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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
 
J Med Chem. Author manuscript; available in PMC 2012 November 10.
Published in final edited form as:
PMCID: PMC3208129
NIHMSID: NIHMS329040

Syntheses and Cellular Investigations of 173-, 152- and 131-Amino Acid Derivatives of Chlorin e61

Abstract

A series of amino acid conjugates of chlorin e6, containing lysine or aspartic acid residues in positions 173, 152 or 131 of the macrocycle were synthesized and investigated as photosensitizers for photodynamic therapy of tumors. All three regioisomers were synthesized in good yields and in 5 steps or less from pheophytin a (1). In vitro investigations using human carcinoma HEp2 cells show that the 152-lysyl regioisomers accumulate the most within cells, and the most phototoxic are the 131 regioisomers. The main determinant of biological efficacy appears to be the conjugation site, probably because of molecular conformation. Molecular modeling investigations reveal that the 173-substituted chlorin e6 conjugates are L-shaped, the 152 and 131 regioisomers assume extended conformations, and the 131 derivatives are nearly linear. It is hypothesized that the 131-aspartylchlorin e6 conjugate may be a more efficient photosensitizer for PDT than the commercial currently used 152 derivative.

Introduction

Photodynamic therapy (PDT) is a binary cancer therapy that relies on the selective uptake of a photosensitizer into cancer cells followed by irradiation with red light, which produces singlet oxygen and other reactive oxygen species.14 Highly cytotoxic singlet oxygen readily reacts with electron-rich biomolecules in its vicinity, such as unsaturated lipids, amino acids and DNA.5 Due to the known limited diffusion of singlet oxygen through tissues the PDT effects are largely localized to the photosensitizer-containing cells, thus reducing potential damage to normal cells in the vicinity of the tumor. The selectivity of the PDT treatment depends both on the tumor-targeting ability of the photosensitizer and the light used to activate it. An ideal photosensitizer should have minimal toxicity in the dark, a high quantum yield of triplet state formation in the presence of light, high selectivity for tumor cells over normal cells, rapid clearance from normal tissues, and a strong absorption peak within the “therapeutic window” (600–800 nm) for optimal light penetration through tissue.

Commercial hematoporphyrin derivative is a porphyrin-based photosensitizer that has been commercially developed and approved in several countries for the PDT treatment of melanoma, early and advanced stage cancer of the lung, digestive tract, genitourinary tract and Barret’s esophagus.36 However, it has only a weak absorption within the therapeutic window and often induces skin photosensitivity in patients. For these reasons, several second generation photosensitizers bearing stronger and red-shifted absorption within the therapeutic window have been investigated in PDT; these include tetra(meta-hydroxyphenyl)chlorin, (mTHPC), HPPH 2-[1-hexyloxyethyl]-2-devinylpyropheophorbide a (HPPH), lutetium(III) texaphyrin (LuTex), mono-L-aspartylchlorin e6, and phthalocyanine 4 (Pc4).36 Chlorins (dihydroporphyrins) are particularly promising photosensitizers for PDT because of their intense absorptions above 640 nm; in particular chlorophyll a derivatives are inherently amphiphilic macrocycles that have been extensively investigated and shown to have low dark toxicities while being able to effectively generate singlet oxygen upon light activation.79 Among these, HPPH and mono-L-aspartylchlorin e6 have been extensively investigated and are currently in advanced-stage clinical trials for oncologic PDT applications;1013 both of these chlorophyll a based compounds have shown superior PDT activity as well as rapid clearance from normal tissue and decreased patient photosensitivity compared with commercial hematoporphyrin derivative.

Chlorophyll a derivatives of the chlorin e6 series possess three carboxylic side chains that can be derivatized in multiple ways to produce novel amphiphilic photosensitizers for PDT investigations. For example, their conjugation to peptides, sugars, lipoproteins and polyamines have been reported.1419 In particular, amino acid residues have been found to improve the biological effects of porphyrin-based compounds, and their nature and position about the macrocycle can have a significant impact on PDT efficacy.5,20,21 In the present study we synthesized and investigated 173, 152 and 131 amino acid-conjugates of chlorin e6 (Figure 1), to evaluate the effect of the nature, conjugation site and position of the amino acid on the PDT efficacy of the conjugates in vitro, using human carcinoma HEp2 cells. The structure of mono-L-aspartylchlorin e6, bearing the aspartyl residue in position 152 (rather than in 173 as initially proposed) has recently been established.22 This study led us to design new syntheses of the 173 and 131 aspartyl regioisomers of mono-L-aspartylchlorin e6, as well as investigate the corresponding cationic lysine derivatives, which could potentially show stronger interactions with negatively charged biological molecules and plasma membranes, and consequently enhanced PDT efficacy.23 Furthermore, we investigated the introduction of a spacer group, ethylene diamine, and of metalation, via insertion of palladium(II). The photosensitizing properties of porphyrin derivatives are known to be modulated by inner coordinated metal ions and associated axial ligands.2426 In particular, Pd(II)-complexes have been shown to be efficient generators of singlet oxygen, and the palladium(II)-bacteriopheophorbide is currently being evaluated in clinical trials for the treatment of prostate cancer.27,28 The work described herein indicates that the site of amino acid conjugation, rather than the presence of Pd(II) and even the nature of amino acid, is the major determinant of phototoxicity.

Figure 1
173, 152 and 131 regioisomers of “R”-substituted chlorin e6.

Results and Discussion

1. Syntheses

The synthetic route to the 173-lysyl conjugate of chlorin e6 (3) is shown in Scheme 1. Pheophytin a (1) was obtained by extraction from Spirulina pacifica alga, an ideal source for chlorophyll a because of the complete absence of chlorophyll b. The selective hydrolysis of the phytyl ester group of pheophytin a (1) using aqueous TFA produced pheophorbide a in high yield, without affecting the β-keto ester of the isocyclic ring.29 This ring serves as a natural protecting group for the 131- and 152-positions during the coupling reactions. Selective hydrolysis of the phytyl ester produced a free 173-carboxylic acid group which was activated with DCC/DMAP and coupled with H-lysine (OtBu) methyl ester to form the lysine (OtBu) methyl ester pheophorbide a (2) in 52% yield.32 1H NMR spectroscopy demonstrated the existence of the isocyclic ring and new peaks for the lysine residue. Subsequent isocyclic ring-opening with sodium methoxide31 in THF produced the 173-lysyl(Boc) chlorin e6 TME in high yield (89%); deprotection of the amine group of the lysine side chain with TFA in CH2Cl2 followed, and yielded the 173-lysylchlorin e6 (3) in 30% overall yield from pheophytin a (1).

Scheme 1
Reaction conditions: a) TFA:H2O 4:1, 0 °C, 1 h, 90%; b) DCC, DMAP, H-Lys(Boc)-OMe•HCl, DIEA, CH2CL2, rt, 14 h, 52%; c) NaOMe, THF, 0 °C, 4 h, 89%; d) TFA, CH2Cl2, thioanisole, 0 °C to rt, 12 h, 72%; e) 5% H2SO4/MeOH, rt, ...

In order to synthesize the 152 and 131 amino acid derivatives of chlorin e6, pheophytin a (1) was converted into methyl pheophorbide a (4) by transesterification of the ester group using sulfuric acid in methanol, as previously reported,31 followed by isocyclic ring-opening, as shown in Scheme 1. An optimized yield of 98% of chlorin e6 trimethyl ester was obtained when 1.1 equiv of freshly prepared sodium methoxide in THF were used in the ring-opening reaction. Chlorin e6 (5) was obtained by hydrolysis of the three methyl esters using an excess of LiI in ethyl acetate under reflux conditions for 24 hours.33 Alternative hydrolysis under basic conditions using NaOH and LiOH gave lower yields of chlorin e6. Under acidic conditions the 131-carboxylic acid of chlorin e6 is deactivated34 allowing for the selective methylation of the 152-and 173-carboxylic acids with 5% H2SO4/MeOH to give chlorin e6 dimethyl ester (6) in quantitative yield.22 Chorin e6 (5) was used as starting material for the preparation of the 152 amino acid derivatives of chlorin e6 (as shown in Scheme 2), whereas the chlorin (6) and methyl pheophorbide a (4) were used to prepare the 131 amino acid derivatives of chlorin e6 (as shown in Schemes 3 and and44).

Scheme 2
Reaction conditions: a) DCC, DMAP, CH2Cl2, rt, 2 h; H-Asp(Boc)2•HCl or H-Lys(Boc)-OMe•HCl, DIEA, CH2Cl2, rt, 14 h; b) CH2N2, CH2Cl2, 30 min; c) TFA, CH2Cl2, thioanisole, 0 °C to rt, 12 h (7a: 45%, 7b: 77%); d) Pd(OAc)2, THF, 60 ...
Scheme 3
Reaction conditions: a) L-Asp(OtBu)2•HCl, DIEA, CH2Cl2, HOBt, TBTU, DMF, rt, 14 h, 66%; b) TFA, CH2Cl2, thioanisole, 0 °C to rt, 12 h, 88%; c) β-Ala(OtBu)•HCl, DIEA, CH2Cl2, HOBt, TBTU, DMF, rt, 12 h, 68%; d) TFA, CH2Cl ...
Scheme 4
Reaction conditions: a) NH2C2H4NH2, CHCl3, rt, 24 h, 91%; b) (Boc)Lys(Boc)-OH, DIEA, CH2Cl2, HOBt, TBTU, DMF, rt, 12 h, 78%; c) TFA, CH2Cl2, thioanisole, 0 °C to rt, 12 h, 81%.

We have previously shown22 that the 152-carboxylic acid of chlorin e6 is the most reactive, regardless of the coupling reagent employed. Therefore, upon activation of chlorin e6 (5) with one equiv of DCC/DMAP, followed by addition of protected amino acid, reaction with diazomethane35 and deprotection under acidic conditions, the 152-aspartyl (7a) and -lysyl (7b) derivatives of chlorin e6 were obtained in overall 36% and 56% yields, respectively (Scheme 2). The use of excess DCC significantly reduced the yields obtained due to the formation of the 173,152-di-amino acid conjugate as a side product.

Insertion of palladium into the chlorin ring was attempted before the deprotection step to simplify the purification process; however low yields of the target metallochlorins were obtained. Therefore, palladium insertion was performed after deprotection, using either palladium(II) acetate or palladium(II) chloride. Quantitative yields for the metal insertion reaction were obtained using 1.2 equiv of palladium(II) acetate in THF at 40 °C, to produce the corresponding Pd(II) complexes (8). As expected, significant blue shifts in the UV-Vis spectrum along with 1H NMR and mass spectrometry confirmed the insertion of palladium. Heavy metal atoms in the core of the tetrapyrroles have been shown to facilitate intersystem crossing and increase the production of singlet oxygen due to the inner heavy atom effect.36,37

Two routes were used to synthesize the 131-amino acid conjugates: (1) Selective esterification of 173- and 152- carboxylic acids of chlorin e6 to produce the monocarboxylic acid (6) followed by coupling34 (Scheme 3), and (2) isocyclic ring-opening of methyl pheoporbide a (4) with a nucleophile (Scheme 4). The 131−carboxylic acid of chlorin (6) was activated using HOBt/TBTU under basic conditions38 and coupled with L-aspartic di(tert)butyl ester. With DCC/DMAP a lower yield was obtained for the coupling reaction. Subsequent deprotection of both carboxylic acids in the aspartic chain provided (9) in 57% overall yield from chlorin e6 (Scheme 3). Coupling of the 131−carboxylic acid of chlorin (6) with β-alanine tert-butyl ester followed by deprotection using TFA gave the 131-β-alanylchlorin e6 dimethyl ester (10) in 64% overall yield. Aspartic di(tert)butyl ester was then coupled under similar conditions, followed by removal of the tert-butyl protecting group to give (11) in 54% yield from (10). β-Alanine provides a 4-atom link between the aspartic acid and chlorin e6 dimethyl ester and allows for direct comparison with the lysine derivative 13 (see Scheme 4). The use of a linker significantly increased the yield of the coupling reaction (from 66 to 87%) and it can also affect the biological properties of the photosensitizer.20,39

The nucleophilic ring-opening of the isocyclic ring in pheophorbide a with ethylenediamine and ethanolamine has already been previously reported.25,40 We took advantage of this high yielding isocyclic ring-opening reaction to develop a potentially high yielding synthetic route to novel chlorin e6 photosensitizers, directly from pheophytin a (1) in four steps. Nucleophilic ring-opening of the isocyclic ring with ethylenediamine produced molecule (12) in 91% yield. Subsequent coupling with protected lysine followed by deprotection gave the desired product (13) in four steps and in 55% overall yield from pheophytin a (1). No absorption quenching of the chlorin macrocycle was observed upon conjugation with the amino acid lysine in comparison with the apsrtic acid derivative (see Figure S1 of the Supporting Information).

2. Molecular Modeling

Conformation analyses for 173-, 152- and 131-lysylchlorin e6 derivatives 3, 7b and 13 were performed in both the gas phase and in water at the HF/6-31G level. These calculations were based on the atom coordinates from the X-ray structure of 152-aspartylchlorin e6 tetramethyl ester.22 The minimum energy conformations found for compounds 3, 7b and 13 in the gas phase, shown in Figure 2a–c, respectively, were very similar to those found in water phase (Figure S2 of the Supporting Information). In addition, the conformation of a mono-cationic 131-lysylchlorin e6 derivative without a linker was also investigated in the gas phase (Figure 2d). As expected, the lysine residue in the 173-lysyl derivative 3 is nearly perpendicular to the macrocyclic plane (Figure 2a), while in the 152-lysyl derivative 7b it forms approximately a 120° angle (Figure 2b). On the other hand the lysine residue extends away from the macrocycle in the 131-lysyl derivatives, with and without the short spacer (Figures 2c,d). Consequently, in the L-shape conformation of the 173-lysylchlorin e6 derivative, the amino acid shelters one face of the chlorin ring, while in the case of the 152- and 131-lysyl derivatives, it extends away from the macrocycle resulting in a nearly linear conformation for the 131 derivatives. The use of a short linker and the presence of two (rather than one) positive charges, as a result from conjugation to the C-terminus rather than the N-terminus of the amino acid, do not appear to have a significant effect upon the preferred conformation; the main determinant of molecule conformation is the site of substitution. While the minimum energy conformations found in water were similar to those in the gas phase, it is possible that in water intermolecular forces (such as π-π stacking) predominate over the intramolecular interactions, in particular for the linear 131-lysylchlorin e6 derivatives.

Figure 2
Energy minimized conformations in gas phase for chlorin e6 derivatives (a) [131 LysChlorin e6TME]+, (b) 13, (c) 7b and (d) 3. Optimization by energy was carried out at HF/6-31G level.

3. Cell Culture Studies

3.1. Time-dependent Cellular Uptake

The results obtained for the time-dependent uptake of chlorin e6 and its derivatives at a concentration of 10 µM in human HEp2 cells are shown in Figure 3 (also see Figure S3 of the Supporting Information). All amino acid conjugates of chlorin e6 were readily taken up by cells and showed uptake kinetics similar to those of unconjugated chlorin e6. Interestingly, the 152-lysylchlorin e6 derivatives 7b and its palladium(II) complex 8b accumulated to a much higher extent than all other compounds, at all time points studied. In comparison with chlorin e6, the lysyl derivatives 7b and 8b showed 18-fold and 4-fold higher cellular uptake, respectively, after 24 h. The observed high uptake for derivatives 7b and 8b is probably due to the lysine residue in position 152 since the corresponding aspartyl derivatives 7a and 8a accumulated to a significantly lower extent within cells. Presumably, the stronger interactions between the positively charged lysine derivatives (compared with the corresponding aspartyl derivatives), with the negatively charged plasma membrane leads to enhanced cellular uptake.20,23 On the other hand a lysine residue in position 173, as in derivative 3, showed a dramatic decrease in cellular uptake compared with the same residue at position 152, suggesting that the molecule conformation plays an important role on the mechanism of cellular uptake. Indeed the extended conformation of the 152-lysylchlorin e6 derivative 7b (Figure 2b) might be the most favored for penetration across the plasma membrane, compared with the L-shape and linear conformations of the 173- and 131-lysyl derivatives (Figures 2a,c), respectively. The presence of a chelated palladium ion, as well as the more linear structure of the 131-lysyl substituent might lead to enhanced π-π stacking of the macrocycles, thereby decreasing cellular uptake.

Figure 3
Time-dependent uptake of chlorin e6 (5, black line) and its derivatives 173-LysChlorin e6TME (3, brown line), 152-AspChlorin e6DME (7a, light blue line), 152-LysChlorin e6TME (7b, red line), 152-AspPdChlorin e6DME (8a, green line), 152-LysPdChlorin e ...

3.2. Cytotoxicity

The dark cytotoxicity and phototoxicity of chlorin e6 and its derivatives was evaluated in HEp2 cells exposed to increasing concentrations of each compound up to 400 µM; the results are shown in Figures 4 and and5,5, respectively, and are summarized in Table 1. Chlorin e6 and its lysyl derivatives 3, 7b and 8b were found to be non-toxic in the dark up to the highest concentration (400 µM) investigated. All other amino acid derivatives showed very low dark cytotoxicities, with IC50 > 320 µM except for the 131-chlorin e6 derivatives 9 and 13, which showed IC50 of 285 and 268 µM, respectively. However, upon exposure to a low light dose (1 J/cm2), all chlorin e6 derivatives were found to be highly toxic to HEp2 cells (Figure 3). The most phototoxic were the 131-chlorin e6 derivatives 9, 11 and 13, with estimated IC50 values of 0.61, 0.82 and 1.34 µM, respectively. Among these, the most promising aspartyl derivatives for PDT applications are compounds 9 and 11, because they have the highest dark cytotoxicity/phototoxicity ratio, > 466:1. The presence of the β-alanine spacer between the 131 carbonyl group and the aspartic acid residue seems to have only a small effect, slightly decreasing compound cytotoxicity. On the other hand the 152-aspartylchlorin e6 derivative 7a was less phototoxic than its 131 regioisomer 9 by approximately 7-fold, and the introduction of palladium(II) further reduced its phototoxicity. The positively charged 173- and 152-lysylchlorin e6 derivatives 3 and 7b were the least phototoxic, and the introduction of palladium increased the phototoxicity of the 152 derivative by about 10-fold. These results show for the first time that the phototoxicity of amphiphilic conjugates of chlorin e6 depends mainly on the site of conjugation, probably as a result of its effect on molecular conformation; the nature of the amino acid, the molecule overall charge, and the presence of palladium(II) also affect phototoxicity, but apparently to a smaller extent. Our results suggest that the most extended, nearly linear conformation of the 131 regioisomers facilitates binding to specific biological substrates, enhancing their toxic effect.

Figure 4
Dark toxicity of chlorin e6 (5, black line) and its derivatives 173-LysChlorin e6TME (3, brown line), 152-AspChlorin e6DME (7a, light blue line), 152-LysChlorin e6TME (7b, red line), 152-AspPdChlorin e6DME (8a, green line), 152-LysPdChlorin e6TME (8b ...
Figure 5
Phototoxicity of chlorin e6 (5, black line) and its derivatives 173-LysChlorin e6TME (3, brown line), 152-AspChlorin e6DME (7a, light blue line), 152-LysChlorin e6TME (7b, red line), 152-AspPdChlorin e6DME (8a, green line), 152-LysPdChlorin e6TME (8b ...
Table 1
Cytotoxicity (HEp2 cells) for chlorin e6 and its derivatives using the Cell Titer Blue assay

3.3. Intracellular Localization

The preferential sites of subcellular localization of this series of chlorin e6 derivatives were evaluated by fluorescence microscopy, upon exposure of HEp2 cells to 10 µM compound concentrations for 18 h. Figure S4 of the Supporting Information shows the fluorescence pattern observed for all compounds and Table 2 summarizes their main sites of subcellular localization. Overlay experiments using the organelle specific fluorescence probes BoDIPY Ceramide (Golgi), LysoSensor Green (Lysosomes), MitoTracker Green (mitochondria), and ER tracker Blue/White fluorescence (ER) were conducted to evaluate the preferential sites of compound localization, as seen in Figures 6, ,77 and and88 for the most phototoxic 131-regioisomers, respectively, and Figures S5–S10 of the Supporting Information. All chlorin e6 derivatives 3, 7a, 7b, 8a, 8b, 9, 11 and 13 were found to localize in the lysosomes and the ER (Table 2). This is not surprising, since the structurally-related 152-aspartylchlorin e6 is a known lysosomal localizer, and HPPH (2-[1-hexyloxyethyl]-2-devinylpyropheophorbide a) localizes preferentially in the ER.41 Photodamage to ER and/or lysosomes has been shown to lead to activation of apoptotic pathways.4143 Furthermore, all the 131-chlorin e6 regioisomers 9, 11 and 13 were also found in Golgi, and in addition the most phototoxic derivative 9 also localized in mitochondria; presumably, the photodamage effect to multiple organelles caused by the 131 derivatives can trigger various apoptotic pathways, leading to more effective cell destruction. The multiple sites of intracellular localization observed for the 131-chlorin e6 derivatives might again be due to their linear conformations that facilitate their binding to various intracellular sites.

Figure 6
Subcellular localization of conjugate 131 AspCe6DME (9) in HEp2 cells at 10 µM for 18 h (a) phase contrast, (b) overlay of conjugate 9 compound and phase contrast, (c) ER tracker Blue/White fluorescence (e) MitoTracker Green fluorescence, (g) ...
Figure 7
Subcellular localization of conjugate 131 β-Ala-AspCe6DME (11) in HEp2 cells at 10 µM for 18 h (a) phase contrast, (b) overlay of conjugate 11 and phase contrast, (c) ER tracker Blue/White fluorescence (e) MitoTracker Green fluorescence, ...
Figure 8
Subcellular localization of conjugate 131 ED-LysCe6DME (13) in HEp2 cells at 10 µM for 18 h (a) phase contrast, (b) overlay of conjugate 11 and phase contrast, (c) ER tracker Blue/White fluorescence (e) MitoTracker Green fluorescence, (g) BoDIPY ...
Table 2
Major (++) and minor (+) subcellular sites of localization for chlorin e6 and its derivatives in HEp2 cells

Conclusions

A series of eight chlorin e6 derivatives, conjugated with either aspartic acid or lysine residues at positions 173, 152 or 131 of the chlorin macrocycle have been synthesized in good yields from pheophytin a (1). In comparison with chlorin e6 (5), all amino acid derivatives readily accumulated within human HEp2 cells, and in particular the 152-lysyl derivatives were taken up by cells to a significantly higher extent than were all other regioisomers. On the other hand, the metal-free 152-lysylchlorin e6 derivative (7b) showed the least phototoxicity, followed by the 173-lysyl regioisomer (3); insertion of palladium into 152-lysychlorin e6 (7b) increased toxicity by approximately 10-fold. However, the most phototoxic compounds were found to be the 131−regioisomers, bearing either an aspartic acid or a lysine residue directly conjugated to position-13 of the chlorin macrocycle, or connected via a β-alanine or ethylene diamine spacer. The most phototoxic compound of this series, and the most promising for PDT applications, is 131-aspartylchlorin e6 (9). Molecular modeling calculations show that the 131-regioisomers assume extended, nearly linear conformations that might facilitate their binding to multiple intracellular components, and subsequent photodamage to multiple cellular sites. Therefore, the site of amino acid conjugation at the chlorin e6 macrocycle is a major determinant of compound phototoxicity, and we hypothesize that the 131-regioisomer of mono-L-aspartylchlorin e6 (9) might be a more efficient photosensitizer for PDT than is the commercial regioisomer (152-mono-L-aspartylchlorin e6).

Experimental Section

1. Chemistry

All air and moisture sensitive reactions were performed in dried and distilled solvents under an argon atmosphere. All solvents and reagents were purchased from commercial sources, unless otherwise stated. Silica gel 60 (230×400 mesh, Sorbent Technologies) was used for column chromatography. Analytical thin-layer chromatography (TLC) was carried out using polyester backed TLC plates 254 (precoated, 200 µm) from Sorbent Technologies. NMR spectra were recorded on an AV-400 spectrometer (400 MHz for 1H, 100 MHz for 13C). The chemical shifts are reported in δ ppm using the following partially deuterated solvents as internal references: CD2Cl2 5.32 ppm (1H), 54 ppm (13C); d-DMSO 2.49 ppm (1H), 39.5 ppm (13C); d-CH3OH 4.78 ppm (1H), 49.0 ppm (13C); CDCl3 7.26 ppm (1H), 77.16 ppm (13C); (CH3)2CO 2.50 ppm (1H), 29.84 ppm (13C). Electronic absorption spectra were measured on an Agilent 8453 UV/Vis spectrophotometer. Mass spectra were obtained on a Bruker Omniflex MALDI Time-of-Flight Mass Spectrometer. Spirulina pacifica alga was purchased as a spray-dried powder from Cyanotech, Hawaii. Pheophytin a (1) was extracted from Spirulina pacifica alga as previously published and its spectroscopic characterization agreed with the published data.29 All compounds synthesized were purified and isolated in ≥ 95% purity, as evidenced by analytical TLC in at least two solvent systems, and confirmed by the absence of extraneous tetrapyrrole resonances in 1H- and 13C-NMR spectra. Energy minimization of all compounds was performed in the framework of Hartree Fock. The restricted-Hartree Fock functional was used at the 6-31G level. All structures were optimized without any symmetry constrains. The solvent effects were accounted for using the Polarizable Continuum Model (PCM). All calculations were performed using the Gaussian 09 program package.

173-Lysyl(Boc)OMe-pheophorbide a (2)

Pheophytin a (1) (250 mg, 0.29 mmol) was selectively hydrolyzed to the 173 carboxylic acid without affecting the 131 carbomethoxy group, as previously reported.31 After evaporation of solvent, 155 mg (0.26 mmol), 90% of pheophorbide a (C35H36N4O5) was obtained. The spectroscopic data obtained for the compound agreed with published data. Pheophorbide a, (100 mg; 0.169 mmol) was dissolved in 10 ml of CH2Cl2. Then DCC (42 mg) and DMAP (10 mg) were added and left it for 15 min. H-Lys(Boc)OMe•HCl (60 mg) and DIEA (0.035 ml) were dissolved in 5 ml of CH2Cl2, added to the reaction mixture and stirred for 4 h. [Note: this reaction is favored by dilute conditions; concentrated conditions favor the formation of the anhydride (bispheophorbide), and will slow the reaction]. The reaction mixture was washed with 10% citric acid, water and brine, dried over anhydrous Na2SO4and purified on a silica gel column (10% acetone in CH2Cl2). After the major brown band was eluted from the column, the solvent was evaporated and the solid was dissolved in ethyl acetate and filtered (DCC precipitated in ethyl acetate.) Evaporation of the filtered ethyl acetate gave 74 mg, 0.089 mmol, 52% yield of lysyl(Boc)OMe-pheophorbide a (C47H58N6O8), 1H NMR [(CD3)2CO, 400 MHz]: δ 9.12 (s, 1H), 8.71 (s, 1H), 8.70 (s, 1H), 7.64 (dd, J = 17.8, 11.63 Hz, 1H), 7.20 (d, J = 7.2 Hz, 1H), 6.33 (s, 1H), 6.02 (d, J = 17.85 Hz, 1H), 5.93 (d, J = 11.61 Hz, 1H), 5.77 (s, 1H), 4.65 (m, 1H), 4.41 (m, 1H), 4.21 (m, 1H), 3.91 (s, 3H), 3.59 (s, 3H), 3.47 (s, 3H), 3.19 (s, 3H) 3.09 (q, J = 7.5 Hz, 2H), 2.92 (3H, s), 2.83 (m, 2H), 2.66 (m, 1H), 2.48 (m, 1H), 2.25 (m, 1H), 1.85-1.62 (br, 4H), 1.86 (d, J = 7.24, 3H), 1.37 (t, J = 7.58, 3H), 1.43 (m, 2H), 1.26 (s, 9H), − 1.95 (s, 1H), −2.23 (s, 1H).

173-Lysyl-chlorin e6 TME (3)

Compound 2 (74 mg, 0.089 mmol) was dissolved in dry 2:1 THF/MeOH (10 ml) and stirred under argon for 10 min. Sodium methoxide (0.17 mL of a 0.5 M solution) was added and the reaction was allowed to stir at 0 °C for 1 h. The reaction was followed using spectrophotometry. The solution turned from brown to green as the isocyclic ring opens. The reaction mixture was then poured into water. The mixture was extracted with CH2Cl2 and the organic layer was washed with water and 5% citric acid, dried over anhydrous Na2SO4 and then evaporated. The residue was dissolved in 2% MeOH/CH2Cl2 and purified on a silica gel plug with the same mobile phase. The solvent was evaporated and 68 mg (0.078 mmol), 89% yield of 173-lysyl(Boc)chlorin e6 TME (C48H62N6O9) was obtained. 1H NMR [(CD3)2CO, 400 MHz]: δ 9.80 (s, 1H), 9.65 (s, 1H), 9.03 (s, 1H), 8.19 (dd, J = 17.87, 11.58 Hz, 1H), 7.44 (d, J = 7.6 Hz, 1H), 6.40 (d, J = 17.87, 1.33 Hz, 1H), 6.13 (d, J = 11.61, 1.36 Hz, 1H), 5.89 (s, 1H), 5.38 (m, 2H), 4.63 (q, J = 7.22 Hz, 1H), 4.55 (dd, J = 10.34, 1.95 Hz, 1H), 4.44 (m, 1H), 4.25 (s, 3H), 3.78 (q, J =7.67, 2H) 3.75 (s, 3H), 3.69 (s, 3H), 3.56 (s, 3H) 3.49 (s, 3H), 3.27 (3H, s), 3.00 (m, 2H), 2.66 (m, 1H), 2.33 (m, 1H), 2.20 (m, 1H), 1.85-1.62 (br, 4H), 1.78 (d, J = 7.24, 3H), 1.69 (t, J = 7.58, 3H), 1.43 (m, 2H), 1.32 (s, 9H), − 1.32 (s, 1H), −1.52 (s, 1H). Lysyl(Boc)chlorin e6 TME (68mg, 0.078 mmol) was dissolved in 2 ml of dry CH2Cl2 in an ice bath under argon. Thioanisole (0.057 mmol) and 1 ml of TFA were added and the reaction mixture was allowed to stir overnight. The reaction mixture was evaporated several times with diethyl ether to remove TFA. The resulting precipitate was washed several times with diethyl ether to remove thioanisole. The precipitate was dissolved in CH2Cl2 and washed three times with H2O and once with 10% NaHCO3 to remove last traces of TFA. The organic layer was dried over anhydrous Na2SO4. Solvent was evaporated and 43 mg (0.056 mmol), 72% yield of 173-lysylchlorin e6 TME (C43H54N6O7) was obtained. UV-Vis (acetone): λmax (ε/M−1cm−1) 664 (65,000), 608 (4871), 528 (4461), 500 (16,100), 400 (213,000); 1H NMR [(CD3)2CO, 400 MHz]: δ 9.79 (s, 1H), 9.63 (s, 1H), 9.03 (s, 1H), 8.17 (dd, J = 17.87, 11.58 Hz, 1H), 7.59 ( d, J = 7.6 Hz, 1H), 6.38 (d, J = 17.87, 1.33 Hz, 1H), 6.12 (d, J = 11.61, 1.36 Hz, 1H), 5.37 (m, 2H), 4.63 (q, J = 7.22 Hz, 1H), 4.55 (dd, J = 10.34, 1.95 Hz, 1H), 4.44 (m, 1H), 4.25 (s, 3H), 3.76 (m, 2H) 3.75 (s, 3H), 3.69 (s, 3H), 3.55 (s, 3H) 3.48 (s, 3H), 3.25 (3H, s), 3.09 (m, 2H), 2.67 (m, 1H), 2.30 (m, 1H), 2.20 (m, 1H), 1.85-1.62 (br, 3H), 1.78 (d, J = 7.24, 3H), 1.68 (t, J = 7.58, 3H), 1.51 (m, 2H), 1.39 (m, 2H), − 1.33 (s, 1H), −1.53 (s, 1H). HRMS (MALDI-TOF) m/z 767.442 [M+H]+, calcd. for C43H55N6O7 767.413.

Chlorin e6 (5)

Chlorin e6 TME was prepared as previously published and its spectroscopic characterization agreed fully with the published data.22 Chlorin e6 TME (50 mg, 0.078 mmol) was dissolved in anhydrous ethyl acetate (10 ml) under argon. Lithium iodide (124 mg, 0.94 mmol) was added. The reaction mixture was refluxed for 48 h under argon. The reaction mixture was diluted with water and adjusted to pH 3 with aqueous citric acid and then washed with CH2Cl2. The solution was evaporated, re-dissolved in acetone and evaporated several times. The solid was washed with water and then dried under vacuum. The residue was dissolved in methanol and purified on a Sephadex LH-20 column to yield 10 mg, 0.017 mmol, 21% yield of chlorin e6 (C34H36N4O6). UV-Vis (MeOH): λmax (ε/M−1cm−1) 666 (45,271), 610 (8,706), 558 (7,835), 530 (9,721), 502 (15,525), 402 (145,100); MS MALDI: m/z 597 (M + H)+; 1H NMR [(CD3)2CO, 400 MHz]: δ 9.73 (s, 1H), 9.51 (s, 1H), 9.04(s, 1H), 8.05 (dd, J = 17.86, 11.61 Hz, 1H), 6.28 (dd, J = 17.85, 1.26 Hz, 1H), 6.02 (dd, J = 11.57, 1.37 Hz, 1H), 5.60 (d, J = 18.94 Hz, 1H), 5.40 (d, J = 18.94 Hz, 1H), 4.65 (m, 1H), 4.55 (m, 1H), 3.64 (m, 2H), 3.63 (s, 3H), 3.42 (s, 3H), 3.15 (s, 3H), 2.72 (m, 1H), 2.35 (m, 1H), 2.26 (m, 1H), 2.06 (m, 1H), 1.75 (d, J = 7.5 Hz, 3H), 1.64 (t, J = 7.5 Hz, 3H), −1.6 (s, 2H)

Chlorin e6 DME (6)

Chlorin e6 DME was prepared as previously published and its spectroscopic characterization agreed with the published data.22

152-Aspartylchlorin e6 DME (7a)

Chlorin e6 (100 mg, 0.168 mmol) was dissolved in dry CH2Cl2. DCC (35 mg) and DMAP (9 mg) were added and allowed to stir until completely dissolved. After 3 h, H-Asp(OtBu)2•HCl (47.2 mg) and DIEA (0.029 ml) were mixed in CH2Cl2 and added to the reaction mixture. The reaction was allowed to stir overnight. The mixture was diluted with CH2Cl2 and then washed with 5% aqueous citric acid, followed by washing with brine and water. It was dried over anhydrous Na2SO4 and then evaporated. The residue was dissolved in CH2Cl2 and treated with ethereal diazomethane. The residue was dissolved in 2% MeOH/CH2Cl2 and purified via silica gel column chromatography using the same mobile phase to afford 65 mg, 0.076 mmol; 45% yield of 152-monoaspartylchlorin e6 di(tert)butyl dimethyl ester (C48H61N5O9). MS (MALDI) m/z 853 (M + H)+; 1H NMR [(CD3)2CO, 400 MHz]: δ 9.64 (s, 1H), 9.32 (s, 1H), 9.02 (s, 1H), 7.81 (dd, J = 11.58, 17.85 Hz, 1H), 7.00 (s, 1H), 6.09 (dd, J = 17.87, 1.26 Hz, 1H), 5.85 (dd, J = 11.60, 1.29 Hz, 1H), 5.38 (s, 2H), 4.6 (br, 3H), 4.32 (s, 3H), 3.62 (s, 3H), 3.54 (s, 3H), 3.52 (q, J = 7.3 Hz, 2H), 3.30 (s, 3H), 3.02 (s, 3H), 2.78 (m, 3H), 2.44 (m, 2H), 1.87 (m, 1H), 1.65 (d, J = 7.3 Hz, 3H), 1.58 (t, J = 7.7 Hz, 3H), 1.26 (s, 9H), 1.16 (s, 9H), −1.42 (s, 1H), −1.53 (s, 1H). The 152-Aspartylchlorin e6 di(tert)butyl dimethyl ester (65 mg, 0.076 mmol) was dissolved in 2 ml of dry CH2Cl2 in an ice bath under argon. Thioanisole(0.006 ml) and 2 ml of TFA were added and the reaction mixture was allowed to stir overnight. The reaction mixture was evaporated several times with diethyl ether to remove TFA. The resulting precipitate was washed several times with diethyl ether to remove thioanisole and then dissolved in CH2Cl2 and washed three times with H2O and once with 10% NaHCO3 to remove TFA. The organic layer was washed with citric acid until all precipitate re-dissolved in the organic layer. The organic layer was dried over anhydrous Na2SO4. Solvent was evaporated and 44 mg, 0.06 mmol; 79% yield of 152-aspartylchlorin e6 DME was obtained (C40H45N5O9). UV-Vis (acetone) λmax (ε/M−1cm−1) 664 (48,400), 609 (6,000), 560 (2,000), 529 (6,280), 500 (14,160), 402 (151,800); MS (MALDI) m/z 841 (M + H)+; 1H NMR [(CD3)2CO, 400 MHz]: δ 9.76 (s, 1H), 9.58 (s, 1H), 9.04 (s, 1H), 8.10 (dd, J = 17.85, 11.59 Hz, 1H), 6.33 (dd, J = 17.87, 0.98 Hz, 1H), 6.07 (dd, J = 11.61, 1.09 Hz, 1H), 5.32 (m, 2H), 4.84 (m, 1H), 4.60 (m, 2H), 4.26 (s, 3H), 3.72 (q, J = 7.3 Hz, 2H), 3.57 (s, 3H), 3.54 (s, 3H), 3.45 (s, 3H), 3.21 (m, 3H), 2.95 (dd, J = 16.85, 5.53 Hz, 1H), 2.88 (dd, J = 16.85, 5.09 Hz, 1H) 2.68 (m, 1H) 2.32 (m, 2H), 1.78 (m, 1H), 1.74 (d, J = 7.3 Hz, 3H), 1.66 (t, J = 7.7 Hz, 3H), −1.39 (s, 1H), −1.58 (s, 1H). HRMS (MALDI-TOF) m/z 740.376 [M+H]+, calcd. for C40H46N5O9 740.330

152-Lysylchlorin e6 TME (7b)

Chlorin e6 (75 mg, 0.12 mmol) was dissolved in dry CH2Cl2 (10 ml). DCC (31.3 mg) and DMAP (11 mg) were added and allowed to stir until completely dissolved. After 3 h, H-lysyl(Boc)OMe•HCl (45 mg) and DIEA (0.022 ml) were mixed in CH2Cl2 and added to the reaction mixture. The reaction was allowed to stir overnight. It was then diluted with CH2Cl2 and washed with 5% aqueous citric acid, followed by a wash with brine and water. It was dried over anhydrous Na2SO4 and then evaporated to dryness. The residue it was dissolved in CH2Cl2 and treated with excess ethereal diazomethane. The residue was dissolved in 12% acetone/CH2Cl2 and purified via silica gel column chromatography with the same mobile phase to afford 84 mg, 0.097 mmol; 77% yield of 152-lysyl(Boc)chlorin e6 TME (C48H62N6O9). MS (MALDI) m/z 868 (M + H)+; 1H NMR [(CD3)2CO, 400 MHz]: δ 9.77 (s, 1H), 9.55 (s, 1H), 9.04 (s, 1H), 8.08 (dd, J = 17.87, 11.58 Hz, 1H), 7.07 (m, 1H), 6.31 (dd, J = 17.87, 1.33 Hz, 1H), 6.06 (dd, J = 11.61, 1.36 Hz, 1H), 5.87 (s, 1H), 5.32 (s, 2H), 4.65 (q, J = 7.22 Hz, 1H), 4.60 (dd, J = 10.34, 1.95 Hz, 1H), 4.49 (m, 1H), 4.26 (s, 3H), 3.71 (q, J = 7.67, 2H) 3.57 (s, 3H), 3.54 (s, 3H), 3.47 (s, 3H) 3.44 (s, 3H), 3.19 (3H, s), 2.97 (m, 2H), 2.71 (m, 1H), 2.37 (m, 2H), 1.80 (m, 3H), 1.77 (d, J = 7.24, 3H), 1.66 (t, J = 7.58, 3H), 1.39 (s, 9H), 1.27 (m, 2H), − 1.35 (s, 1H), −1.56 (s, 1H). The 152-lysyl(Boc)chlorin e6 TME (84 mg 0.097 mmol) was dissolved in 3 ml of dry CH2Cl2 in an ice bath under argon. Thioanisole (0.01 ml) and 1 ml of TFA were added and the reaction mixture was allowed to stir overnight. The reaction mixture was evaporated several times with diethyl ether to remove residual TFA. The resulting precipitate was washed several times with diethyl ether to remove thioanisole. Then the precipitate was dissolved in CH2Cl2 and washed three times with H2O and once with 10% NaHCO3 to remove TFA. The organic layer was washed with citric acid until the precipitate is re-dissolved in the organic layer. The organic layer was dried over anhydrous Na2SO4 and then was evaporated to give 56 mg, 0.071 mmol; 73% yield of 152-lysylchlorin e6 TME (C43H54N6O7). UV-Vis (acetone) λmax (ε/M−1cm−1) 664 (57,800), 608 (4,600), 529 (4,900), 500 (14,800), 400 (187,200); 1H NMR [(CD3)2CO, 400 MHz]: δ 9.68 (s, 1H), 9.42 (s, 1H), 9.02 (s, 1H), 7.95 (dd, J = 17.87, 11.58 Hz, 1H), 7.12 (d, J = 6.8 Hz, 1H), 6.20 (dd, J = 17.87, 1.33 Hz, 1H), 5.95 (dd, J = 11.61, 1.36 Hz, 1H), 5.35 (s, 2H), 4.65 (q, J = 7.22 Hz, 1H), 4.61 (dd, J = 10.34, 1.95 Hz, 1H), 4.53 (m, 1H), 4.27 (s, 3H), 3.59 (m, 2H) 3.55 (s, 6H), 3.48 (s, 3H) 3.37 (s, 3H), 3.09 (3H, s), 2.99 (m, 3H), 2.73 (m, 1H), 2.38 (m, 2H), 1.80 (m, 3H), 1.78 (d, J = 7.24, 3H), 1.61 (t, J = 7.58, 3H), 1.43 (m, 2H), − 1.40 (s, 1H), −1.57 (s, 1H). HRMS (MALDI-TOF) m/z 767.399 [M+H]+, calcd. for C43H55N6O7 767.413

Palladium(II) 152-Aspartylchlorin e6 DME (8a)

152-Aspartylchlorin e6 DME (44 mg, 0.06 mmol) was dissolved in 2 ml of dry THF. Palladium(II) acetate (14.2 mg, 0.063 mmol) was dissolved in THF and added to the reaction vessel and allowed to stir at 40 °C for 3 h. The reaction was followed by spectrophotometry. The solution turned from green to bluish green as the complex formed. After evaporation of solvent, the residue was dissolved in methanol and purified via Sephadex LH-20 chromatography using the same mobile phase to afford 50 mg, 0.059 mmol; 98% yield of palladium 152-lysylchlorin e6 TME (C40H43N5O9Pd). UV-Vis (acetone): λmax (ε/M−1cm−1) 619 (31,240), 579 (6,210), 489 (4,035), 393 (44,160); MS (MALDI) m/z 845 (M + H)+; 1H NMR [(CD3)2CO, 400 MHz]: δ 9.53 (s, 1H), 9.54 (s, 1H), 8.93 (s, 1H), 8.03 (dd, J = 17.82, 11.54 Hz, 1H), 6.17 (d, J = 17.79 Hz, 1H), 6.00 (dd, J = 11.61, 1H), 5.19 (d, J = 18.77 Hz, 1H), 5.04 (d, J = 18.59 Hz, 1H), 4.92 (m, 1H), 4.63 (m, 2H), 4.20 (s, 3H), 3.62 (s, 3H), 3.57 (m, 2H), 3.57 (s, 3H), 3.37 (s, 3H), 3.07 (m, 3H), 2.68 (m, 2H) 2.38 (m, 1H) 2.12 (m, 1H), 1.78 (m, 2H), 1.74 (d, J = 7.3 Hz, 3H), 1.50 (t, J = 7.7 Hz, 3H). HRMS (MALDI-TOF) m/z 843.109 [M]+, calcd. for C40H43N5O9Pd 843.210.

Palladium(II) 152-Lysylchlorin e6 TME (8b)

152-Lysylchlorin e6 TME (56 mg) was dissolved in 5 ml of dry THF. Palladium(II) acetate (53 mg) was dissolved in THF and added to the reaction vessel and stirred at 60 °C for 3 h. The reaction was followed by spectrophotometry. The solution turned from green to bluish green as the complex formed. After evaporation of solvent, the residue was dissolved in methanol and purified via Sephadex LH-20 chromatography using the same mobile phase to afford 61 mg, 0.07 mmol; 99% yield of palladium(II) 152-lysylchlorin e6 TME (C43H53N6O7Pd). UV-Vis (acetone): λmax (ε/M−1cm−1) 620 (97,520), 581 (14,500), 490 (7,600), 394 (140,900); 1H NMR [(CD3)2CO, 400 MHz] all the peaks were broad due to partial paramagnetic nature of the compound. HRMS (MALDI-TOF) m/z 872.337 [M+2H]+, 767.450 [M+H-Pd]+ Calcd. for C43H54N6O7Pd 871.301, C43H55N6O7 767.413.

131-Aspartylchlorin e6 DME (9)

Chlorin e6 dimethyl ester (55 mg, 0.088 mmol) was dissolved in dry CH2Cl2. A mixture of HOBt (12 mg), TBTU (29 mg) and DIEA (0.017 ml) in DMF was added and the mixture was allowed to stir for 30 min. H-Asp(OtBu)2•HCl (60 mg) and DIEA (0.033 ml) were mixed in CH2Cl2 and added to this reaction mixture. The mixture was stirred overnight. It was diluted with CH2Cl2 and then washed with 5% aqueous citric acid, followed by a wash with brine and water. It was dried over anhydrous Na2SO4 and then evaporated. The residue was dissolved in 5% acetone/CH2Cl2 and purified via silica gel column chromatography using the same mobile phase to afford 50 mg, 0.058 mmol; 66% yield of 131-aspartylchlorin e6 di(tert)butyl dimethyl ester (C48H61N5O9). MS (MALDI) m/z 852 (M + H)+; 1H NMR [(CD3)2CO, 400 MHz]: δ 9.79 (s, 1H), 9.70 (s, 1H), 9.10 (s, 1H), 8.39 (d, J = 7.93 Hz, 1H), 8.19 (dd, J = 11.58, 17.85 Hz, 1H), 6.37 (dd, J = 17.85, 1.23 Hz, 1H), 6.10 (dd, J = 11.87, 1.26 Hz, 1H), 5.71 (d, J = 18.95 Hz, 1H), 5.30 (m, 2H), 4.67 (q, J = 7.22 Hz, 1H), 4.49 (dd, J = 10.34, 1.95 Hz, 1H), 3.76 (q, J = 7.3 Hz, 2H), 3.74 (s, 3H), 3.64 (s, 3H), 3.61 (s, 3H), 3.50 (s, 3H), 3.27 (s, 3H), 3.16 (dd, J = 5.8, 0.78 Hz, 2H), 2.73 (m, 1H), 2.32 (m, 2H), 1.79 (m, 1H), 1.69 (m, 6H), 1.64 (s, 9H), 1.53 (s, 9H), −1.57 (s, 1H), −1.85 (s, 1H). The 131-Aspartylchlorin e6 di(tert)butyl dimethyl ester (50 mg, 0.058 mmol) was dissolved in 2 ml of dry CH2Cl2 in a ice bath under argon. Thioanisole (0.005 ml) and 2 ml of TFA were added and the reaction mixture was stirred overnight. The reaction mixture was evaporated several times with diethyl ether to remove residual TFA. The resulting precipitate was washed several times with diethyl ether to remove thioanisole. Then the precipitate was dissolved in CH2Cl2 and washed three times with H2O and once with 10% NaHCO3 to remove TFA. The organic layer was dried over anhydrous Na2SO4 and then evaporated to give 38 mg, 0.051 mmol; 88% yield of 131-aspartylchlorin e6 DME (C40H45N5O9). UV-Vis (acetone): λmax (ε/M−1cm−1) 663 (126,800), 607 (9,000), 528 (7,450), 500 (31,540), 399 (385,800); 1H NMR [(CD3)2CO, 400 MHz]: δ 9.79 (s, 1H), 9.70 (s, 1H), 9.12 (s, 1H), 8.50 (d, J = 7.93 Hz, 1H), 8.19 (dd, J = 11.58, 17.85 Hz, 1H), 6.37 (d, J = 18.85 Hz, 1H), 6.10 (d, J = 11.87 Hz, 1H), 5.71 (d, J = 18.95 Hz, 1H), 5.45 (dd, J = 13.76, 5.80 Hz, 1H), 5.30 (d, J = 18.93 Hz, 1H), 4.67 (q, J = 7.22 Hz, 1H), 4.50 (dd, J = 10.34, 1.95 Hz, 1H), 3.76 (q, J = 7.3 Hz, 2H), 3.71 (s, 3H), 3.63 (s, 3H), 3.60 (s, 3H), 3.51 (s, 3H), 3.31 (dd, J = 5.76, 3.47 Hz, 2H), 3.26 (s, 3H), 2.70 (m, 2H), 2.31 (m, 2H), 1.79 (m, 1H), 1.71 (d, J = 7.24, 3H), 1.67 (t, J = 7.58, 3H), −1.57 (br s, 1H), −1.87 (s, 1H). HRMS (MALDI-TOF) m/z 740.344 [M+H]+, Calcd. for C40H46N5O9 740.330

131-β-Alanylchlorin e6 DME (10)

Chlorin e6 DME (55 mg, 0.088 mmol) was dissolved in dry CH2Cl2. A mixture of HOBt (12 mg), TBTU (29 mg) and DIEA (0.017 ml) in DMF was added and the mixture was stirred for 30 min. β-Alanyl(OtBu)•HCl (18 mg) and DIEA (0.017 ml) were mixed in CH2Cl2 and added to the reaction mixture. The mixture was then allowed to stir for overnight before being diluted with CH2Cl2 and then washed with 5% aqueous citric acid, followed by a wash with brine and water. It was dried over anhydrous Na2SO4 and then evaporated to dryness. The residue was dissolved in 5% acetone/CH2Cl2 and purified via silica gel column chromatography using the same mobile phase to afford 45 mg, 0.06 mmol; 68% yield of 131- β-alanylchlorin e6 (tert)butyl dimethyl ester (C43H53N5O7). 1H NMR [(CD3)2CO, 400 MHz]: δ 9.70 (s, 1H), 9.63 (s, 1H), 9.01 (s, 1H), 8.17 (t, J = 5.71 Hz, 1H), 8.11 (dd, J = 11.58, 17.85 Hz, 1H), 6.31 (d, J = 18.85 Hz, 1H), 6.04 (d, J = 11.87 Hz, 1H), 5.65 (d, J = 18.95 Hz, 1H), 5.38 (d, J = 19.06 Hz, 1H), 4.66 (q, J = 7.22 Hz, 1H), 4.52 (dd, J = 10.34, 1.95 Hz, 1H), 4.02 (m, 1H), 3.09 (m, 1H), 3.77 (s, 3H), 3.68 (q, J = 7.3 Hz, 2H), 3.61 (s, 3H), 3.51 (s, 3H), 3.46 (s, 3H), 3.21 (s, 3H), 2.71 (m, 2H), 2.31 (m, 2H), 1.79 (m, 1H), 1.72 (d, J = 7.24, 3H), 1.65 (t, J = 7.58, 3H), 1.54 (s, 9H), −1.62 (s, 1H), −1.91 (s, 1H). The 131- β-alanylchlorin e6 (tert)butyl dimethyl ester (45 mg, 0.059 mmol) was dissolved in 1.5 ml of dry CH2Cl2 in a ice bath under argon. Thioanisole (0.005 ml) and 1.5 ml of TFA were added and the reaction mixture was stirred overnight before being diluted with CH2Cl2 and washed three times with H2O and once with 10% NaHCO3. The organic layer was dried over anhydrous Na2SO4 and the solvent was evaporated to give 39 mg, 0.056 mmol; 95% yield of 131- β-alanylchlorin e6 DME (C39H45N5O7). 1H NMR [(CD3)2CO, 400 MHz]: δ 9.74 (s, 1H), 9.72 (s, 1H), 9.10 (s, 1H), 8.22 (dd, J = 11.58, 17.85 Hz, 1H), 8.15 (t, J = 5.71 Hz, 1H), 6.39 (d, J = 18.85 Hz, 1H), 6.12 (d, J = 11.87 Hz, 1H), 5.65 (d, J = 18.95 Hz, 1H), 5.37 (d, J = 19.06 Hz, 1H), 4.66 (q, J = 7.22 Hz, 1H), 4.52 (dd, J = 10.34, 1.95 Hz, 1H), 4.07 (m, 1H), 3.95 (m, 1H), 3.77 (s, 3H), 3.68 (q, J = 7.3 Hz, 2H), 3.61 (s, 3H), 3.53 (s, 3H), 3.52 (s, 3H), 3.28 (s, 3H), 2.69 (m, 2H), 2.31 (m, 2H), 1.79 (m, 1H), 1.72 (d, J = 7.24, 3H), 1.65 (t, J = 7.58, 3H), −1.62 (s, 1H), −1.91 (s, 1H).

131-β-Alanyl-aspartylchlorin e6 DME (11)

Chlorin e6 DME (39 mg, 0.056 mmol) was dissolved in dry CH2Cl2. A mixture of HOBt (8 mg), TBTU (18 mg) and DIEA (0.017 ml) in DMF was added and stirred for 30 min. H-Asp(OtBu)2•HCl (40 mg) and DIEA (0.025 ml) were mixed in CH2Cl2 and added to the reaction mixture. The mixture was stirred overnight before being diluted with CH2Cl2 and then washed with 5% aqueous citric acid, followed by a wash with brine and water. It was dried over anhydrous Na2SO4and then evaporated. The residue was dissolved in 10% acetone/CH2Cl2 and purified via silica gel column chromatography using the same mobile phase to afford 45 mg, 0.048 mmol; 87% yield of 131- β-alanyl-aspartylchlorin e6 di(tert)butyl dimethyl ester. 1H NMR [(CD3)2CO, 400 MHz]: δ 9.74 (s, 1H), 9.70 (s, 1H), 9.10 (s, 1H), 8.19 (dd, J = 11.58, 17.85 Hz, 1H), 8.11 (t, J = 5.66 Hz, 1H), 7.64 (d, J = 8.11 Hz, 1H), 6.37 (d, J = 19.18 Hz, 1H), 6.10 (d, J = 12.94 Hz, 1H), 5.66 (d, J = 19.03 Hz, 1H), 5.38 (d, J = 19.09 Hz, 1H), 4.77 (dt, J = 8.14, 5.74 Hz, 1H), 4.66 (q, J = 7.22 Hz, 1H), 4.52 (dd, J = 10.34, 1.95 Hz, 1H), 4.07 (m, 1H), 3.95 (m, 1H), 3.75 (s, 3H), 3.76 (q, J = 7.3 Hz, 2H), 3.60 (s, 3H), 3.53 (s, 3H), 3.50 (s, 3H), 3.27 (s, 3H), 2.80 (d, J = 2.57 Hz, 1H), 2.79 (d, J = 3.06 Hz, 1H), 2.31 (m, 2H), 1.79 (m, 1H), 1.71 (d, J = 7.24, 3H), 1.68 (t, J = 7.58, 3H), 1.43 (s, 9H), 1.42 (s, 9H), −1.61 (s, 1H), −1.91 (s, 1H). The 131- β-alanyl-aspartylchlorin e6 di(tert)butyl dimethyl ester (45 mg, 0.048 mmol) was dissolved in 2 ml of dry CH2Cl2 in a ice bath under argon. Thioanisole (0.004 ml) and 2 ml of TFA were added and the reaction mixture was stirred overnight before being evaporated several times with diethyl ether to remove residual TFA. The resulting precipitate was washed several times with diethyl ether to remove more residual thioanisole. The precipitate was dissolved in CH2Cl2 and washed three times with H2O and once with 10% NaHCO3 to remove TFA. The organic layer was dried over anhydrous Na2SO4 and the solvent was evaporated to give 38 mg, 0.046 mmol; 97% yield of 131-aspartylchlorin e6 DME (C43H50N6O10). UV-Vis (acetone): λmax (ε/M−1cm−1) 663 (77,125), 607 (3,621), 528 (1,831), 500 (17,200), 399 (237,100); 1H NMR [(CD3)2CO, 400 MHz]: δ 9.72 (s, 1H), 9.70 (s, 1H), 9.10 (s, 1H), 8.20 (dd, J = 11.58, 17.85 Hz, 1H), 8.11 (br t, J = 5.66 Hz, 1H), 7.64 (br d, J = 8.11 Hz, 1H), 6.32 (d, J = 19.18 Hz, 1H), 6.10 (d, J = 12.94 Hz, 1H), 5.63 (d, J = 19.03 Hz, 1H), 5.37 (d, J = 19.09 Hz, 1H), 4.92 (dt, J = 8.14, 5.74 Hz, 1H), 4.66 (q, J = 7.22 Hz, 1H), 4.51 (dd, J = 10.34, 1.95 Hz, 1H), 4.07 (m, 1H), 3.95 (m, 1H), 3.74 (s, 3H), 3.75 (q, J = 7.3 Hz, 2H), 3.60 (s, 3H), 3.51 (s, 3H), 3.50 (s, 3H), 3.25 (s, 3H), 2.96 (d, J = 5.27 Hz, 1H), 2.69 (d, J = 3.06 Hz, 1H), 2.31 (m, 2H), 1.79 (m, 1H), 1.71 (d, J = 7.24, 3H), 1.66 (t, J = 7.58, 3H), −1.61 (s, 1H), −1.91 (s, 1H). HRMS (MALDI-TOF) m/z 811.381 [M+H]+, calcd. for C43H51N6O10 811.367.

131-Ethylenediaminylchlorin e6 DME (12)

Methyl pheophorbide a, (100 mg, 0.164 mmol) was dissolved in dry CHCl3 and stirred under argon for 10 min. 0.2 ml of ethylenediamine was added to the solution and the mixture stirred for 24 h. The reaction was monitored by spectrophotometry. The reaction mixture was evaporated and the residue was dissolved in 2.5% MeOH/CH2Cl2 and then chromatographed on a short silica gel column using the same mobile phase to remove by-products, and then the product was eluted using 50% MeOH/CH2Cl2 to afford 100 mg, 0.150 mmol: 91% yield of 131-ethylenediaminylchlorin e6 DME (C38H46N6O5). 1H NMR [(CD3)2CO, 400 MHz]: δ 9.69 (s, 1H), 9.63 (s, 1H), 9.09 (s, 1H), 8.09 (dd, J = 17.78, 11.58 Hz, 1H), 8.07 (br s, 1H), 6.29 (d, J = 18.85 Hz, 1H), 6.02 (d, J = 11.87 Hz, 1H), 5.67 (d, J = 19.08 Hz, 1H), 5.39 (d, J = 19.11 Hz, 1H), 4.66 (q, J = 7.22 Hz, 1H), 4.51 (dd, J = 10.34, 1.95 Hz, 1H), 4.01 (m, 1H), 3.87 (m, 1H), 3.75 (s, 3H), 3.66 (q, J = 7.3 Hz, 2H), 3.61 (s, 3H), 3.50 (s, 3H), 3.45 (s, 3H), 3.20 (s, 3H), 2.71 (m, 1H), 2.31 (m, 2H), 1.95 (br m, 1H), 1.80 (m, 1H), 1.72 (d, J = 7.24, 3H), 1.64 (t, J = 7.58, 3H), −1.64 (s, 1H), −1.93 (s, 1H). 13C NMR [(CD3)2CO, 100 MHz] δ 173.1, 173.0, 169.2, 168.3, 167.7, 153.6, 149.1, 144.4, 138.1, 136.0, 135.3, 134.7, 133.9, 130.1, 129.8, 129.3, 120.9, 103.2, 100.7, 98.4, 93.9, 53.1, 51.4, 50.8, 50.2, 48.8, 41.2, 37.2, 30.7, 29.6, 22.6, 19.0, 17.2, 11.3, 11.0, 10.3. HRMS (MALDI-TOF) m/z 667.395 [M+H]+, calcd. for C38H47N6O5 667.361.

131-Ethylenediaminyl-lysylchlorin e6 DME (13)

(Boc)Lysine(Boc)OH (55 mg, 0.082 mmol) was dissolved in dry CH2Cl2 (10 ml) A mixture of HOBt (15 mg), TBTU (33 mg) and DIEA (0.025 ml) in DMF was added and then stirred for 1 h. 131-Ethylenediaminylchlorin e6 DME (50 mg, 0.075 mmol) was added to the reaction mixture, which was stirred overnight. The mixture was diluted with CH2Cl2 and then washed with 5% aqueous citric acid, followed by a wash with brine and water. It was dried over anhydrous Na2SO4 and then evaporated. The residue was dissolved in 2.5% MeOH/CH2Cl2 and purified via silica gel column chromatography using the same mobile phase to afford 65 mg, 0.065 mmol; 78% yield of 131-ethylenediaminyl(Boc)lysyl(Boc)chlorin e6 DME (C54H74N8O10). 1H NMR [(CD3)2CO, 400 MHz]: δ 9.54 (s, 1H), 9.53 (s, 1H), 9.07 (s, 1H), 8.14 (br s, 1H) 7.94 (dd, J = 17.78, 11.58 Hz, 1H), 7.78 (br s, 1H), 6.18 (d, J = 18.85 Hz, 1H), 6.16 (br s, 1H), 5.93 (d, J = 11.87 Hz, 1H), 5.89 (br s, 1H), 5.63 (d, J = 19.08 Hz, 1H), 5.39 (d, J = 19.11 Hz, 1H), 4.67 (q, J = 7.22 Hz, 1H), 4.52 (dd, J = 10.34, 1.95 Hz, 1H), 4.17 (m, 1H), 3.85 (m, 1H), 3.75 (s, 3H), 3.66 (m, 3H), 3.61 (s, 3H), 3.52 (br q, J = 7.14 Hz, 2H), 3.41 (s, 3H), 3.39 (s, 3H), 3.11 (s, 3H), 3.00 (m, 2H), 2.71 (m, 1H), 2.32 (m, 2H), 1.95 (br m, 1H), 1.84 (m, 2H), 1.72 (d, J = 7.24, 3H), 1.67 (m, 1H), 1.59 (t, J = 7.58, 3H), 1.41 (m, 3H), 1.37 (s, 9H), 1.34 (s, 9H), −1.66 (s, 1H), −1.94 (s, 1H). The 131- ethylenediaminyl(Boc)lysyl(Boc)-chlorin e6 DME (65 mg, 0.065 mmol) was dissolved in 3 ml of dry CH2Cl2 in a ice bath under argon. Thioanisole (0.003 ml) and 2 ml of TFA were added and the reaction mixture was stirred overnight. The reaction mixture was evaporated several times with diethyl ether to remove TFA. The resulting precipitate was washed several times with diethyl ether to remove thioanisole. Then the precipitate was dissolved in CH2Cl2 and washed three times with H2O and once with 10% NaHCO3 to remove TFA. The organic layer was dried over anhydrous Na2SO4 and the solvent was evaporated to give 42 mg, 0.052 mmol; 81% yield of 131-ethylenediaminyl-lysylchlorin e6 DME (C44H58N8O6). UV-Vis (acetone): λmax (ε/M−1cm−1) 663 (76,420), 607 (1810), 527 (657), 500 (14,560), 399 (242,100); 1H NMR [(CD3)2CO, 400 MHz]: δ 9.76 (s, 1H), 9.69 (s, 1H), 9.10 (s, 1H), 8.29 (br s, 1H) 8.17 (dd, J = 17.78, 11.58 Hz, 1H), 7.86 (br s, 1H), 6.35 (d, J = 18.85 Hz, 1H), 6.07 (d, J = 11.87 Hz, 1H), 5.64 (d, J = 19.08 Hz, 1H), 5.40 (d, J = 19.11 Hz, 1H), 4.66 (q, J = 7.22 Hz, 1H), 4.52 (dd, J = 10.34, 1.95 Hz, 1H), 3.92 (m, 2H), 3.75 (br m, 5H), 3.74 (s, 3H), 3.60 (s, 3H), 3.53 (s, 3H), 3.49 (s, 3H), 3.26 (s, 3H), 2.96 (br t, 2H), 2.70 (m, 1H), 2.29 (m, 2H), 1.95 (br m, 1H), 1.84 (m, 2H), 1.72 (d, J = 7.24, 3H), 1.68 (t, J = 7.58, 3H), 1.45 (m, 2H), 1.31 (m, 2H) −1.60 (s, 1H), −1.89 (s, 1H). HRMS (MALDI-TOF) m/z 795.492 [M+H]+, calcd. for C44H59N8O6 795.456.

2. Cell Studies

Human carcinoma HEp2 cells were maintained in a 50:50 mixture of DMEM:AMEM (Invitrogen) supplemented with 5% FBS (Invitrogen), 1% Primocin antibiotic (Invitrogen) in a humidified, 5% CO2 incubator at 37 °C. The cells were sub-cultured twice weekly to maintain sub-confluent stocks. The 4th to 15th passage cells were used for all experiments.

2.1. Time-Dependent Cellular Uptake

The HEp2 cells were plated at 7500 cells per well in a Costar 96-well plate and allowed to grow for 48 h. Compound stock solutions were prepared at 32 mM in DMSO and Cremophor (10% of Cremophor in DMSO). Further dilution into the cells of the 96-well plate gave a final concentration of 10 µM with maximum DMSO concentration of 1% and Cremophor concentration of 0.1%. For the uptake test, the compounds were diluted to 20 µM (2× stock solution) and added to the 96-well to give a final concentration of 10 µM at 0, 1, 2, 4, 8, 12 and 24 h interval. The uptake was terminated by removing the loading medium and washing the wells with 200 µL PBS buffer. Cells and compounds were then solubilized using 100 µL of 0.25% Triton X-100 in PBS buffer. The compound concentration was measured using intrinsic fluorescence as measured with a BMG FLUOstar plate reader equipped with a 355 nm excitation and a 650 nm emission filter. The cells were measured using a CyQuant Cell proliferation assay (Invitrogen) as per manufacturer’s instructions, as previously reported.20,21,24

2.2. Dark Cytotoxicity

The HEp2 cells were plated as described above for the uptake experiment. The compounds were diluted into media to give 400 µM solution concentrations. Two-fold serial dilutions were then prepared from 400 to 50 µM, and the cells were incubated for 18 h. The loading medium was removed and the cells were fed new medium, followed by 20 µL CellTiter blue per 100 µL medium, and incubated for 4 h. Cell toxicity was measured using Promega’s CellTiter Blue Viability Assay Kit as per manufacturer’s instructions; untreated cells were considered 100% viable and cells treated with 0.2% saponin as 0% viable, as previously reported.20,21,24

2.3. Phototoxicity

The cells were prepared as described above with compound concentration range from 6.25–100 µM. After loading overnight, the medium was replaced with medium containing 50 mM HEPES pH 7.2. The cells were exposed to a 100 W halogen lamp filtered through a 610 nm long pass filter to provide approximately 1 Jcm−2 light dose. The cells were kept cool by filtering the IR radiation through 10 mm of water and placing the culture in an ice-water bath. After exposure to light for 20 min, the plate was incubated in the 37 °C, 5% CO2 incubator overnight. Cell viability was then measured as described above using CellTiter blue Viability Assay Kit, as previously reported.20,21,24

2.4. Microscopy

The cells were incubated in a glass bottom 6-well plate (MatTek) and allowed to grow for 48 h. The cells were then exposed to 10 µM of each compound for 18 h. Organelle tracers were obtained from Invitrogen and used at the following concentrations: LysoSensor Green 50 nM, MitoTracker Green 250 nM, ER Tracker Blue/white 100 nM, and BODIPY FL C5 ceramide 1 mM. After 30 min incubation in the 37 °C, 5% CO2 incubator, both the media and the organelle tracers were removed and washed with PBS buffer for 3 times. Images were acquired using a Leica DMRXA microscope with 40× NA 0.8 dip objective lens and DAPI, GFP and Texas Red filter cubes (Chroma Technologies).

Supplementary Material

1_si_001

Acknowledgments

This work was supported by the National Institutes of Health, grant numbers CA132861 (KMS) and CA139385 (MGHV).

Footnotes

1Abbreviations: PS, photosensitizer; PDT, photodynamic therapy; DMF, dimethylformamide; DMSO, dimethylsulfoxide; THF, tetrahydrofuran; DME, dimethyl ester; TME, trimethyl ester; DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; DCC, 1,3-dicyclohexylcarbodiimide; TFA, trifluoroactic acid; DIEA, N,N-diisopropylethylamine; TBTU, 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate; DMAP, 4-dimethylaminopyridine; HOBt, 1-hydroxybenzotriazole; PBS, phosphate buffered saline; FBS, fetal bovine serum; MEM, modified eagle medium; ER, endoplasmic reticulum.

Supporting Information Available: Normalized absorption spectra, energy minimized conformations in water, time-dependent uptake, and subcellular localization microscopy figures (S1–S10). Proton NMR spectra for compounds 2–13 and other synthetic intermediates, carbon- 13 NMR shifts, carbon-13 NMR spectra (21 pages). This material is available free of charge via the internet at http://pubs.acs.org.

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