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Replacing the typical β-alkyl substituents of -semirubin and -oxosemirubin, two intramolecularly hydrogen-bonded bilirubin analogs, with methoxy groups produces amphiphilic dipyrrinones. Synthesized from the respective 9H-dipyrrinones prepared by base-catalyzed condensation of 3,4-dimethoxypyrrolin-2-one with the appropriate pyrrole α-aldehyde, the 2,3-dimethoxy and 2,3,7,8-tetramethoxy analogs of -semirubin are yellow-colored dipyrrinones that form intramolecularly hydrogen-bonded monomers in CDCl3, as deduced from 1H-NMR NH chemical shifts. They are monomeric in CHCl3, as determined by vapor pressure osmometry. In contrast, in the solid, X-ray crystallography reveals supramolecular ribbons of intermolecularly hydrogen-bonded (dipyrrinone to dipyrrinone and acid to acid) 2,3,7,8-tetramethoxy--semirubin. The latter is approximately 20 times more soluble in water than the parent -semirubin with four β-methyl groups.
Bilirubin (Fig. 1A), the yellow pigment of jaundice and the end product of heme metabolism,1 is very insoluble in water (Ksp ~4 × 10−15 M at pH 7 at 37°C),2 which makes it unexcretable except by formation of glucuronide conjugates, the main pathway for its elimination. The pigment’s poor aqueous solubility is explained by a ridge-tile conformation,3–5 wherein the propionic acids are tucked inward and firmly hydrogen bonded to the opposing dipyrrinones, thereby presenting a hydrocarbon-like periphery (Fig. 1B). Insightful studies of bilirubin chemistry, photobiology and metabolism have been advanced from its dipyrrinone6 models, inter alia. Among such analogs of bilirubin, -semirubin (Fig 1C)7 was synthesized in order to evaluate carboxylic acid to amide hydrogen bonding. -Semirubin, like bilirubin, is lipophilic and insoluble in water.
Interest in improving the aqueous solubility of bilirubin, and thereby facilitating its elimination led us to synthesize a bilirubin with a MW 2200 polyethylene glycol unit attached at the exo-vinyl group. The bilirubin derivative was soluble in both water and CHCl3 but was present as an aggregate in water (presumably with bilirubin molecules aggregated inside a polyether micelle).8 Shorter, single polyethylene glycol chains led to decreased aqueous solubility. To prepare a water-soluble pigment that was not aggregated, we considered the possibility that multiple, short polyethylene glycol units might produce the desired aqueous solubility without aggregation. Merz et al.,9a and Sessler9b and Schmuck9c counteracted the intrinsic aqueous insolubility of porphyrins and pyrroles by attaching short polyether chains, e.g., diethylene glycol, at the pyrrole β-positions which suggested that replacing some of the pyrrole β-substituents of bilirubin might produce a similar salutary effect by improving the pigment’s aqueous solubility and avoiding aggregation. While considering the feasibility of synthesizing bilirubinoids and model dipyrrinones with di- or triethylene glycol β-substituents, we decided to explore whether even the smallest β-ether (OCH3) substituent might improve the aqueous solubility of the pigment. In the current study we focused on the model for one half of bilirubin, the dipyrrinone -semirubin, in order to learn to what extent replacing its lactam and pyrrole β-substituents with methoxy groups might (1) enhance the pigment’s aqueous solubility, and (2) avoid aggregation in water. In the following we describe the syntheses, solution structures and solubilities of: tetramethoxy (1) and dimethoxy (2) analogs (Fig. 1D) of -semirubin. We also report the syntheses of the corresponding 91-oxo analogs (3 and 4) and compare their properties to -oxosemirubin (Fig. 1C). X-Ray crystallographic structures of 1 and the ethyl ester (4e) of 4 were obtained.
The key intermediates for the synthesis of methoxylated -semirubins (1–4) are 9H-dipyrrinones: tetramethoxy 5 and dimethoxy 6, which were available from earlier work.10 Reaction of the latter under Friedel-Crafts acylation conditions using the half ester-acid chloride of adipic acid afforded -oxosemirubin analogs 3e and 4e, from 5 and 6, respectively. The esters were saponified to the corresponding acids (3 and 4). Reduction of 3e and 4e using NaBH4 + boron trifluoride etherate afforded the corresponding ethyl esters 1e and 2e. Reduction of 3e and 4e using NaBH4 in 2-propanol followed by saponification in the same reaction pot gave semirubin analogs 1 and 2.
The constitutional structures of 1–4 and 1e-4e follow from the 9H-dipyrrinone precursors 5 and 6 and comparison of their 13C-NMR spectral data (Table 1) with those of the parent -semirubin and -oxosemirubin and their esters.6 The methoxy carbons at C(2) and C(3) appear in the expected ranges, with the endo OCH3 (at C(3)) more shielded than the exo (at C(2)); however, C(4) of 1 and 2 is shifted 4–8 ppm upfield relative to 3 and 4. The latter show signals near 190 ppm for the 91-oxo group. The presence of the semirubin acids is evident by the deshielding of the CO2H carbon (~180 ppm) compared to that of the esters (~174 ppm).
Using vapor pressure osmometry (VPO) to determine molecular weights in solution, all of the acids (1–4) were shown to be monomers in both CHCl3 (Table 2). The data are consistent with intramolecularly hydrogen bonded species, found in the parent -semirubin and -oxosemirubin.7 As expected from earlier studies of dipyrrinones,6,7,11 esters 1e and 2e were dimeric in CHCl3, but oxosemirubin esters 3e and 4e were monomeric. The latter is attributed to the oxo group being oriented in such a way that the ester chain blocks formation of intermolecular hydrogen bonding between the dipyrrinones.11
Intramolecular hydrogen bonding in -semirubin (Fig. 1C) in CDCl3 was firmly established previously7 by 1H-NMR and Nuclear Overhauser Effect (NOE) measurements. NOEs in CDCl3 in 1–4 found between the lactam and pyrrole NHs (Fig. 2), and between the C(5)-H and the neighboring OCH3/CH3 groups confirm the syn-Z configuration of the dipyrrinone. Weak NOEs between the CO2H and the lactam NH confirm intramolecular hydrogen bonding. In addition, 1 and 2 exhibit deshielded NH chemical shifts commensurate with hydrogen bonding to the hexanoic acid carbonyl (Table 3) and not much different from those found in -semirubin.7 The OCH3 groups cause small upfield shifts of the NH signals. As in -semirubin the relevant NOEs support the intramolecularly hydrogen bonded 4Z-configuration structure. Similarly, at least some intramolecular hydrogen bonding is detected in the -oxosemirubin analogs 3 and 4, as has been seen in -oxosemirubin itself.7
Despite the large number of known dipyrrinones, only a few X-ray crystal structures of them have been obtained.6,12 They all have the common feature of in-plane dipyrrinone to dipyrrinone hydrogen bonding. Until the present work there have been no crystal structures published of a semirubin, despite earlier attempts. Figures 3 and and44 present the crystal structure drawings from crystals of 1 and 4e grown from diethyl ether-1,2-dichloroethane and CHCl3-n-hexane, respectively. The constitutional structures of 1 and 4e are fully confirmed in their crystals structures. In the unit cells, there are two distinct (A and B) molecules of 1 (Fig. 3A); for 4e there is but one molecule (Fig. 4A). Based on all previous and current studies of -semirubin, we had fully expected an intramolecularly hydrogen bonded structure for 1. Yet in the crystal it is found to be intermolecularly hydrogen bonded, dipyrrinone to dipyrrinone and carboxylic acid to carboxylic acid, to form stacked, parallel streams of supramolecular ribbons (Fig. 3B) with the parallel stacks of ribbons separated by 7.8 Å. The dipyrrinones are nearly planar, with relevant torsion angles, N(1)-C(4)=C(5)-C(6) = 3.3° in (A) and 0.3° in (B), and C(4)=C(5)-C(6)-C(6)-N(2) = 4.2° in (A) and 0.9° in (B), which confirm a planar syn-Z configuration. All dipyrrinone bond lengths and bond angles of 1 (Fig. 5A) conform to those measured in previous crystal structures12 and can thus be viewed as “normal,” with alternating double and single bonds.
In contrast to 1, the oxosemirubin ester (4e) was not expected to be intramolecularly hydrogen bonded. In the crystal 4e is intermolecularly hydrogen bonded, dipyrrinone to dipyrrinone; however the dipyrrinone units of the hydrogen-bonded pair do not lie co-planar but are orthogonal (Fig. 4B). The dipyrrinones of 4e are only slightly less planar than in 1, as indicated by the relevant torsion angles: N(1)-C(4)=C(5)-C(6) = 4.9° and C(4)=C(5)-C(6)-N(2) = 8°. The dipyrrinone conformation is thus again shown to be syn-Z, and the 91-oxo group is found to be syn to the pyrrole nitrogen. The presence of the 91-oxo induces some interesting changes in the dipyrrinone bond angles and bond lengths (Fig. 5B) relative to those of 1. Thus, while the alternating double and single bonds of the pyrrole ring of 1 (Fig. 5A) are blurred into a single value near 1.4 Å, they remain distinct in the lactam ring, with somewhat (0.15–0.20 Å) lengthened N(1)-C(1), C(1)-C(2), and C(3)-C(4) bond lengths in 4e relative to 1. Similarly, C(5)-C(6) is lengthened by 0.15 Å in 4e relative to 1, but the C(2)=C(5) and C(4)=C(5) double bond lengths remain essentially unchanged. The significant changes in bond angles indicate a widening of the N(1)-C(4)=C(5)-C(6)-N(2) cavity of 4e relative to 1, possibly to accommodate the orthogonal hydrogen bonding motif in the former. Again, other differences in bond angles lie mainly in the pyrrole ring.
The characterization of methoxylated-dipyrrinones 1–4 indicates great similarity in solution structure and hydrogen bonding to the parent -semirubin and -oxo-semirubin parents. The acids (1–4) and their esters (1e-4e) are more soluble in CHCl3 and in CH3OH than the parents. In H2O, where the parent (tetramethyl) semirubins are very insoluble, the methoxylated analogs show better solubility. (Comparison of 1 and 1e to 2 and 2e, as well as 3 and 3e to 4 and 4e is shown in Table 4.) Thus, we examined their aqueous solubility as well as their solubility in CH3OH (as a control). UV-visible spectroscopy was used to determine the concentrations relative to standard ~1 × 10−5 M solutions. The CH3OH control experiment shows that the solubility of the pigment at 1–3 × 10−5 M in pure CH3OH is almost exactly as that in CH3OH-2% CHCl3 (vol/vol) in which the pigment is freely soluble. All of the pigments are also freely soluble in a reference standard: H2O-2% (CH3)2SO (vol/vol). Comparing pure H2O to this reference (Table 4), one finds that 1 is approximately 10 times more soluble in water than 2 and that while 1 is ~30% more soluble than its ester, 1e, and 1e is 15 times more soluble than 2e at saturation.
The data reveal, as expected, that the presence of four methoxyl groups improves the aqueous solubility more than the two methoxyl groups of 2, but that while the presence of a 91-oxo group does not improve the solubility (of 3) relative to 1, the 91-oxo groups in 4 and 4e have a major effect on improving (4–6 times) the aqueous solubility of 2 and 2e.
UV-visible spectroscopy of 1–4 (Table 5) reveal several interesting facts. In general, the long wavelength dipyrrinone absorption of the tetramethoxy analogs (1 and 3) are shifted 5–6 nm hypsochromic relative to the dimethoxy analogs (2 and 4). This trend generally carries over to their esters, 1e and 3e vs 2e and 4e. The oxosemirubin long wavelength absorption is generally centered at a shorter λmax than the corresponding semirubin analogs. Chloroform solutions, of 1–4, in which the semirubins are intramolecularly hydrogen bonded produce bathochromically shifted long wavelength absorptions relative to CH3OH, (CH3)2SO, and H2O solutions, and the λmax and εmax in the latter three solvents are nearly the same. In contrast, esters 1e and 2e exhibit somewhat shorter wavelength λmax in CHCl3 relative to CH3OH, (CH3)2SO and H2O solvents, while the reverse is found for the oxosemirubin esters 3e and 4e.
The presence of methoxyl groups on the lactam and pyrrole β-positions of -semirubin and -oxosemirubin (to give 1, 2 and 3, 4, Fig. 1C) improves their water solubility, with four methoxyl groups (of 1) providing up to 0.06 M solutions (1 and 3) and up to 0.04 M solutions of the corresponding ethyl esters. However, the aqueous solubility was insufficient to analyze for aggregation by VPO. Nonetheless, the studies indicate that with di-, tri- or tetraethylene glycol chains, the semirubins should be sufficiently water soluble for analysis by VPO. The acids (1–4) form intramolecularly hydrogen bonded monomers in CHCl3 solvent; whereas the -semirubin esters 1e and 2e are intermolcularly hydrogen bonded dimers. In contrast, in the crystal, 1 is intermolecularly hydrogen bonded in supramolecular ribbons.
All nuclear magnetic resonance (NMR) spectra were obtained on a Varian 500 MHz (1H) and 125 MHz (13C), respectively, in deuteriochloroform unless otherwise indicated. Chemical shifts were reported in ppm referenced to the residual chloroform proton signal at 7.26 ppm and 13C-NMR signal at 77.23 ppm unless otherwise noted. Melting points were taken on a Mel-Temp capillary apparatus and are corrected. Combustion analyses were performed by Desert Analytics, Tucson, AZ and gave results within ±0.4% of theoretical values. All ultraviolet-visible spectra were recorded on a Perkin-Elmer Lambda-12 spectrophotometer. Vapor pressure osmometry (VPO) measurements were performed on an OSMOMAT 070-SA instrument (Gonotech GmbH, Germany) in HPLC grade CHCl3 (Fisher) at 45°C. Analytical thin layer chromatography (TLC) was carried out on J.T. Baker silica gel IB-F plates (125 μm layer). For final purification, radial chromatography was carried out on Merck silica gel PF254 with calcium sulfate binder, preparative layer grade. All solvents were reagent grade obtained from Fisher-Acros. Spectral data were obtained in spectral grade solvents (Aldrich or Fisher). The dipyrrinones 5 and 6 were available from earlier work.10
To a solution of oxosemirubin 3 (100 mg, 0.23 mmol) dissolved in 2-propanol, NaBH4 (50 mg, 1.3 mmol) was added and the mixture was heated at reflux for 2.5 h. The reaction was then poured into 80 mL of cold water and acidified carefully to pH 4 to produce a yellow precipitate, which was extracted by CH2Cl2, dried and evaporated in vacuo (rotovap). The crude solid was purified by radial chromatography (95:5 CH2Cl2:CH3OH by vol eluent) to yield 48 mg (50%) yield of pure 1. It had mp 92–94°C; 1H-NMR, δ: 1.39 (2H, m), 1.62 (2H, m), 1.70 (2H, m), 2.49 (2H, t, J = 7.2 Hz), 2.68 (2H, t, J = 7.2 Hz), 3.78 (3H, s), 3.88 (3H, s), 3.90 (s, 3H), 4.11 (s, H), 6.23 (1H, s), 8.38 (1H, brs), 10.11 (1H, brs) ppm; 13C-NMR data are in Table 1.
Anal. Calcd for C19H26N2O7 (394.5): C, 57.86; H, 6.64; N, 7.10
Found: C, 57.97; H, 6.60; N, 7.13
To a solution of oxosemirubin ester 3e (300mg, 0.75 mmol) dissolved in ~10 mL of dry THF at 0°C (ice-salt bath), ~60 mg NaBH4 was added followed by addition of 3 mL of boron trifluoride etherate (BF3·OEt2O) in ~8 mL of dry THF over a period of 10 minutes. The reaction was stirred at 0°C for 1 h and then quenched by the dropwise addition of methanol. The entire solution was extracted with CH2Cl2, and the organic extracts were dried and evaporated in vacuo (roto-vap). The crude product was purified using radial chromatography (98:2 CH2Cl2:CH3OH by vol. eluent) to afford 183 mg (61%) yield of 1e. It had mp 70–71°C; 1H-NMR, δ: 1.24 (3H, t, J = 7.5 Hz), 1.37 (2H, m), 1.63 (4H, m), 2.31 (2H, t, J = 7.2 Hz), 2.60 (2H, t, J = 7.2 Hz), 3.77 (3H, s), 3.91 (3H, s), 4.02 (3H, s), 4.11 (3H, s), 4.12 (2H, q, J = 7.5 Hz), 6.00 (1H, s), 8.26 (1H, brs), 9.31 (1H, brs) ppm; 13C-NMR data are in Table 1.
Anal. Calcd for C21H30N2O7 (422.5): C, 59.70; H, 7.16; N, 6.63
Found: C, 59.52; H, 7.10; N, 6.29
To a solution of oxosemirubin 4 (50 mg, 0.12 mmol) dissolved in 2-propanol, NaBH4 (50 mg, 1.3 mmol) was added and the mixture was heated at reflux for 2.5 h. After work-up similar to that for 1 and purification of the crude yellow solid by radial chromatography (95:5 CH2Cl2:CH3OH by vol eluent), 40 mg (92%) of pure 2 was obtained. It had mp 159–161°C; 1H-NMR, δ: 1.39 (2H, m), 1.58 (2H, m), 1.71 (2H, m), 1.93 (3H, s), 2.07 (3H, s), 2.48 (2H, t, J = 7.2 Hz), 2.70 (2H, t, J = 7.2 Hz), 3.90 (3H, s), 4.13 (3H, s), 6.23 (1H, s), 8.79 (1H, brs), 10.10 (1H, brs) ppm; 13C-NMR data are in Table 1.
Anal. Calcd for C19H26N2O5 (362.5): C, 62.97; H, 7.23; N, 7.73
Found: C, 62.80; H, 6.97; N, 7.73
To a solution of oxosemirubin ester 4e (200 mg, 0.75 mmol) dissolved in ~10 mL of dry THF at 0°C (ice-salt bath), ~39 mg of NaBH4 was added, followed by addition of 2 mL of (BF3·OEt2O) in ~8 mL of dry THF over a period of 10 min. The reaction mixture was stirred at room temperature for 1.5 h, then quenched by the dropwise addition of methanol. The product was isolated as per the work-up for 1e and after radial (98:2 CH2Cl2:CH3OH by vol. eluent) chromatography afforded 105 mg (53%) yield of 2e. It had mp 139–140°C; 1H-NMR, δ: 1.22 (2H, t, J = 7.5 Hz), 1.37 (2H, m), 1.63 (2H, m), 1.65 (2H, m), 1.94 (3H, s), 2.09 (3H, s), 2.26 (2H, t, J = 7.2 Hz), 2.73 (2H, t, J=7.2 Hz), 3.85 (3H, s), 4.11 (2H, q, J = 7.5 Hz), 4.14 (3H, s), 6.27 (1H, s), 9.70 (1H, brs), 10.45 (1H, brs) ppm; 13C-NMR data are in Table 1.
Anal. Calcd for C21H30N2O5 (390.5): C, 64.60; H, 7.74; N, 7.17
Found: C, 64.96; H, 7.15; N, 6.98
To a solution of oxosemirubin 3e (0.23 gm, 0.53 mmol) in 100 mL of dry THF and 10 mL of CH3OH, 0.7 g NaOH was added. The solution was heated at reflux for 2 h under N2. The reaction mixture was poured into 200 mL of ice-water. The aqueous solution was then acidified with 10 % HCl to pH ~4, and then extracted with CH2Cl2 until the organic phase became clear. The combined organic phases were dried over anhyd. Na2SO4 and evaporated in vacuo (roto-vap). The crude product was purified by radial chromatography using 3%-5% gradient of CH3OH in CH2Cl2. The pure fractions were evaporated in vacuo (roto-vap) and recrystallized from CH2Cl2-hexane to obtain 183 mg (83%) of 3. It had mp 190–191°C; 1H-NMR, δ: 1.74 (4H, m), 2.41 (2H, t, J = 7.2 Hz), 2.84 (2H, t, J = 7.2 Hz), 3.82 (3H, s), 3.95 (3H, s), 4.02 (3H, s), 4.14 (3H, s), 5.96 (1H, s), 8.56 (1H, brs), 9.54 (1H, brs) ppm; 13C-NMR data are in Table 1.
Anal. Calcd for C19H24N2O8 (408.4); C, 55.88; H, 5.92; N, 6.86
Found: C, 55.95; H, 5.60; N, 6.84
To a cooled mixture of half ethyl ester acid chloride of adipic acid (0.3 g, 1.57 mmol) and SnCl4 (1.03 g, 3.93 mmol) in 50 mL CH2Cl2, a solution of 9-H tetramethoxydipyrrinone 5 (110 mg, 0.39 mmol) in 50 mL of CH2Cl2 was added dropwise. The solution was stirred at room temperature for 7 h with constant checking by TLC. The reaction mixture was poured into 100 gm ice-water and stirred for 2 h. The organic layer was separated and aqueous layer was washed with CH2Cl2 (2 × 100 mL). The combined organic layers were washed with 5% aq. NaHCO3 then with water, dried over anhyd. Na2SO4 and evaporated in vacuo (roto-vap). The crude product was purified by flash column chromatography (98:2 CH2Cl2:CH3OH by vol. eluent) followed by radial chromatography (98:2 CH2Cl2:CH3OH by vol. eluent) to obtain pure 3e in 47% yield. It had mp 98–100°C 1H-NMR, δ: 1.74 (4H, m), 2.34 (2H, t, J = 7.2 Hz), 2.84 (2H, t, J = 7.2 Hz), 3.83 (3H, s), 3.95 (3H, s), 4.02 (3H, s), 4.10 (3H, s), 5.99 (1H, s), 8.50 (1H, brs), 9.55 (1H, brs) ppm; 13C-NMR data are in Table 1.
Anal. Calcd for C21H28N2O8 (436.5): C, 57.79; H, 6.47; N, 6.42
Found: C, 57.69; H, 6.39; N, 6.40
To a solution of oxosemirubin 4e (0.25 gm, 0.62 mmol) in 100 mL of dry THF and 10 mL of CH3OH, was added 0.7 g NaOH, and the mixture was heated at reflux for 2 h under N2. The reaction mixture was treated as in the synthesis of 3 and worked up in the same way to give pure fractions which were evaporated in vacuo (roto-vap) and recrystallized from CH2Cl2-hexane to obtain 201 mg (87%) of 4. It had mp 162–163°C; 1H-NMR, δ: 1.82 (2H, m), 1.85 (2H, m), 2.04 (3H, s), 2.29 (3H, s), 2.46 (2H, t, J = 7.2 Hz), 2.82 (2H, t, J = 7.2 Hz), 3.92 (3H, s), 4.14 (3H, s), 6.16 (1H, s), 9.25 (1H, brs), 9.97 (1H, brs) ppm; 13C-NMR data are in Table 1.
Anal. Calcd for C19H24N2O6·1/2 H2O (385.4): C, 59.21; H, 6.54; N, 7.27
Found: C, 59.88; H, 6.36; N, 7.03
To a cooled mixture of half ethyl ester acid chloride of adipic acid (0.12g, 0.65 mmol) and SnCl4 (0.29g, 1.13 mmol) in 50 mL of CH2Cl2, a solution of 9H-dimethoxydipyrrinone 6 (40 mg, 0.16 mmol) in 50 mL of CH2Cl2 was added dropwise. The solution was stirred at room temperature for 12 h and then poured into 100 gm of ice-water and stirred for 2 h. The organic layer was separated, and the aqueous layer was washed with CH2Cl2 (2 × 100 mL). The combined organic layers were washed with aq. NaHCO3 then with water, dried over anhyd. Na2SO4 and evaporated in vacuo (roto-vap). The crude product was purified by flash column (95:5 CH2Cl2:CH3OH by vol. eluent) then radial (98:2 CH2Cl2:CH3OH by vol. eluent) chromatography to obtain pure 4e in 60% yield. It had mp 158–159°C; 1H-NMR, δ: 1.25 (3H, t, J = 7.4 Hz), 1.75 (4H, m), 2.05 (3H, s), 2.28 (3H, s), 2.36 (2H, t, J = 7.2 Hz), 2.79 (2H, t, J = 7.2 Hz), 4.02 (3H, s), 4.11 (2H, q, J = 7.4 Hz), 4.33 (3H, s), 6.07 (1H, s), 7.62 (1H, brs), 10.76 (1H, brs) ppm; 13C-NMR data are in Table 1.
Anal. Calcd for C21H28N2O6 (404.5): C, 62.36; H, 6.98; N, 6.93
Found: C, 62.29; H, 6.70; N, 6.85
Crystals of 1 were grown by slow diffusion of diethyl ether into a solution of CH2Cl2. Crystals of 4e were grown by slow diffusion of diethyl ether into a solution of CHCl3. A crystal of 1 (0.51 × 0.13 × 0.09 mm3) and a crystal of 4e (0.36 × 0.14 × 0.06 mm3) were placed onto the tips of 0.1 mm diameter glass capillaries and mounted on a Bruker SMART Apex system for data collection at 100(2) K. A preliminary set of cell constants was calculated from reflections harvested from three sets of 20 frames for 1 and 4e. These initial sets of frames were oriented such that orthogonal wedges of reciprocal space were surveyed (final orientation matrices determined from global least-squares refinement of 7646 reflections for 1 and 3591 reflections for 4e. The data collection was carried out using Mo Kα radiation (0.71073 Å graphite monochromator) with a frame time of 40 s for 1 and 30 s for 4e and a detector distance of 4.94 cm. A randomly oriented region of reciprocal space was surveyed to the extent of two hemispheres and to a resolution of 0.66 Å. Four major sections of frames were collected with 0.3° steps in ω at 600 different settings and a detector position of 36° in 2θ for 1 and 4e. The intensity data were corrected for absorption and decay (SADABS).13 Final cell constants were calculated from the xyz centroids of strong reflections from the actual data collection after integration (SAINT 6.45, 2003).14 Crystal data and refinement information for 1 and 4e are provided in Tables 6 and and7,7, respectively.
The structures were solved and refined using SHELXLT-L.15 The monoclinic space groups P2(1) and P2(1)/n were determinedfor 1 and 4e, respectively, based on systematic absences and intensity statistics. A direct-methods solution was calculated, which provided non-hydrogen atoms from the E-map. Full-matrix least squares/difference Fourier cycles were performed for structure refinement. All non-hydrogen atoms were refined with anisotropic displacement parameters unless stated otherwise. Hydrogen atom positions were placed in ideal positions and refined as riding atoms with relative isotropic displacement parameters (a C-H distance fixed at 0.96 Å and a thermal parameter 1.2 times the host carbon atom). Tables of atomic coordinates, bond lengths and angles, anisotropic displacement parameters, hydrogen coordinates, and isotropic displacement parameters have been deposited at the Cambridge Crystallographic Data Centre, CCDC No. 703014 for 1 and 703013 for 4e.
We thank the U.S. National Institutes of Health (HD 17779) for generous support of this research. We also thank the National Science Foundation (CHE-0226402 and CHE-0521191) for providing funding to purchase the X-ray diffractometer used in this work and acquiring a 400 MHz NMR spectrometer and upgrading an existing unit. We thank Prof. T.W. Bell for use of the vapor pressure osmometer.
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