Selective Sensing of Metal Ions and Nitro Explosives by Efficient Switching of Excimer-to-Monomer Emission of an Amphiphilic Pyrene Derivative.

An amphiphilic pyrene derivative exhibiting unusually stable excimer emission due to strong aggregation is presented. The aggregated system served as an intelligent sensor for metal ions and nitro explosives in aqueous media. The excimer displayed excellent selectivity toward Cu2+ among the tested cations. The observation was interpreted on the basis of chelation of metal ions involving the hydroxyl and amino groups of two molecules, leading to the ligand-to-metal charge-transfer (CT) process. The excimer was further applied for the cell imaging of Cu2+ ions. Also, while treating the excimer with various nitro explosives, it displayed efficient 2,4,6-trinitrophenol sensing, corroborating mainly the CT process from pyrene to the analyte due to intercalation of the analyte within pyrene.

Introduction

Pyrene is a unique fluorophore that exhibits distinct monomer (λmono = 370–400 nm) and excimer fluorescence (λexc = 420–600 nm) depending on the distance (d > 1 nm and 0.4 nm < d < 1 nm, respectively) between two pyrene moieties.[1−3] Either the formation or the destruction of the excimer in the presence of external inputs rendered pyrene a versatile fluorophore for probing specific noncovalent interactions in biology (e.g., protein conformation,[4,5] binding,[6] etc.) and chemistry[7,8] (e.g., chemosensors for cations,[9−12] anions,[13,14] nitro explosives,[15−17] etc.). Sensors, which exhibit excimer fluorescence in their latent form, are designed mostly via covalent linking of two or more pyrene moieties to a recognition domain. Such a connection of pyrenes ensures intramolecular aggregation of the fluorophore, resulting in strong and stable excimer emission. Over the past several years, numerous chemosensors have been developed, applying the pyrene excimer for detecting different species of interest.[18−23] There are very few multifunctional sensors reported which can detect more than one type of analyte;[24] however, they use the same recognition site.[25,26] Chemosensors that employ different recognition sites for the detection of two distinct classes of analytes are rare. Kaur and co-workers have reported a phenanthro[4,5-fgh]pyrido[2,3-b]quinoxaline derivative that displayed red-shifted excimer emission in the presence of Hg2+ ions via coordination with the aromatic ring nitrogen.[27] The probe was also able to sense nitroaromatics through electron energy transfer from the probe to the analyte, resulting in the quenching of excimer emission.[28] The research group of Cao reported 1,3,6,8-tetrabromopyrene and 1,3,5-tris(4-bromophenyl)benzene as monomers to synthesize a series of porous covalent organic polymers (COPs) displaying strong emission above 500 nm. These COPs were able to sense metal ions and nitroaromatics through quenching of fluorescence.[29] Most of these chemosensors typically work in organic media. Other examples of pyrene-based chemosensors capable of sensing two different classes of analytes are (1) conjugated polymer-based nanospongy continuous structures reported by Kim and co-workers for detecting transition-metal ions and nitroaromatics,[30] (2) a pyrene-linked thiourea derivative reported by Velmathi and co-workers for detecting metal ions and picric acid,[31] (3) a ferrocene-triazole derivative reported by Oton and co-workers for detecting ion pairs,[32] (4) a metal-organic framework reported by Moorthy and co-workers for sensing solvent polarity and nitroaromatics,[33] and so forth.

Herein, we report an intermolecular pyrene-aggregation-based strong excimer that is unusually stable in aqueous media. The system is an intelligent sensor that utilizes two different recognition sites for sensing two distinct classes of analytes, viz., metal ions and nitroaromatics, through different sensing mechanisms. The design of such a sensor is challenging because no clear notion is available for (1) controlling the efficiency and stability of the excimer and (2) inculcating two different working mechanisms in the detection of distinct classes of analytes. Figure A illustrates the structural aspects of pyrene-linked amphiphile 1. At the polar domain of the molecule, the 3-aminopropane-1,2-diol moiety was introduced. Beyond their contribution to the solvent-dependent self-assembly process, the vicinal hydroxyl and amino groups were also envisaged to play a significant role in metal-ion recognition.[34] Two hydrophobic decyl chains were introduced as self-adjustive tails to favor aggregation in the polar solvent (e.g., H2O) environment (Figure B). In the milieu of these consummate partners, a pyrene moiety was tethered as the signaling domain for either intermolecular aggregation-induced emission enhancement or the sensing process.

Figure 1

Structure of amphiphilic pyrene derivative 1 and illustration of its various domains (A). Solvent-polarity-dependent aggregation model of 1 (B).

Results and Discussion

Synthesis of the Amphiphilic Pyrene Derivative

Synthesis of compound 1 was carried out from (R)-2,2-dimethyl-1,3-dioxolane-4-carbaldehyde (2) following the aldehyde–amine–aldehyde three-component coupling (A3-coupling)[35] protocol established by us.[36,37] Aldehyde 2 upon reaction with didecylamine 3 and pyrene alkyne 4 under CuBr catalytic conditions provided compound 5 in 70% yield. This product upon deprotection of the ketal group gave the desired compound, 1, in 87% yield (Scheme ). The structure of the product was confirmed by NMR spectroscopy (see Figures S1–S4), crystallography (Figure S5), and mass spectrometry (Figure S19).

Scheme 1

Synthesis of Compound 1

Excimer Formation

Amphiphile 1 was soluble in a range of organic solvents (e.g., heptane, toluene, CHCl3, THF, CH3CN, MeOH, EtOH, and dimethyl sulfoxide), and from a compatibility point of view, we selected EtOH as the solvent for the aggregation studies. The UV–visible and fluorescence spectra in EtOH suggested the existence of 1 exclusively in the monomeric form (Figures S6 and S7). Upon gradual addition of H2O, the absorption and emission characteristics of 1 remained unaffected up to the water percentage fw ≤ 50% (Figures A and S8A). At fw ≥ 60%, bathochromic shifts of the absorption bands to 333, 350, and 370 nm were observed, indicating the onset of aggregation of compound 1 through strong π–π stacking interactions between pyrene moieties. Indeed, the respective fluorescence spectra of 1 in EtOH/H2O (fw ≥ 60%) showed the appearance of an intense band at 498 nm, typical of pyrene excimer emission (Figure A). Cuvette images (under the handheld UV lamp, with λex = 365 nm) of freshly prepared samples of 1 also indicated strong cyan excimer emission when the solvent polarity was increased to fw ≥ 50% (inset of Figures B and S8B).

Figure 2

Fluorescence spectra (A) and quantum yield values (B) of 1 in EtOH/H2O (fw = 0–90%). Inset: cuvette images of 1 in EtOH and EtOH/H2O (fw = 60%).

Stepwise addition of EtOH (100–700 μL) to 1 (33.3 μM, 2 mL) prepared in EtOH/H2O (fw = 60%) resulted in a decrease of the excimer intensity and enhancement of monomer signals (Figure S9A). The monomer–excimer structural change was reversible, as indicated by the images of samples captured after the sequential addition of EtOH and H2O to the excimer of 1 (Figure S9B). This study confirmed that the formation of an excimer is a solvent-polarity-dependent feature of 1. Dynamic light scattering (DLS) studies of 1 also confirmed weak aggregation in EtOH (diameter < 10 nm), and the size of the aggregated materials increased to a diameter of 400–800 nm in EtOH/H2O (fw = 60%) (Figure S10). Subsequent increase in the fw resulted in the disintegration of the self-assembly, which is possibly due to the increase in solvent polarity. To analyze the effect of solvent polarity on the emission of the aggregated material, quantum yield, Φ, values at different fw’s were recorded. This data also provided low Φ = 0.01–0.04 (standard: quinine sulfate in 0.5 M H2SO4)[38] in the polarity range fw = 0–50% (Figure B). An abrupt increase in Φ from 0.04 to 0.32 was observed with an increase in the H2O percentage from fw = 50 to 60%. However, a further increase in fw resulted in a slow decrease of Φ until fw = 80% and a subsequent rapid decrease to Φ = 0.2 at fw = 90%. Note that the freshly prepared so-called static excimer[39] of 1 exhibited an excellent excimer-to-monomer intensity ratio, Fexc/Fmono > 20, and was stable for more than 1 month (retention of 20% of the initial excimer emission and optically detectable) under ambient conditions (Figure S11). The excimer was also stable at pH = 6–12 (Figure S12). The excimer stability in the long pH range endowed us to explore the chemosensing applications of excimer 1 at physiological pH. On the basis of these intriguing results, the chelating ability of the free vicinal hydroxyl and amino groups with metal ions were evaluated to explore the application of 1 as a metal-ion sensor. On the other hand, the π–π stacking ability of pyrene with electron-deficient aromatic systems was exploited through the sensing of nitroaromatic explosives.

Detection of Metal Ions

The metal-ion sensing abilities of excimer 1 (33.3 μM) were investigated in EtOH/H2O (fw = 60%) by screening Na+, Ba2+, Ca2+, Cd2+, Co2+, Cu2+, Hg2+, Mn2+, Ni2+, Pb2+, Zn2+, Cr3+, and Fe3+ (70 μM each). Upon the addition of each cation, the observed quenching efficiencies were significantly lower for Na+, Ba2+, Ca2+, Cd2+, Co2+, Hg2+, Mn2+, Ni2+, Pb2+, Zn2+, Cr3+, and Fe3+ (Figures A and S15). The addition of Fe3+ to the excimer of 1 yielded a maximum of 45% excimer fluorescence quenching. Interestingly, the addition of Cu2+ resulted in an excellent reduction in excimer fluorescence by up to 94%. To further strengthen the selectivity of Cu2+-ion sensing, we have tested a bi-analyte system upon sequential addition of Zn2+ and Cu2+ ions. Indeed, our probe exhibited a remarkable selectivity toward the Cu2+ ion (Figure S16).

Figure 3

Fluorescence quenching efficiency at λ = 498 nm (λex = 343 nm) upon addition of various metal ions (70 μM) in EtOH/H2O (fw = 60%) (A). Inset: cuvette images of 1 in the absence and presence of Cu2+ ions. Job’s plot analysis of 1 for the interaction with Cu2+ (B).

Titration of the excimer (33.3 μM) with Cu2+ ions (0–70 μM) yielded a stepwise decrease in the excimer band at 498 nm, with a concomitant enhancement of signals at 387 and 405 nm and an isosbestic point at 425 nm, indicating that only one product was generated from 1 upon binding to Cu2+ ions (Figure S17). This study established the application of 1 as a ratiometric chemosensor[40−42] for Cu2+ ions. Quenching of the excimer and appearance of blue fluorescence were also evident from the cuvette images captured before and after the addition of Cu2+ ions (70 μM) to 1 (33.3 μM, fw = 60% in EtOH) (Figure B, inset). A typical Job’s analysis (Figure B) of the fluorometric titrations data (Figure S18) was done to determine the stoichiometry of the binding of Cu2+ ions with ligand 1. The stoichiometric ratio for Cu2+–1 was estimated to be 1:2, which is very much consistent with earlier reports on the fluorescence quenching of pyrene-based ligands (either N-donor or O-donor) upon complexation with ions.[43,44] matrix-assisted laser desorption ionization tandem time-of-flight (MALDI-TOF-TOF) mass spectrometric analysis of the mixture containing 1 and Cu(NO3)2·3H2O also provided the signals corresponding to the 1:2 complexation[45] (Figure S19). UV–visible data obtained after the addition of Cu2+ ions strongly indicated that the pyrene moieties were in the aggregated state (Figure S14), which was further supported by DLS data (Figure S32A). This study confirmed a predominant ligand-to-metal charge-transfer (LMCT) process for Cu2+-ion sensing by quenching of the excimer emission.[46]

We further examined the quantitative response of excimer 1 toward Cu2+ by fluorometric titrations. A stepwise decrease in the excimer fluorescence band (λex = 498 nm) was observed (Figure S17) when titrations were carried out by the addition of increasing concentrations of Cu(NO3)2·3H2O (0–70 μM) to probe 1 (33 μM, fw = 60% in EtOH). When the fluorescence intensities at 498 nm were plotted against the concentrations of Cu2+, an excellent linear correlation was observed up to 8 μM (regression factor, R2 = 0.9959). A detection limit of 0.394 μM was calculated for probe 1 during the detection of Cu2+, on the basis of the signal-to-noise ratio, S/N = 3 (Figure S20).

Among various transition-metal ions, copper is the third most abundant in the human body and plays a significant role in diverse physiological processes.[47] Therefore, sensitive and reliable detection of copper[41,42,48,49] in environmental[50] samples and inside living cells is needed. Cell-imaging studies were carried out to demonstrate the Cu2+-ion-sensing ability of 1 in the biological system. In the study using the HeLa cell line, strong cyan excimer fluorescence was observed when HeLa cells were incubated with only 1 (15 μM, 10% ethanol, pH = 7.4) at 37 °C for 45 min (Figure A). However, further treatment of these preincubated cells with Cu(NO3)2·3H2O (10 μM) at 37 °C for 1 min resulted in quenching of the cyan fluorescence (Figure B). These experiments strongly demonstrate the cell-membrane permeability of amphiphile 1, its stable aggregation in the intracellular environment, and its ability to sense intracellular Cu2+ ions.

Figure 4

Confocal laser scanning microscopy images of HeLa cells (A) after incubation with 1 (15 μM) for 45 min and (B) upon incubation with 1, followed by treatment with Cu(NO3)2·3H2O (10 μM) for 1 min.

Detection of Nitroaromatic Explosives

For the second application, typical aromatic (e.g., 1,3-dinitrobenzene (1,3-DNB), 2,4-dinitrotoluene (2,4-DNT), 2,6-dinitrotoluene (2,6-DNT), 2,4,6-trinitrotoluene (TNT), 4-nitrophenol (4-NP), 2,4-dinitrophenol (2,4-DNP), and 2,4,6-trinitrophenol (TNP)) and nonaromatic nitro explosives (e.g., nitromethane (NM) and 1,3,5-trinitroperhydro-1,3,5-triazine (RDX)) were used. In each experiment, the response of 1 (33.3 μM, fw = 60% in EtOH) toward an analyte (0–39.6 μM) was evaluated by recording the fluorescence spectrum (λex = 343 nm) immediately after mixing (Figures S22–S30). Among the tested aromatic explosives, TNP displayed an excellent reduction of excimer fluorescence up to 98% (Figure A), with a concomitant increase in monomer bands (Figure S22). The cyan-to-blue fluorescence change upon addition of TNP (62.5 μM) to 1 (33.3 μM, fw = 60% in EtOH) was also confirmed optically from the corresponding cuvette images (Figure A, inset). Quenching of excimer fluorescence by TNP under the competing environment of other analytes established the selectivity of 1 toward this nitro explosive (Figure B). We attribute the excellent TNP selectivity of the excimer to the charge-transfer (CT) complex[51−53] formation between electron-rich pyrene and electron-deficient TNP (evident from the pronounced CT band in Figure S21) through complementary intermolecular π-stacking interactions. However, the contribution from the resonance energy transfer process[54−56] could not be ignored (evident from the spectral overlap between the absorption of an analyte and emission of 1, Figure S31). These π-stacking interactions further enhanced the aggregation behavior of 1, which was supported by the DLS measurement after the addition of TNP (Figure S32B).

Figure 5

Percentage quenching of the excimer fluorescence (at λem = 498 nm with λex = 343 nm) of 1 (33.3 μM) in EtOH/H2O (fw = 60%) upon addition of different nitro explosives. Inset: cuvette images of 1 in the absence and presence of TNP (A). Normalized fluorescence intensity upon the addition of different nitro explosives (20 μL of the 1 mM analyte during each addition) followed by TNP (20 μL of the 1 mM analyte during each addition) to 1 (B).

We further examined the quantitative response of probe 1 toward TNP by fluorometric titrations. A stepwise decrease in the fluorescence (λex = 498 nm) of the excimer band was observed (Figure S22) when the titrations were carried out by the addition of increasing concentrations of TNP (0–39.6 μM) to probe 1 (33 μM, fw = 60% in EtOH). When the fluorescence intensities at 498 nm were plotted against the concentrations of TNP, an excellent linear correlation (regression factor, R2 = 0.95534) was observed. A detection limit of 155 nM was calculated for probe 1 during the detection of TNP, on the basis of a signal-to-noise ratio, S/N = 3 (Figure S33). Notably, the sensing of nitro explosives has been a matter of growing interest in the recent past. These sensors include dispersions of relatively expensive metal-organic frameworks,[55,57,58] as well as quantum dots,[59−61] although most of the experiments were performed in nonaqueous solvents and few in aqueous solvents.[23,62−64] However, the use of the inexpensive pyrene derivatives for the sensing of nitroaromatics in aqueous media appears closer to practical applications.

Mechanisms of Sensing

On the basis of the above studies, mechanisms for Cu2+ ion and TNP-sensing applications of 1 were proposed (Figure ). The observed selectivity for Cu2+ was attributed to complex formation, involving the hydroxyl oxygen and amino nitrogen atoms coordinating with the soft Cu2+ ion (Figure , path A).[43,44] Sensing of TNP, on the other hand, was correlated to the intercalation of the nitroaromatic compounds between pyrenes (i.e., π–π stacking interactions), leading to a CT process from pyrene to the analyte (Figure , path B).

Figure 6

Proposed models for the detection of metal ions and nitro explosives by the excimer of 1.

Conclusions

In summary, a simple and smart amphiphilic pyrene compound was developed for strong and stable excimer formation in aqueous media. Sensing applications of the excimer in the aqueous media were demonstrated in the present work through detections of metal ions and nitro explosives. The suitably decorated pyrene derivative (with a polar vicinal diol moiety and hydrophobic alkyl chains) displayed blue fluorescence in EtOH, which switched to strong cyan fluorescence (excimer) upon increasing the H2O percentage. In 40:60 EtOH/H2O, the aggregation resulted in particles of size 400–800 nm, and a significant amount of the excimer was retained even after 1 month. During the screening of metal ions, the amphiphile displayed selectivity toward Cu2+ ions, as evident from the quenching of the cyan excimer fluorescence and appearance of the LMCT band in the absorption spectrum. A chelation model involving the hydroxyl and amino groups of two molecules of the amphiphile was also established. Imaging of intracellular Cu2+ ions by the excimer demonstrated the applicability of the amphiphile in biological systems. The aggregate also exhibited a better TNP-sensing ability when compared to that of other nitro explosives. Almost complete quenching of the excimer fluorescence, observed during TNP sensing, was associated with an effective CT process (from electron-rich pyrene to electron-deficient TNP) through complementary intermolecular π-stacking interactions. Our synthetic approach of combining a polar head, nonpolar tails, and a responsive moiety in a single entity could provide a platform for generating self-assembled multifunctional molecule-based materials.

Experimental Section

Materials

All chemicals for the synthesis were purchased from commercial sources and used as received, unless stated otherwise. All reactions were conducted under a nitrogen atmosphere. The solvents, petroleum ether, ethyl acetate (EtOAc), dichloromethane, and methanol (MeOH), were distilled prior to thin-layer chromatography (TLC) and column chromatography. Column chromatographic purifications were performed on Merck silica gel (100–200 mesh). TLC was carried out with E. Merck silica gel 60-F-254 plates. The analytes, 1,3-DNB, 2,4-DNT, 2,6-DNT, 4-NP, 2,4-DNP, TNP, and NM, were purchased from commercial sources. TNT and RDX were collected from the High Energy Materials Research Laboratory, Pune.

Methods

1H and 13C NMR spectra were recorded on either a 400 MHz Jeol ECS-400 (or 100 MHz for 13C) or 400 MHz Bruker AV400 (or 100 MHz for 13C) spectrometer using either residual solvent signals as an internal reference or internal tetramethylsilane on the δ scale (CDCl3: δH, 7.24 ppm, δC 77.0 ppm). The following abbreviations are used to describe peak patterns where appropriate: b, broad; s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet. Coupling constants are reported in hertz (Hz). High-resolution mass spectra (HRMS) were recorded either on an electron spray ionization (ESI)-TOF or on a MALDI-TOF-TOF mass spectrometer. Melting points were measured with a micro melting point apparatus. Photophysical studies were conducted on UV–visible and steady-state spectrophotometers. DLS studies were performed using a Nano ZS-90 apparatus utilizing a 633 nm red laser (at 90° angle) from Malvern Instruments. At 90°, scattered fluctuations were detected to generate correlation function [g2(t)]; from this function, the diffusion coefficient (D) was calculated using the cumulant method. By applying the Stokes–Einstein equation, the particle diameter was calculated. The reproducibility of the data was checked at least three times using independent solutions of 1.

Synthesis of N-Decyl-N-((R)-1-((S)-2,2-dimethyl-1,3-dioxolan-4-yl)-3-(pyren-1-yl)prop-2-yn-1-yl)decan-1-amine (C44H61NO2) 5

To a solution of (R)-2,2-dimethyl-1,3-dioxolane-4-carbaldehyde (2; 570 mg, 4.38 mmol) in dry toluene (10.0 mL) were added didecylamine 3 (1.30 g, 4.38 mmol), 4 Å molecular sieves (2.00 g), CuBr (32 mg, 0.22 mmol), and pyrene alkyne 4 (1.00 g, 4.38 mmol). The reaction mixture was stirred at room temperature for 48 h. After reaction completion, the reaction mixture was filtered through a celite bed and washed with Et2O (2 × 20 mL). The combined filtrate was concentrated under reduced pressure to obtain a liquid, which was further purified by column chromatography over silica gel (eluent: 1% EtOAc in petroleum ether) to furnish 5 (1.95 g, 70%) as a yellow thick oil. IR (KBr) ν (cm–1): 3021, 2364, 1647, 1427, 1368, 1214; [α]D25 = −6.60 (c = 1.0, CHCl3); 1H NMR (400 MHz, CDCl3) δ (ppm): 8.60 (d, J = 9.1 Hz, 1H), 8.27–8.03 (m, 8H), 7.56 (d, J = 7.3 Hz, 4H), 7.36 (t, J = 7.4 Hz, 4H), 7.29–7.25 (m, 2H), 4.56 (q, J = 6.3 Hz, 1H), 4.22 (dd, J = 8.4, 6.3 Hz, 1H), 4.17 (dd, J = 8.3, 6.2 Hz, 1H), 4.11–4.06 (m, 3H), 3.76 (d, J = 13.9 Hz, 2H), 1.42 (s, 3H), 1.38 (s, 3H); 13C NMR (100 MHz, CDCl3) δ (ppm): 139.5 (2C), 129.1 (4C), 128.6 (4C), 128.4 (2C), 127.3, 127.2, 125.8, 124.6, 117.5, 109.9, 90.2, 85.9, 76.6, 67.7, 56.7, 55.9 (2C), 26.7, 25.7; HRMS (ESI): calcd for C44H62NO2 636.4781 [M + H]+; found, 636.4781.

Synthesis of (2S,3R)-3-(Didecylamino)-5-(pyren-1-yl)pent-4-yne-1,2-diol (C41H57NO2) 1

To a solution of 5 (1.70 g, 2.68 mmol) in THF (15 mL) was added 2 M HCl (3 mL), and the mixture was stirred for 12 h at room temperature. After completion of the reaction, the solution was neutralized with solid K2CO3 and the reaction mixture was concentrated. The reaction mixture was diluted by adding H2O (20 mL), followed by extraction of the product in EtOAc (2 × 20 mL). The combined organic layer was dried over Na2SO4 and concentrated in vacuo. The crude product was purified by column chromatography over silica gel (eluent: 10% EtOAc in petroleum ether) to furnish 1 (1.38 g, 87%) as a pale yellow solid. mp: 68–70 °C; IR (KBr) ν (cm–1): 3436, 3096, 2925, 2855, 2313, 1461, 1405, 1261, 1187, 1094, 1023; [α]D25 = −32.0 (c = 1.0, CHCl3); 1H NMR (400 MHz, CDCl3) δ (ppm): 8.49 (d, J = 9.1 Hz, 1H), 8.22 (t, J = 7.0 Hz, 2H), 8.17 (d, J = 9.1 Hz, 1H), 8.12–8.07 (m, 3H), 8.03 (dd, J = 8.1, 7.3 Hz, 2H), 4.12 (dd, J = 11.8, 2.9 Hz, 1H), 4.03 (d, J = 9.9 Hz, 1H), 3.92 (dd, J = 11.8, 2.9 Hz, 1H), 3.83 (dt, J = 9.9, 3.0 Hz, 1H), 2.83–2.61 (m, 4H), 1.65–1.54 (m, 4H), 1.44–1.23 (m, 28H), 0.87 (t, J = 6.8 Hz, 6H); 13C NMR (100 MHz, CDCl3) δ (ppm): 132.1, 131.4, 131.3, 131.1, 130.1, 128.6, 128.3, 127.3, 126.4, 125.8, 125.3, 124.5, 124.4, 117.3, 89.8, 86.5, 70.7, 63.2, 56.2, 52.0, 32.0, 29.8, 29.7, 29.5, 28.3, 27.6, 22.8, 14.3; HRMS (ESI): calcd for C41H58NO2 596.4468 [M + H]+; found, 596.4468.