Synthesis and Characterization of Positively Charged Pentacationic [60]Fullerene Monoadducts for Antimicrobial Photodynamic Inactivation

We designed and synthesized two analogous pentacationic [60]fullerenyl monoadducts, C60(>ME1N6+C3) (1) and C60(>ME3N6+C3) (2), with variation of the methoxyethyleneglycol length. Each of these derivatives bears a well-defined number of cationic charges aimed to enhance and control their ability to target pathogenic Gram-positive and Gram-negative bacterial cells for allowing photodynamic inactivation. The synthesis was achieved by the use of a common synthon of pentacationic N,N′,N,N,N,N-hexapropyl-hexa(aminoethyl)amine arm (C3N6+) having six attached propyl groups, instead of methyl or ethyl groups, to provide a well-balanced hydrophobicity–hydrophilicity character to pentacationic precursor intermediates and better compatibility with the highly hydrophobic C60 cage moiety. We demonstrated two plausible synthetic routes for the preparation of 1 and 2 with the product characterization via various spectroscopic methods.


Introduction
Broad-spectrum one-photon based photodynamic therapy (1γ-PDT)-mediated killing of pathogenic Gram-positive (e.g., Staphylococcus aureus) and Gram-negative (e.g., Escherichia coli) bacterial targets using a conventionally accessible light source is an emerging medical approach to treat infectious diseases, especially, those caused by multi-antibiotic-resistant bacteria [1][2][3][4][5]. The efficacy of 1γ-PDT depends on several parameters, including photophysical characteristics of the photosensitizer, the ability of the photosensitizer to target bacteria, the method of administration, and the availability of an appropriate light source. Fullerenes are highly photostable molecules suitable for single-dose multiple-treatments applications. Nearly quantitative efficiency of intersystem crossing from the excited singlet state of [60]fullerene ( 1 C 60 *) to its triplet excited state ( 3 C 60 *) readily allows intermolecular triplet energy transfer from 3 C 60 * to molecular oxygen leading to the production of singlet oxygen ( 1 O 2 ) [6,7], which is highly reactive toward biological substrates producing subsequent cell damage. This photochemical mechanism serves as the basis of photodynamic cytotoxicity against pathogenic microorganisms, including multi-antibiotic-resistant bacteria. However, chemical functionalization of C 60 is necessary to enhance its solubility in water. In general, attachment of multiple hydroxyl, carboxylic acid, and glycolic oxide addend groups may serve the purpose. Water-solubility of these derivatives increases as the number of hydrophilic groups increases, whether these functional groups are located in either the same addend group or different addend moieties. The latter case leads to the synthesis of fullerenyl multiadducts that may change significantly the molecular orbital configuration of the fullerene cage and, thus, its HOMO-LUMO energy gap level and effectiveness in the production of 1 O 2 . The 1γ-PDT efficiency can be optimized by performing only a limited number of addition reactions to the fullerenyl olefinic bonds to preserve the low HOMO-LUMO energy gap level of the cage. This restriction suggests that [60]fullerene monoadducts using hydrophilic or amphiphilic groups would be suitable candidates. However, since only one addend group is able to be attached to the cage in a monoadducts, sufficient hydrophilicity is required to allow compatibility of the resulting derivative with water.
It has been demonstrated that polycationic photosensitizers exhibited high activity as 1γ-PDT agents for targeting and photokilling against both Gram-positive and Gram-negative bacterial species [8,9]. These reported findings revealed the importance of cell surface interactions between multicationic drug molecules and anionic peptide residues in the cell wall. Specifically, several factors including differences in physiology, cell wall, and cytoplasmic membrane structures between Gram-positive and Gram-negative bacteria [10−12] affect the properties of particular functional groups to be attached on the fullerene cage to allow effective targeting selectivity, drug-delivery, and photodynamic inactivation.
Accordingly, we considered the structural modification of C 60 to allow the increase of its solubility in physiologic media and its compatibility in an environment of bacterial disease in tissue. This led to our design and synthesis of [60]fullerenyl monoadducts bearing a well-defined high number of cationic charges that hitherto had remained challenging and has rarely been reported to date. In this paper, we describe a rational linkage of water-soluble quaternary alkylammonium multi-salts and ester-amide functional groups to a well-defined pentacationic arm together with an efficient synthetic method for its attachment on a C 60 nanocage. The synthesis led to the preparation of new pentacationic [60]fullerene-based nano-photosensitizers 1 and 2, as shown in Schemes 1 and 2. Scheme 1. The first synthetic steps of C 60 (>ME 1 N 6 + C 3 ) 1 and C 60 (>ME 3 N 6 In these structures, arm moieties each bearing a high number of cationic charges and an amide moiety are capable of inducing the H-bonding in the vicinity of the fullerene cage.

Results and Discussion
Enhancement of the hydrophilicity of fullerene derivatives can be achieved by incorporation of the oligo(ethylene glycol) unit [13][14][15], an aminoacid moiety [16], or ionic functional groups [17][18][19] as addend attachments of C 60 cage. The resulting amphiphilic derivatives have been reported to undergo different forms of solid aggregation in aqueous solution if the hydrophilic moiety of the addend is insufficiently large to overcome the high hydrophobicity of the fullerene cage. To circumvent this solid aggregation problem, we undertook the effort to synthesize a well-defined water-soluble pentacationic N, N′,N,N,N,N-hexapropyl-hexa(aminoethyl)amine arm moiety C 3 N 6 + , as a charged C 3 N 6 (6), with the number of charge being fixed at five per arm and used as the common synthon in the preparation of [60]fullerene monoadducts, as shown in Scheme 1. One example was given by the combination of a water-compatible ethylene glycol unit with a C 3 N 6 + arm to a single addend, such as the arm precursors ME 1 N 6 + C 3 (9) and ME 3 N 6 + C 3 (10), to enhance the water-solubility. Synthesis of 9 and 10 began with the reaction of either 2-methoxyethanol or triethylene glycol monomethyl ether with 2,2-dimethyl-1,3-dioxane-4,6-dione (3, Meldrum's acid) at 90-95 °C for a period of 12 h to afford malonic acid methoxyethyleneglycol ester, ME 1 (4), or malonic acid methoxytriethyleneglycol ester, ME 3 (5), in 95 or 90% yield, respectively. Amidation reaction of 4 and 5 was carried out by the treatment with N-hydroxysuccinamide and N,N′-dicyclohexyl carbodiimide (DCC) in anyhydrous THF at ambient temperature over a period of 12 h, followed by the removal of insoluble byproduct of N,N′-dicyclohexyl urea and the further treatment with N,N′,N,N,N,N-hexapropyl-hexa(aminoethyl)amine, C 3 N 6 (6), for an additional period of 12 h. These reactions resulted in the corresponding products of methoxyethyleneglycol-[N,N′,N,N,N,N-hexapropyl-hexa(aminoethyl)-amino]malonamide ester, ME 1 N 6 C 3 (7), and methoxy-tri(ethyleneglycol)-[N,N′,N,N,N,N-hexapropyl-hexa(aminoethyl)amino]malonamide ester, ME 3 N 6 C 3 (8), in 82 and 80% yield, respectively. Quaternization reaction of mono-and tri(ethoxylated) hexaaminomalonamide precursors 7 and 8 using methyl iodide as the methylation agent at 45-50 °C for a period of 3.0 days afforded the corresponding methoxyethyleneglycol-[N,N′,N,N,N,N-hexapropyl-hexa(aminoethyl)amino]malonamide ester methyl quaternary ammonium salt, ME 1 N 6 + C 3 (9), and its tri(ethyleneglycolated) analogue ME 3 N 6 + C 3 (10) in 94 and 92% yield, respectively. Attachment of a C 60 cage on the quaternary ammonium salts of malonamide was accomplished by the treatment of 9 and 10 in DMF with predissolved C 60 in toluene in the presence of 1.8-diazabicyclo [5.4.0]-undec-7-ene (DBU) at ambient temperature for a period of 8.0 h. In this reaction, carbon tetrabromide was applied as the bromination agent for the replacement of malonyl α-proton in situ. To minimize the possible formation of partial fullerenyl byproducts containing multiaddends, an excess amount (2.5 equiv.) of C 60 was applied. At the end of fullerenation, an excessive amount of C 60 molecules was recovered and removed by repeatedly washing the crude products with toluene until the observation of a clear toluene solution in washings. The reaction procedure led to the isolation of methoxyethyleneglycol-(20-oxo-4,7,10,13,16-pentapropyl-4,7,10,13,16,19-hexaaza-nonadecan-19-yl)[60]fullerenyl malonate quaternary methyl ammonium salt, C 60 (>ME 1 N 6 + C 3 ) (1), and its tri(ethyleneglycolated) analogue C 60 (>ME 3 N 6 + C 3 ) (2) in 55 and 50% yield, respectively.

Figure 1.
FT-IR spectra of (a) ME 3 N 6 C 3 8, (b) ME 3 N 6 + C 3 10, and (c) C 60 (>ME 3 N 6 Mass spectroscopic data collection of both 1 and 2 was proven to be difficult due to their polycationic nature and facile fragmentations occurring at the conjunction of the C 60 cage and the pentacationic malonate arm, giving mainly the highly detectable C 60 ion mass fragment at m/z 721, as displayed in MALDI−TOF mass spectra using sinapic acid as the matrix. All fragmented high mass ions were very weak in intensity including peaks at m/z 1930 (M + -I − ) (supporting information) and 2019 (MH + -I − ) as the molecular ion of 1 and 2, respectively. The most pronounced mass fragmentation ions at m/z 874 and 876 in both spectra of 1 and 2 were assigned to the substructure of C 60 [>H(C=O)NHCH 2 CH 2 N + -PrMe 2 ], indicating a main malonylamide moiety being attached on a C 60 cage, consistent with the [60]fullerenyl monoadduct structure.
In the case 1 H-NMR spectroscopic analyses, we first well-characterized the arm structures ME 1 (t-C 4 ) (11, Figure 2a) and ME 3 (t-C 4 ) (12) by identification of all protons (H α , H e , H f , H g , and H i indicated in Scheme 2) with their assignments to peaks shown in the spectra. Attachment of 11 on C 60 leading to a monoadduct C 60 [>ME 1 (t-C 4 )] (13) was evident by the disappearance of a H α proton peak with the chemical shift at δ 3.33. It was also accompanied with up-fielded shifts of two proton peaks to δ 3.06 and 3.26, corresponding to the chemical shift of terminal methoxy protons H g and ethylene oxide 4000 3500 3000 2500 2000 1500 1000 500 protons H f , in toluene-d 8 from δ 3.34 and 3.56 of 11 in CDCl 3 , respectively, reflecting partly the solvent effect. Hydrolysis of 13 to C 60 (>ME 1 H) (15) was also apparent by the loss of t-butyl protons at δ 1.55, as shown in Figures 2b and 2c, accompanied with down-fielded shifts of proton peaks (H g , H f , and H e ) to δ 3.34, 3.71, and 4.59, respectively, in THF-d 8 showing a larger solvent effect. Monoadduct structures of 13 and 14 were also confirmed by the detection of well-defined characteristic fullerenyl sp 2 carbon peaks in their 13 C-NMR spectra (Figure 3b) showing a group of 26 and 27 peaks, respectively, each accounted for two carbons and a group of 6 single carbon peaks for 13 (two of these single carbon peaks may be derived from the slightly unsymmetrical environment around the malonate moiety) and   In addition, the spectra of both compounds 15 and 16 displayed a group of 27 peaks each accounted for two sp 2 fullerenyl carbons and four single sp 2 carbon peaks in the region of  135-145 provided the further confirmation of a C v molecular symmetry of C 60 (>ME 1 H) and C 60 (>ME 3 H), respectively, as monoadducts without change during the chemical reaction. Attachment of hexa(aminoethyl)amine C 3 N 6 + arm to the malonic acid moiety of C 60 (>ME 1 H) leading to the structure of C 60 (>ME 1 N 6 + C 3 ) 1′ and subsequently 1 was confirmed by the analyses of both infrared and 1 H-NMR spectra. The latter of 1′ showed proton peaks corresponding to the C 3

Spectroscopic Measurements
Infrared spectra were recorded as KBr pellets on a Thermo Nicolet Avatar 370 FT-IR spectrometer. 1 H-NMR and 13 C-NMR spectra were recorded on a Bruker Avance Spectrospin-500 spectrometer.
UV-Vis spectra were recorded on a Perkin Elmer Lambda 750 UV-Vis-NIR Spectrometer. MALDI-mass spectra were recorded on a WATERS Micromass MALDI-TOF mass spectrometer. Elemental analysis was taken by Galbraith Laboratories, Inc. (Knoxville, TN, USA).

Synthesis of Malonic Acid Methoxytriethyleneglycol Ester, ME 3 (5)
A mixture of triethylene glycol monomethyl ether (1.0 g, 6.1 mmol) and 2,2-dimethyl-1,3-dioxane-4,6-dione (3, Meldrum's acid, 0.9 g, 6.2 mmol) was stirred under Ar atmosphere over a period of 12 h at 90 °C. The reaction mixture was cooled to room temperature, treated with aqueous sodium carbonate solution (5%), and washed with diethyl ether. The resulting aqueous layer was subsequently treated with dil. HCl (2.0 N) and extracted with ethyl acetate (50 mL). The ethyl acetate solution was dried over Na 2 SO 4 and concentrated on rotary evaporator to give the product ME 3 (5) in 90% yield

Synthesis of Methoxyethyleneglycol-[N,N′,N,N,N,N-hexapropyl-hexa(aminoethyl)amino]malonamide Ester, ME 1 N 6 C 3 (7)
A mixture of malonic acid methoxyethyleneglycol ester 4 (0.5 g, 3.08 mmol), N-hydroxysuccinamide (0.35 g, 3.08 mmol), and N,N′-dicyclohexyl carbodiimide (DCC, 0.63 g, 4.0 mmol) in anyhydrous tetrahydrofuran (20 mL) were stirred under N 2 atmosphere over a period of 12 h at ambient temperature. The resulting white solids of N,N′-dicyclohexyl urea byproduct were filtered off and the filtrate was taken into a second round-bottom flask containing N,N′,N,N,N,N-hexapropyl-hexa(aminoethyl)amine 6 (1.49 g, 3.08 mmol). The mixture was stirred under N 2 atmosphere for an additional period of 12 h. At the end of the reaction, the solvent was removed on rotavap. To this residue, ice-cold hexane-dichloromethane (1:1, 15 mL) was added followed by filtration to remove further white solids of N-hydroxysuccinamide. The filtrate was washed with aqueous sodium carbonate (5%) solution (10 mL). The organic layer was then dried and concentrated to give ME 1 N 6 C 3 (7) N,N′,N,N,N,N-hexapropylhexa(aminoethyl)amine 6 (1.94 g, 3.9 mmol). The mixture was stirred under N 2 atmosphere for an additional period of 12 h. At the end of the reaction, the solvent was removed on rotavap. To this residue, ice-cold hexane-dichloromethane (1:1, 20 mL) was added followed by filtration to remove white solids of N-hydroxysuccinamide. The filtrate was washed with aqueous sodium carbonate (5%) solution (10 mL). The organic phase was then dried and concentrated to give ME 3 N 6 C 3 (8)     Finely divided [60]fullerene (0.94 g, 1.30 mmol, more than two-fold excess to allow the formation of monoadduct only) was taken into a round bottom flask and added anhydrous toluene (700 mL) under nitrogen. The solution was stirred for 12 h at ambient temperature to ensure complete dissolution of C 60 . To the resulting purple-colored solution added carbon tetrabromide (0.19 g, 0.57 mmol) followed by a solution of the compound 9 (0.70 g, 0.52 mmol) in anhydrous DMF (100 mL). The solution mixture was stirred for an additional 30 min and added slowly 1.8-diazabicyclo [5.4.0]-undec-7-ene (DBU, 0.17 g, 1.15 mmol) over a period of 45 min. The color of solution slowly turns into brown in a reaction period of 8.0 h. The solution was then concentrated on rotavap to roughly 100 mL. Upon the addition of methanol to this concentrated solution, the crude product was precipitated as brown solids which were collected via centrifugation. Unreacted C 60 in the crude solids was removed by repeated washings with toluene (5 × 100 mL) until no color in the washing solution or filtrate. The remaining product of C 60 (>ME 1 N 6 + C 3 ) (1) was obtained as brown solids in 55% yield (0.59 g, after recovered C 60 ). Finely divided [60]fullerene (1.0 g, 1.40 mmol, more than two-fold excess to allow the formation of monoadduct only) was taken into a round bottom flask and added anhydrous toluene (700 mL) under nitrogen. The solution was stirred for 12 h at ambient temperature to ensure complete dissolution of C 60 . To the resulting purple-colored solution added carbon tetrabromide (0.17 g, 0.51 mmol) followed by a solution of compound 10 (0.65 g, 0.45 mmol) in anhydrous DMF (100 mL). The solution mixture was stirred for an additional 30 min of stirring and added slowly 1.8-diazabicyclo [5.4.0]-undec-7-ene (DBU, 0.15 g, 0.98 mmol) over a period of 45 min. The color of solution slowly turns into brown in a reaction period of 8 h. The solution was then concentrated on rotavap to roughly 100 mL. Upon the addition of methanol to this concentrated solution, the crude product was precipitated as brown solids which were collected via centrifugation. Unreacted C 60 in the crude solids was removed by repeated washings with toluene (5 × 100 mL) until no color in the washing solution or filtrate. The remaining product of C 60 (>ME 3   3.3.11. Synthesis of tert-Butyl (2-methoxyethyl)[60]fullerenyl Malonate, C 60 [>ME 1 (t-C 4 )] (13) Finely divided [60]fullerene (1.23g, 1.70 mmol) was taken into a round bottom flask and added anhydrous toluene (850 mL) and 1,2-dichlorobenzene (30 mL) under nitrogen. The solution was stirred for 1.0 h at ambient temperature to ensure complete dissolution of C 60 . To the resulting purplecolored solution was added carbon tetrabromide (0.50 g, 1.51 mmol) followed by a solution of tertbutyl(2-methoxyethyl)malonate (11, 0.30 g, 1.37 mmol) in anhydrous toluene (10 mL). The solution mixture was stirred for an additional 30 min and added slowly 1.8-diazabicyclo [5.4.0]-undec-7-ene 3.3.13. Synthesis of 2-[60]Fullerenyl-3-(2-methoxyethoxy)-3-oxopropanoic Acid, C 60 (>ME 1 H) (15) The compound of [60]fullerenyl malonate 13 (0.5 g, 0.64 mmol) was taken into a round bottom flask containing anhydrous dichloromethane (50 mL) and purged with N 2 for 15 minutes at ambient temperature. To this reaction mixture was added trifluoroacetic acid (30 mL, excess) and stirred for overnight at room temperature. At the end of the reaction, dichloromethane was removed on rotavap. Additional dichloromethane (3 × 15 mL) was added and removed on rotavap in order to fully eliminate an excessive amount of trifluoroacetic acid. The resulting residue was then washed with diethyl ether (2 × 15 mL) to afford C 60 (>ME 1 H) (15) The compound of [60]fullerenyl malonate 14 (0.75 g, 0.73 mmol) was taken into a round bottom flask containing anhydrous dichloromethane (50 mL) and purged with N 2 for a period of 15 minutes at ambient temperature. To this reaction mixture was added trifluoroacetic acid (50 mL) and stirred for overnight at room temperature. At the end of the reaction, dichloromethane was removed on rotavap. Additional dichloromethane (3 × 20 mL) was added and removed on rotavap in ordered to fully eliminate an excessive amount of trifluoroacetic acid. The resulting residue was then washed with diethyl ether (3 × 20 mL) to afford C 60 (>ME 3 H) (16) as brown solids in 87% yield (0.62 g).

Conclusions
We have designed and synthesized two analogous pentacationic [60]fullerenyl monoadducts, C 60 (>ME 1 N 6 + C 3 ) (1) and C 60 (>ME 3 N 6 + C 3 ) (2), with variation of the methoxyethyleneglycol length. Each of these derivatives bears a well-defined number of cationic charges aimed to enhance and control their ability to target pathogenic Gram-positive and Gram-negative bacterial cells. The intrinsic nature of the high charge number and increased water-solubility of the precursor arm intermediates hindered the efficiency of their reactions with a highly hydrophobic C 60 cage having low compatibility in polar solvents. Furthermore, consecutive ethylamino group linkage in a structure of N,N′,N,N,N,Nhexapropyl-hexa(aminoethyl)amine (C 3 N 6 ) largely increased its electron-donating capability that gave complication in forming an insoluble partial charge-transfer complex with C 60 during the reaction and workup procedures. After many attempts, we found a circumventive solution by the use of partially quaternized C 3 N 6 prior to the reaction with C 60 coupled with the modification of C 3 N 6 arm using propyl groups, instead of methyl or ethyl groups, to provide a well-balanced hydrophobicityhydrophilicity character of pentacationic precursor intermediates and better compatibility with the C 60 cage moiety.