Water-Soluble Rylene Dyes as High-Performance Colorants for the

Biomacromolecules 2005, 6, 68-79

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Water-Soluble Rylene Dyes as High-Performance Colorants for the Staining of Cells Tanja Weil,*,†,‡ Moustafa A. Abdalla,† Claudia Jatzke,‡ Jan Hengstler,§ and Klaus Mu¨llen† Max-Planck-Institut fu¨r Polymerforschung, Ackermannweg 10, 55128 Mainz, Germany, Merz Pharmaceuticals, Eckenheimer Landstrasse 100, 60318 Frankfurt, Germany, and University of Leipzig, Haertelstrasse 16-18, 04107 Leipzig, Germany Received June 4, 2004; Revised Manuscript Received September 1, 2004

The synthesis of perylene and terrylene chromophores carrying a single poly(ethylene oxide) chain is presented. These chromophores reveal a strong solvatochromic behavior: High fluorescence in nonpolar solvents and weak fluorescence in polar solvents which is mainly attributed to aggregation. Therefore, such chromophores are attractive candidates as sensitive fluorescent probes reflecting the polarity of their environment. In particular, their suitability for the staining of cellular membranes is presented in detail. Introduction Fluorescence spectroscopy has proven to be a powerful tool for determining the relative position and the dynamic behavior of fluorescent probes incorporated in a single macromolecule1,2 or for tracing individual molecules in living systems such as cellular structures.3-6 Since the technique is noninvasive and fluorescence can be detected with high sensitivity and signal specificity it is particularly well suited for the understanding of biological processes in the living cell.7 For in vivo staining experiments, the spectral properties of the fluorescent probe in aqueous media, its chemical stability with respect to cell metabolism, as well as low or no toxicity toward the cells are crucial criteria. Adequate chromophores combine absorption and emission maxima above 500 nm due to the background noise of fluorescent impurities and the auto-fluorescence of the cell as well as high fluorescence quantum yields in aqueous solution.8,9 Furthermore, if sophisticated photophysical investigations such as single molecule spectroscopy are intended, a sufficient photostability of the chromophore becomes a key concern. Up to now, a lot of work has been dedicated toward the synthesis of water-soluble chromophores combining the above-mentioned characteristics.10,11 However, in particular, the synthesis of chromophores with strongly red-shifted absorption and emission envelopes is difficult to achieve since extended aromatic scaffolds generally show low watersolubility. Therefore, there are only few chromophores available that are fluorescent in water and display emission envelopes above 600 nm.12 Oligo(peri)naphthylene chromophores (rylene chromophores, Figure 1a) are characterized by an exceptional thermal and photochemical stability as well as fluorescence quantum yields close to unity in organic solvents.13-15 As a key * To whom correspondence should be addressed. Fax: ++49-691503100. E-mail: [email protected]. † Max-Planck-Institut fu ¨ r Polymerforschung. ‡ Merz Pharmaceuticals. § University of Leipzig.

feature, the optical properties of such chromophores can be fine-tuned via the extension of their aromatic scaffold by additional naphthylene groups leading to perylene (n ) 2, Figure 1b), terrylene (n ) 3, Figure 1b), and quaterrylene (n ) 4, Figure 1b) chromophores.16,17 In this way, the rylene chromophore BTI (n ) 3, Figure 1b) already displays emission envelopes in the near-infrared region. Therefore, with regard to these unique features, such chromophores should be attractive candidates as fluorescent probes in a biological environment. However, due to their hydrophobic nature, the introduction of further hydrophilic substituents is essential to obtain water-solubility. Unfortunately, it has been shown in the past that the fluorescence quantum yield of perylene chromophores bearing polar substituents is strongly diminished in aqueous solution18-20 since intermolecular chromophore interactions open up efficient energy relaxation pathways.21 In this article, we will describe the synthesis of watersoluble perylene and terrylene dyes via the attachment of a poly(ethylene oxide) (PEO) chain. PEO is a biodegradable and biocompatible polymer that has already been approved by the Food and Drug Administration which allows its applicability for in vivo experiments. We chose PEO as a hydrophilic substituent since the attachment of this polymer induces solubility in a wide variety of solvents ranging from very polar to nonorganic solvents. Thereafter, the suitability of such rylene chromophores as polarity-sensitive probes is investigated via a staining of different cells lines. In particular, their amphiphilic character with a lipophilic chromophore scaffold (headgroup) should facilitate cellular uptake via endocytosis which enables their application as a contrast agent for the in vivo staining of cellular membranes. Results and Discussion Synthesis of Water-Soluble Rylene Dyes. The synthesis of water-soluble rylene chromophores necessitates the attachment of at least one hydrophilic substituent. For this purpose, a rylene chromophore with one single functionality,

10.1021/bm049674i CCC: $30.25 © 2005 American Chemical Society Published on Web 12/08/2004

Water-Soluble Rylene Dyes

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Figure 1. (a) General structure of rylene chromophores as oligo(peri-naphthylenes); (b) chemical structures of unsubstituted rylenediimide chromophores perylenedicarboxmonoimide (PMI), perylenetetracarboxdiimide (PDI), and benzoylterrylen-3,4-dicarboximid (BTI). Scheme 1. Synthesis of 6

(i) 1.5 equiv. 2, 3 equiv. potassium acetate, 5% Pd(dppf), dioxane, 12 h., 75 °C, argon atmosphere. (ii) 1.5 equiv. 4, 2M potassium carbonate, toluene, ethanol, 12 h, 80 °C, argon atmosphere. (iii) 5, 2 M potassium hydroxide, tetrahydrofuran, 24 h, 80 °C.

preferably a carboxy or an amino group, is required. Since the introduction of a bromine substituent into the aromatic scaffold of PMI (Figure 1b) as well as BTI (Figure 1b) chromophores has already been described before,22,23 we focused on such chromophores as starting material. The bromo substituent of 1 was converted into the boronic ester 3 via a Suzuki reaction23-25 by applying the commercially available bispinacolato diboron 2 in high yields (Scheme 1).26 Thereafter, 3 was reacted with 4-bromobenzoic acid methyl ester (4) leading to the PMI chromophore 5 in moderate yield followed by ester cleavage under caustic conditions. The resulting PMI chromophore 6 carries a single benzoic acid group which is suitable for the introduction of a PEO chain. The functionalization of PDI and BTI chromophores generally requires the introduction of bulky substituents as a first step to increase their solubility which facilitates purification via column chromatography (e.g., Scheme 2, step ii). According to the reaction sequence shown in Scheme 2, the palladium(0)-catalyzed coupling of BTI-Br22,23 7 with the commercially available 4-pinacolatoboronic benzoic acid methyl ester (8) gave 9 (38%). Hydrolysis of 9 under caustic conditions lead to BTI chromophore 10 carrying a single benzoic acid group. The synthesis of a PDI chromophore with only one functional group has not been reported before and, therefore,

will be discussed herein in slightly greater detail. 1,6,7,12Tetrachloroperylen-3,4,9,10-tetracarbonic acid dianhydride (11, Scheme 3) reacted with 4-amino-benzoic acid tert.-butyl ester (12) and 4-bromo aniline (13) in propionic acid for 20 h at 160 °C leading to a statistic product mixture comprising 14 as one of the byproducts. Due to the low solubility of the product mixture in common organic solvents, a separation of 14 was not achieved. Then, 4-(1,1,3,3-tetramethyl-butyl)phenol (15) and potassium carbonate were added to the crude product and heated for 12 h at 90 °C. Cleavage of the tert.butyl group of 16 with trifluoro acetic acid yielded 17 carrying a bromine group as well as a carboxylic ester in the imide structure (32%). A second strategy toward PDI carrying a PEO chain as well as a polar carboxyl group is given in Scheme 4. First, the reaction of 11 with 4-bromo-2,6-diisopropyl aniline (18) gave 19 in high yield.27,28 Subsequent introduction of four 1-phenoxy-4-(1,1,3,3-tetramethyl-butyl)-benzene groups into the bay region of the chromophore proceeded at 90 °C and 12 h reaction time leading to 20 carrying two bromine groups (66%). A palladium(0) catalyzed coupling of 20 and 21 under caustic conditions yielded 22 (93%), and thereafter, the cleavage of both methyl ester groups of 22 proceeded under caustic conditions and 24 h reaction time. The target molecule 23 bears two carboxylic acid groups in the imide structure of the PDI chromophore and was obtained in 96% yield.

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Scheme 2. Synthesis of 10

(i) 2.5 equiv. 8, potassium carbonate (2M), toluene, methanol, 0.0625 equiv. tetrakis(triphenylphosphin) palladium (0) catalyst, 75 °C, 15 h, argon atmosphere, exclusion of light. (ii) 1 equiv. potassium hydroxide, tetrahydrofuran, water, 24 h, 80 °C.

Scheme 3. Synthesis of 17

(i) 2.5 equiv. 4-bromo-anilin (13), 2.5 equiv. 4-amino-benzic acid tert. butyl ester (12), propionic acid, 20 h, 160 °C, argon atmosphere. (ii) 4-(1,1,3,3tetramethyl-butyl)-phenol, N-methylpyrrolidone, potassium carbonate, 12 h, 90 °C. (iii) dichloromethane, trifluoroacetic acid (1:1), 2 h, RT.

All chromophores reported so far (6, 10, 17, and 23) reveal no or only a poor solubility in water. Therefore, a PEO chain was attached to each of these chromophores (Scheme 5). For this purpose, commercially available poly(ethylene oxide) monomethyl ether (PEO-OH, Mn ) 5000 g/mol) was reacted with chromophore 6, whereas methoxy poly(ethylene oxide) amine (PEO-NH2, Mn ) 5000 g/mol) was reacted with 10, 17, and 23, respectively, by applying N′-(3dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC) and N,N-dimethylaminopyridine (DMAP) in a solvent mixture of methylene chloride and dimethylformamide. The crude product was purified via dialysis in N,N-dimethylformamide (DMF) leading to 24 (Scheme 5) and 10 (17, 23) yielded 25 (26, 27), respectively. Characterization. All compounds reported herein were characterized by applying NMR spectroscopy as well as FDor MALDI-ToF mass spectrometry. The characterization of nonpolymeric compounds usually required special NMR experiments such as 2D-NMR or NOE experiments. Figure

2 shows a H,H-COSY two-dimensional NMR spectrum of 6 which is essential to distinguish the protons of the chromophore scaffold. In the case of chromophores 24-27 carrying a PEO chain, NMR spectroscopy as well as GPC analysis and MALDITof spectrometry have been applied. The dispersity of the chromophores was found to be in the range of 1.1 up to 1.2. The MALDI-Tof spectra of 26 (Figure 3b) and PEO (Figure 3a) are given in Figure 3. With respect to PEO-NH2 (Mn ) 5000 g/mol) which was used as starting material, an increase in the average molecular weight by about 1447 g/mol was detected for 26 (Mn ) 6700 g/mol) which is in accordance with the expected increase in the average molecular weight for PEO functionalized with a single PDI chromophore. All PEO-substituted chromophores 24-27 are well soluble in organic solvents such as methylene chloride, tetrahydrofuran, methanol or ethanol and they are soluble in water. Figure 4 depicts the absorption and emission spectra of 24



Scheme 4

(i) 5 equiv. 4-bromo-2,6-diisopropyl-phenylamine (18), propionic acid, 20 h, 160 °C. (ii) 10.7 equiv. 4-(1,1,3,3-tetramethyl-butyl)-phenol (15), 5 equiv. potassium carbonate, N-methylpyrrolidone, 12 h, 90 °C, argon atmosphere. (iii) equiv. 4-methoxycarbonyl-phenyl-boronic acid (21) toluene, methanol (1:6), potassium carbonate (2 M), 0.2 equiv. tetrakis(triphenylphosphin) palladium (0) catalyst, 0.0476 mmol), 75 °C, 15 h, argon atmosphere, exclusion of light. (iv) 20 equiv. potassium hydroxide, water, tetrahydrofuran, 24 h, 80 °C.

Scheme 5. Synthesis of 24, 25, 26 and 27

(i) Poly(ethylene oxide) monomethyl ether (PEO-OH), 3 equiv. EDC, 1.5 DMAP, dimethylformamide, dichloromethane (3:1), 4 d, RT, exclusion of light, argon atmosphere. (ii) Methoxy poly(ethylene oxide) amine (PEO-NH2), 4.4 equiv. EDC, 3 equiv. DMAP, dimethylformamide, 5 d, RT, argon atmosphere, exclusion of light. (iii) Methoxy poly(ethylene oxide) amine (PEO-NH2), 4 equiv. EDC, 3 equiv. DMAP, dimethylformamide, 5 d, RT, argon atmosphere, exclusion of light. (iv) Methoxy poly(ethylene oxide) amine (PEO-NH2), 4 equiv. EDC, 3 equiv. DMAP, dimethylformamide, 5 d, RT, argon atmosphere, exclusion of light.

in chloroform and in water. A strong solvatochromic behavior becomes evident: In chloroform, 24 shows similar absorption and emission spectra as 1 carrying no PEO chain.29 However, in aqueous solution, 24 displays broad and unstructured

spectra with a bathochromic shift of the emission maximum (λmax,em) of about 98 nm (Table 1). The fluorescence quantum yield (FQY) of 24 is quantitative in chloroform whereas in water a reduction down to 15% is observed. This behavior

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Figure 2. 500 MHz1H NMR spectrum of 6 in d8-THF at 298 K.

is mainly attributed to the high tendency of the hydrophobic PMI chromophore headgroup of 24 to undergo strong intramolecular π interaction which generally results in a spectral broadening and diminished FQYs.30 Nevertheless, other fluorescence quenching processes that are not related to aggregation such as a photoinduced electron transfer31 or a vibrational relaxation32 might also occur. A visualization of aggregated PMI chromophores is given as a sketch in Figure 4. By investigating chromophores 26 and 27, the same trend e.g. high FQY in toluene and low FQY in water is evident (Table 1). However, in water, these chromophores display sharper absorption and emission envelopes in comparison to 24 in the same solvent (Figure 5a/b) which could be attributed to a weaker tendency to form aggregates. Obviously, the four bulky substituents of the aromatic chromophore scaffold of 26 and 27 lead to a protection of the chromophore core thus reducing π-interactions. Furthermore, the absorption and emission spectra of 26 are shifted according to the polarity of the solvent (Figure 5a,b). In this way, an emission maximum of 605 nm is found in toluene, whereas in chloroform and water, a bathochromic shift of 11 and 28 nm, respectively, is detected. This behavior

could be due to an increase in aggregation with increasing polarity of the solvent. In the case of 27, similar absorption spectra are obtained in toluene and water, whereas in chloroform, a bathochromic shift of 9 nm is evident. By investigating the emission of 27, a bathochromic shift of 10 nm is found in toluene, whereas in water and chloroform, no spectral shift occurs. A comparison of 26 and 27 reveals for the latter case a bathochromically shifted emission spectrum (5 nm) in toluene and hypsochromically shifted absorption (14 nm) and emission spectra (12 nm) in water. This observation could be related to the substituent of the PDI chromophore. In the nonpolar solvent toluene, 26 bearing a bromine substituent should possess a higher solubility and thus a lower tendency to form aggregates than in an aqueous solution. On the other hand, the introduction of a hydrophilic carboxyl group should result in a better solubilization of 27 in water compared with 26, and in this way, the tendency to form aggregates is less strongly expressed. Table 2 reveals the FQY of 24 and 26 in a variety of solvents ranging from very polar to nonpolar solvents. Thereby, 24 and 26 show considerable changes. The more polar the solvent, the lower the FQY: In chloroform, the



Figure 3. (a) MALDI-TOF mass spectrum of poly(ethylene oxide) monomethyl ether (PEO-OH) and a dithranol-matrix; MALDI-TOF mass spectrum of 26 with n ) 5000 and a dithranol-matrix.

Figure 4. Absorption and emission spectra of 24 in chloroform (λexc. ) 488 nm, small line) and water (λexc. ) 488 nm, bold line).

FQY of 24 is nearly quantitative, in DMF or THF it is reduced by about 40%, whereas in water a FQY of only 15% was obtained. We can only speculate that this stepwise decrease of the FQY with increasing polarity of the solvent is directly connected to their increasing tendency to form aggregates in polar solvents. Recently, the optical behavior of PDI chromophores in water has been investigated in a systematic way.11 However, up to now, all quenching processes that are involved in aqueous solution are still not

completely understood, and therefore, further detailed photophysical investigations will be necessary to explain this matter. However, the significant solvatochromic behavior of the water-soluble chromophores reported herein suggest that they should be attractive fluorescent probes reflecting a hydrophobic environment with a high contrast since they only reveal elevated FQY in nonpolar media. In the following, the applicability of water-soluble chromophores 24-27 as


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Table 1. Absorption and Emission Maxima of 24-27 in Water, Chloroform, and Toluenea toluene

chloroform

water

λmax,abs [nm]

λmax,em [nm]

λmax,abs [nm]

λmax,em [nm]

λmax,abs [nm]

λmax,em [nm]

φF, toluene† chloroform‡ [%]

φF, water [%]

24b

n.s.

n.s.

561

499

613

99‡

15

25c

n.s.

n.s.

742

650

n.d.

26‡

n.d.

26d

447 538 578 453 542 580

605

496 515 (643)** 699 453 549 585 452 545 587

622

460 553 592 448 540 578

633

86†

16

621

86†

19

27d

a

610

620

n.s. ) not soluble, n.d. )not detectable. b PMI: λExc. ) 488 nm. c BTI: λExc. ) 590 nm. d PDI: λExc. ) 540 nm.

Table 2. Fluorescence Quantum Yields of 24 and 26 in Different Solventsa solvent compound 24 26 a

Figure 5. (a) Normalized absorption spectra of 26 in toluene, dimethylformamide, methanol and water; (b) normalized emission spectra of 26 (λexc. ) 540 nm) in toluene, dimethylformamide, methanol and water.

polarity-sensitive colorants for the in vitro staining of cells is investigated. Staining of Different Cell Lines. Cell staining experiments were performed by using chromophores 24-26 each dissolved in dimethyl sulfoxide (DMSO) in a separate vial

water

methanol

DMF†/ DMSO‡

15 ( 0.1 61 ( 0.1 73 ( 0.1‡ 16 ( 0.1 66 ( 0.1 64 ( 0.1†

chloroform

toluene

98 ( 0.1 86 ( 0.1

no soluble 87 ( 0.1

24: λExc. ) 488 Nm, 26: λExc. ) 540 Nm.

with a millimolar concentration (stock solution, cchromophore ) 10-3 M). Thereafter, the stock solution was diluted by applying a phosphate buffer (PBS buffer33) down to a concentration of the chromophore of 10-4, 10-5, and 10-6 M each with a final DMSO concentration of 1%. Then, different cell types (Chinese hamster ovarian cell line CHO, bronchial carcinoma cell line LU1, and ovarian carcinoma cell line EFO2) that were grown on a glass plate were incubated with the chromophore solution for a time period of 30 min at 37 °C. After removal of the staining solution, the cells were rinsed carefully with PBS buffer. Figure 6a-d shows cells of an ovarian carcinoma cell line EFO2 after 30 min incubation with 24 (10-5 M) in the transmission mode (A) and under red fluorescence (B, filter 580 nm). A homogeneous staining of the cells and a high contrast to the surrounding media is clearly evident. An enlargement of this picture is given in Figure 6 (C). Obviously, the cytoplasma as well as the peri-nuclear region of the cells reveal a particularly intense staining whereas the nucleus is spared out. By reducing the incubation time down to 5 min (D), it becomes evident that already after this very short period of time these cells are homogeneously stained suggesting a very fast diffusion process of 24 through the cellular membrane. Since 24 is highly fluorescent in a hydrophobic environment but bears only a low fluorescence quantum yield in water, especially hydrophobic compartments are visualized under the fluorescence microscope. Further more, even after one month the cells still retained their brilliant color. By applying 26 and 27 at different concentrations ranging from 10-4 to 10-6 M to CHO cells, only few cells were stained. Furthermore, these chromophores were found to be cell-toxic at higher concentrations (10-4-10-5 M), and therefore, a possible diffusion through the cellular membrane after a longer period of time could not be observed. In contrast to this behavior, incubation of CHO cells with 25


Figure 6. (a) Ovarian carcinoma cell line EFO2 after 30 min incubation with 24 (10-5 M) in the transmission mode; b) under red fluorescence (filter 580 nm); c) enlargement of picture b); d) under red fluorescence (filter 580 nm), 5 min; e) Chinese hamster ovarian cell line (CHO) after 30 min incubation with 25 (10-5M).

over 30 min lead to a homogeneous staining of the cell where especially the outer membrane of the cells was stained (E).It further seemed that the diffusion of 25 into the cells is somewhat slower compared with 24. The differences of chromophores 24-27 with regard to their ability to penetrate the cellular membrane which is followed by a staining of the interior of the cell might be due to differences in the three-dimensional shape of the chromophores. The aromatic scaffold of 24 is planar and bears no substituents which should facilitate the diffusion through the cellular membrane. In contrast to this, 25 carries two and 26 and 27 carry four bulky substituents, and in the case of 26 and 27, the chromophore scaffold is twisted with an angle of about 28°.34 Since cellular uptake via passive diffusion is usually favored in the case of planar and less bulky molecules, we believe that the number of substituents at the chromophore scaffold plays a crucial role which in consequence leads to the very limited staining of cells by applying the twisted chromophores 26 and 27. Conclusion and Outlook We have presented the synthesis of water-soluble rylene chromophores, e.g., PMI, PDI, and BTI chromophores, that were obtained after the attachment of a single PEO chain to the chromophore scaffold. All of these chromophores reveal a strong solvatochromic behavior. In nonpolar solvents, their optical spectra are well structured and high fluorescence quantum yields are found. With increasing polarity of the solvent, a significant decrease in the fluorescence quantum yields down to about 15% in water for PMI chromophore


24 and down to 20% for PDI chromophores 26 and 27 is observed, whereas in the case of BTI chromophore 25, no fluorescence is found. With respect to 24, this behavior has mainly been attributed to the high tendency of this chromophore to form aggregates in water. Thereafter, the suitability of these chromophores for the staining of cellular membranes has been presented. 24 and 25 appear to pass the outer cellular membrane which is evidenced by the staining of inner cellular structures. In particular, the application of 24 lead to a homogeneous staining of different cell types where a high contrast between hydrophobic parts such as membranes and their hydrophilic surrounding is achieved. In the case of 24, long-term staining experiments have been performed that reveal high chemical stability as well as nontoxicity of this chromophore. The chromophore 25 is characterized by an emission wavelength above 700 nm and, due to a lack of fluorescence in water, has been shown to stain cellular membranes with a high contrast to the surrounding medium. In particular, 24 and 25 are attractive candidates as sensitive fluorescent probes reflecting the polarity of their environment and, therefore, allow investigations of membranes in the living cell over a long period of time. Future work will focus on detailed photophysical investigations including those at the single molecule level in order to investigate the location of the chromophores inside the cell as well as their diffusion through the cellular membrane. Experimental Section General Information. The solvents used were of commercial grade; tetrahydrofuran (THF) was dried over potassium, and toluene was distilled from sodium. 1H NMR and 13 C NMR spectra were recorded on a Bruker DRX 500 (500 and 125 MHz, respectively) or Bruker AMX 300 (300 and 75 MHz, respectively). Mass spectra were recorded on a Bruker MALDI-TOF spectrometer. UV-vis data were obtained on a Perkin-Elmer Lambda 9, and fluorescence spectra were measured on a SPEX Fluorolog 2 Type 212 spectrometer. Tetrakis(triphenylphosphine)-palladium (0) (Pd(PPh3)4) and dichloro[1,1′-bis(diphenylphosphino)ferrocene]palladium(II) dichloromethane adduct (Pd(dppf)) catalysts were purchased from ABCR. 3.26 A mixture of N-(2,6-diisopropylphenyl)-9-bromoperylene-3,4-dicarboximide (1.1 g, 0.2 mmol), bispinacolato diboron (558 mg, 2.5 mmol) and potassium acetate (588 mg, 5.3 mol) was dissolved in 20 mL of dioxane under an argon atmosphere. Pd-dppf catalyst (44 mg, 0.1 mmol) was added, and the resulting mixture was stirred for 16 h at 70 °C. Thereafter, the product mixture was washed with water (dest.) and dichloromethane. The dichloromethane layer was separated and dried over MgSO4, and the crude product was purified by column chromatography eluting with dichloromethane to afford 15 (0.9 g, 75%) as a red solid. mp > 200 °C; MS (FD, 8kV) m/z (%) ) 607.4 (M+, 100%); 1H NMR (250 MHz, d2-CH2Cl2, 25 °C): δ ) 8.86 (d, J ) 8.25 Hz, 1H), 8.62 (dd, 2H), 8.43 (m, 4H), 8.16 (d, J3 (H,H) ) 7.57 Hz, 1H), 7.63 (t, 7.59 Hz, 1H), 7.50 (t, 7.74 Hz, 1H), 7.35 (d, J3 (H,H) ) 7.58 Hz, 2H), 2.79 (h, 2H), 1.46 (s,

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12H), 1.15 (d, 12H); 13C NMR (62.5 MHz, CD2Cl2, 25 °C): δ ) 165.85, 165.80, 147.94, 139.57, 138.93, 137.83, 133.59, 133.46, 133.39, 133.17, 132.13, 131.07, 130.55, 129.31, 128.73, 128.50, 125.80, 125.46, 124.60, 123.02, 122.56, 122.47, 122.04, 86.09, 30.69, 26.60, 25.56, 25.53; UV/vis (CHCl3): λmax () ) 241 (28159), 265 (30569), 485(36712), 515 (36897); IR (KBr pellet): ν (cm-1) ) 2970, 2862, 1708, 1667, 1594, 1512, 1470, 1429 1367, 1336, 1243, 1144, 1113, 963, 865, 818, 762. Anal. Calcd. for C40H38NO4B: C, 79.08; H, 6.30; N, 2.31; Found: C, 78.53; H, 6.91; N, 2.25. 5. 4-Bromo-benzoic acid methyl ester (4, 0.18 g, 0.549 mmol) and 3 (0.5 g, 0.823 mmol) were dissolved in 23 mL of toluene and 1.7 mL of ethanol. Then, 9 mL of a solution of 2.71 g K2CO3 in H2O was added, and the reaction mixture was flushed with argon. Pd(PPh3)4 catalyst (47.5 mg, 0.041 mmol) was added, and the reaction mixture was stirred at 80 °C for 12 h. The resulting solution was washed three times with water and chloroform, and thereafter, the organic layer was separated and dried over magnesium sulfate. The crude product was purified by chromatography eluting with dichloromethane to give 5 as a red solid (0.213 g; 0.346 mmol, 63%). MS (FD, 8kV) m/z (%) ) 615 (M+, 100%); (calcd. C42H33NO4 ) 615.74 g/mol); 1H NMR (500 MHz, d8-THF, 298 K): δ (ppm) ) 8.69-8.58 (m, 6 H), 8.21 (d, 3J ) 7.95 Hz, 2 H), 7.95 (d, 3J ) 7.95 Hz, 1 H), 7.66-7.60 (m, 4 H), 7.38 (t, 3J ) 7.3 Hz, 1 H), 7.29 (d, 3J ) 7.95 Hz, 2 H), 2.80 (septet, 3J ) 6.70 Hz, 2 H), 1.15 (d, 3J ) 6.70 Hz, 12 H). 13 C NMR (125 MHz, d8-THF, 298 K): δ (ppm) ) 167.53 (q), 164.51 (q), 147.a¨ (q), 145.46 (q), 143.36 (q), 138.52 (q), 138.28 (q), 133.60 (q), 132.97 (q), 131.80 (q), 131.60 (q), 131.05 (t), 130.90 (t), 130.78 (q), 130.33 (q), 129.78 (t, broad), 129.57 (q), 129.43 (t), 129.28 (t), 128.40 (t), 128.26 (t), 128.09 (q), 124.46 (t), 122.66 (q), 130.19 (t), 24.43 (t). Anal. Calcd. for C42H33NO4: C, 81.93; H, 5.40; N, 2.27; found: C, 81.88; H, 5.47; N, 2.27. 6. 5 (0.2 g, 0.325 mmol) was dissolved in tetrahydrofuran (20 mL). Then, potassium hydroxide (182 mg, 3.25 mmol) dissolved in water (2 mL) was added. The reaction mixture was first flushed with argon several times, and thereafter, it was stirred for 42 h at 80 °C. After 16 and 19 h, each time water (1 mL) was added to keep the product in solution. After cooling to RT, the crude product was precipitated in hydrochloric acid (150 mL, 2 M), and the precipitate was filtered, washed, and dried at 80 °C under vacuum to give 6 as a red solid (172 mg, 0.286 mmol, 88%); mp ) 91 °C. MS (FD, 8kV) m/z (%) ) 601 (M+), (calcd. C41H31NO4 ) 601.71); 1H NMR (500 MHz, d8-THF, 298 K): δ (ppm) ) 8.69-8.58 (m, 6 H), 8.21 (d, 3J ) 7.95 Hz, 2 H), 7.95 (d, 3J ) 7.95 Hz, 1 H), 7.66-7.60 (m, 4 H) 7.38 (t, 3J ) 7.3 Hz, 1 H), 7.29 (d, 3J ) 7.95 Hz, 2 H), 2.80 (septet, 3J ) 6.70 Hz, 2 H), 1.15 (d, 3J ) 6.70 Hz, 12 H). 13C NMR (125 MHz, d8-THF, 298 K): δ (ppm) ) 167.53 (q), 164.51 (q), 147.11 (q), 145.46 (q), 143.36 (q), 138.52 (q), 138.28 (q), 133.60 (q), 132.97 (q), 131.80 (q), 131.60 (q), 131.05 (t), 130.90 (t), 130.78 (q), 130.33 (q), 129.78 (t, broad), 129.57 (q), 129.43 (t), 129.28 (t), 128.40 (t), 128.26 (t), 128.09 (q), 124.46 (t), 122.66 (q), 30.19 (t), 24.43 (t). Anal. Calcd. for C41H31NO4: C, 81.64; H, 5.19; N, 2.33; found: C, 81.35; H, 5.10; N, 2.31.

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9. 7 (104 mg, 0.096 mmol), 4-methoxycarbonyl-phenylboronic acid (44 mg, 0.24 mmol), was dissolved in toluene (30 mL) and methanol (5 mL). Then, potassium carbonate (5 mL, 2 M) and tetrakis(triphenylphosphin) palladium (0) catalyst ((Pd(PPh3)4, 7 mg, 0.006 mmol) were added. The reaction mixture was flushed with argon and stirred at 75 °C for 15 h under the exclusion of light. After cooling to RT, the reaction mixture was washed with water several times, and the organic phase was separated and dried over magnesium sulfate. Purification was achieved via column chromatography eluting with dichloromethane and hexane (2:1) to give 9 as a dark red solid (42 mg, 0.0369 mmol, 38%). MS (FD, 8kV) m/z (%) ) 1139.4.0 (100%, M+); (calcd. C79H63NO7 ) 1138,35); 1HNMR (250 MHz, d8-THF, 298 K): δ (ppm) ) 9.21 (d, 3J ) 9.15 Hz, 1 H), 8.79 (d, 3J ) 8.85 Hz, 1 H), 8.54 (d, 3J ) 8.2 Hz, 1 H), 8.44 (s, 1 H), 8.39 (d, 3J ) 8.3 Hz, 1 H), 8.27 (s, 1 H), 8.21 (m, 3 H), 8.05 (d, 3J ) 8.22 Hz, 2 H), 7.93 (s, 1 H), 7.52-7.43 (m, broad, 6 H), 7.4 (d, 3J ) 8.85 Hz, 2 H), 7.32 (m, 2 H), 7.26 (d, 3J ) 7.25 Hz, 2 H), 7.17 (d, 3J ) 8.85 Hz, 2 H), 7.02 (d, 3 J ) 8.52 Hz, 2 H), 4.21 (s, 3H), 3.06 (septet, 3J ) 6.78 Hz, 2 H), 1.36 (s, 9 H), 1.333 (s, 9 H), 1.21 (d, 3J ) 6.32 Hz, 12 H). 13CNMR (75 MHz, d8-THF, 323 K): δ (ppm) ) 185.19, 164.8, 162.13, 158.12, 157.52, 154.91, 154.51, 153.34, 152.57, 149.57, 146.25, 145.11, 141.7, 140.4, 139.16, 138.16, 136.69, 135.73, 135.15, 134.48, 134.08, 133.88, 133.69, 133.41, 132.88, 132.75, 132.54, 132.01, 131.88, 131.74, 131.46, 131.34, 131.28, 131.08, 130.87, 130.74, 130.61, 130.14, 129.74, 129.67, 129.6, 129.48, 129.41, 129.07, 128.87, 128.6, 128.34, 128.13, 128.02: 127.93, 127.8, 127.74, 127.2, 126.93, 126.8, 126.33, 125.04, 120.38, 119.91, 39.96, 38.95, 33.8, 31.72, 24.81. 10. 9 (30 mg, 0.0263 mmol) was dissolved in tetrahydrofuran (15 mL). Then, potassium hydroxide (14.7 mg, 0.262 mmol) dissolved in water (1.5 mL) was added, and the reaction mixture was flushed with argon. After stirring for 24 h at 80 °C, the reaction mixture was cooled to RT and hydrochloric acid (300 mL, 2 N) was added. The precipitate was filtered and dried under vacuum to give 10 as a blue solid (18 mg, 0.016 mmol). MS (FD, 8kV) m/z (%) ) 1124.6 (100%, M+) (calcd. C78H61NO7 ) 1124,32) 1H NMR (250 MHz, d8-THF, 298 K): δ (ppm) ) 9.29 (d, 3J ) 9.15 Hz, 1 H), 8.86 (d, 3J ) 8.85 Hz, 1 H), 8.59 (d, 3J ) 8.2 Hz, 1 H), 8.48 (s, 1 H), 8.43 (d, 3J ) 8.3 Hz, 1 H), 8.3 (s, 1 H), 8.26 (m, 3 H), 8.09 (d, 3J ) 8.22 Hz, 2 H), 7.99 (s, 1 H), 7.58-7.49 (m, broad, 6 H), 7.46 (d, 3J ) 8.85 Hz, 2 H), 7.39 (m, 2 H), 7.3 (d, 3J ) 7.25 Hz, 2 H), 7.22 (d, 3J ) 8.85 Hz, 2 H), 7.02 (d, 3J ) 8.52 Hz, 2 H), 2.85 (septet, 3J ) 6.78 Hz, 2 H), 1.35 (s, 9 H), 1.32 (s, 9 H), 1.16 (d, 3J ) 6.32 Hz, 12 H). 13C NMR (75 MHz, d8-THF, 323 K): δ (ppm) ) 185.12, 164.76, 162.07, 158.11, 157.49, 154.9, 154.5, 153.31, 152.56, 149.54, 146.22, 145.09, 141.71, 140.38, 139.13, 138.15, 136.67, 135.73, 135.16, 134.41, 134.08, 133.82, 133.66, 133.41, 132.91, 132.75, 132.54, 132.02, 131.86, 131.74, 131.45, 131.34, 131.27, 131.06, 130.9, 130.73, 130.6, 130.14, 129.71, 129.64, 129.57, 129.46, 129.38, 129.07, 128.87, 128.61, 128.36, 128.13, 128.01: 127.92, 127.81, 127.71, 127.2, 126.93, 126.77, 126.31, 125.01, 120.32, 119.84, 38.79, 33.81, 31.72, 24.83.


17. 4-{9-(4-Bromo-phenyl)-1,3,8,10-tetraoxo-5,6,12,13tetrakis-[4-(1,1,3,3-tetramethyl-butyl)-phenoxy]-3,8,9,10-tetrahydro-1H-anthra[2,1,9-def;6,5,10-d′e′f′]diisoquinolin-2-yl}benzoic acid (11; 1 g, 1.886 mmol), 4-bromo-anilin (13, 0.811 g, 4.716 mmol), and 4-amino-benzic acid tert. butyl ester (0.911 g, 4.716 mmol) were suspended in propionic acid (40 mL). Then, the reaction mixture was flushed with argon and stirred for 20 h at 160 °C. After cooling to RT, the crude product containing 14 was precipitated from water (80 mL), filtered, and dried at 80 °C under vacuum. The separation of 14 from byproducts that were formed during the reaction was not successful due to the low solubility of these chromophores. The crude product was then dissolved in N-methylpyrrolidone (80 mL) and stirred for 15 min at 80 °C. Thereafter, 4-(1,1,3,3-tetramethyl-butyl)-phenol (15, 4.65 g, 22.53 mmol) and potassium carbonate (0.527 g, 9.4 mmol) were added, and the reaction mixture was stirred for 12 h at 90 °C. After cooling to RT, hydrochloric acid was added (2N, 800 mL) and the precipitate was stirred for an additional 2 h. At this stage, the crude product contained 16 as well as the already deprotected chromophore 17. Therefore, dichloromethane and trifluoroacetic acid (1:1, 100 mL) were added to the crude product, and the reaction mixture was stirred for 2 h at RT. Then, the reaction mixture was evaporated to dryness and purified via column chromatography eluting with dichloromethane. 17 was obtained as a violet solid (900 mg, 0.607 mmol, 32%). MS (FD, 8kV) m/z (%) ) 1483.4 (100%, M+); (calcd. C93H97BrN2O10 ) 1482,68); 1H NMR (250 MHz, d2CD2Cl2, 298 K): δ (ppm) ) 8.23 (d, 3J ) 8.52 Hz 2H), 8.12 (s, 4 H), 7.66 (d, 3J ) 8.85 Hz, 2 H), 7.41 (d, 3J ) 8.52, 2 H) 7.32 (d, 3J ) 8.82 Hz, 8 H), 7.16 (d, 3J ) 8.52 Hz, 2 H), 6.91 (d, 3J ) 8.82 Hz, 8H), 1.72 (s, 8 H), 1.34 (s, 24 H), 0.73 (s, 36 H). 13C NMR (125 MHz, d8-THF, 298 K): δ (ppm) ) 167.78 (q), 162.98 (q), 156.83 (q), 153.96 (q), 147.10 (q), 139.98 (q), 135.90 (q), 133.73 (q), 132.54 (t), 131.69 (t), 130.71 (t), 129.48 (t), 128.42 (t), 123.94 (q), 123.81 (q), 122.84 (q), 120.84 (q), 120.74 (q), 120.37 (q), 120.07 (t), 119.85 (t), 57.58 (q), 38.84 (q), 32.84 (q), 32.06 (t). Anal. Calcd. for C93H97BrN2O10: C, 75.34; H, 6.59; N, 1.89; found: C, 73.98; H, 7.47; N, 2.11. 19. 11 (1.5 g, 2.82 mmol) and 4-bromo-2,6-diisopropylphenylamine (18, 3.61 g, 14.1 mmol) were suspended in propionic acid (80 mL). Then, the reaction mixture was flushed with argon and stirred for 20 h at 160 °C. After cooling to RT, the crude product containing 14 was precipitated from water (120 mL), filtered, and dried under vacuum. The separation of 14 from byproducts that were formed during the reaction was not successful due to the low solubility of this chromophore. MS (FD, 8kV) m/z (%) ) 1006.1 (100%, M+) (calcd. C48H36Br2Cl4N2O4 ) 1006.45) 20. 19 (500 mg, 0.497 mmol) was dissolved in Nmethylpyrrolidone and stirred at 80 °C for 15 min. Then, 4-(1,1,3,3-tetramethyl-butyl)-phenol (15, 1.1 g, 5.33 mmol) and potassium carbonate (0.345 g, 2.5 mmol) were added, and the reaction mixture was stirred for 12 h at 90 °C under argon atmosphere. After cooling to RT, hydrochloric acid (800 mL, 2 N) was added and the reaction mixture was stirred for additional 2 h. The precipitate was filtered under


vacuum and dried. Purification was achieved via flash chromatography by applying dichloromethane as an eluent to give 20 as an orange solid (553 mg, 0.316 mmol, 65.9%). MS (FD, 8kV) m/z (%) ) 1686 (100%, M+) (calcd. C104H120Br2N2O8 ) 1685,88); 1H NMR (250 MHz, d2-CD2Cl2, 298 K): δ (ppm) ) 8.11 (s, 4 H), 7.41 (s, 4 H), 7.36 (d, 3J ) 8.85 Hz, 8 H), 6.94 (d, 3J ) 8.52 Hz, 8 H), 2.72-2.57(septet, 3J ) 6.62 Hz, 4 H), 1.73 (s, 8 H), 1.36 (s, 24 H), 1.08 (d, 3J ) 6.65 Hz, 24 H), 0.75 (s, 36 H). 13C NMRSpectrum, (62.8 MHz, d8-THF, 298 K): δ (ppm) ) 163.54 (q), 157.28 (q), 153.77 (q), 149.53 (q), 147.50 (q), 134.06 (q), 131.51 (q), 128.80 (t), 127.97 (t), 124.15 (q), 123.84 (q), 121.06 (q), 120.87 (q), 120.46 (t), 119.91 (t), 57.87 (q), 39.11 (q), 33.07 (q), 32.27 (t), 32.05 (t), 30.07 (t), 23.98 (t). Anal. Calcd. for C104H120Br2N2O8: C, 74.09; H, 7.17; N, 1.66; found: C, 74.27; H, 7.26; N, 1.61. 22. 20 (400 mg, 0.237 mmol) and 4-methoxycarbonylphenyl-boronic acid (21, 256 mg, 1.422 mmol) were dissolved in a solvent mixture containing toluene (60 mL) and methanol (10 mL). Then, potassium carbonate (10 mL, 2 M) and tetrakis(triphenylphosphin) palladium (0) catalyst (Pd(PPh3)4, 55 mg, 0.0476 mmol) were added. The reaction mixture was flushed with argon and stirred at 75 °C for 15 h under the exclusion of light. After cooling to RT, the reaction mixture was washed with water several times, the organic phase was separated and dried over magnesium sulfate. Purification was achieved via column chromatography eluting with dichloromethane and low boiling petrol ether (2:1) to give 22 as a dark red solid (395 mg, 0.22 mmol, 93%). MS (FD, 8kV) m/z (%) ) 1796.0 (100%, M+) (calcd. C120H134N2O12 ) 1796,35); 1H NMR (250 MHz, d2-CD2Cl2, 298 K): δ (ppm) ) 8.14 (d, 3J ) Hz, 4 H), 8.10 (s, 4 H), 7.77 (d, 3J ) 8.2 Hz, 4 H), 7.54 (s, 4 H), 7.37 (d, 3J ) 8.52 Hz, 8 H), 6.97 (d, 3J ) 8.82 Hz, 8 H), 3.92 (s, 6 H), 2.842.71 (septet, 3J ) 6.65 Hz, 4 H), 1.74 (s, 8 H), 1.37 (s, 24 H), 1.17 (d, 3J ) 6.62 Hz, 24 H), 0.75 (s, 36 H). 13C NMR (65 MHz, d8-THF, 298 K): δ (ppm) ) 166.84 (q), 163.62 (q), 157.22 (q), 153.81 (q), 147.47 (q), 147.37 (q), 146.59 (q), 141.59 (q), 134.05 (q), 132.46 (q), 130.58 (t), 130.10 (q), 128.72 (t), 127.95 (t), 123.93 (q), 123.55 (t), 121.03 (q), 120.89 (q), 120.4 (t), 119.91(t), 57.83 (q), 52.04 (t), 39.05 (q), 33.03 (q), 32.22 (t), 32.01 (t), 30.10 (t), 24.18 (t). Anal. Calcd. for C120H134N2O12: C, 80.23; H, 7.52; N, 1.56; found: C, 80.19; H, 7.67; N, 1.41. 23. 22 (180 mg, 0.1 mmol) was dissolved in tetrahydrofuran (15 mL) and potassium hydroxide (114 mg, 2.026 mmol) dissolved in water (2.5 mL) was added. The reaction mixture was flushed with argon and stirred for 24 h at 80 °C. After cooling to RT, hydrochloric acid was added (300 mL, 2 N), and the precipitate was filtered and dried under vacuum. 23 was obtained as a dark red solid (169 mg, 0.095 mmol, 96%). MS (FD, 8kV) m/z (%) ) 1767.6 (100%, M+); (calcd. C118H130N2O12 ) 1768,30); 1H NMR (250 MHz, d8THF, 298 K): δ (ppm) ) 8.18 (s, 4 H), 8.15 (d, 3J ) 8.52 Hz, 4 H), 7.78 (d, 3J ) 8.2 Hz, 4 H), 7.56 (s, 4 H), 7.41 (d, 3 J ) 8.52 Hz, 8 H), 6.99 (d, 3J ) 8.52 Hz, 8 H), 2.87 (septet, 3 J ) 6.62 Hz, 4 H), 1.72 (s, 8 H), 1.37 (s, 24 H), 1.17 (d, 3J ) 6.62 Hz, 24 H), 0.76 (s, 36 H). 13C NMR (62.8 MHz, d8-THF, 298 K): δ (ppm) ) 167.46 (q), 163.62 (q), 157.21

78


(q), 153.8 (q), 147.41 (q), 146.26 (q), 141.77 (q), 134.05 (q), 132.32 (q), 130.85 (t), 130.77 (q), 128.71 (t), 127.81 (t), 123.92 (q), 123.54 (t), 121.02 (q), 120.88 (q), 120.39 (t), 119.91 (t), 57.82 (q), 39.04 (q), 33.02 (q), 32.21 (t), 32.01 (t), 30.09 (t), 24.18 (t). Anal. Calcd. for C118H130N2O12: C, 80.15; H, 7.41; N, 1.58; found: C, 77.44; H, 7.69; N, 1.61. 24. 6 (100 mg, 0.166 mmol), poly(ethylene oxide) monomethyl ether (PEO-OH, MN ) 5253, 300 mg), N′-(3dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC, 70 mg, 0.5 mmol) and N,N-dimethylaminopyridine (DMAP, 30 mg, 0.25 mmol) were dissolved in dimethylformamide and dichloromethane (3:1, 3 mL). The reaction mixture was flushed with argon and stirred for 4 days at RT under the exclusion of light. Thereafter, the reaction mixture was washed several times with dichloromethane and water, the organic phase was separated and dried over magnesium sulfate. The crude product was purified by chromatography eluting with tetrahydrofuran, water, and acetic acid (8/8/1) to give 6 as a red solid (300 mg). 1H NMR (500 MHz, d8THF, 298 K): δ (ppm) ) 8.67-8.56 (m), 8.25 (d, 3J ) 7.95 Hz), 7.98 (d, 3J ) 7.95 Hz), 7.67-7.66 (m) 7.1 (t, 3J ) 7.4 Hz), 7.35 (d, 3J ) 7.95 Hz), 3.67-3.55 (m, broad), 2.76 (septet, 3J ) 6.70 Hz), 1.15 (d, 3J ) 6.70 Hz). 13C NMR (125 MHz, d2-CH2Cl2, 298 K): δ (ppm) ) 166.44 (q), 164.37 (q), 146.47 (q), 144.90 (q), 142.56 (q), 138.02 (q), 137.77 (q), 132.70 (q), 132.25 (t), 131.93 (q), 130.92 (q), 130.49 (t), 130.18 (q), 130.11 (t), 129.77 (q), 129.61 (t), 129.39 (q), 128.71 (q), 128.67 (t), 127.71 (t), 127.28 (q), 124.33 (t), 121.40 (q), 72.84 (sec.), 72.25 (sec.), 69.52 (sec.), 70.86 (sec., broad), 58.96 (t), 29.40 (t), 24.05 (t). 25. 10 (9.27 mg, 0.0082 mmol), methoxy-poly(ethylene oxide) amine (PEO-NH2, MN ) 5253, 29 mg), EDC (7 mg, 0.036 mmol), and DMAP (3 mg, 0.024 mmol) were dissolved in dimethylformamide (3 mL) and reacted for 5 days at RT under argon atmosphere and under the exclusion of light according to the procedure described for 26. The reaction mixture was evaporated to dryness under vacuum, dissolved in water, and filtered. The aqueous solution was then purified in dimethylformamide via dialysis to give 25 as a dark red solid (38 mg). MALDI-TOF: m/z ) 6200-6800 g mol-1; 1 H NMR (700 MHz, d2-C2H2Cl4, 403 K): δ (ppm) ) 9.56 (d, 3J ) 9.15 Hz, 1 H), 9.02 (d, 3J ) 8.85 Hz, 1 H), 8.65 (d, 3 J ) 8.2 Hz, 1 H), 8.53 (s, 1 H), 8.4 (d, 3J ) 8.3 Hz, 1 H), 8.32 (s, 1 H), 8.27 (m, 3 H), 8.16 (d, 3J ) 8.22 Hz, 2 H), 8.02 (s, 1 H), 7.91-7.56 (m, broad, 6 H), 7.45 (d, 3J ) 8.85 Hz, 2 H), 7.38 (m, 2 H), 7.22 (d, 3J ) 7.25 Hz, 2 H), 7.15 (d, 3J ) 8.85 Hz, 2 H), 7.01 (d, 3J ) 8.52 Hz, 2 H), 3.693.52 (m, broad), 3.11 (septet, 3J ) 6.78 Hz, 2 H), 1.33 (s, 9 H), 1.28 (s, 9 H), 1.11 (d, 3J ) 6.32 Hz, 12 H). 13C NMR (175 MHz, d2-C2H2Cl4, 403 K): δ (ppm) ) 185.02, 164.69, 161.91, 158.03, 157.43, 154.91, 154.39, 153.31, 152.49, 149.42, 146.17, 145.03, 141.77, 140.32, 139.07, 138.11, 136.56, 135.64, 135.08, 134.39, 133.94, 133.78, 133.55, 133.32, 132.78, 132.66, 132.54, 131.92, 131.77, 131.63, 131.35, 131.28, 131.21, 131.06, 130.79, 130.7, 130.58, 130.07, 129.62, 129.56, 129.49, 129.42, 129.34, 129.01, 128.83, 128.52, 128.27, 128.11, 128.02: 127.87, 127.75, 127.68, 127.12, 126.85, 126.72, 126.24, 125.02, 120.29, 119.8, 70.97, 39.89, 38.86, 33.69, 31.63, 24.68.

Weil et al.

26. 17 (25 mg, 0.0168 mmol) methoxy-poly(ethylene oxide) amine (PEO-NH2, MN ) 5253, 63 mg), EDC (13 mg, 0.068 mmol), and DMAP (6.2 mg, 0.05 mmol) were dissolved in dimethylformamide (4 mL). The reaction mixture was flushed with argon and stirred for 5 days at RT under the exclusion of light. Thereafter, the reaction mixture was evaporated to dryness, dissolved in water, and filtered. The solution was then purified in dimethylformamide via dialysis to give 26 as a violet solid (71 mg). MALDI-TOF: m/z ) 6300-7000 g mol-1, 1H NMR (250 MHz, d2-CD2Cl2, 298 K): δ (ppm) ) 8.41 (s, 4 H), 8.28 (d, 3J ) 8.52 Hz 2 H), 7.94 (d, 3J ) 8.85 Hz, 2 H), 7.7 (d, 3J ) 8.52 Hz, 8 H), 7.62 (d, 3J ) 8.2 Hz, 2 H), 7.52 (d, 3J ) 8.52 Hz, 2 H), 7.27 (d, 3J ) 8.52 Hz, 8 H), 3.95-3.84 (m, broad), 2.08 (s, 8 H), 1.67 (s, 24 H), 1.07 (s, 36 H). 13C NMR (75 MHz, CD2Cl2, 298 K): δ (ppm) ) 166.82 (q), 163.71 (q), 156.84 (q), 153.31 (q), 147.4 (q), 138.75 (q), 135.74 (q), 135.24 (q), 133.59 (q), 132.98 (t), 131.1 (t), 129.5 (q), 128.56 (t), 128.34 (t), 123.1 (q), 121.07 (q), 120.22 (t), 120.02 (t), 71.08 (q), 57.46 (q), 38.82 (q), 32.82 (q), 32.13 (t), 31.92 (t). 27. 23 (40 mg, 0.0226 mmol), methoxy-poly(ethylene oxide) amine (PEO-NH2, MN ) 5253, 100 mg), EDC (17.3 mg, 0.09 mmol) and DMAP (8 mg, 0.0678 mmol) were dissolved in dimethylformamide (3 mL) and reacted for 5 days at RT under argon atmosphere and under the exclusion of light according to the procedure described for 26. The reaction mixture was evaporated to dryness, dissolved in water, and filtered. The solution was then purified in dimethylformamide via dialysis to give 27 as a dark red solid (110 mg). MALDI-TOF: m/z ) 6400-7200 g mol-1, 1H NMR (250 MHz, d8-THF, 298 K): δ (ppm) ) 8.47 (s, 4 H), 8.34 (d, 3J ) 8.52 Hz, 2 H), 8.26 (d, 3J ) 8.22 Hz, 2 H), 8.10 (d, 3J ) 8.52 Hz, 2 H), 8.03 (d, 3J ) 8.22 Hz, 2 H), 7.83 (s, 4 H), 7.7 (d, 3J ) 8.85 Hz, 8 H), 7.28 (d, 3J ) 8.85 Hz, 8 H), 3.89-3.77 (m, broad), 3.17 (septet, 3J ) 6.62 Hz, 4 H), 2.06 (s, 8 H), 1.67 (s, 24 H), 1.46 (d, 3J ) 6.62 Hz, 24 H), 1.05 (s, 36 H). 13C NMR (75 MHz, d8-THF, 298 K): δ (ppm) ) 167.46 (q), 164.7 (q), 158.21 (q), 154.96 (q), 148.42 (q), 146.26 (q), 141.77 (q), 134.05 (q), 132.32 (q), 130.85 (t), 130.77 (q), 128.71 (t), 127.81 (t), 123.92 (q), 123.54 (t), 121.02 (q), 120.88 (q), 120.39 (t), 119.91 (t), 72.59 (q), 58.72 (q), 40.14 (q), 34.04 (q), 33.27 (t), 33.03 (t), 31.15 (t), 25.23 (t). Acknowledgment. The authors thank the European Associated Laboratory (LEA) and the BMBF nano-center for financial support. We would like to thank Tanja Bauer (Merz Pharmaceuticals) for her valuable assistance in performing the fluorescence microscopy experiments. References and Notes (1) Vosch, T.; Hofkens, J.; Cotlet, M.; Ko¨hn, F.; Fujiwara, H.; Gronheid, R.; Best, K. V. D.; Weil, T.; Herrmann, A.; Mu¨llen, K.; Mukamel, S.; Auweraer, M. V. d.; Schryver, F. C. D. Angew. Chem. 2001, 113, 4779-4784. (2) Lor, M.; De, R.; Jordens, S.; Belder, G. D.; Schweitzer, G.; Hofkens, J.; Weil, T.; Mu¨llen, K.; Auweraer, M. V. D.; Schryver, F. C. D. J. Phys. Chem. A 2002, 106, 2083-2090. (3) Moerner, W. E.; Orrit, M. Science 1999, 283, 1670. (4) Nguyen, D. C.; Keller, R. A.; Jett, J. H.; Martin, J. C. Anal. Chem. 1987, 59, 2158. (5) Peck, K.; Stryer, L.; Glazer, A. N.; Mathies, R. A. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 4087.

Water-Soluble Rylene Dyes (6) Weiss, S. Science 1999, 283, 1676. (7) Bastiaens, P. I. H.; Squire, A. Trends Cell Biol. 1999, 9, 48-52. (8) Hope-Ross, M.; Yannuzzi, L. A.; Gragoudas, E. S.; Guyer, D. R.; Slakter, J. S.; Sorenson, A. Ophthalmology 1994, 101, 529-533. (9) Mordon, S.; Devoisselle, J. M.; Soulie-Begu, S.; Desmettre, T. MicroVasc. Res. 1998, 55, 146-152. (10) Buschmann, V.; Weston, K.; Sauer, M. Bioconjug. Chem. 2003, 14, 195-204. (11) Kohl, C.; Weil, T.; Qu, J.; Mu¨llen, K. Chemistry 2004, in press. (12) Haugland, R. P. Handbook of Fluorescent Probes and Research Chemicals; Molecular Probes Inc.: Eugene, OR, 1989. (13) Nagao, Y.; Misono, T. Dyes Pigm. 1984, 5, 171. (14) Rademacher, A.; Markle, S.; Langhals, H. Chem Ber. 1982, 115, 2927. (15) Zollinger, H. Color Chemistry; VCH Verlagsgesellschaft: Weinheim, Germany, 1987. (16) Herbst, W.; Hunger, K. Industrial Organic Pigments; VCH: Weinheim, Germany, 1993. (17) Nagao, Y. Prog. Org. Coatings 1997, 31, 43. (18) Langhals, H. German Patent; DE-3703513, Germany, 1987. (19) Schnurpfeil, G.; Stark, J.; Wo¨hrle, D. Dyes Pigm. 1995, 27, 339350. (20) Langhals, H.; Jona, W.; Einsiedl, F.; Wohnlich, S. AdV. Mater. 1998, 10, 1022-1024. (21) Quante, H.; Schlichting, P.; Rohr, U.; Geerts, Y.; Mu¨llen, K. Macromol. Chem. Phys. 1996, 197, 4029. (22) Quante, H.; Mu¨llen, K. Angew. Chem. 1995, 107, 1487.

Biomacromolecules, Vol. 6, No. 1, 2005 79 (23) Weil, T.; Wiesler, U. M.; Herrmann, A.; Bauer, R.; Hofkens, J.; Schryver, F. C. D.; Mu¨llen, K. J. Am. Chem. Soc. 2001, 123, 81018108. (24) Ishiyama, T.; Murata, M.; Miyaura, N. J. Org. Chem. 1995, 60, 7508. (25) Miyaura, N.; Suzuki, A. Chem. ReV. 1995, 95, 2457-2483. (26) Weil, T.; Reuther, E.; Beer, C.; Mu¨llen, K. Chemistry 2004, 10, 6, 1398-1414. (27) Becker, S., Ph.D. Thesis, Johannes Gutenberg University, Mainz, 2001. (28) Klok, H.-A.; Hernandez, J. R.; Becker, S.; Mu¨llen, K. J. Polym. Sci. A 2001, 39, 1572. (29) Weil, T., Ph.D. Thesis, Johannes Gutenberg University, Mainz, 2002. (30) Herrmann, A.; Weil, T.; Sinigersky, V.; Wiesler, U.-M.; Vosch, T.; Hofkens, J.; Schryver, F. C. De.; Mu¨llen, K. Chemistry 2001, 7, 4854-4862. (31) Lor, M.; Thielemans, J.; Viaene, L.; Cotlet, M.; Hofkens, J.; Weil, T.; Hampel, C.; Mu¨llen, K.; Auweraer, M. V. d.; Schryver, F. C. D. J. Am. Chem. Soc. 2002, 124, 9918-9925. (32) Kavarnos, G. J.; Turro, N. J. Chem. ReV. 1986, 86, 401-449. (33) Wilbur, D. S.; Pathare, P. M.; Hamlin, D. K.; Frownfelter, M. B.; Kegley, B. B.; Leung, W. Y.; Gee, K. R. Bioconjugate Chem. 2000, 11, 584-598. (34) Hofkens, J.; Vosch, T.; Maus, M.; Ko¨hn, F.; Cotlet, M.; Weil, T.; Herrmann, A.; Mu¨llen, K.; Schryver, F. C. De. Chem. Phys. Lett. 2001, 333, 255-263.

BM049674I

Water-Soluble Rylene Dyes as High-Performance Colorants for the

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