shutter �les generated by the QYDS

Figure 8: Shutter �le of the cyclization of compound 1a

Figure 9: Shutter �le of the cyclization of compound 1b

49

5 APPENDIX

Figure 10: Shutter �le of the cyclization of compound 1c

Figure 11: Shutter �le of the cyclization of compound 11

50

5 APPENDIX

Figure 12: Shutter �le of the cycloreversion of compound 1a

Figure 13: Shutter �le of the cycloreversion of compound 1c

51

5 APPENDIX

Figure 14: Shutter �le of the cycloreversion of compound 1b

Figure 15: Shutter �le of the cycloreversion of compound 11

52

5 APPENDIX



53

Chemical CommunicationsAll photophysical experiments were performed in quartz glass cuvettes (d = 1...

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Transcript of Chemical CommunicationsAll photophysical experiments were performed in quartz glass cuvettes (d = 1...