Bioconjugate Chem. 2008, 19, 705–713
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Mitochondria Targeting by Guanidine- and Biguanidine-Porphyrin Photosensitizers Martha Sibrian-Vazquez, Irina V. Nesterova, Timothy J. Jensen, and M. Graça H. Vicente* Department of Chemistry, Louisiana State University, Baton Rouge, Louisiana 70803. Received October 23, 2007; Revised Manuscript Received December 3, 2007
We report the syntheses of three new amphiphilic porphyrin derivatives, containing a guanidine, a biguanidine, or an MLS peptide, that were designed to target the cell mitochondria. The guanidine- and biguanidine-porphyrins are poorly soluble in water, forming J-type aggregates in aqueous solutions. On the other hand, the porphyrin-MLS peptide conjugate bearing a low molecular weight PEG spacer is highly water-soluble and does not aggregate in aqueous media. The fluorescence quantum yields determined for all porphyrins were higher at low pH (98% as determined by HPLC on an analytical Delta Pak C18 (3.9 × 150 mm, 5 µm) column. HPLC tr ) 9.44 min. UV–vis (CH3OH) λmax (/M-1 cm-1) 415 (295 100), 513 (16 900), 547 (10 500), 588 (8000), 645 (6700). LRMS (MALDI) m/z (M + H+), 4183.55, calculated for C203H310N51O43S 4182.33. Photophysical Studies. All absorption spectra were acquired using a Perkin-Elmer Lambda 25 UV–visible Spectrophotometer with 10 mm path length quartz cuvettes. Corresponding diluents were utilized as reference solutions. Emission spectra were obtained using a Florolog 3 spectrofluorimeter. The optical density of solutions used for emission studies was below 0.05 at the excitation wavelength to eliminate inner filter effects. All the measurements were performed within 3 h of solution preparation. Stock solutions (10 mM) of porphyrins 2 and 3 were prepared in DMSO and for porphyrin-peptide conjugate 4 water was used. The aqueous solutions of porphyrins 3 and 4 were prepared by spiking 1.5–2 µL of the corresponding DMSO stock solutions into 1 mL of 100 mM phosphate buffer. The quantum yields were calculated using a secondary standard method (28, 29). Methylene blue was used as a secondary standard. According to the approach, the integrated fluorescence intensity of the analyte (I) and standard (IR), the optical density of the analyte (OD) and the standard (ODR), and the refractive index of analyte solvent (n) and standard solvent (nR) are related to the quantum yield of the analyte (Q) as I ODR n2 (1) Q ) QR IR OD n2 R
1 Abbreviations: HOBt, 1-hydroxybenzotriazole; DMF, N,N-dimethylformamide; CCA, R-cyano-4-hydroxycinnamic acid; TFA, trifluoroaectic acid; DIEA, N,N-diisopropylethylamine; TBTU, 2-(1Hbenzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate; DMSO, dimethyl sulfoxide; Boc, t-butoxy carbonyl; HEPES, 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid; PBS, phosphate buffer saline; FBS, fetal bovine serum; DMEM, Dulbecco’s Modified Eagle’s Medium; MEM, Minimal Essential Medium; PEG, poly(ethylene glycol); TIS, tri-isopropylsilane; Fmoc, 9-fluorenylmethoxycarbonyl; TCA, tricarboxylic acid cycle; TFE, trifluoroethanol; SDS, sodium dodecylsulfonate.
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Scheme 1a
a Conditions: (a) CNNH2/HCl/CH3OH/DMF, 70 °C, 24 h (42%). (b) Dicyanamide/HCl/ isopropanol/DMF, 90 °C, 18 h (61%). (c) Diglycolic anhydride, DMF, rt, 24 h (100%). (d) HOBt/TBTU/DIEA, NH2CH2CH2(OCH2CH2)5OCH2CO2tBu, rt, 48 h (64%). (e) TFA, rt, 4 h (100%). (f) HOBt/TBTU/DIEA, peptidyl resin, rt, 24 h. (g) TFA/H2O/Phenol/TIS, 88/5/5/2, rt, 4 h (23%).
where QR is a quantum yield of the reference standard (0.03 for Methylene Blue). To account for incomplete spectra, the bands were extrapolated using appropriate fitting models whenever necessary. Cell Culture. All tissue culture media and reagents were obtained from Invitrogen. Human HEp2 cells were obtained from ATCC and maintained in a 50:50 mixture of DMEM: Advanced MEM containing 5% FBS. The cells were subcultured biweekly to maintain subconfluent stocks. Time-Dependent Cellular Uptake. HEp2 cells were plated at 10 000 per well in a Costar 96 well plate and allowed to grow for 36 h. Porphyrin stocks were prepared in DMSO or water at a concentration of 10 mM and then diluted into medium to final working concentrations. The cells were exposed to 10 µM of each conjugate for 0, 1, 2, 4, 8, and 24 h. At the end of the incubation time the loading medium was removed and the cells were washed with PBS. The cells were solubilized upon addition of 100 µL of 0.25% Triton X-100 (Calbiochem) in PBS. To determine the porphyrin concentration, fluorescence emission was read at 410/650 nm (excitation/emission) using a BMG FLUOstar plate reader. The cell numbers were quantified using the CyQuant reagent (Molecular Probes). Dark Cytotoxicity. The HEp2 cells were plated as described above and allowed 36 h to attach. The cells were exposed to increasing concentrations of porphyrin up to 100 µM and incubated overnight. The loading medium was then removed and the cells fed medium containing Cell Titer Blue (Promega) as per manufacturer’s instructions. Cell toxicity was then measured by reading the fluorescence at 520/584 nm using a BMG FLUOstar plate reader. The signal was normalized to 100% viable (untreated) cells and 0% viable (treated with 0.2% saponin from Sigma) cells. Phototoxicity. The HEp2 cells were prepared as described above for the dark cytotoxicity assay and treated with porphyrin concentrations of 0, 0.625, 1.25, 2.5, 5, and 10 µM. After compound loading, the medium was removed and replaced with
medium containing 50 mM HEPES pH 7.4. The cells were then placed on ice and exposed to light from a 100 W halogen lamp filtered through a 610 nm long pass filter (Chroma) for 20 min. An inverted plate lid filled with water to a depth of 5 mm acted as an IR filter. The total light dose was approximately 1 J/cm2. The cells were returned to the incubator for 24 h and assayed for toxicity as described above for the dark cytotoxicity experiment. Microscopy. The HEp2 cells were plated on LabTek 2 chamber coverslips and incubated overnight, before being exposed to 10 µM of porphyrin. Since the porphyrin macrocycle fluorescences in the red region of the optical spectrum, the images were acquired using standard Texas Red filters. For the colocalization experiments, the cells were incubated for 24 h concurrently with porphyrin and one of the following organelle tracers for 30 min: MitoTracker Green (Molecular Probes) 250 nM, LysoSensor Green (Molecular Probes), BODIPY FL C5ceramide at 50 nM (Molecular Probes) and 50 nM, DiOC6(Molecular Probes) 5 µg/mL. The slides were washed three times with growth medium and new medium containing 50 mM HEPES pH 7.4 was added. By utilizing green fluorescing organelle tracers, it becomes possible to identify the intracellular compartments associated with the porphyrins; when the red and green images are overlaid, areas of colocalization appear as orange-yellow regions. Fluorescent microscopy was performed using a Zeiss Axiovert 200 M inverted fluorescent microscope fitted with standard FITC and Texas Red filter sets (Chroma). The images were aquired with a Zeiss Axiocam MRM CCD camera fitted to the microscope.
RESULTS Synthesis. The new series of potentially mitochondria-targeting photosensitizers, guanidinium-porphyrin 2, biguanidinium-porphyrin 3, and porphyrin-MLS conjugate 4 were synthesized from monoaminoporphyrin 1, as shown in Scheme 1. The reaction of protonated aminoporphyrin 1 with cyanamide at 70
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Figure 1. CD spectra in the amide region of conjugate 4 at 20 µM, (full line) TFE/H2O, pH 7.00, (dash line) 50 mM NaH2PO4, pH 5.00, (dot line) 100 mM SDS/H2O. The ellipticity is given in deg cm2 dmol-1, the cell path was 0.1 cm, and the temperature 25 °C.
°C in a mixture of DMF/methanol, gave guanidinium-porphyrin 2 in 42% yield. Using a similar procedure, although at 90 °C in a mixture of DMF/isopropanol and in the presence of dicyanamide, biguanidinium-porphyrin 3, was obtained in 61% yield. The aminoporphyrin 1 was found to be unreactive in the presence of other guanidinylation reagents, including 1Hpyrazole-[N,N′-bis(tert-butoxycarbonyl)]-carboxamidine, N,N′di-Boc-N′-triflyguanidine, and N,N′-bis(tert-butoxycarbonyl) thiourea, probably as a result of the low nucleophilicity of the amino group of 1, as we have previously observed (30); the use of Mukaiyama’s reagent (31) in conjunction with N,N′bis(tert-butoxycarbonyl)thiourea gave the corresponding Nalkylated derivative rather than the targeted guanidiniumporphyrin 2. The porphyrin-MLS conjugate 4 was prepared using solidphase methodology as we have previously reported (19). Briefly, aminoporphyrin 1 was converted to the corresponding carboxylate by reaction with diglycolic anhydride followed by pegylation, after deprotection of the terminal carboxylate and activation as the hydroxybenzotriazole ester. The activated pegylated porphyrin was conjugated to the peptidyl resin containing the sequence MSVLTPLLLRGLTGSARRLPVPRAKIHSL, and after cleavage and deprotection from the solid support, conjugate 4 was isolated in 23% yield. All porphyrins were characterized by 1H NMR, UV–vis, and MS, and in the case of conjugate 4, also by CD (Vide infra). Porphyrins 2-4 are all soluble in polar solvents, such as CH3OH, DMSO, and DMF. However, while the porphyrin-peptide conjugate 4 was highly water-soluble, the guanidinium- and biguanidinium-substituted porphyins 2 and 3 were only partially soluble in water. CD Studies. Conformational studies of the MLS peptide sequence in conjugate 4 were conducted in different media, including aqueous and membrane mimicking environments (in the presence of TFE or SDS). In aqueous solution at pH 5.0, the porphyrin-MLS peptide conjugate 4 shows negative Cotton effects at 198–207 nm as shown in Figure 1, which are characteristic of a random coil conformation. For example, for polylysine at pH 7, [121] ) -41 900 deg cm2 dmol-1 at 197 nm (32). In membrane mimicking environments, an R-helix structure characterized by two negative bands at 205 and 221 nm, associated with a positive band at 193 nm is induced for conjugate 4; this conformational change is more clearly observed in the presence of SDS. For example, for polylysine at pH 7, [θ] ) 76 900 deg cm2 dmol-1 at 191 nm, [θ] ) -32 600 deg cm2 dmol-1 at 208 nm, and [θ] ) -35 700 deg cm2 dmol-1 at 222 nm (32). These results show that the MLS sequence in conjugate 4 adopts different conformations depending on the
Figure 2. (a) Absorption spectra of 20 µM of porphyrin 2 in DMSO (short dot), and in aqueous solution at different pH. (b) Emission spectra of 2.8 µM porphyrin 2 at different pH (λex) 600 nm). pH ) 8.00 (full line), pH ) 7.4 (dash-dot-dot), pH ) 7.00 (dash-dot-dash), pH ) 6.00 (large dot), pH ) 5.00 (dash).
nature of the environment, as a result of the number and distribution of the hydrophobic amino acids in the MLS sequence. Photophysical Studies. The absorption and emission spectra in DMSO and in aqueous solutions at different pH values for porphyrins 2 and 3 and peptide conjugate 4 are shown in Figures 2, 3, and 4, respectively. The shape of the absorption spectra for porphyrins 2 and 3 (i.e., wide, poorly resolved bands in aqueous media compared with DMSO) indicate ground-state aggregation of the molecules. The slight red-shift of the Soret band on the absorption spectra (aqueous vs DMSO) and observed fluorescence suggest J-type aggregate formation (33). In contrast, the shape of the absorption spectra of the porphyrin-MLS conjugate 4 does not indicate any aggregation in aqueous media or in DMSO. No significant effect of pH on the position and shape of the absorption bands were observed for all porphyrins. The fluorescence quantum yields were found to be pH-dependent as shown in Figure 5. The highest quantum yield determined for porphyrins 2 and 3 and conjugate 4 were 0.0006 (pH ) 5.00), 0.0012 (pH ) 6.00), and 0.012 (pH ) 5.00), respectively. At physiological pH (7.4), the fluorescence of all porphyrins is quenched as determined by the quantum yields determined at this pH. The reduction in quantum yields at pH 7.4 from the maxima observed for porphyrins 2 and 3 and conjugate 4 are 33%, 42%, and 66%, respectively. Cell Studies. The time-dependent uptake of porphyrins 2 and 3 and conjugate 4 was investigated at a concentration of 10 µM in human HEp2 cells, and the results obtained are shown in Figure 6. The porphyrin-peptide conjugate 4 bearing an amphiphilic MLS sequence accumulated the most within cells at all time points studied, followed by porphyrin 2. The biguanidinium-porphyrin 3 was the least accumulated within cells (about half as much as porphyrin 2) of this series of compounds. All porphyrins showed similar uptake kinetics in the first 1–2 h but while porphyrin 2 and conjugate 4 continued
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Figure 5. Estimated fluorescence quantum yields (%) at different pH. Porphyrins 2 (dash line), 3 (dot line) and 4 (full line).
Figure 3. (a) Absorption spectra of 20 µM of porphyrin 3 in DMSO (short dot), and in aqueous solution at different pH. (b) Emission spectra of 2.8 µM porphyrin 3 at different pH (λex) 580 nm). pH ) 8.00 (full line), pH ) 7.4 (dash-dot-dot), pH ) 7.00 (dash-dot-dash), pH ) 6.00 (large dot), pH ) 5.00 (dash). Figure 6. Time-dependent uptake of porphyrins 2 (dash line), 3 (dot line), 4 (full line), at 10 µM by HEp2 cells.
Figure 7. Dark toxicity of porphyrins 2 (dash line), 3 (dot line), and 4 (full line), toward HEp2 cells using the Cell Titer Blue assay.
Figure 4. (a) Absorption spectra of 15 µM porphyrin conjugate 4 in DMSO (short dot), and in aqueous solution at different pH. (b) Emission spectra of 4.0 µM porphyrin conjugate 4 at different pH (λex) 590 nm). pH ) 8.00 (full line), pH ) 7.4 (dash-dot-dot), pH ) 7.00 (dashdot-dash), pH ) 6.00 (large dot), pH ) 5.00 (dash).
to accumulate steadily over time, the biguanidinium-porphyrin 3 exhibited slower uptake. The dark cytotoxicity and phototoxicity of the new porphyrin conjugates were evaluated upon exposure of HEp2 cells to increasing concentrations of each porphyrin for 24 h. The results obtained are shown in Figures 7 and 8, respectively. While the biguanidinium-porphyrin 3, the least accumulated within cells, shows no toxicity at concentrations up to 100 µM in the dark,
Figure 8. Phototoxicity of porphyrins 2 (dash line), 3 (dot line), 4 (full line) toward HEp2 cells using 1 J/cm2 dose light.
the guanidinium-porphyrin 2 and conjugate 4 have estimated IC50 ) 84 and 86 µM, respectively, in the dark (Figure 7). The most phototoxic compound with estimated IC50 ) 4.8 µM at 1 J cm-2 was found to be the guanidinium-porphyrin 2, followed
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Figure 9. Subcellular localization of porphyrin 2 in HEp2 cells at 10 µM for 24 h. (a) phase contrast, (b) overlay of porphyrin 2 fluorescence and phase contrast, (c) BODIPY Ceramide fluorescence, (e) LysoSensor Green fluorescence, (g) MitoTracker Green fluorescence, (i) DiOC6 fluorescence, (d), (f), (h), (j) overlays of organelle tracers with porphyrin 2 fluorescence. Scale bar: 10 µm.
Figure 10. Subcellular localization of porphyrin 3 in HEp2 cells at 10 µM for 24 h. (a) phase contrast, (b) overlay of porphyrin 3 fluorescence and phase contrast, (c) BODIPY Ceramide fluorescence, (e) LysoSensor Green fluorescence, (g) MitoTracker Green fluorescence, (i) DiOC6 fluorescence, (d), (f), (h), (j) overlays of organelle tracers with porphyrin 3 fluorescence. Scale bar: 10 µm.
by the biguanidinium-porphyrin 3 (IC50 ) 8.2 µM at 1 J cm-2) and conjugate 4 (IC50 ) 9.8 µM at 1 J cm-2), as shown in Figure 8. The subcellular localization of the new porphyrin derivatives was investigated in HEp2 cells after 24 h, using fluorescence microscopy. Figures 9, 10, and 11 show the fluorescent patterns observed for porphyrins 2-4, respectively, and their overlay with the organelle specific fluorescent probes BODIPY FL C5ceramide (Golgi network), LysoSensor Green (lysosomes), Mitotracker Green (mitochondria), and DiOC6 (ER). The preferential sites of intracellular localization for porphyrin 2 were found to be the mitochondria and the ER (Figure 9h,j). A small amount of 2 was also found in the cell lysosomes. The biguanidinium-porphyrin 3 distributed within cell membranes with a large component in vesicles that correlated to some extent with the lysosomes and, upon longer exposures, in mitochondria and the cytosolic membrane (Figure 10f,h). On the other hand,
conjugate 4 was found to localize mainly within the cell lysosomes (Figure 11f).
DISCUSSION We used two general strategies in the design of mitochondriatargeting photosentizers. The first approach was based on the introduction of a functional group (i.e., guanidine or biguanidine) with a delocalized positive charge, in order to promote preferential accumulation via the high membrane potential across the inner mitochondrial membrane. The guanidinium and biguanidinium groups were chosen because they are strong bases (pKa ∼ 13) that are protonated at physiological pH (34–36), and that can form multiple hydrogen bonds and/or electrostatically interact with biological substrates. Since the positive charge is delocalized, guanidinium and biguanidinium groups attached to hydrophobic moieties (such as a porphyrin) form amphiphatic molecules with potentially enhanced affinity for crossing cellular
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Figure 11. Subcellular localization of conjugate 4 in HEp2 cells at 10 µM for 24 h. (a) phase contrast, (b) overlay of conjugate 4 fluorescence and phase contrast, (c) BODIPY Ceramide fluorescence, (e) LysoSensor Green fluorescence, (g) MitoTracker Green fluorescence, (i) DiOC6 fluorescence, (d), (f), (h), (j) overlays of organelle tracers with conjugate 4 fluorescence. Scale bar: 10 µm.
membranes, for example, via formation of bidentate hydrogen bonds with phosphate groups (37). Several methodologies have been developed for the preparation of guanidines in high yield, under mild conditions, typically involving the treatment of an amine with an electrophilic amidine species. The most commonly used reagents include derivatives of pyrazole-1-carboxamidine (38), S-alkylisothioureas (39), protected thiourea derivatives in conjunction with mercury salts or the Mukaiyama reagent (31), and protected triflylguanidines (40). However, aminoporphyrin 1 was either unreactive to the above guanidinylation reagents or afforded the corresponding N-alkylated derivative. We therefore prepared porphyrins 2 and 3 as shown in Scheme 1, by reaction of the anilinium salt of 1 with cyanamide or dicyanamide in DMF/alcohol, using a previously reported procedure (34, 41), in 42–61% yields. Our second approach involved the conjugation of a MLS peptide sequence to porphyrin 1 via a low molecular weight PEG spacer using a published procedure (19), in order to exploit
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the mitochondrial import machinery. We chose the peptide sequence MSVLTPLLLRGLTGSARRLPVPRAKIHSL, isolated from the subunit VII of human cytochrome c oxidase (42), which is encoded in a commercially available expression vector (pShooter) that targets a recombinant protein to the mitochondria of mammalian cells. The PEG spacer was used not only to improve the water solubility of the conjugate, but also to reduce aggregation (43, 44) and other possible interactions between the porphyrin macrocycle and the peptide sequence (30). The time-dependent uptake of porphyrins 2 and 3 and conjugate 4 was found to depend significantly on the nature of the substituents at the porphyrin periphery. Conjugate 4 bearing a MLS peptide accumulated the most within cells at all time points studied, while the biguanidinium-porphyrin 3 accumulated the least. The high uptake observed for conjugate 4 is probably due to the presence of the amphiphilic peptide sequence, the low molecular weight PEG spacer, and the low tendency of this conjugate to form aggregates in aqueous media. This result is in agreement with our previous studies showing that conjugation of a peptide to a porphyrin or phthalocyanine macrocycle via a short PEG generally enhances cellular uptake and decreases both intra and intermolecular interactions (19, 45). Surprisingly, biguanidinium-porphyrin 3 exhibits significantly lower uptake than guanidinium-porphyrin 2; this may be attributed to the structural differences between these two groups and their ability to interact with negatively charged groups (e.g., phosphates) on the cell membrane. While the guanidinium group exhibits a planar structure and acts as a bidentate ligand, structural studies on biguanidinium salts indicate that it adopts a nonplanar conformation in order to reduce allylic strain, while still permitting a significant degree of conjugation. Furthermore, structural analyses by X-ray diffraction indicate that arylbiguanidines and their salts can form unique networks bonded by characteristic patterns of hydrogen bonds (34), which might have lower affinity for membrane crossing. It is interesting to note that the guanidinium-porphyrin 2 showed similar uptake by the HEp2 cells to that of the peptide conjugate 4. All porphyrins showed low dark cytotoxicity, in particular the biguanidinium-porphyrin 3 which was the least taken up by the HEp2 cells. The most phototoxic compound is porphyrin 2, followed by 3 and the porphyrin peptide conjugate 4, as a result of both the extent of their uptake and their subcellular distribution. Whereas the preferential sites of intracellular localization of porphyrin 2 are the mitochondria and the ER, porphyrin 3 was mainly found within cellular membranes, the lysosomes, and mitochondria. In contrast, the porphyrin conjugate 4 bearing a MLS peptide sequence localized mainly in the lysosomes rather than in mitochondria, in agreement with our previous studies on porphyrin-peptide conjugates (19, 30). Our results show, in agreement with previous reports (46), that the nature of the substituents at the porphyrin periphery play an important role in determining their mechanism(s) of cellular uptake and subcellular distribution. Although SAR investigations on mitochondria-targeting molecules (47, 48) have indicated that (i) positively charged amphiphilic molecules are delivered into the mitochondria in response to its highly negative membrane potential, and (ii) lipophilic compounds containing delocalized positive charge(s) tend to reduce the free energy change when moving from an aqueous to an hydrophobic environment, not all cationic porphyrin-based sensitizers localize preferentially in mitochondria. We believe that the partial localization in mitochondria observed for porphyrins 2 and 3 is due to the presence of the guanidinium and biguanidinium groups, which play an important role on the lysosomal escape of these photosensitizers as a result of membrane rupture, probably by the proton sponge effect; these photosensitizers are therefore released into the cytoplasm and subsequently delivered into other
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organelles, such as the ER and mitochondria (49–52). The porphyrin-peptide conjugate 4 containing an MLS peptide sequence was not found in mitochondria, probably as a result of its trapping within endosomes and lysosomes as a result of an endocytotic mechanism of uptake. It is possible that the MLS peptide in conjugate 4 might be cleaved off and degraded to some extent by lysosomal enzymes, as we have recently reported for other porphyrin-peptide conjugates (53), before the release of the conjugate from these compartments. As a result of partial mitochondria targeting, porphyrins 2 and 3 were found to be more phototoxic than the peptide conjugate 4. The highest phototoxicity observed for the guanidinium-porphyrin 2 is probably due to its higher uptake and preferential localization in the mitochondria and ER, as photodamage to these sensitive organelles can lead to rapid cell death via apoptosis (54–56).
ACKNOWLEDGMENT The authors thank Ms. Martha Juban for the MLS peptide sequence synthesis. This work was partially supported by the National Science Foundation, grant number CHE-304833. Supporting Information Available: HPLC trace for pophyrin-MLS conjugate 4 and conditions. This material is available free of charge via the Internet at http://pubs.acs.org.
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