Synthesis and in Vitro Testing of a Pyropheophorbide-a-Fullerene

Jun 6, 2007 - Bioconjugate Chem. , 2007, 18 (4), pp 1078–1086. DOI: 10.1021/bc0603337. Publication Date (Web): ... In this paper, we report on the s...
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Bioconjugate Chem. 2007, 18, 1078−1086

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Synthesis and in Vitro Testing of a Pyropheophorbide-a-Fullerene Hexakis Adduct Immunoconjugate for Photodynamic Therapy Fiorenza Rancan,*,† Matthias Helmreich,‡ Andreas Mo¨lich,§ Eugeny A. Ermilov,| Norbert Jux,*,‡ Beate Ro¨der,| Andreas Hirsch,‡ and Fritz Bo¨hm† Universita¨tsklinikum Charite´, Hautklinik, Photobiologisches Labor, Berlin, Germany, Institut fu¨r Organische Chemie, Universita¨t Erlangen-Nu¨rnberg, Erlangen, Germany, Institut fu¨r Biologie, Tierphysiologie, Humboldt Universita¨t, Berlin, Germany, and Institut fu¨r Physik, Photobiophysik, Humboldt-Universita¨t zu Berlin, Berlin, Germany. Received October 24, 2006; Revised Manuscript Received February 13, 2007

The employment of carriers to enhance drug selectivity is one of the strategies to increase the efficacy and reduce the side effects of antitumor therapy. The concept of a modular carrier system (MCS) was developed to construct a complex drug having a high efficacy and selectivity. An MCS employs diverse units or modules: beside the therapeutic unit, an addressing unit (e.g., an antibody) serves to direct the drug to its target, and a multiplying unit has the role of increasing the number of biological active moieties the system can carry. In this paper, we report on the synthesis of a modular carrier system in which the role of multiplying unit is given to a [5:1]fullerene hexakis adduct. This fullerene hexaadduct has five malonate spacers which can bind two therapeutic units (the photosensitizer pyropheophorbide-a) each, for a total of ten, and a longer malonate spacer which serves for the conjugation to the addressing unit, the monoclonal antibody rituximab. Confocal microscopy studies using Epstein-Barr virus-transformed B-lymphocytes and Jurkat cells showed that the antibody conjugate conserves the affinity for its receptor (CD20) and its selectivity toward CD20 positive B-lymphocytes. On the contrary, the antibody-free complex did not show any bounding or intracellular uptake.

INTRODUCTION The enhancement of drug selectivity is the most important task of modern investigations directed to improve the efficacy and reduce the side effects of anticancer therapy. The idea of binding a drug to an addressing unit with specificity for tumor tissue was formulated a long time ago (1). Several carrier systems have been used for this purpose, like liposomes (2), lipoproteins (3), dendrimers (4), or polymers (5), but antibodies or antibody fragments were the most selective molecules (6, 7). One inconvenience of using tumor-specific antibodies is the loss of their biological activity when many drug units are covalently bound to their peptide backbone (8). The concept of the modular carrier system (MCS) was developed as a solution to this problem (9). In an MCS, diverse units or modules are combined to construct a complex drug with diverse specific functions. Besides the therapeutic unit, an addressing unit (e.g., an antibody) serves to direct the drug to the target, and a multiplying unit has the role of increasing the number of drug moieties the system can carry. In this way, a number of drug units can be loaded on a tumor-specific antibody. Dendrimers and polymers were proposed as multiplying units (10, 11). In particular, dendrimers became of rising interest in different fields of biophysical research due to their highly branched and welldefined architecture having a high number of functional end * Corresponding authors: Fiorenza Rancan, Universita¨tsklinikum Charite´, Photobiologisches Labor, Ziegelstr. 5-9, 10117 Berlin, Germany. Tel. +49 30 450518085, Fax. +49 30 450513941, E-mail: [email protected]. Dr. Norbert Jux, Institut fu¨r Organische Chemie, Universita¨t Erlangen-Nu¨rnberg, Henkestr. 42, D-91054 Erlangen, Germany. Tel. +49 9131 8522976, Fax. +49 9131 8526864, E-mail: [email protected]. † Universita ¨ tsklinikum Charite´. ‡ Universita ¨ t Erlangen-Nu¨rnberg. § Institut fu ¨ r Biologie, Tierphysiologie, Humboldt Universita¨t. | Institut fu ¨ r Physik, Photobiophysik, Humboldt-Universita¨t zu Berlin.

groups (12). C60-fullerene adducts are good candidates to be used as the core of multiplying units. Fullerene hexakis adducts have an octahedral addition pattern, which allows the six substituents occupying spread positions with minimal steric interactions (13). So-called [5:1]hexaadducts are well-known in fullerene chemistry. In such adducts, one position can be occupied by the addressing unit, while the other five positions are suitable for spacers bearing drug moieties. The fullerene’s ability to scavenge free radicals (14) and its spherical shape, which could adapt to active sites of enzymes (15), make the use of C60 in medical research particularly interesting. Recently, a communication appeared reporting the conjugation of fullerene derivatives with the antibody ZME-18 (16). The authors used water-soluble fullerenes and reached C60/antibody molar ratios of 42:1 and 15:1 without the loss of antigen binding ability. Although one of the two water-soluble derivatives had no activated group for covalent binding to the monoclonal antibody (mAb), it still showed similar results to the derivative with a binding site. The authors therefore concluded that in such immunoconjugates the fullerene moieties are not necessarily connected to the antibody through a covalent bond. In this paper, we report the synthesis of a modular carrier system in which the role of multiplying unit is given to a [5:1]fullerene hexakis adduct. This fullerene hexaadduct has five malonate addends, which can bind two pyropheophorbide-a 2 units each, and a longer malonate spacer with an activated ester moiety, which serves for the conjugation to one addressing unit, the mAb rituximab. Rituximab is a genetically engineered chimeric murine/human mAb directed against the CD20 antigen found on the surface of normal and malignant B lymphocytes (17). The antibody is an IgG1 κ immunoglobulin containing murine light- and heavy-chain variable region sequences and human constant region sequences. Rituximab is composed of two heavy chains of 451 amino acids and two light chains of 213 amino acids (based on cDNA analysis) and has an

10.1021/bc0603337 CCC: $37.00 © 2007 American Chemical Society Published on Web 06/06/2007

Fullerene Immunoconjugate as Carrier System

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Figure 1. [5:1]Fullerene hexaadduct 3 and fullerene hexaadduct 4 carrying 6 and 12 pyropheophorbide-a moieties, respectively.

approximate molecular weight of 145 kDa. Rituximab has a binding affinity for the CD20 antigen of approximately 8.0 nM (17). This monoclonal antibody is an already approved drug and is indicated for the treatment of patients with relapsed or refractory, low-grade or follicular, CD20-positive, B-cell nonHodgkin’s lymphoma (NHL). The therapeutic unit, pyropheophorbide-a (2) (18) is a photosensitizer (PS) which might be used in photodynamic therapy (PDT). This is a treatment of cancerous and noncancerous diseases (19), which involves the administration of a PS and light. By irradiation of the region to be treated, the PS is activated and can induce the generation of singlet molecular oxygen followed by a sequence of reactions generating other reactive oxygen species (20). 2 has optimal photophysical properties for its use as PS: it has an absorption maximum at 668 nm with high extinction coefficient (668 nm ) 4 × 104 M-1 cm-1) and an emission maximum at 672, and the singlet oxygen quantum yield in ethanol using pheophorbide-a as reference was estimated to be 0.52 (21). Moreover, it has a free carboxylic group which can be used for attaching it to the multiplying unit. In previous works, in which we employed antibodies as carriers for photosensitizers, we found that the dye’s ability to produce singlet oxygen and the antibody’s ability to recognize its antigen strongly decrease when a high number of dye molecules are covalently bound to the antibody (8). We concluded that the use of a multiplying unit may be necessary to increase the local concentration of drugs without a decrease of the antibody’s biological activity. The choice of a [5:1]fullerene hexakis adduct as the multiplying unit came after several studies with different fullerene adducts carrying a different number of photosensitizers. First, we prepared fullerene mono- and hexaadducts carrying two pyropheophorbide-a (2) moieties. The investigation of the photophysical properties of these derivatives showed that in the fullerene monoadduct an efficient photoinduced electron transfer occurs between the exited pyropheophorbide-a (2) moieties and the fullerene core.

This event results in a dramatic reduction of PSs singlet oxygen quantum yield. On the contrary, in the fullerene hexaadduct, the π-system is broken up and C60 cannot act as a strong electron-acceptor anymore (22-24). As a direct consequence, the in vitro phototoxicity of the fullerene monoadduct was significantly inferior to that of the fullerene hexaadduct carrying the same number of PSs (25). Successively, we synthesized fullerene hexaadducts carrying six (3) (26, 27) and twelve (4) (27, 28) pyropheophorbide-a moieties (Figure 1). It was found that dye molecules covalently linked to one fullerene moiety could interact with each other, forming energy traps. A very fast and efficient delivery of the excitation energy to a trap can therefore occur. As a result, the fluorescence as well as the singlet oxygen quantum yields of the different complexes were reduced with increasing number of dye molecules per complex (26-28). This was confirmed by in vitro cell experiments (36): While the fullerene adduct loaded with six PS moieties (3) still has a significant phototoxic activity against lymphoid T-cells, the [6:0] adduct loaded with twelve PSs (4) is not phototoxic. A [5:1]fullerene adduct analogous to 4 and carrying ten pyropheophorbide-a moieties (5) has been prepared for the coupling with the mAb. 5 has an activated carboxylic ester group at the end of a long alkyl chain, which serves for the covalent binding to an amino group of the antibody. The reason we chose 5 for the coupling with rituximab is that the analogous compound 4 turned out to be photostable even under high-energy irradiation while other carriers (diaminobutane dendrimers) were completely fragmented under the same conditions (28). In this way, we avoid a possible overlap of the conjugate biological activity and light-caused effects like disconnection of the antibody from the fullerene adduct.

EXPERIMENTAL PROCEDURES Chemicals and Characterization of the Samples. C60 was obtained from Hoechst AG/Aventis and separated from higher

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fullerenes by a plug filtration process (29, 30). The monoclonal antibody rituximab was bought as pharmaceutical preparation Mabthera, (Roche, Germany). The antibody concentration in the preparation is 10 mg/mL. Chemicals and solvents were used as received unless otherwise noted. Solvents were dried using standard procedures (31). Pyropheophorbide-a-NHS ester and malonate 6 and malonate 7 were prepared according to literature procedures (26). Column chromatography was performed on silica gel 32-63, 60 Å, MP Biomedicals. 1H and 13C NMR spectra were recorded on JEOL JMM EX 400 and JEOL GX 400 instruments; an asterisk indicates a resonance of a pyropheophorbide-a proton or carbon atom; the atom indices are given according to the literature (32). FAB mass spectrometry was performed with Micromass Zabspec and Varian MAT 311A instruments; MALDI-TOF mass spectrometry was done on an AUTOFLEX instrument from Bruker Daltonics GmbH. Standard UV/vis spectra were recorded on a Shimadzu UV-3102 PC UV/vis NIR scanning spectrophotometer. IR spectra were either taken with a Bruker Vector 22 spectrometer or an ASI React IR-1000 spectrometer. HPLC was performed with a Shimadzu Liquid Chromatograph LC-10AT equipped with an SCL-10AVP system controller, LC-8A preparative liquid chromatographs, a diode array detector, and a UV/vis detector, on a Nucleosil 100-5 column. All compounds decomposed prior to melting. All reactions were performed in nitrogen atmosphere. Synthesis. 20-Hydroxyeicosyl Methylmalonate 9. A solution of 1,20-dihydroxyeicosane 8 (4.13 g, 13 mmol) and pyridine (4 mL, 52 mmol) in dry THF (500 mL) was cooled in an ice bath under nitrogen. Malonyl dichloride (1.4 mL, 13 mmol) was diluted in dry THF (20 mL) and added dropwise over a period of 1 h via a dropping funnel. The reaction mixture was stirred for 6 h at room temperature. CH2Cl2 (200 mL) was added, and the mixture was washed with water (150 mL, three times). After drying over MgSO4, the solvent was removed in vacuo. Flash chromatography on silica (1. CHCl3 2. CHCl3/ethyl acetate 95: 5) yielded 9 as a white solid (yield: 1.96 g, 37%). 1H NMR (400 MHz, CDCl3, 25 °C, TMS): δ ) 4.11 (t, 3J ) 6.7 Hz, 2H, COOCH2), 3.72 (s, 3H, CH3), 3.61 (t, 3J ) 6.6 Hz, 2H, CH2O), 3.36 (s, 2H, OOCCH2COO), 2.61 (tt, 3J ) 6.8 Hz, 3J ) 6.8 Hz, 2H, CH2CH2O), 1.63 (tt, 3J ) 6.8 Hz, 3J ) 6.7 Hz, 2H, COOCH2CH2), 1.39 (bs, 32H, CH2). 13C NMR (100 MHz, CDCl3, 25 °C, TMS): δ ) 176.1 (CO), 166.6 (CO), 65.7, 63.1, 52.5, 41.4, 32.8, 29.7, 29.6, 29.5, 29.4, 29.2, 28.4, 26.2, 25.7, 25.7, 23.5. IR (ATR): ν ) 3331 cm-1, 2918, 2849, 1737 (CO), 1463, 1339, 1200, 1154, 1061, 1031, 729. MS (FAB, NBA): m/z ) 416 [M+]. Anal. calcd (%) for C24H46O5 (414.34): C 69.52, H 11.18. Found: C 69.74, H 10.98. [1-(Methoxycarbonyl)-1-(20-hydroxyeicosyl)oxycarbonyl]1,2-methano [60]-fullerene 10. C60 (2.5 g, 3.47 mmol) was dissolved in dry toluene (1200 mL), resulting in a dark purple solution. The solution was stirred until the C60 was completely dissolved. Afterward, CBr4 (1.7 g, 5.1 mmol) and the malonate 9 (1.4 g, 3.37 mmol) were added. DBU (800 µL, 5.2 mmol) was diluted in toluene (50 mL) and added dropwise over a period of 1 h to the stirred solution at room temperature. After the solution was stirred for another 12 h, the reaction mixture was controlled by thin layer chromatography to reveal unreacted C60, the monoadduct 10, as well as traces of bis- and trisadducts. The toluene was almost completely removed in vacuo and the remaining solution transferred to a flash chromatography column. Flash chromatography on silica gel with toluene yielded unreacted C60 as first fraction (0.98 g, 39%). 10 was obtained as a brown powder which was dried in vacuo (yield: 1.65 g, 42%). 1H NMR (400 MHz, CDCl3, 25 °C, TMS): δ ) 4.48 (t, 3J ) 6.5 Hz, 2H, COOCH ), 4.07 (s, 3H, CH ), 3.61 (t, 3J ) 2 3 6.7 Hz, 2H, CH2O), 1.81 (tt, 3J ) 6.6 Hz, 3J ) 6.7 Hz, 2H, COOCH2CH2), 1.54 (m, 2H, CH2CH2O), 1.44 (m, 2H, CH2-

Rancan et al.

CH2CH2O), 1.22 (bs, 30H, CH2). 13C NMR (100 MHz, CDCl3, 25 °C, TMS): δ ) 164.5 (CO), 163.9 (CO), 145.4, 145.4, 145.4, 145.1, 144.9, 144.9, 144.1, 143.3, 143.3, 142.4, 142.2, 142.1, 141.2, 139.4, 139.1 (58C, C60 sp2), 71.5 (2C, C60 sp3), 67.5, 63.2, 53.9, 52.4, 32.6, 29.5, 29.4, 29.3, 29.0, 28.4, 25.8, 25.58. IR (KBr): ν ) 2921 cm-1, 2851, 1741 (CO), 1462, 1430, 1267, 1233, 1187, 1060, 749. MS (FAB, NBA): m/z ) 1132 [M+]+, 720 [C60+]. UV/vis (CH2Cl2): λmax () ) 257 nm (113 000 mol-1 dm3 cm-1), 325 (35 000), 425 (2100). Anal. calcd (%) for C84H44O5 × 0.5CHCl3 (1191.3): C 85.08, H 3.76. Found: C 85.58, H 3.63. [5:1]Hexakis Adduct 11. Monoadduct 10 (1.37 mg, 1.2 mmol) was dissolved in degassed, dry toluene (250 mL). 9,10Dimethylanthracene (2.50 mg, 12 mmol) was added and the mixture stirred for 12 h at room temperature under protection from light. Malonate 7 (6.10 g, 12.1 mmol) and CBr4 (4.03 g, 12.1 mmol) were added subsequently to the reaction flask. DBU (3.18 mL, 21.0 mmol) was diluted in dry toluene (20 mL) and added dropwise over a period of 1 h. Precleaning on silica gel (CH2Cl2/ethyl acetate 60:40) and subsequent purification by preparative HPLC (Nucleosil 5 µm, CH2Cl2/ethyl acetate 74: 26) gave 11 as a yellow solid (yield: 1.07 g, 24.1%). 1H NMR (400 MHz, CDCl3, 25 °C, TMS): δ ) 4.76 (bs, 10H, NH), 4.22 (t, 3J ) 6.3 Hz, 20H, COOCH2), 4.22 (t, 3J ) 6.3 Hz, 2H, COOCH2), 3.83 (s, 3H, OCH3), 3.60 (t, 3J ) 6.6 Hz, 2H, CH2O), 3.06 (m, 20H, NCH2), 1.66 (m, 22H, COOCH2CH2), 1.54 (m, 2H, CH2CH2O), 1.40 (s, 90H, CH3), 1.50-1.20 (m, 94H, CH2). 13C NMR (100 MHz, CDCl , 25 °C, TMS): δ ) 164.3 (CO), 3 163.8 (CO), 156.0 (NHCO), 145.7 (C60 sp2), 141.1 (C60 sp2), 78.9 (10C, OC(CH3)3), 69.1 (C60 sp3), 66.9, 63.0, 53.6, 45.5, 40.5, 32.8, 29.9, 29.7, 29.6, 29.4, 28.4, 28.3, 26.4, 25.7, 25.6. IR (ATR): ν ) 2975 cm-1, 2930, 2856, 1743, 1689, 1516, 1365, 1244, 1212, 1165, 714. MS (FAB, NBA): m/z ) 3635 [M+], 3579 [M - tBu+], 3536 [M+ - Boc], 3479 [M+ - Boc-tBu], 3435 [M+ - 2×Boc], 3336 [M+ - 3×Boc], 3235 [M+ 4×Boc], 3135 [M+ - 5×Boc]. UV/vis (CH2Cl2): λmax () ) 244 nm (93 600 mol-1 dm3 cm-1), 280 (73 100), 496 (500). Anal. calcd for C209H264N10O45 × 2CH2Cl2 (3801.8): C 66.58, H 7.10, N 3.68. Found: C 66.65, H 7.08, N 3.67. [5:1]Hexaadduct Tosylate 12. The hexaadduct 11 (460 mg, 0.126 mmol), trimethylamine hydrochloride (102 mg, 1.07 mmol), and triethyamine (2 mL, 14 mmol) were dissolved in dry CH2Cl2 (150 mL). The reaction mixture was cooled to 0 °C. Tosyl chloride (182 mg, 1 mmol) was dissolved in CH2Cl2 (30 mL) and added dropwise over a period of 1 h, keeping the temperature below 5 °C. After the mixture was stirred for 12 h at room temperature, the solvent was evaporated. Flash chromatography on silica (1. CH2Cl2, 2. CH2Cl2/ethyl acetate 70: 30) yielded 12 as an orange solid (yield: 380 mg, 79%). 1H NMR (400 MHz, CDCl3, 25 °C, TMS): δ ) 7.75 (d, 3J ) 8.2 Hz, 2H, aryl-H), 7.31(d, 3J ) 8.3 Hz, 2H, aryl-H), 4.75 (bs, 10H, NH), 4.22 (t, 3J ) 6.3 Hz, 20H, COOCH2), 3.98 (t, 3J ) 6.5 Hz, 2H, CH2OSO2), 3.82 (s, 3H, OCH3), 3.06 (m, 20H, NCH2), 2.42 (s, 3H, aryl-CH3), 1.65 (m, 22H, COOCH2CH2), 1.61 (m, 2H, CH2CH2OSO2), 1.40 (s, 90H, CH3), 1.50-1.20 (m, 94H, CH2). 13C NMR (100 MHz, CDCl3, 25 °C, TMS): δ ) 164.3 (CO), 163.8 (CO), 156.0 (NHCO), 145.7 (C60 sp2), 144.6, 141.1 (C60 sp2), 133.2, 129.7, 127.8, 78.9 (OC(CH3)3), 70.7, 69.1, 69.0 (C60 sp3), 67.1, 66.8, 53.5, 45.5, 40.5, 29.9, 29.7, 29.6, 29.5, 29.4, 29.2, 28.9, 28.8, 28.4, 28.3, 28.1, 27.9, 26.4, 25.7, 25.6, 25.3, 21.6 (1C, 27). IR (ATR): ν ) 2976 cm-1, 2930, 2857, 1742, 1692, 1515, 1365, 1242, 1214, 1167, 1043, 753, 714. MS (FAB, NBA): m/z ) 3789 [M+], 3733 [M+ tBu], 3690 [M+ - Boc], 3633 [M+ - Boc-tBu], 3490 [M+ 3×Boc], 3390 [M+ - 4×Boc], 3289 [M+ - 5×Boc], 3188 [M+ - 6×Boc]. UV/vis (CH2Cl2): λmax () ) 244 nm (90 100 mol-1 dm3 cm-1), 280 (69 000), 496 (420). Anal. calcd

Fullerene Immunoconjugate as Carrier System

for C216H270N10O47S × 2CH2Cl2 (3955.8): C 66.11, H 6.97, N 3.54, S 0.81. Found: C 65.83, H 7.14, N 3.56, S 0.47. [5:1]Hexaadduct Azide 13. The hexaadduct 12 (202 mg, 0.053 mmol) and sodium azide (60 mg, 0.9 mmol) were dissolved in 4 mL of dry DMF and stirred for 16 h at room temperature. The solvent was removed in vacuo. Flash chromatography on silica (CH2Cl2/ethyl acetate 70:30) yielded 13 as an orange solid (yield: 160 mg, 82%). 1H NMR (400 MHz, CDCl3, 25 °C, TMS): δ ) 4.76 (bs, 10H, NH), 4.22 (t, 3J ) 6.5 Hz, 20H, COOCH2), 4.14 (t, 2H, COOCH2), 3.83 (s, 3H, OCH3), 3.22 (t, 3J ) 6.9 Hz, 2H, CH2N3), 3.06 (m, 20H, NHCH2), 1.66 (m, 20H + 2H, COOCH2CH2), 1.56 (tt, 2H, CH2CH2N3), 1.40 (s, 90H, CH3), 1.45-1.20 (m, 94H, CH2). 13C NMR (100 MHz, CDCl3, 25 °C, TMS): δ ) 164.3 (CO), 163.8 (CO), 156.0 (NHCO), 145.7 (C60 sp2), 141.1 (C60 sp2), 78.9 (OC(CH3)3), 69.1 (C60 sp3), 66.9, 53.6, 51.5, 45.4, 40.5, 29.9, 29.7, 29.7, 29.6, 29.6, 29.5, 29.4, 29.2, 28.8, 28.3, 28.3, 26.7, 26.4, 25.7, 25.6. IR (ATR): ν ) 2976 cm-1, 2930, 2856, 2097, 1743, 1691, 1514, 1365, 1244, 1213, 1166, 753, 715. MS (FAB, NBA): m/z ) 3682 [M + Na+], 3659 [M+], 3603 [M+ - tBu], 3561 [M+ - Boc], 3504 [M+ - Boc-tBu], 3359 [M+ - 2×Boc], 3259 [M+ - 3×Boc], 3160 [M+ - 4×Boc]. UV/vis (CH2Cl2): λmax () ) 243 nm (95 400 mol-1 dm3 cm-1), 270 (70 900), 280 (72 500). Anal. Calcd for C209H263N13O44×CH2Cl2 (3742.82): C 67.33, H 7.13, N 4.86. Found: C 67.36, H 7.30, N 5.00. Decakis Pyropheophorbide-a[5:1]fullerene Hexaadduct Azide 14. The hexakis adduct 13 (160 mg, 0.04 mmol) was dissolved in methanol (30 mL) and hydrochloric acid (5 mL, 4 N in 1,4dioxane) was added. After stirring for 3 h at room temperature, the solvent was removed in vacuo. The orange decakis ammonium salt was used without any further purification for the next transformation. The deprotected hexakis adduct (55 mg, 0.018 mmol) was dissolved in brine (50 mL). CHCl3 (50 mL) and triethylamine (200 µL) were added, and the organic layer was washed twice with brine. After this solution was dried over Na2SO4, pyropheophorbide-a-NHS ester 6 (170 mg, 0.27 mmol) was added and the solution stirred for 72 h at room temperature. Evaporation of the solvent and subsequent size exclusion chromatography (1. Bio-Beads SX3 2. Bio-Beads SX1, CHCl3) yielded a black-green solid. Additional flash chromatography with silica (CHCl3/methanol, 93.5:7.5) yielded 14 as a dark green solid (yield: 85 mg, 74%). 1H NMR (400 MHz, CDCl3, 25 °C, TMS): δ ) 8.83* (m, 10H, β), 8.54* (m, 10H, R), 8.20* (m, 10H, δ), 7.48* (m, 10H, 2a), 6.21* (bs, 2H + 1H, CONH, NHCO-C4H8), 6.11* (bs, 8H, CONH), 5.86* (m, 20H, 2b), 4.91* (m, 10H, 10), 4.64* (m, 10H, 10), 4.22* (m, 10H, 7), 3.96* (m, 10H, 8), 3.96 (m, 20H + 2H, COOCH2), 3.64 (s, 3H, OCH3), 3.25-2.60* (m, 30H + 30H + 20H + 30H + 20H + 2H, 1a, 3a, 4a, 5a, NHCH2, CH2N3), 2.40* (m, 10H, 7a/b), 2.10* (m, 20H, 7a/b), 1.88* (m, 10H, 7a/b), 1.54* (m, 30H, 8a), 1.35* (m, 30H + 20H + 2H, 4b, COOCH2CH2), 1.241.03 (m, 94H, CH2), -0.15* (bs, 10H, NH), -2.16* (s, 10H, NH). 13C NMR (100 MHz, CDCl3, 25 °C, TMS): δ ) 195.9, 172.4, 171.5, 164.1, 163.6, 160.3, 154.7, 150.2, 148.4, 146.0, 144.5, 141.0, 140.8, 137.1, 135.7, 135.5, 131.1, 129.5, 129.5, 128.7, 127.2, 121.9, 107.9, 105.5, 105.5, 103.2, 96.5, 92.6, 69.0, 67.6, 66.7, 51.6, 51.4, 49.6, 49.4, 47.8, 45.4, 39.1, 32.8, 31.9, 30.2, 29.6, 29.5, 29.4, 29.1, 28.7, 28.1, 26.6, 26.2, 25.3, 23.9, 22.7, 18.9, 17.2, 11.8, 11.2, 11.1, 10.9. IR (ATR): ν ) 2961 cm-1, 2923, 2855, 2095, 1743, 1688, 1617, 1498, 1367, 1260, 1218, 1057, 978, 793. MS (Maldi-TOF, 2,5-dihydroxybenzoic acid): m/z ) 7833 [M+] (calcd 7826.55). MS (FAB, NBA): m/z ) 7821 [M+]. UV/vis (CH2Cl2): λmax () ) 281 nm (184 600 mol-1 dm3 cm-1), 324 (220 900), 400 (623 200), 413 (621 300), 509 (708 000), 540 (60 600), 613 (55 900),

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669 (287 600). Anal. Calcd for C489H503N53O44×1.5CHCl3 (7997.75): C 73.59, H 6.35, N 9.27. Found: C 73.42, H 6.44, N 9.90. Decakis Pyropheophorbide-a[5:1]fullerene Hexaadduct Adipinic Acid ActiVe Ester 5. To a solution of hexakis adduct 14 (25 mg, 0.003 mmol) in THF (2 mL) and water (0.2 mL), trimethyl phosphine (1 mL, 1 mol solution in toluene, 1 mmol) was added. After stirring for 16 h at room temperature, the solvent was removed in vacuo. The dark green residue 15 was used without any further purification in the next transformation. The amino hexakis adduct 15 (25 mg, 0.003 mmol) was dissolved in dry CH2Cl2 (5 mL) and adipinic acid bis(hydroxysuccinimide) ester 16 (50 mg, 0.14 mmol) was added. After stirring for 16 h at room temperature and subsequent size exclusion chromatography (Bio-Beads SX3 CH2Cl2), 5 was obtained as a black-green solid (yield: 22 mg, 91%). 1H NMR (400 MHz, CDCl3, 25 °C, TMS): δ ) 8.83* (m, 10H, β), 8.53* (m, 10H, R), 8.19* (m, 10H, δ), 7.48* (m, 10H, 2a), 6.18* (bs, 2H, CONH), 6.10* (bs, 8H, CONH), 5.81* (m, 10H, 2b), 4.91* (m, 10H, 10), 4.63* (m, 10H, 10), 4.21* (m, 10H, 7), 3.96* (m, 10H, 8), 3.96 (m, 20H + 2H, COOCH2), 3.64 (m, 3H, OCH3), 3.30-2.60* (m, 30H + 30H + 20H +30H + 20H + 2H + 4H + 4H, 1a, 3a, 4a, 5a, NHCH2, adipinic OOCCH2, succinyl-CH2), 2.40* (m, 10H, 7a/b), 2.10* (m, 20H, 7a/b), 1.88* (m, 4H + 10H, adipinic OOCCH2CH2, 7a/b), 1.54* (bs, 30H, 8a), 1.35* (bs, 52H, 4b, COOCH2CH2), 1.24-1.03 (m, 98H, CH2), -0.14* (bs, 10H, NH), -2.18* (s, 10H, NH). 13C NMR (100 MHz, CDCl3, 25 °C, TMS): δ ) 195.9, 172.3, 171.4, 163.6, 160.3, 154.7, 150.2, 148.4, 145.6, 144.5, 141.0, 137.1, 135.7, 135.5, 135.1, 131.1, 129.6, 129.5, 128.7, 127.2, 121.9, 105.6, 103.2, 96.6, 92.7, 69.1, 66.7, 51.6, 49.7, 47.8, 45.5, 39.1, 32.9, 30.2, 29.5, 29.4, 29.1, 28.1, 26.2, 25.4, 22.8, 19.0, 17.2, 11.8, 11.3, 10.9. IR (ATR): ν ) 2963 cm-1, 2921, 2865, 1741, 1685, 1617, 1497, 1410, 1259, 1218, 1082, 1014, 866, 792. MS (Maldi-TOF, 2,5-dihydroxybenzoic acid): m/z ) 8028.57 [M+] (calcd 8025.76), 7803, 7394. UV/vis (CH2Cl2): λmax () ) 279 (175 900 mol-1 dm3 cm-1), 322 (211 900), 399 (592 980), 413 (588 100), 510 (67 200), 540 (57 500), 613 (53 100), 669 (273 300). Rit-5. The conjugation of 5 with the antibody Rituximab Rit was performed by dissolving 5 (5.5 mg, ∼0.7 µmol) in a little DMF (∼3 mL), which was then added to a stirred solution of Rit (10 mg/mL in 100 mg solution, ∼0.7 µmol) at pH 9. The concentration of DMF in the final reaction mixture did not exceed 35%. After 12 h at 4 °C, the crude mixture was purified by size exclusion chromatography (Bio-Gel P-60 eluent: PBS/ DMF 80:20) to give a light green fraction of Rit-5. Unreacted 5 was recovered in the second fraction. The yield of the antibody conjugate Rit-5 was calculated to be about 90% by adjusting the antibody’s protein absorption to that before the reaction. Thus, the sample contained approximately 10% of nonreacted antibody. Cell Culture. Epstein-Barr virus (EBV) transformed human B-lymphocytes, B-lymphoma cells (Ramos), and human Tlymphocytes (Jurkat cells: clone E 6-1) were used. Jurkat and Ramos cells were purchased from ACACC. EBV transformed B-lymphocytes were kindly provided by Dr. S. Lobitz (OttoHeubner-Centrum fu¨r Kinder- and Jugendmedizin, Charite´, Berlin). Cell lines were cultivated in 50 mL flasks in 5 mL RPMI 1640 medium containing Glutamax-I, supplemented with 10% fetal calf serum (FCS), 100 µg/mL streptomycin, and 100 IE/mL penicillin. Cells were cultivated at 37 °C in 100% humidity and 5% CO2 and were seeded in new medium every 2-3 days. Laser Scan Microscopy. The ability of the conjugate to bind the antigen CD20 was proven using a confocal laser scanning microscope (CLSM 510, Zeiss) equipped with a Helium-Neon

1082 Bioconjugate Chem., Vol. 18, No. 4, 2007 Scheme 1. Synthesis of the [5:1]Fullerene Hexaadduct Azide 13

laser. Pictures of the fluorescence of pyropheophorbide-a (2) were taken by exciting the PS at λexc ) 633 nm and capturing the fluorescence at an emission wavelength λem > 650 nm using a long-pass filter (LP 650). Cells were incubated 24 h with the conjugate Rit-5 (0.5 µmol as determined from the intensity of the last Q-band of the absorption spectrum of Rit-5), with the fullerene adduct 5 (0.5 µmol) and with compound 3 (0.8 µmol), which served as the positive control (30). The final concentration of the pyropheophorbide-a moieties was approximately 5 µmol (equivalent concentration). Cells incubated with the solvent were used as the negative control. After incubation, the cells were washed twice with phosphate buffered solution (PBS) and observed at the CLSM. Pictures were taken of transmitted light as well as fluorescence light. Transmitted and fluorescent light were then merged using the Paint Shop Pro 7 software. Cell Vitality and Proliferation. Cell vitality and proliferation were measured by the XTT test (Roche). The assay is based on the conversion of the yellow tetrazolium salt XTT into an orange formazane dye by the activity of mitochondria dehydrogenase in metabolically active cells. The amount of formazane product was analyzed spectrophotometrically at the absorbance of 492 nm. The reference wavelength was at 650 nm. The blank optical density (OD) was subtracted from the samples OD, and the percentage of cell vitality and proliferation was determined as follows: (ODsample/ODreference) × 100. Bars represent the standard deviations for three experiments.

RESULTS AND DISCUSSION Synthesis. After establishing a protocol for the synthesis of multi-pyropheophorbide-a conjugates (26, 27), we applied it to the preparation of the synthetically much more demanding system 5 (Scheme 2). 5, which is a fullerene [5:1]hexakis adduct, has a lower symmetry than the previously synthesized C60

Rancan et al.

hexakis adduct 4 (26); even more challenging is the attachment of an activated ester for coupling of the whole carrier system to an antibody. Due to the various reaction conditions during the synthesis of 5 and the inherent sensitivity of the pyropheophorbide-a moieties, very mild methods had to be used for generating the active ester unit. It also should be introduced at a very late point in the synthesis. We decided to make an azide derivative which can be reduced easily to an amine and then coupled to a diester derivative, preferably the bis-active ester of an aliphatic dicarboxylic acid. The size of monoclonal antibodies, typically around 130-150 kDa, as well as the dimensions of the fullerene-pyropheophorbide-a conjugate 5, requires the introduction of a long spacer unit between fullerene and active ester to allow for an efficient coupling reaction. Starting from 1,20-dihydroxyeicosane 8, the coupling with methyl malonylchloride yielded the corresponding malonate 9 in 37%; given the fact that 8 is poorly soluble, this yield is quite acceptable. The malonate 9 was attached to C60 by the well-established Bingel reaction (33) giving the monoadduct 10 in good yields. Subsequent coupling of five BOC-protected malonates 7 (26) to the monoadduct 10 by using the template activation method with 9,10-dimethyl anthracene (34) yielded the [5:1]hexakis adduct 11. Here, purification with preparative HPLC was necessary to remove the pentakis adduct impurities. The overall yield after the purification steps was 24%. The spectroscopic properties of 11 clearly show its constitution. Of particular importance are the resonances of the sp3-carbon atoms of the fullerene core at 69.1 ppm. The typical C60 hexakis adduct bands at 271 and 281 nm in the UV/vis spectrum adds further support to the [5:1] structure. Tosylation of the free alcohol functionality of 11 gave the fullerene tosylate 12 which, after substitution of the tosyl group with sodium azide in DMF, gave the azido compound 13 in high yields (Scheme 1). The azide stretching vibration at 2095 cm-1 together with the NMR data unambiguously prove the formation of 13. It should be pointed out that 13 not only is a very useful precursor for the project presented here, but may also be employed as a carrier/docking unit for other drugs. After removal of the BOC protecting groups with HCl in dioxane, which liberates a decaamino compound (not isolated), and subsequent coupling with the NHS-active ester of pyropheophorbide-a 6 (26), the decapyropheophorbide-a [5:1]fullerene hexaadduct 14 with an intact azide group was obtained. The reduction of the azido group of 14 to an amino group was performed by a modified Staudinger reaction (35) using trimethylphosphine in THF/water (Scheme 2). The resulting amine 15 could not be isolated or characterized. Nevertheless, the MALDI-TOF mass spectrum of the crude mixture shows clearly the molecular ion peak of the amino compound 15 at 7807.20 (calculated 7800.56) as the main signal. The molecular ion peak of the adduct 14 appears as a small signal at 7836.22 (calculated 7826.55) (see Supporting Information). Compounds 6-14 were fully characterized by standard spectroscopic and analytical methods. The reaction of 15 with a large excess of adipinic acid-bisNHS ester 16 gave compound 5 with one remaining activated carboxylic acid group. This active ester group is the aforementioned coupling unit which provides the covalent bond between the fullerene-pyropheophorbide-a residue and the monoclonal antibody Rituximabsor, in the future, other proteins. The 1H NMR spectrum of 5 (see Supporting Information) shows quite well resolved signals, which is in contrast to the spectrum of the symmetrical dodecakis pyropheophorbide-a fullerene conjugate 4 (26) where all signals were rather broad. The signals of the chlorin moiety can be clearly assigned as well as those of the malonate spacer groups. Unfortunately, the resonances

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Scheme 2. Synthesis of the Decakis Pyropheophorbide-a [5:1]Fullerene Hexaadduct 5 with Active Ester Docking Unit

of the active ester unit cannot be resolved, because they are hidden under the much more intense signals of the other groups. Fortunately, the successful generation of the active ester 5 can be proven with the MALDI-TOF mass spectrum (see Supporting Information). The main signal at 8028.82 can be assigned to the molecular ion peak (calculated 8025.76), whereas the smaller signal at 7802.34 can be assigned to either the unreacted amino

compound 15 (calculated 7800.56) or a fragmentation product. This proves clearly the formation of compound 5 as the main component. Synthesis of the Antibody Conjugate. We used the monoclonal antibody Rituximab (Rit) which was obtained from Hoffmann La Roche and is distributed under the commercial name MabThera. MabThera is a sterile, clear, colorless,

1084 Bioconjugate Chem., Vol. 18, No. 4, 2007

Figure 2. UV-vis spectrum of a mixture of compound 5 with the pure antibody and of the antibody conjugate (Rit-5).

preservative-free liquid concentrate for intravenous administration, supplied at a concentration of 10 mg/mL in 100 mg (10 mL) single-use vials. The product is formulated for intravenous administration in 9.0 mg/mL sodium chloride, 7.35 mg/mL sodium citrate dihydrate, 0.7 mg/mL polysorbate 80, and sterile water for injection. The pH is adjusted to 6.5. The conjugation of 5 with the antibody Rit was performed by dissolving 5 in as little DMF as possible to allow solubility but to prevent denaturation of Rit. This solution was then added to the aforementioned solution of the antibody adjusted to pH 9, keeping the total amount of DMF below 35%. The separation from unreacted 5 and other small-molecule impurities was achieved by size exclusion chromatography (Bio-Gel P-60 eluent: PBS/DMF 80/20) to give a light green fraction of Rit5. Figure 2 reports the UV-vis absorption spectra of the immunoconjugate Rit-5 and a solution of 5 and Rituximab (1:1). In order to estimate the antibody/C60 ratio, we adjusted the conjugate’s protein absorption band at 270 nm to that of the unreacted antibody. By integration over the last Q-band of 5 which stems from the pyropheophorbide a units, and comparison of this value with that one of Rit-5, we determined that the antibody/C60 ratio is approximately 1:0.9. It has to be noted that the Q-band of the antibody-fullerene complex is much broader compared to that of 5. This means that the antibody strongly affects the pyropheophorbide-a moiety even in the ground state. We are convinced that we have achieved a covalent attachmentsand not an electrostatic interactionsof the fullerene derivative 5 to the antibody Rit, because (i) we observed clear bands on the chromatography column in contrast to broad or tailing bands in case of a breakup of electrostatically coupled components, and (ii) 5 does not contain charged groups or highly polarizable substituents, which could form coulomb complexes. In our opinion, it is more convenient that the drug complex is covalently bonded to the addressing unit. If a drug is merely absorbed on the mAb, once the carrier system is administered, it could be released or sequestered from other plasma proteins (i.e., albumin) before it has reached the target tissue. Antigen Binding Ability. The monoclonal antibody rituximab Rit binds to the membrane receptor (CD20), which is exclusively expressed by normal B-lymphocytes or cancer B-cells, i.e., non-Hodgkin’s lymphoma. The binding of the antibody conjugate Rit-5 to the complement protein CD20 was tested on transformed B-lymphocytes by detecting the fluorescence of pyropheophorbide-a moieties with a confocal fluorescence microscope. The selectivity of the immunoconjugate Rit-5 for the targeted B-cells was tested by comparison with

Rancan et al.

its binding efficiency to cells which do not express the CD20 receptor (Jurkat cells). The uptake of C60 derivatives 5 and 3 was also tested on both cell lines. The comparison of the uptake of 5 with that of the conjugate Rit-5 should give an idea of the efficiency of the antibody as addressing unit. 3, a [5:1]fullerene hexaadduct loaded with six pyropheophorbide-a moieties, which was shown to be taken up by Jurkat cells, served as positive control for cellular internalization. All dyes were applied in concentrations as such that the equivalent concentration of pyropheophorbide-a in each sample was approximately 5 µmol. Finally, cells incubated without any photosensitizer or antibody conjugate were used as reference to distinguish the autofluorescence of the cells. Fluorescence pictures were taken 24 h after incubation. Four different fields were observed with a confocal laser scan microscope. Pictures of both transmitted and fluorescence light were taken for each sample. Superimposed images were then created to clearly show the localization of the fluorescence on cell membranes or in the cell. Figure 3 reports a summary of the analyzed cells. The T-lymphocytes used as reference did not show any autofluorescence, while a weak autofluorescence was detected in B-lymphocytes. The fluorescence of bound or taken-up photosensitizer (i.e., cells incubated with 3, Figure 3C) had higher intensity than the autofluorescence of the cells and was clearly discernible from it. The hexakis pyropheophorbide-a [5:1]fullerene hexaadduct 3 was taken up by both cell types, and its fluorescence was detected mainly in the cytoplasm and in the perinuclear region. Compound 5 was not taken up by Jurkat and B-cells, which has obviously nothing to do with the size of the fullerene adduct; otherwise, 3 would not have been taken up either. The long lipophilic chain of 5 may have a detrimental influence on its intracellular uptake. The fact that 5 is not taken up makes the enhancement of delivery efficiency that can be reached by the use of an addressing unit even more remarkable. In fact, B-cells incubated with the antibody conjugate Rit-5 had a bright fluorescence on a restricted region of the plasma membrane. The pictures clearly show that the immunoconjugate binds to surface receptors and that it is not internalized by the cells. These results demonstrate that the conjugation with the fullerene adduct 5 does not negatively influence the affinity of the antibody for its antigen. No fluorescence was detected when the same immunoconjugate was incubated with Jurkat cells. This means that Rit-5 selectively recognizes B-lymphocytes without binding to T-cells which do not express the CD20 membrane receptor. In cytotoxicity experiments, the Rit-5 preparation exhibited dark toxicity toward Ramos cells (CD20+ B-lymphoma cells) but not toward Jurkat cells (Figure 4). The immunoconjugate preparation gave the same cytotoxicity as the pure antibody. This means that the immunocojugate Rit-5 has the same affinity for the CD20 receptor as the pure antibody and, furthermore, the same biological activity. However, it has to be considered that the immunoconjugate preparation may contain unreacted rituximab together with Rit-5, which may be the cause for the dark cytotoxicity. Further experiments considering dark and phototoxicity dependent on the incubation concentration of the Rit-5 preparation will better demonstrate the biological activity of the immunoconjugate. Our results show that fullerene hexakis adducts are useful multiplying units for modular carrier systems to be used in photodynamic therapy and can be potentially useful in any therapy in which a higher selectivity for the target is required. The special structure and chemistry of C60 and the use of long spacers allow the coupling of many drug moieties to a monoclonal antibody avoiding the inactivation of the antibody antigen-specific site. Confocal microscopy studies showed that the antibody conjugate Rit-5 conserves its affinity for the CD20

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Figure 4. Cytotoxicity of immunoconjugate Rit-5 toward Ramos cells (CD20+) and Jurkat cells (CD20-). Cells were incubated 24 h with 0.1 µmol Rit-5, fullerene adducts 5 and 3, and the pure antibody rituximab (Rit). Cells incubated without any compound were used as reference. Cell vitality and proliferation were detected with the XTT test 24 h after incubation. Percentages were calculated with respect to reference cells.

In our future work, the introduction of a photounstable bond at the PS side will be pursued. In this way, once the modular carrier system has reached the target cells, the drug moieties can be released through irradiation and successively reach those intracellular compartments where the phototoxic effect is mostly efficient.

ACKNOWLEDGMENT The authors thank the Deutsche Forschungsgemeinschaft (DFG) for financial support (F.R., F.B.: grant no. BO 1353/ 2-1,2; E.A.E., B.R.: grant no. RO 1042/9-3,4; E.A.E., B.R.: grant no. RO 1042/11-1,2; A. H., M. H., N. J.: grant no. HI 468/11-1). We also thank Mrs. Gisela Wo¨hlecke (Berlin) for technical assistance and Priv.-Doz. Dr. Andreas Humeny (Erlangen) for performing the MALDI-TOF experiments. Supporting Information Available: 1H NMR spectrum of 5 and MALDI-TOF spectra of 5 and of crude 15. This material is available free of charge via the Internet at http://pubs.acs.org/ BC.

LITERATURE CITED

Figure 3. Binding of the conjugated antibody Rit-5 to CD20 positive (CD20+) B-lymphocytes, and CD20 negative (CD20-) Jurkat cells. Both cells lines were incubated with the conjugated antibody (A, A′) and with the fullerene complexes 5 (B, B′) and 3 (C, C′) at a final pyropheophorbide-a equivalent concentration of ∼5 µmol. Cells incubated without any PS were used as reference (D, D′). After incubation, cells were washed twice and observed at the confocal laser scanning microscope (CLSM 510, Zeiss) using λexc ) 633 nm and λem > 650 nm.

receptor and its selectivity toward CD20 positive B-lymphocytes. On the contrary, the antibody-free complex 5 did not show any bounding or intracellular uptake.

(1) Ehrlich, P. (1906) Studies in Immunity (Wiley, J. & Sons, Eds.) Plenum Press, New York. (2) Harashima, H., Shinohara, Y., and Hiroshi, K. (2001) Intracellular control of gene trafficking using liposomes as drug carriers. Eur. J. Pharm. Sci. 13, 85-89. (3) Matsumura, Y., and Maeda, H. (1986) A new concept for macromolecular therapeutics in cancer chemotherapy: mechanism of tumoritropic accumulation of proteins and the antitumor agent smancs. Cancer Res. 46, 6387-6392. (4) Svenson, S., and Tomalia, D. A. (2005) Dendrimers in biomedical applications-reflections on the field. AdV. Drug DeliVery ReV. 57, 2106-2129. (5) Putnam, D., and Kopecˇek, J. (1995) Polymer conjugates with anticancer activity. AdV. Polym. Sci. 122, 55-123. (6) Guillemard, V., and Saragovi, H. U. (2001) Taxane-antibody conjugates afford potent cytotoxicity, enhanced solubility, and tumor target selectivity. Cancer Res. 61, 694-699. (7) Patri, A. K., Myc, A., Beals, J., Thomas, T. P., Bander, N. H., and Baker, J. R., Jr. (2004) Synthesis and in vitro testing of J591 antibody-dendrimer conjugates for targeted prostate cancer therapy. Bioconjugate Chem. 15, 1174-1181. (8) Dressler, C., Mo¨ller, U., Lewald, T., Berlien, H. P., Ro¨der, B., and Risse, H. J. (1992) Introduction of a simple model for testing immunoconjugates with photosensitizers. Laser Med. Sci. 7, 164168.

1086 Bioconjugate Chem., Vol. 18, No. 4, 2007 (9) Ro¨der, B. (2000) Photodynamic therapy. In Encyclopedia of Analytical Chemistry (Meyers, A. Ed.) pp 302-320, John Wiley & Sons, Chichester, UK. (10) Patri, A. K., Majoros, I. J., and Baker, J. R., Jr. (2002) Dendritic polymer macromolecular carriers for drug delivery. Curr. Opin. Chem. Biol. 6, 466-471. (11) van Vlerken, L. E., and Amiji, M. M. (2006) Multi-functional polymeric nanoparticles for tumor-targeted drug delivery. Expert Opin. Drug DeliVery 3, 205-216. (12) Gillies, E. R., and Fre´chet, J. M. J (2005) Dendrimers and dendritic polymers in drug delivery. Drug DiscoVery Today 10, 35-43. (13) Herzog, A., Hirsch, A., and Vostrowsky, O. (2000) Dendritic mixed hexakis adducts of C60 with a Th symmetrical addition pattern. Eur. J. Org. Chem. 171-180. (14) Dugan, L. L., Turetsky, D. M., Du, C., Lobner, D., Wheeler, M., Almli, C. R., Shen, C. K.-F., Luh, T.-Y., Choi, D. W., and Lin, T.-S. (1997) Carboxyfullerenes as neuroprotective agents. Proc. Natl. Acad., Sci. U.S.A. 94, 9434-9439. (15) Nakamura, E., and Isobe, H. (2003) Functionalized fullerenes in water, the first 10 years of their chemistry, biology, and nanoscience. Acc. Chem. Res. 36, 807-815. (16) Ashcroft, J. M., Tsyboulsky, D. A., Hartman, K. B., Zakharian, T. Y., Marks, J. W., Weisman, R. B., Rosenblum, M. G., and Wilson, L. J. (2006). Chem. Commun. 3004-3006. (17) (a) Reff, M. E., Carner, C., Chambers, K. S., Chinn, P. C., Leonard, J. E., Raab, R., Hanna, N., and Anderson, D. R. (1994) Depletion of B cells in vivo by a chimeric mouse human monoclonal antibody to CD20. Blood 83, 435-445. (b) Demidem, A., Lam, T., Alas, S., Hariharan, K., Hanna, N., and Bonavida, B. (1997) Chimeric anti-CD20 (IDEC-C2B8) monoclonal antibody sensitizes a B cell lymphoma cell line to cell killing by cytotoxic drugs. Cancer Biother. Radiopharm. 12, 177-186. (c) Berinstein, N. L., Grillo-Lo´pez, A. J., White, C. A., Bence-Bruckler, I., Maloney, D., Czuczman, M., Green, D., Rosenberg, J., McLaughlin, P., and Shen, D. (1998) Association of serum Rituximab (IDEC-C2B8) concentration and anti-tumor response in the treatment of recurrent low-grade or follicular non-Hodgkin’s lymphoma. Ann. Oncol. 9, 995-1001. See also: http://www.gene.com/gene/products/information/oncology/rituxan/insert.jsp. (18) Shimokawa, K., Hashizume, A., and Shioi, Y. (1990) Pyropheophorbide-a, a catabotite of ethylene-induced chlorophyll a degradation. Phytochemistry 29, 2105-2106. (19) Levy, J. G., and Obochi, M. (1996) New applications in photodynamic therapy introduction. Photochem. Photobiol. 64, 737739. (20) Dougherty, T. J., Gomer, C. J., Henderson, B. W., Jori, G., Kessel, D., Korbelik, M., Moan, J., and Peng, Q. (1998) Photodynamic therapy. J. Natl. Cancer Inst. 90, 889-905. (21) Spiller, W., Kliesch, H., Wo¨hrle, D., Hackbarth, S., Ro¨der, B., and Schnurpfeil, G. (1998) Singlet oxygen quantum yields of different photosensitizers in polar solvents and micellar solutions. J. Porphyrins Phthalocyanines 2, 145-158. (22) Ermilov, E. A., Al-Omari, S., Helmreich, M., Jux, N., Hirsch, A., and Ro¨der, B. (2004) Photophysical properties of fullerenedendron-pyropheophorbide supramolecules. Chem. Phys. 301, 2731. (23) Ermilov, E. A., Al-Omari, S., Helmreich, M., Jux, N., Hirsch, A., and Ro¨der, B. (2004) Steady-state and time-resolved studies on

Rancan et al. the photophysical properties of fullerene-pyropheophorbide a complexes in polar and nonpolar solvents. Opt. Commun. 234, 245252. (24) Al, Omari, S., Ermilov, E. A., Helmreich, M., Jux, N., Hirsch, A., and Ro¨der, B. (2004) Transient absorption spectroscopy of a monofullerene C60-bis-(pyropheophorbide a) molecular system in polar and nonpolar environments. Appl. Phys. B 79, 617-622. (25) Rancan, F., Helmreich, M., Mo¨lich, A., Jux, N., Hirsch, A., Ro¨der, B., Witt, C., and Bo¨hm, F. (2005) Fullerene-pyropheophorbide a complexes as sensitizer for photodynamic therapy: uptake and photoinduced cytotoxicity on Jurkat cells. J. Photochem. Photobiol., B 80, 1-8. (26) Helmreich, M., Ermilov, E. A., Meyer, M., Jux, N., Hirsch, A., and Ro¨der, B. (2005) Dissipation of electronic excitation energy within a C60 [6:0]-hexaadduct carrying twelve pyropheophorbide a moieties. J. Am. Chem. Soc. 127, 8376-8385. (27) Ermilov, E. A., Hackbarth, St., Al-Omari, S., Helmreich, M., Jux, N., Hirsch, A., and Ro¨der, B. (2005) Trap formation and energy transfer in the hexapyropheophorbide a-fullerene C60 hexaadduct molecular system. Opt. Commun. 250, 95-104. (28) Ro¨der, B., Ermilov, E. A., Hackbarth, St., Helmreich, M., and Jux, N. (2006) Trap formation and energy transfer in pheophorbide a-DAB-dendrimers and pyropheophorbide a-fullerene C60 hexaadduct molecular systems. SPIE 6192, 495-507. (29) Reuther, U. (2001) Evaluation of synthetic pathways for the heterofullerene C58N2 and introduction of the first concept for a completely regioselective control of multiple adduct formation of C60. Ph.D. Dissertation, University of Erlangen-Nu¨rnberg, Germany. (30) Isaacs, L., Wehrsig, A., and Diederich, F. (1993) Improved purification of C60 and formation of σ- and π-homoaromatic methano-bridged fullerenes by reaction with alkyl diazoacetates. HelV. Chim. Acta 76, 1231-1250. (31) Perrin, D. D., and Amarego, W. L. F. (1988) Purification of laboratory chemicals, Pergamon Press, Oxford, UK. (32) (a) Closs, G. L., Katz, J. J., Pennington, F. C., Thomas, M. R., and Strain, H. H. (1963) Nuclear magnetic resonance spectra and molecular association of chlorophylls a and b, methyl chlorophyllides, pheophytins, and methyl pheophorbides. J. Am. Chem. Soc. 85, 3809-3821. (b) Smith, K. M., and Unsworth, J. F. (1975) Carbon-13 NMR spectra of some chlorins and other chlorophyll degradation products. Tetrahedron 31, 367-375. (33) Bingel, C. (1993) Cyclopropanation of fullerenes. Chem. Ber. 126, 1957-1959. (34) Lamparth I., Maichle-Mo¨ssmer C., and Hirsch, A. (1995) Reversible template-directed activation of equatorial double bonds of the fullerene framework: regioselective direct synthesis, crystal structure, and aromatic properties of Th-C66(COOEt)12. Angew. Chem., Int. Ed. Engl. 34, 1607-1609. (35) (a) Staudinger, H., and Meyer, J. (1919) New organic compounds of phosphorus. III. Phosphinemethylene derivatives and phosphinimines. HelV. Chim. Acta 2, 635-646. (b) Garcia, J., Urpı´, F., and Vilarrasa, J. (1984) New synthetic tricks. Triphenylphosphinemediated amide formation from carboxylic acids and azides. Tetrahedron Lett. 25, 4841-4844. (36) Rancan, F., Helmreich, M., Mo¨lich, A., Jux, N., Hirsch, A., Ro¨der, B., Bo¨hm, F. Unpublished results. BC0603337