Synthesis and Characterization of Positively Charged Porphyrin

Bioconjugate Chem. 2005, 16, 852−863

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Synthesis and Characterization of Positively Charged Porphyrin-Peptide Conjugates Martha Sibrian-Vazquez, Timothy J. Jensen, Frank R. Fronczek, Robert P. Hammer, and M. Grac¸ a H. Vicente* Department of Chemistry, Louisiana State University, Baton Rouge, Louisiana 70803. Received February 28, 2005; Revised Manuscript Received May 26, 2005

The total syntheses of 14 porphyrin conjugates containing one to four positively charged amino acids and two distinct linkers are described. All conjugates were fully characterized using spectroscopic methods, and the X-ray structure of a porphyrin isothiocyanate precursor was obtained. In vitro studies using HEp2 cells show that these conjugates have low cytotoxicity (IC50 > 250 µM) and that the extent of their cellular uptake depends significantly on the number, nature, and sequence of amino acids in the peptide, and on the presence of a centrally chelated metal ion. Metal-free conjugates bearing three consecutive arginine residues accumulated the most within cells. On the other hand, the preferential sites of subcellular localization were found to be independent from the number, nature, and sequence of amino acids in the conjugate, the linker, and coordinated metal ion; it is suggested, based on theoretical calculations, that the peptides in these conjugates fold over the porphyrin macrocycle in order to maximize intramolecular hydrophobic interactions.

INTRODUCTION

The biomedical applications of porphyrin-type macrocycles have been the subject of intense research in the past decades because of their unique photophysical and pharmacokinetic properties. In particular porphyrin derivatives have been investigated for use as sensitizers in the photodynamic therapy (PDT) and in the boron neutron capture therapy (BNCT) of tumors (1, 2). PDT and BNCT are binary therapies for cancer treatment that involve the activation of a tumor-localized porphyrin sensitizer with light (in PDT) or low energy neutrons (in BNCT). These therapies have the potential to be highly localized to neoplastic tissue, since the cytotoxic species generated in PDT (mainly 1O2) and BNCT (4He2+ and 7 Li3+) have short distances of travel through tissue. In fact the preferential subcellular site(s) of localization of porphyrin sensitizers are considered to be an important factor in determining their biological efficacy, as they define the primary site(s) of damage and can thus determine the mechanism(s) of cell death (3, 4). Among the sensitizers, positively charged molecules are particularly interesting because they potentially target the most vulnerable intracellular sites, such as the cell mitochondria and nuclei (4-6) and have shown to cause effective DNA and RNA damage (7-9). Furthermore, cationic porphyrins are active in the photoinactivation of bacteria (10-14) and viruses (15, 16). With the aim to increase the biological effectiveness of porphyrin sensitizers and to broaden their application as fluorescent bioimaging agents, their conjugation to various proteins (17-20), peptides (21-23), oligonucleotides (24, 25), and monoclonal antibodies (26, 27) have been investigated in recent years. Several conjugation techniques have been reported, and the resulting bioconjugates have shown improved cell target ability and enhanced biological efficacy. In our continuing studies * Corresponding author. Phone: (225) 578 7405, Fax: (225) 578 3458, E-mail: [email protected].

of structure-activity relationships in porphyrin macrocycles, we prepared 14 conjugates of a model porphyrin (1) to various positively charged amino acids and peptides containing lysine and arginine residues and investigated their cytotoxicity, cellular uptake, and sites of intracellular localization. Arginine (side chain pKa ∼ 12.5) and lysine (pKa ∼ 9.4) are amphiphilic amino acids that are protonated at physiological pH. Whereas lysine has a localized positive charge on its amino group, the guanidinium group has delocalized charge and is capable of forming multiple hydrogen bonds. Furthermore, the planar geometry and charge distribution of the guanidinium group is ideal for binding negatively charged groups, such as phosphates and carboxylates. Herein we report that porphyrin conjugates containing one to four lysine or arginine amino acid residues have low dark cytotoxicity and localize mainly in cellular vesicles and lysosomes, and that their cellular uptake depends significantly on the nature, number, and sequence of amino acid residues and the presence of a centrally chelated metal ion. Furthermore, we hypothesize that in such conjugates bearing small linkages between the porphyrin and peptide molecules, intramolecular forces favor a foldover conformation of the conjugates, which might in part explain their similar sites of subcellular localization. EXPERIMENTAL PROCEDURES

Syntheses. Unless otherwise indicated, all commercially available starting materials were used directly without further purification. Reactions under anhydrous conditions were performed in dried and distilled solvents under an argon atmosphere. All reactions were monitored by TLC using Sorbent Technologies 0.25 mm silica gel plates with or without UV indicator (60F-254). Silica gel Sorbent Technologies 32-63 µm was used for flash column chromatography. 1H and 13C NMR were obtained on either a DPX-250 or a ARX-300 Bruker spectrometer. Chemical shifts (δ) are given in ppm relative to CDCl3 (7.26 ppm, 1H; 77.00 ppm, 13C) unless otherwise indi-

10.1021/bc050057g CCC: $30.25 © 2005 American Chemical Society Published on Web 07/02/2005

Positively Charged Porphyrin−Peptide Conjugates

cated. Electronic absorption spectra were measured on a Perkin-Elmer Lambda 35 UV-vis spectrophotometer, and fluorescence spectra were measured on a PerkinElmer LS55 spectrometer. Mass spectra were obtained on an Applied Biosystems QSTAR XL, a hybrid QqTOF mass spectrometer with a MALDI ionization source using CCA as the matrix. HPLC separation and analysis were carried out on a Dionex system including a P680 pump and a UVD340U detector. Semipreparative HPLC was carried out using a Luna C18 100 Å, 5 µm, 10 × 250 mm (Phenomenex) column and a stepwise gradient; analytical HPLC was carried out using a Delta Pak C18 300 Å, 5 µm, 3.9 × 150 mm (Waters) column and a stepwise gradient (deprotected conjugates) or under isocratic conditions (protected conjugates). The samples were isolated and analyzed using the following solvents: buffer A (5% acetonitrile, 0.1% TFA, H2O), buffer B (5% H2O, 0.1% TFA, acetonitrile), and acetonitrile. The Gasteiger-Huckel computational model and the Tripos Force Field parameters incorporated into the software package SYBYL 7.0 (Tripos, St. Louis, MO) were used for the energy minimization calculations. After initial minimization for each molecule, annealing was performed by heating the molecules to 500 K and then cooled to 0 K over a period of 1000 fs for 10 cycles. Following annealing, minimization was performed using the Powell descent series method. Solvation studies were performed by explicitly including water molecules in the calculation. The solute was solvated with one layer of water (TIP3P model) in a periodic box (46.46 × 46.46 × 46.46 Å), using the appropriate routine within SYBYL, and minimized within that box. Conjugates 2a and 2b. Boc-L-Lys(Boc)OH (0.030 g, 0.0869 mmol) was dissolved in 0.5 mL of DMF.1 To this solution were added HOBt (0.0133 g, 0.0869 mmol), DMAP (0.0010 g, 0.0086 mmol), Et3N (0.0087 g, 0.0869 mmol), porphyrin 1 (0.050 g, 0.079 mmol), and EDCI (0.0166 g, 0.0869 mmol). The reaction mixture was stirred at room temperature for 48 h, after which 10 mL of distilled water and 5 mL of EtOAc were added. The two phases were separated, the organic phase was washed with distilled water (2 × 10 mL), dried over anhydrous Na2SO4, filtered, and the solvent was evaporated under vacuum. The protected conjugate was isolated by flash chromatography on silica gel using dichloromethane/ methanol in the ratio 99/1. The protected conjugate 2a was obtained in 96% yield as purple crystals. UV-vis (CHCl3) λmax (/M-1 cm-1) 419 (371,400), 515 (16,400), 551 (7,900), 590 (5,100), 646 (3,700). 1H NMR (CDCl3, 300 MHz): δ 8.79-8.97 (9H, m), 8.13-8.25 (8H, m), 8.00 (2H, d, J ) 8.49 Hz), 7.66-7.82 (9H, m), 5.46 (1H, d, J ) 6.94 Hz), 4.71-4.73 (1H, m), 4.45 (1H, s), 3.22-3.27 (2H, m), 1.27-1.65 (24H, m), -2.76 (2H, s). 13C NMR (CDCl3, 62.5 MHz): δ 170.99, 156.39, 142.13, 138.05, 137.65, 135.10, 134.52, 131.03, 127.61, 126.65,120.08, 119.56, 118.05, 55.40, 39.59, 31.44, 29.69, 28.49, 28.42, 22.70. LRMS (ESI) m/z 958.40 (M + H+), calculated for C60H59N7O5 1 HOBt, 1-hydroxybenzotriazole; DMF, N,N-dimethylformamide; DMAP, 4-(dimethylamino)pyridine; Et3N, triethylamine; CCA, R-cyano-4-hydroxycinnamic acid; EDCI, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; DCC, 1,3-dicyclohexylcarbodiimide; TFA, trifluoroactic acid; DIEA, N,Ndiisopropylethylamine; TBTU, 2-(1H-benzotriazole-1-yl)-1,1,3,3tetramethyluronium tetrafluoroborate; TIS, triisopropylsilane; DMSO, dimethyl sulfoxide; NHS, N-hydroxysuccinimide; Boc, tert-butoxy carbonyl; Z, benzyloxy carbonyl; Fmoc, fluorenylmethoxy carbonyl; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; PBS, phosphate-buffered saline; FBS, fetal bovine serum; MEM, modified eagle medium.

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958.45. Deprotection was accomplished in quantitative yield using TFA at room temperature for 4 h, following by evaporation of the TFA and washings with Et2O. Conjugate 2b was obtained as purple crystals, UV-vis (CHCl3) λmax (/M-1 cm-1) 420 (282,300), 516 (16,300), 552 (7,900), 590 (5,100), 646 (3,800). 1H NMR (CDCl3, 300 MHz): δ 8.83-8.86 (8H, m), 8.80-8.22 (8H, m), 7.93 (2H, d, J ) 8.1 Hz), 7.66-7.74 (9H, m), 3.60 (1H, s), 3.21 (4H, s), 2.94 (2H, s), 1.29-1.46 (6H, m), -2.74 (2H, s). HRMS (ESI) m/z 758.3575 (M + H+), calculated for C50H44N9OS 758.3607. 5-(4-Isothiocyanatophenyl)-10,15,20-triphenyl-porphyrin (5). To a stirred solution of porphyrin 1 (0.050 g, 0.0793 mmol) in freshly distilled dichloromethane (20 mL) was added 1,1′-thiocarbonyldi-2(1H)-pyridone (0.0369 g, 0.1587 mmol). The resulting mixture was stirred at room temperature for 2 h under an argon atmosphere, before being concentrated under vacuum. The residue was dissolved in dichloromethane and the product was isolated by flash chromatography on silica gel using dichloromethane as the eluent. The title product was isolated as purple crystals in 84% yield. HPLC tR ) 13.36 min. UV-vis (CHCl3) λmax (/M-1 cm-1) 414 (336,000), 515 (10,500), 548 (5,600), 589 (3,500), 645 (2,600). 1H NMR (CDCl3, 300 MHz): δ 8.89-8.91 (6H, m), 8.81 (2H, d, J ) 4.7 Hz), 8.19-8.26 (8H, m), 7.73-7.84 (9H, m), 7.62 (2H, d, J ) 8.3 Hz), -2.74 (2H, s). 13C NMR (CDCl3, 62.5 MHz): δ 142.49, 141.90, 136.89, 135.90, 135.02, 131.56, 128.26, 127.18, 124.53, 121.06, 120.88, 118.65. HRMS (ESI) m/z 672.2238 (M + H+), calculated for C45H30N5S 672.2222. The crystal structure of the hemi-CH2Cl2 solvate of porphyrin 5 was determined, using data collected at T ) 105 K with Mo KR radiation on a Nonius KappaCCD diffractometer. Crystal data: C45H29N5S‚ 0.5CH2Cl2, triclinic space group P-1, a ) 7.020(6), b ) 10.789(8), c ) 24.120(16) Å, R ) 87.55(3), β ) 85.04(3), γ ) 85.86(7)° V ) 1814(2) Å3, Z ) 2, R ) 0.086 (F2 > 2σ), Rw ) 0.245 (all F2) for 5075 unique data and 487 refined parameters. The solvent molecule is disordered about an inversion center. Crystallographic data (excluding structure factors) for the structure in this paper have been deposited with the Cambridge Crystallographic Data Centre as supplementary publication no. CCDC 263394. Copies of the data can be obtained, free of charge, on application to CCDC, 12 Union Road, Cambridge, CB2 1EZ, UK, (fax: +44- (0)1223-336033 or e-mail: deposit @ccdc.cam.ac.uk). 4-[4-(10,15,20-Triphenyl-porphyrin-5-yl)-phenylcarbamoyl]-butyric Acid (6). Porphyrin 1 (0.1 g, 0.159 mmol) was added to a solution of glutaric anhydride (0.0543 g, 0.4769 mmol) in 2 mL of dry DMF at room temperature, and the final mixture was stirred at room temperature for 48 h. Distilled water (10 mL) was added, followed by 10 mL of EtOAc. The two phases were separated, the organic phase was washed with distilled water (3 × 10 mL), dried over anhydrous Na2SO4, and filtered, and the solvent was removed under vacuum. The title compound was purified on a silica gel plug using dichloromethane/methanol (99/1 to 90/10 ratios) for elution, in 100% yield. HPLC tR ) 21.34 min. UV-vis (CHCl3) λmax (/M-1 cm-1) 419 (336,000), 516 (16,900), 551 (7,900), 590 (5,100), 646 (3,800). 1H NMR (CDCl3, 300 MHz): δ 8.8 (7H, s), 8.43 (7H, s); 7.71 (2H, s), 7.60 (10H, s), 2.63 (4H, s), 2.21 (2H, s), -2.79 (2H, s). HRMS (ESI) m/z 744.2865 (M + H+), calculated for C49H38N5O3 744.2896. Conjugate 10. H-L-Lys(Boc)OtBu‚HCl (0.0158 g, 0.0468 mmol) was dissolved in 500 µL of dry DMF under argon. To this solution was added Et3N (0.0047 g, 0.0468 mmol),

854 Bioconjugate Chem., Vol. 16, No. 4, 2005

followed by porphyrin 5 (0.031 g, 0.0468 mmol). The reaction mixture was stirred at room temperature for 24 h, and then 10 mL of distilled water and 5 mL of EtOAc were added. The two phases were separated, the organic phase was washed with distilled water (2 × 10 mL), dried over anhydrous Na2SO4, and filtered, and the solvent was removed under vacuum. The title compound was purified by flash chromatography on silica gel using dichloromethane/methanol 98/2 for elution, in 93% yield. HPLC tR ) 6.94 min. UV-vis (CHCl3) λmax (/M-1 cm-1) 420 (301,600), 516 (14,000), 552 (7,000), 590 (4,500), 646 (3,400). 1H NMR (CDCl3, 250 MHz): δ 8.83-8.88 (6H, m), 8.20-8.24 (9H, m), 7.71-7.77 (11H, m), 5.19 (1H, s), 4.62 (1H, s), 3.16 (2H, d, J ) 6.1 Hz), 1.27-1.63 (24H, m), -2.76 (2H, s). 13C NMR (CDCl3, 62.5 MHz): 180.10, 171.45, 156.07, 142.14, 140.69, 136.04, 134.54, 131.25, 127.75, 126.69, 122.13, 120.42, 120.29, 118.49,117.49, 32.12, 29.74, 28.40, 28.10, 22.30, LRMS (ESI) m/z 973.37 (M+), calculated for C63H57N7O4S 973.43. Conjugate 11. The title compound was synthesized as described above for conjugate 10, in 45% yield. HPLC tR ) 7.15 min. UV-vis (CHCl3) λmax (/M-1 cm-1) 419 (511,300), 516 (14,600), 552 (7,000), 590 (4,500), 646 (3,400). 1H NMR (CDCl3, 300 MHz): δ 8.85-8.86 (8H, s), 8.23-8.26 (7H, m), 7.73-7.76 (10H, m), 7.25 (1H, s), 5.31 (1H, s), 4.89 (1H, s), 4.61 (1H, s), 3.22 (4H, s), 1.372.01 (39H, m), -2.72 (2H, s). 13C NMR (CDCl3, 62.5 MHz): δ 180.30, 172.70, 156.07, 143.63, 137.34, 136.14, 132.77, 129.29, 128.25, 123.82, 121.84, 83.84, 80.76, 59.81, 41.77, 33.26, 31.27, 30.07, 29.67, 24.12. LRMS (ESI) m/z 1202.43 (M + H+), calculated for C71H79N9O7S 1202.50. Conjugate 12. The title compound was synthesized as described above for conjugate 10, in 35% yield. HPLC tR ) 9.78 min. UV-vis (CHCl3) λmax (/M-1 cm-1) 420 (343,700), 516 (19,600), 552 (9,600), 590 (6,000), 642 (4,700). 1H NMR (CDCl3, 300 MHz): δ 8.86-8.94 (8H, m), 8.22-8.28 (8H, m), 8.06 (1H, d, J ) 7.87 Hz), 7.767.77 (10H, m), 5.04 (1H, s), 4.66-4.83 (2H, m), 4.16 (1H, s), 3.19 (6H, s), 1.28-1.92 (54H, m), -2.74 (2H, s). 13C NMR (CDCl3, 62.5 MHz): δ 181.21, 174.00, 171.77, 156.77, 142.60, 134.99, 131.56, 128.13, 127.11, 122.58, 121.65, 120.54, 82.17, 40.95, 40.30, 39.44, 31.48, 29.62, 28.52, 28.00, 22.82, 22.42, 21.73. HRMS (ESI) m/z 1430.7433 (M + H+), calculated for C82H100N11O10S 1430.7375. Conjugate 13. Compound 10 (0.0442 g, 0.043 mmol) was added to a 10 mL round-bottom flask, previously purged with argon for 5 min, followed by 4 mL of TFA. The reaction mixture was stirred at room temperature for 4 h. TFA was evaporated under vacuum, 5 mL of Et2O was added, and the suspension was transferred to a centrifuge tube. After centrifugation, the residue was washed with Et2O (6 × 10 mL) to remove residual TFA and dried under vacuum. Yield 100%. HPLC tR ) 22.66 min. UV-vis (CHCl3) λmax (/M-1 cm-1) 419 (384,500), 515 (16,500), 551 (7,800), 590 (5,300), 646 (3,800). 1H NMR (CDCl3, 250 MHz): δ 8.83-8.88 (6H, m), 8.20-8.24 (9H, m), 7.71-7.77 (11H, m), 5.19 (1H, s), 4.62 (1H, s), 3.143.19 (2H, m), 1.27-1.63 (6, m), -2.76 (2H, s). 13C NMR (CDCl3, 62.5 MHz): δ 180.10, 172.45, 156.07, 142.14, 140.69, 135.96, 134.96, 130.99, 127.71, 126.66, 122.29, 120.42, 120.29, 30.29, 29.69, 22.30, LRMS (ESI) m/z 817.34 (M+), calculated for C51H43N7O2S 817.32. Conjugate 14. The title compound was deprotected as described above for conjugate 13 in quantitative yield. HPLC tR ) 15.71 min. UV-vis (CHCl3) λmax (/M-1 cm-1) 419 (276,300), 516 (24,100), 551 (11,500), 590 (7,600), 646 (5,600). 1H NMR (CD3OD, 300 MHz): δ 8.50 (8H, s),

Sibrian-Vazquez et al.

8.20-8.28 (9H, m), 7.76 (12H, s), 3.31-3.5 (4H, m), 1.251.96 (12H, m), -2.78 (2H, s). LRMS (ESI) m/z 945.48 (M+), calculated for C57H55N9O8S 945.41. Conjugate 15. The title compound was deprotected as described for conjugate 13 in quantitative yield. HPLC tR ) 10.38 min. UV-vis (2:1 MeOH/CHCl3) λmax (/M-1 cm-1) 414 (280,600), 513 (15,700), 549 (8,700), 589 (5,900), 645 (4,600). 1H NMR (CD3OD, 300 MHz): δ 8.82-8.95 (7H, m), 8.55-8.57 (7H, m), 8.27-8.38 (2H, m), 8.05 (8H, s), 4.4-4.5 (1H, m), 3.99-4.02 (1H, m), 3.82-3.86 (1H, m), 2.94-3.01 (6H, m), 1.56-1.97 (18H, m). LRMS (ESI) m/z 1073.54 (M+), calculated for C63H67N11O4S 1073.51. Conjugate 16. To a solution of porphyrin 6 (0.030 g, 0.043 mmol) in 500 µL of dry DMF were added DIEA (0.032 g, 0.2419 mmol), TBTU (0.0129 g, 0.0403 mmol), and HOBt (0.0062 g, 0.0403 mmol), followed by H-L-Lys(Boc)OtBu‚HCl (0.0136 g, 0.0403 mmol). The reaction mixture was stirred at room temperature for 24 h, diluted with 10 mL of EtOAc, and washed with water (3 × 10 mL). The organic extracts were combined, dried over anhydrous Na2SO4, and filtered, and the solvent was evaporated under vacuum. The protected conjugate was purified by flash chromatography on silica gel using 1% methanol/dichloromethane for elution. Yield 71%. HPLC tR ) 7.91 min. UV-vis (CHCl3) λmax (/M-1 cm-1) 417 (359,200), 516 (12,200), 552 (6,900), 590 (4,400), 646 (3,300). 1H NMR (CDCl3, 300 MHz): δ 9.12 (1H, s), 8.86-8.92 (7H, m), 8.16-8.24 (8H, m), 8.02 (2H, d, J ) 8.29 Hz), 7.72-7.77 (9H, m), 6.43 (2H, d, J ) 7.34 Hz), 4.65 (1H, s), 4.57 (1H, s), 3.15 (2H, t, J ) 6.1 Hz), 2.62 (2H, t, J ) 4.7, 5.9 Hz), 2.51 (2H, t, J ) 5.86, 6.71 Hz), 2.25 (2H, m), 1.37-1.91 (24H, m), -2.74 (2H, s). 13C NMR (CDCl3, 62.5 MHz): δ 172.88, 172.48, 171.66, 156.33, 142.13, 138.30, 137.53, 135.06, 134.53, 131.06, 127.68, 126.66, 120.01, 119.12, 117.76, 82.51, 79.51, 52.78, 39.83, 36.11, 34.74, 31.51, 29.81, 28.43, 28.05, 22.45, 22.01. HRMS (MALDI) m/z 1028.5081 (M+), calculated for C64H66N7O6 1028.5074. Deprotection as described above for conjugate 13 gave the title compound in quantitative yield. HPLC tR ) 16.28 min. UV-vis (9:1 MeOH/DMF) λmax (/M-1 cm-1) 414 (284,400), 513 (11,500), 551 (5,900), 587 (3,700), 645 (2,800). 1H NMR (d6-DMSO, 300 MHz): δ 8.79-8.90 (9H, m), 8.198.21 (10H, m), 7.79 (9H, m), 2.72 (2H, s), 2.43 (2H, s), 2.08 (2H, s), 1.3-1.93 (8H, m), -2.91 (2H, s). HRMS (ESI) m/z 872.3959 (M + H+), calculated for C55H50N7O4 872.3924. Conjugate 17. Protected conjugate 17 was synthesized as described above for protected 16 and purified by flash chromatography on silica gel using 1% methanol/dichloromethane for elution. Yield 53%. HPLC tR ) 9.25 min. UV-vis (CHCl3) λmax (/M-1 cm-1) 420 (294,800), 516 (15,700), 552 (8,000), 590 (4,800), 646 (3,700). 1H NMR (CDCl3, 300 MHz): δ 8.77-8.91 (7H, m), 8.04-8.24 (9H, m), 7.68-7.82 (9H, m), 6.84 (1H, d, J ) 7.82 Hz), 6.72 (1H, d, J ) 5.45 Hz), 5.03 (1H, s), 4.74 (1H, t, J ) 6.05 Hz), 4.46-4.55 (2H, m), 3.13 (4H, s), 2.60 (2H, t, J ) 6.21 Hz), 2.24 (2H, m), 1.18-1.90 (41H, m), -2.75 (2H, s). 13C NMR (CDCl3, 62.5 MHz): δ 173.48, 172.42, 171.71, 171.29, 156.41, 142.15, 138.55, 137.42, 134.99, 134.51, 131.05, 127.66, 126.65, 120.06, 119.89, 117.82, 82.28, 79.31, 53.77, 52.90, 40.06, 39.39, 35.87, 34.60, 31.73, 31.22, 28.46, 28.43, 22.53, 22.31, 22.08. HRMS (ESI) m/z 1278.6389 (M + Na+), calculated for C75H85N9O9Na 1278.6368. Deprotection as described above for 13 afforded the title conjugate in quantitative yield. HPLC tR ) 12.82 min. UV-vis (MeOH) λmax (/M-1 cm-1) 414 (336,000), 515 (10,500), 548 (5,600), 589 (3,500), 645 (2,600).1H NMR (CD3OD, 300 MHz): δ 8.82 (6H, s), 8.15


(7H, d, J ) 6.76 Hz), 8.04 (2H, d, J ) 8.20 Hz), 7.78 (8H, d, J ) 7.45 Hz), 4.46 (1H, d, J ) 6.74 Hz), 4.37-4.39 (1H, m), 3.01 (4H, s), 2.64 (2H, t, J ) 7.03 Hz), 2.51 (2H, t, J ) 7.01 Hz), 2.16 (2H, t, J ) 7.21 Hz), 1.31-1.85 (12H, m). HRMS (MALDI) m/z 1000.4878 (M + H+), calculated for C61H61N9O5 1000.4796. Conjugate 18. Protected conjugate 18 was synthesized as described above for protected 16 and obtained in 72% yield. HPLC tR ) 9.34 min. UV-vis (CHCl3) λmax (/M-1 cm-1) 420 (241,600), 516 (15,000), 551 (7,300), 590 (4,600), 647 (3,500). 1H NMR (CDCl3, 300 MHz): δ 8.51-8.90 (7H, m), 8.04-8.23 (8H, m), 7.74-7.76 (9H, m), 7.00 (1H, s), 4.97 (2H, s), 4.51 (2H, s), 3.14 (6H, s), 2.55-2.63 (4H, m), 2.24 (2H, s), 1.21-1.66 (54H, m), -2.75 (2H, s). 13C NMR (CDCl3, 62.5 MHz): δ 173.48, 172.42, 171.71, 171.29, 171.03, 156.27, 142.16, 138.55, 134.98, 134.52, 131.01, 127.67, 126.65, 120.06, 119.90, 117.92, 79.18, 53.75, 52.93, 40.08, 39.7, 31.70, 29.67, 28.44, 27.95, 22.43. HRMS (MALDI) m/z 1484.8077 (M + H+), calculated for C85H106N11O12 1484.8022. Deprotection as described above for 13 afforded the title conjugate in quantitative yield. HPLC tR ) 8.70 min. UV-vis (MeOH) λmax (/M-1 cm-1) 415 (225,100), 512 (12,100), 547 (6,200), 589 (3,900), 645 (3,000). 1H NMR (CD3OD, 300 MHz): 8.88 (7H, s), 8.21 (8H, d, J ) 6.44 Hz), 8.06 (2H, d, J ) 7.91 Hz), 7.82 (9H, d, J ) 6.79 Hz), 4.32-4.37 (2H, m), 2.93-3.01 (6H, m), 2.65 (2H, t, J ) 7.22 Hz), 2.51 (2H, t, J ) 7.44 Hz), 2.17 (2H, t, J ) 7.18 Hz), 1.2-1.83 (20H, m). 13C NMR (CD3OD, 62.5 MHz): δ 174.82, 174.25, 156.95, 143.33, 136.16, 135.69, 129.64, 119.61, 40.60, 35.81, 32.38, 28.42, 28.11, 23.72. HRMS (MALDI) m/z 1127.5726 (M+), calculated for C67H73N11O6 1127.5745. Zn(II) Conjugate 19. Under an argon atmosphere, conjugate 18 (0.005 g, 0.0044 mmol) and ZnCl2 (0.0024 g, 0.0177 mmol) were dissolved in 2 mL of DMF. To this solution were added 0.1 mL of MeOH and 0.150 mL of pyridine. The final mixture was stirred at room temperature for 18 h, the solvent was removed under vacuum, and the residue was filtered through a silica gel plug. The title compound was obtained in quantitative yield. HPLC tR ) 20.52 min. UV-vis (MeOH) λmax (/M-1 cm-1) 422 (190,000), 557 (5,000), 595 (2,800). 1H NMR (CD3OD, 300 MHz): 8.88 (7H, s), 8.21 (8H, d, J ) 6.44 Hz), 8.06 (2H, d, J ) 7.91 Hz), 7.82 (9H, d, J ) 6.79 Hz), 4.324.37 (2H, m), 2.93-3.01 (6H, m), 2.65 (2H, t, J ) 7.22 Hz), 2.51 (2H, t, J ) 7.44 Hz), 2.17 (2H, t, J ) 7.18 Hz), 1.20-1.83 (20H, m). LRMS (MALDI) m/z 1190.082 (M+), calculated for C67H71N11O6Zn 1190.488. Sn(IV) Conjugate 20. Protected conjugate 18 (0.025 g, 0.0168 mmol) and SnCl2 (0.083 g, 0.0437 mmol) were dissolved in 1 mL of pyridine. The reaction mixture was refluxed for 2 h, cooled to room temperature, and diluted with 10 mL of chloroform. The mixture was washed with water (3 × 20 mL), the organic extracts were combined and dried under anhydrous Na2SO4, and the solvent was evaporated under vacuum to give the protected Sn(IV) conjugate in quantitative yield. UV-vis (CHCl3) λmax (/ M-1 cm-1) 429 (308,000), 562 (12,700), 603 (9,400). 1H NMR (CDCl3, 300 MHz): δ 9.17-9.27 (7H, m), 8.23-8.33 (7H, m), 8.12 (2H, d, J ) 8.20 Hz), 7.81-7.89 (9H, m), 6.98 (1H, s), 5.50 (2H, s), 4.92 (2H, s), 3.12 (6H, s), 2.53 (4H, m), 2.23-2.27 (3H, m), 1.27-1.83 (54H, m). LRMS (ESI) m/z 1636.30 (M+), calculated for C85H103N11O12SnCl 1636.11. Deprotection as described above for conjugate 18 gave the title conjugate in quantitative yield. HPLC tR ) 9.91 min. UV-vis (MeOH) λmax (/M-1 cm-1) 422 (341,600), 557 (9,300), 597 (6,400). 1H NMR (CD3OD, 300 MHz): δ 9.21-9.43 (7H, m), 8.18-8.22 (8H, m), 8.048.08 (2H, m), 7.81 (9H, s), 4.23-4.32 (3H, m), 2.85 (6H,


m), 2.52 (2H, t, J ) 7.37 Hz), 2.37 (2H, t, J ) 7.62 Hz), 2.01-2.07 (2H, m), 1.18-1.62 (18H, m). LRMS (MALDI) m/z 1280.84, 1245.27, calculated for C67H71N11O6SnCl 1280.8988, C67H71N11O6Sn 1245.46. Solid-Phase Synthesis of Porphyrin Conjugates. Method A. Peptidyl resin (0.025 mmol) was added to a glass synthesizer, washed with DMF, and left to stand for 1 h, before being filtered. To the resin was added a 500 µL solution containing 0.05 mmol of porphyrin 6, 0.150 mmol of DIEA, 0.05 mmol of HOBt, and 0.05 mmol of TBTU. The reaction mixture was stirred overnight at room temperature and then filtered to afford a dark purple resin. A fresh solution of porphyrin 6 (0.025 mmol) in DIEA, HOBt, and TBTU was added and the mixture stirred for an additional 12 h. After filtration, the resin was washed repeatedly with DMF until the filtrate was colorless and then with dichloromethane and methanol, before being dried under vacuum. Cleavage and deprotection was accomplished with 3 mL of a mixture of TFA/ phenol/TIS/H2O in 88/5/2/5 ratio, at room temperature for 4 h. The resin was filtered and washed with TFA (3 × 2 mL), and then the filtrates were combined and evaporated under vacuum to give a green residue. Addition of 5 mL of cold Et2O gave a fine green precipitate which was centrifuged, washed successively with Et2O, and dried under vacuum. Purification of the conjugates was accomplished by reverse phase HPLC using a Luna C18 semipreparative column (10 × 250 mm, 5 µm) and a buffer system of water/acetonitrile, both containing 0.1% TFA with a stepwise gradient from 40 to 75%. The fraction containing the conjugate was collected and lyophilized to yield pure conjugate. The purity of the peptides was >98% as obtained by HPLC on an analytical Delta Pak C18 column (3.9 × 150 mm, 5 µm). Method B. Peptidyl resin (0.050 mmol) was added to a glass synthesizer, washed with DMF, and left to stand for 1 h, before being filtered. To the resin was added a 500 µL solution containing 0.150 mmol of porphyrin 5 and 0.300 mmol of Et3N. The reaction mixture was stirred overnight at room temperature and filtered, a fresh solution of 0.050 mmol of 5 in Et3N was added, and the mixture was stirred for another 12 h. After filtration, the resin was washed with DMF, dichloromethane, and methanol as described above and dried under vacuum. Cleavage and deprotection was carried out as described in Method A. The purification of the conjugates was accomplished by reverse phase HPLC using a Luna C18 semipreparative column and a water/acetonitrile buffer containing 0.1% TFA with a stepwise gradient from 40 to 95%. The fraction containing the conjugate was collected and lyophilized to yield the pure conjugate. The purity of the peptides was >97% as obtained by HPLC on an analytical Delta Pak C18 column. Conjugate 21 was prepared as described in Method B. Yield 38%. HPLC tR ) 15.25 min. UV-vis (MeOH) λmax (/M-1 cm-1) 414 (205,300), 512 (11,300), 547 (5,000), 590 (3,400), 645 (2,300). 1H NMR (CD3OH, 250 MHz): δ 8.72-8.77 (8H, m), 8.33 (1H, d, J ) 7.42 Hz), 8.198.25 (10H, m), 8.03-8.09 (2H, m), 7.77-7.80 (9H, m), 7.42 (2H, s), 7.06-7.09 (3H, m), 3.15-3.19 (6H, m), 3.03-3.08 (2H, m), 1.4-1.6 (18H, m), -0.033 (3H, s). LRMS (MALDI) m/z 1284.72 (M+), calculated for C69H80N20O4S, 1284.63. Conjugate 22 was prepared as described in Method A. Yield 32%. HPLC tR ) 8.53 min. UV-vis (MeOH) λmax (/M-1 cm-1) 414 (236,400), 515 (6,600), 548 (3,300), 588 (2,300), 645 (2,000). 1H NMR (CD3OH, 250 MHz): 8.718.76 (8H, m), 8.20-8.26 (10H, m), 8.03 (2H, t, J ) 8.59 Hz), 7.69-7.80 (16H, m), 2.82-2.85 (6H, m), 2.50 (2H, t,


J ) 6.85, 7.36 Hz), 2.35 (2H, t, J ) 6.81, 7.95 Hz), 1.91 (2H, t, J ) 7.97 Hz), 1.39-1.62 (19H, m), -0.04 (2H, s). HRMS (MALDI) m/z 1127.5897, calculated for C67H75N12O51127.5893. Conjugate 23 was prepared as described in Method A. Yield 52%. HPLC tR ) 12.56 min. UV-vis (MeOH) λmax (/M-1 cm-1) 414 (227,300), 512 (9,900), 547 (6,600), 590 (3,300), 645 (2,300). 1H NMR (CD3OH, 250 MHz): δ 8.72-8.76 (8H, m), 8.28-8.32 (2H, m), 8.20 (10H, s), 8.11 (1H, d, J ) 7.08 Hz), 8.00 (2H, d, J ) 8.07 Hz), 7.687.76 (9H, m), 7.56 (1H, s), 7.37-7.43 (3H, m), 2.51 (2H, t, J ) 6.84 Hz), 2.37 (2H, t, J ) 6.9 Hz), 2.00 (2H, t, J ) 7.38, 7.04 Hz), 1.40-1.63 (12H, m), -0.028 (2H, s). HRMS (MALDI) m/z 1211.6175 (M + H+), calculated for C67H75N18O5, 1211.6168. Conjugate 24 was prepared as described in Method A. Yield 45%. HPLC tR ) 9.13 min. UV-vis (MeOH) λmax (/M-1 cm-1) 414 (235,900), 512 (7,000), 547 (3,500), 588 (2,400), 645 (2,100). 1H NMR (CD3OH, 250 MHz): δ 8.72-8.77 (8H, m), 8.32-8.34 (1H, d, J ) 7.11 Hz), 8.198.25 (10H, m), 7.99-8.06 (2H, m), 7.78-7.90 (9H, m), 7.39 (1H, s), 7.06-7.12 (3H, m), 3.09-3.17 (6H, m), 3.06-3.09 (2H, m), 2.51 (2H, t, J ) 7.91 Hz), 2.37 (2H, t, J ) 7.40 Hz), 1.91 (2H, t, J ) 6.45 Hz), 1.4-1.6 (18H, m), -0.03 (2H, s). HRMS (MALDI) m/z 1339.7168 (M+), calculated for C73H86N20O6, 1339.7117. Conjugate 25 was prepared as described in Method A. Yield 54%. HPLC tR ) 9.65 min. UV-vis (MeOH) λmax (/M-1 cm-1) 414 (263,300), 512 (7,800), 548 (3,800), 588 (2,500), 644 (2,000). 1H NMR (7% D2O/H2O, 400 MHz): δ 8.68 (4H, d, J ) 8.89 Hz), 8.43-8.56 (5H, m), 8.22 (2H, d, J ) 9.31 Hz), 7.67 (2H, s), 7.55 (2H, broad s), 7.147.23 (4H, m), 4.28-4.33 (3H, m), 3.19-3.27 (6H, m), 2.98-3.02 (3H, m), 2.70 (2H, t, J ) 8.44 Hz), 2.54 (2H, t, J ) 9.40 Hz), 2.09-2.17 (2H, m), 1.25-1.87 (18H, m), -0.04 (2H, s). HRMS (MALDI) m/z 1340.7141 (M + H+), calculated for C73H87N20O6 1340.6264. Cell Culture. The HEp2 cell line was purchased from ATCC. The cells were maintained in a 50:50 mixture of MEM:Advanced MEM (Gibco) and 5% FBS (Gibco) in a humidified, 5% CO2 incubator at 37 °C. PBS, FBS, Triton X100, and trypsin were purchased from Gibco. All conjugate solutions were filter sterilized using a 0.22 µm Pal syringe filter. Cytotoxicity. HEp2 cells were plated at a concentration of 10,000 cells per well on a Costar 96-well plate and allowed to attach overnight. Conjugate stock solutions were prepared in DMSO at a concentration of 50 mM. These stocks were diluted to give a final concentration of 500 µM in medium at 1% DMSO. Serial 2-fold dilutions were then made into medium containing 1% DMSO to give a concentration range of 500 µM down to 7.8 µM and maintaining the 1% DMSO concentration. The cells were then incubated 18 h in the dark. Cytotoxicity was measured using the Promega Cell Titer Blue assay, reading the fluorescent signal using a FluoStar Optima plate reader from BMG at 520 nm excitation and 594 nm emission. Cellular Uptake. HEp2 cells were seeded as described above and then exposed to 10 µM conjugate in medium for 25, 12, 8, 4, 2, and 1 h time periods. At the end of the loading period, the medium was removed, and the cells were washed three times with PBS and solubilized with 0.25% Triton ×100 in PBS. The intracellular accumulation of conjugate was determined by measuring the porphyrin’s fluorescence emission on a BMG FluoStar Optima plate reader using excitation/emission wavelengths of 410 and 650 nm, respectively. Cell numbers


were measured using the CyQuant Cell Proliferation Assay (Molecular Probes). Intracellular Localization. HEp2 cells were seeded onto Lab-Tek II, two-chamber coverglass and incubated for 72 h. Conjugate was then added from a 10 mM stock solution in DMSO to reach a final concentration of 10 µM (the DMSO concentration never exceeded 1%). The cells were incubated overnight (18 h) and then washed with drug-free medium and given medium containing 50 mM HEPES pH 7.2. The coverslips were examined using a Zeiss Axiovert 200M inverted fluorescent microscope fitted with standard FITC and Texas Red filter sets (Chroma Technology Corp.). For colocalization experiments LysoSensor Green DND-189, MitoTracker Green FM and Hoechst 33342 (Molecular Probes) were used. The tracer compounds were diluted into medium to a final concentration of 50 nM (LysoSensor Green), 250 nM (MitoTracker Green) or 1 mg/mL (Hoechst), 10 min before microscopy. The cells were incubated concurrently with conjugate for 30 min (LysoSensor Green and MitoTracker Green) or 10 min (Hoechst) before washing and viewed by microscopy. In addition, three lipid mixtures of conjugates 22 or 23 were added to the cells prior to microscopy. Avanti transfection lipids, Cardiolipin (Avanti Polar Lipids) and Lipofectamine Transfection Reagent (Invitrogen) were used as the delivery vehicles. Since the Avanti lipids were provided in chloroform, the solvent was first removed under nitrogen before resuspension in sterile distilled water at 2 mg/mL. Lipofectamine was supplied in water and used as received. A mixture containing 2.5 µL of the lipid and 25 µL of serum-free medium was incubated for 15 min. Conjugates 22 and 23 (1 µL of 10 mM stock solution) were diluted into 25 µL serum-free medium, mixed with each lipid, and incubated for 45 min. The lipid mixtures were then diluted to 600 µL using serum-free medium, added to cells grown on Lab Tek chamber coverslips, incubated for 2.5 h, before adding 600 µL of medium containing 10% FBS (2×) and the cells incubated overnight. Microscopy was performed as described above. RESULTS

The coupling of mono-aminoporphyrin 1 with protected amino acids and peptides was performed in either solution- or solid-phase, as described below. Mono-aminoporphyrin 1 was used as the starting material, because of its high solubility in organic solvents and easy synthesis on a multigram scale from meso-tetraphenylporphyrin (28). Porphyrins bearing meso-carboxyphenyl groups have also been used in such conjugation reactions (e.g. 29) but these molecules are considerably less soluble than the amino derivatives and generally give low yields of the target conjugates. The Boc- and Z-protected peptides were obtained by either solution- or solid-phase syntheses, as previously reported (30, 31). All compounds were completely characterized by spectroscopic methods, as described in the Experimental Section. Solution-Phase Synthesis of Porphyrin Conjugates. Direct conjugation of 1 to Boc-L-Lys(Boc)OH using DCC/DMAP in dichloromethane (32) gave compound 2a in 65% yield (Scheme 1). Under the same conditions, conjugates 3 and 4 were obtained in 40 and 18% yields, respectively. These yields could be improved to 96, 65, and 45%, respectively, when DMF was used as the solvent and HOBt/EDCI as the coupling agents (33, 34). To further increase the conjugation efficiency, the amino group of 1 was converted into an isothiocyanate (18, 35) by reaction of 1 with 1,1′-thiocarbonyldi-(2H)-pyridone,


Positively Charged Porphyrin−Peptide Conjugates Scheme 1a

a Conditions: (a) Boc-protected amino acid or peptide, DCC, DMAP, CH Cl , rt, 48 h (18-65%) or HOBt, EDCI, DMAP, Et N, 2 2 3 DMF, rt, 48 h (45-96%). (b) TFA, rt, 4 h (100%). (c) 1,1′-Thiocarbonyldi-(2H)-pyridone, CH2Cl2, Ar, rt, 2h (84%). (d) Glutaric anhydride, DMF, rt, 24h (100%).

Table 1. Reaction Conditions for Coupling Porphyrin Isothiocyanate 5 to Protected Peptides entry 1 2 3 4 5 6 7 8

Figure 1. Molecular structure of porphyrin isothiocyanate 5.

affording 5 in 84% yield (Scheme 1). The molecular structure of porphyrin 5 is shown in Figure 1. The porphyrin core is reasonably planar, having mean deviation of 24 atoms from their best plane only 0.062 Å, and maximum deviation 0.151(7) Å. The phenyl planes form dihedral angles with the porphyrin plane in the range 61.6(1)-82.4(2)° and that involving the ring carrying the isothiocyanate group is in the middle of that range, 76.1(1)°. Reaction of porphyrin 1 with glutaric anhydride gave carboxylic acid 6 in quantitative yield (Scheme 1). The use of porphyrin 6 in the conjugation reactions allowed us to study the effect of a five-carbon chain between the porphyrin and the positively charged peptides. We investigated the conjugation of isothiocyanate 5 with various protected peptides under different reaction conditions and using both free amino terminus Z- and Boc-protected peptides (Table 1). No reaction took place in aqueous solution due to the poor solubility and limited stability of porphyrin 5 in water (Table 1, entry 1).

protected peptide

solvent

DMSO/pH ) 9.2, 0.1 M carbonate buffer HLys(Z)OBz THF HLys(Z)Lys(Z)OBz THF HLys(Z)Lys(Z)OBz DMF HLys(Z)Lys(Z)Lys (Z)OBz DMF/CHCl3, 2/1 HLys(Boc)OtBu DMF HLys(Boc)Lys(Boc)OtBu DMF HLys(Boc)Lys(Boc)Lys(Boc)OtBu DMF HLys(Z)OH

% yield no rxn 90 69 67 63 93 45 35

Although high yields of the conjugates were obtained with the Z-protected peptides (Table 1, entries 2-5), the deprotection to the corresponding free peptide conjugates was problematic. Indeed several deprotection methodologies were investigated, including catalytic hydrogenation (36), transfer hydrogenation (37), and dealkylation using Me3SiI (38), but all were unsuccessful giving mixtures of partially deprotected conjugates. On the other hand, the Boc groups were easily cleaved in quantitative yield using TFA at room temperature (Scheme 2). The thusobtained deprotected conjugates 13-15 were insoluble in water and mainly soluble in polar organic solvents such as CHCl3, DMF, and DMSO. Conjugates of porphyrin carboxylic acid 6 to Bocprotected peptides were synthesized in good yields using HOBt/TBTU in DMF, as shown in Scheme 3. A similar procedure has been recently reported for the coupling of a carboxy-functionalized porphyrin with the free amino terminus of a cyclic pentapeptide (39). Removal of the Boc group as described above produced conjugates 1618 in quantitative yield. In contrast to the porphyrin isothiocyanate conjugates, compounds 16-18 are partially soluble in aqueous solutions and their water solubility increases with the number of amino acid residues on the peptide chain. In addition, the metalloconjugates 19 and 20 were prepared using a similar route in quantitative yields, by either direct insertion of Zn(II) into conjugate 18 using ZnCl2 in DMF or by reaction



Scheme 2a

a Conditions: (a) Protected amino acid or peptide, DMF, rt, 48 h (35-93%). (b) TFA, rt, 4 h (100%). (c) Free amino terminus Boc-protected amino acid or peptide, Et3N, DMF, rt, 24 h. (d) 88:5:2:5 TFA/phenol/TIS/H2O, rt, 4 h (38% from 5).

Scheme 3a

a Conditions: (a) free amino terminus Boc-protected amino acid or peptide, HOBt, TBTU, DIEA, DMF, rt, 48 h (53-72%). (b) TFA, rt, 4 h (100%). (c) free amino terminus Boc-protected peptidyl resin, HOBt, TBTU, DIEA, DMF, rt, 24 h. (d) 88:5:2:5 TFA/ phenol/TIS/H2O, rt, 4h (32-54% from 6).

of protected conjugate 18 with SnCl2 in pyridine, followed by deprotection with TFA. Solid-Phase Synthesis of Porphyrin Conjugates. The peptidyl PAL-PEG-PS resins used in the solid-phase synthesis of conjugates 21-25 reacted with porphyrin isothiocyanate 5 or with carboxylic acid 6 in the presence of, respectively, Et3N or HOBt/TBTU/DIEA, as shown in Schemes 2 and 3 (40, 41). Using this methodology, the deprotection and cleavage of the porphyrin-peptide conjugates from the solid support was achieved in a single step upon treatment with a mixture of TFA, phenol, TIS, and water in the ratio 88:5:2:5. The resulting conjugates

Figure 2. Dark cytotoxicity of conjugates 17 (green), 20 (blue), and 23 (red) toward HEp2 cells using a Cell Titer Blue assay.

21-25 were obtained as the C-terminally amidated peptides, in 32-54% overall yields. Cellular Studies. The dark cytotoxicity and cellular uptake of representative porphyrin conjugates were investigated in human HEp2 cells (Figures 2 and 3). The cytotoxicity was assayed using a Cell Titer Blue assay


Figure 3. Time-dependent uptake of conjugates 15 (brown), 17 (green), 20 (blue), 23 (red), 24 (black), and 25 (orange) at 10 µM by HEp2 cells.

to evaluate the cytotoxicity of conjugates 17, 20, and 23, at concentrations up to 500 µM, 18 h after exposure (42). The conjugates studied were the most water-soluble, and all showed low dark toxicity (IC50 > 250 µM) toward HEp2 cells (Figure 2). The time-dependent uptake of conjugates 15, 17, 20, 23, 24, and 25 at a concentration of 10 µM in HEp2 cells was investigated. At this concentration all conjugates were nontoxic to the cells, as evaluated using the Cell Titer Blue assay (Supporting Information). Significant differences in the cellular uptake were observed, as seen in Figure 3, indicating that the number, nature, and sequence of amino acids, as well as the presence of a chelated metal ion affect the uptake


of the conjugates. The di- and tri-lysine conjugates 17 and 15 show similar uptake kinetics, and at short time points (up to 4 h) there was no significant difference in their uptake by HEp2 cells. However after 25 h, the amount of tri-lysine conjugate 15 accumulated within cells is about 25% higher than that of the di-lysine derivative 17. The Sn(IV) complex 20 is taken-up faster than 15 and 17, reaching a plateau after about 2 h exposure time to cells. At longer time points (>4 h), the tri-lysine metal complex 20 is clearly the least accumulated within cells of all conjugates studied. On the other hand, the arginine-containing conjugates (23-25) were taken-up by HEp2 cells to a much larger amount (>50%) than the lysine derivatives at short time points (up to 4 h). After 25 h, conjugate 24 was clearly the one that accumulated the most within cells, followed by 23, suggesting that at least three consecutive arginine residues are necessary for high cellular uptake. The subcellular localization of all conjugates and starting porphyrins was also investigated in HEp2 cells. Figure 4 shows the fluorescent patterns observed for starting porphyrin 1 and conjugates 14-16, 19, 21-24 (supplementary data is available for all conjugates in the Supporting Information). All compounds were found to localize into vesicles, suggesting similar mechanisms of cellular uptake for all conjugates studied. Colocalization experiments were performed for all compounds using the organelle-specific fluorescent probes LysoSensor Green (lysosomes), MitoTracker Green (mitochondria), and Hoechst 33342 (nuclei). As shown in Figure 5 for conjugate 14, the preferential sites of intracellular localization of 14 were the cell lysosomes. Similar observations were obtained for all other conjugates and for starting por-

Figure 4. Subcellular fluorescence of (a) porphyrin 1, and conjugates (b) 14, (c) 15, (d) 16, (e) 19, (f) 21, g) 22, (h) 23, (i) 24, in HEp2 cells at 10 µM for 18 h. Scale bar: 10 µm.



and the porphyrin inner N-H. Furthermore, additional stabilization via hydrophobic interactions between the aliphatic chain of the lysine residue and the porphyrin aromatic system was observed (e.g. Figure 6b). Our results suggest that hydrogen bonding and hydrophobic interactions of the peptide chain with the porphyrin platform reduce the conformational entropy (by as much as 60 kcal/mol) as compared to the linear conformation. The water effects were evaluated only for conjugate 18, and the minimum energy conformation found was similar to the vacuum structure shown in Figure 6b, with only a slight change on the position of the amine group of the lysine residues (see Supporting Information). It is, however, likely that in the presence of water, intermolecular forces (such as π-π stacking of porphyrin macrocycles) predominate over the intramolecular interactions. Although the NMR chemical shifts of the CR peptidyl protons can also be useful in the assignment of the secondary structure of peptides, we were not able to use this technique because of the overlap of these protons with those of the solvent. DISCUSSION

Figure 5. Subcellular localization of conjugate 14 in HEp2 cells at 10 µM for 18 h. (a) Phase contrast, (b) 14 fluorescence, (c) LysoSensor Green fluorescence, (e) MitoTracker Green fluorescence, (g) Hoechst fluorescence, (d, f, h) overlays of organelle tracers with 14 fluorescence. Scale bar: 10 µm.

phyrins 1 and 5 (Supporting Information). To investigate the effect of the route of delivery on cellular localization, three different transfection lipids, Avanti, Cardiolipin, and Lipofectamine, were used as delivery vehicles for conjugates 22 and 23. Fluorescent microscopy revealed that all lipid mixtures transported the conjugates to the mitochondria of HEp2 cells (Supporting Information). We have previously observed that the use of liposomes as delivery vehicles can have a dramatic effect on the subcellular sites of accumulation of photosensitizers (42). Molecular Modeling. Conformation analysis for conjugates 15, 18, 23, 24, and 25 was performed in a vacuum and, in the case of conjugate 18, also in the presence of water. Only the minimum energy conformations were selected and those determined under vacuum are shown in Figure 6. All structures show a bend conformation of the peptides over the porphyrin ring, which is more pronounced in the case of the lysine derivatives bearing the five-carbon linker. In some cases (e.g. Figure 6a) additional stabilization was obtained via formation of an intramolecular bond between the N-H of a lysine residue

The coupling of peptides to aminophenylporphyrin 1 via amide bond formation using traditional coupling reagents (DCC or EDCI) requires several days, due to the weak nucleophilicity of the aromatic amino group in pophyrin 1 and to steric factors, as previously observed (43). For example, while the coupling of porphyrin 1 with Boc-glycine produced the corresponding conjugate in 93% yield, the coupling with Boc-lysine under the same conditions resulted in 65% yield, and no conjugate was obtained in the presence of activated Boc-lysine with NHS. The reaction yields decreased with the increasing number of amino acid residues on the protected peptide, even under optimized coupling conditions (HOBt/EDCI/ TBTU/DIEA in DMF). Slightly higher yields of the target conjugates were obtained upon conversion of the amino group of porphyrin 1 to a reactive isothiocyanate functionality (18, 35), although steric factors still significantly influenced the reaction yields (Table 1). Using isothiocyanate porphyrin 5, conjugates 13-15 were obtained in solution phase, in 35-93% yields. The free amino terminus Boc-protected peptides resulted in higher yields of the target conjugates, due to the easier cleavage of this protecting group compared with the Z-protected peptides. This result can be due, in the case of catalytic hydrogenation, to the poisoning of the palladium catalyst by the sulfur on the isothiourea group. The solution-phase conjugation of Boc-protected peptides to carboxylic acid porphyrin 6 containing a five-carbon spacer resulted in high yields of the target conjugates 17 and 18 that were independent from the size of the reacting peptide. The Zn(II)- and Sn(IV)-porphyrin conjugates were also prepared to investigate the effect of a centrally chelated diamagnetic metal ion on the cellular uptake and subcellular localization. In complexes 19 and 20 the amino groups on the lysine residues can potentially interact with the metal ion, especially in the case of the Zn(II) complex. The solid-phase synthesis of C-amidated conjugates 21-25 proceeded considerably faster although lower yields were obtained compared with the solution-phase reactions. While the solution-phase conjugations are more time-consuming because they require rigorous isolation, purification, and characterization of the target compounds following each synthetic step, the conjugation to biomolecules built on a solid support allows a stepwise assembly of the conjugates with no isolation of interme-



Figure 6. Energy-minimized conformations in a vacuum for conjugates (a) 15, (b) 18, (c) 23, (d) 24, and (e) 25.

diates. The yields of the latter route are, however, lower because of poor solvation, increased intra- and intermolecular interactions in the growing peptide chains, and to porphyrin- or peptide-polymer adsorptions. Furthermore, in solid-phase synthesis various byproducts from either incomplete reactions, side reactions, or impure reagents usually accumulate on the resin during chain assembly, contaminating the final product and requiring HPLC purification; lower reactions rates are therefore observed as a result of secondary structures or aggregates formed with other peptide chains or with the polymer support. The dark cytotoxicity of the most water-soluble porphyrin conjugates bearing two or three lysine or arginine residues was evaluated at concentrations up to 500 µM (Figure 2). All conjugates showed low dark toxicity (IC50 > 250 µM), and the least toxic was the Sn(IV) complex 20, which was also the least accumulated within HEp2 cells at long exposure times (>2 h) (Figure 3). The cellular uptake of the conjugates depended significantly on the number, nature, and sequence of the amino acid residues, the chelated metal ion, and the C-termination (OH vs NH2), i.e., on the overall molecular charge and its distribution. For all compounds studied, the metal-free conjugates showed similar uptake kinetics, and the amount of conjugate accumulated within cells was higher for those containing three consecutive arginine residues (Figure 3). It has been previously observed that peptides containing cationic amino acids, in particular argininerich peptides, have a unique ability to cross cellular

membranes (44-47). This property is attributed to the unique structure of the positively charged guanidinium group and its ability to form bidentate hydrogen bonds with, for example, membrane-containing phosphate groups. Lysine-rich peptides usually show lower uptake compared with arginine peptides, and this uptake is dependent on the number of residues, suggesting that the overall charge is important but certainly not the only factor (47). In agreement with these studies our results show that a tri-lysine conjugate (15) is taken-up to a higher extent than a di-lysine derivative (17), that arginine-containing conjugates (e.g. 23 and 24) rapidly and most effectively accumulate within cells, and that the amino acid sequence (LysArgArgArg vs ArgArgLysArg, i.e., conjugates 24 and 25) plays an important role on the cellular uptake of these compounds. Furthermore, the presence of a centrally chelated metal ion (as in 20) significantly changes the uptake kinetics, promoting higher uptake at low exposure times (

Synthesis and Characterization of Positively Charged Porphyrin

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