Variable-Temperature 1H-NMR Studies on Two C-Glycosylflavones

Two known C-glycosylflavones, swertisin and embinoidin, were isolated from the leaves of Anthurium aripoense, and characterized by room temperature 1D and 2D NMR experiments. At this temperature, the 1H- and 13C-NMR spectra of these C-glycosylflavones revealed doubling of signals, which suggested the presence of two rotamers in solution. Variable-temperature (VT) 1H-NMR studies supported this hypothesis. The T-ROESY data, in addition to the theoretical (MM2) calculations utilizing the Chem3D Pro software, confirmed the hypothesis that the two rotamers interchange via rotation about the C-glycosidic bond.


Introduction
Anthurium is a genus of evergreen, climbing or epiphytic herbs which belongs to the Araceae [1], and has reported folkloric uses, which include hallucinogens, insecticides, oral contraceptives, rheumatoid arthritis treatments and skin care agents [2][3][4]. Anthurium aripoense is a terrestrial or short climbing herb found in Trinidad and Venezuela [5,6]. A survey of the literature revealed that there has been no phytochemical investigation of this species. Thus, the chemical constituents of this plant were investigated.

OPEN ACCESS
Two known C-glycosylflavones, swertisin and embinoidin [7], were isolated from the leaves of Anthurium aripoense. Both compounds showed duplication of signals in the 1 H-and 13 C-NMR spectra at room temperature. Similar observations had been made earlier on another C-glycosylflavone, spinosin, and attributed to the presence of two rotamers which were slowly interconverting via rotation about the C-glycosidic bond [8].
Rotational isomerism describes the phenomenon of rotation about a single bond in a molecule. Rotamers result when rotation is hindered by a rotational energy barrier [9]. Some interactions that influence the stability of rotamers include double bond character due to resonance, intramolecular hydrogen bonding and steric repulsions between adjacent atoms [10]. For the purpose of this study, steric hindrance proved to be the dominating effect.
In this investigation, the structure of the two compounds was confirmed with the aid of 2D NMR experiments while the origin of signal doubling was investigated using variable-temperature (VT) 1 H-NMR studies in combination with theoretical calculations.

Results and Discussion
The structures of swertisin (1) and embinoidin (2) were confirmed and spectra assigned by a combination of 1 H-1 H COSY [11], HSQC [12] and HMBC [13] spectra. T-ROESY spectra [14] were obtained to aid in interpretation of the origins of the spectral doubling. Complete spectral assignments for the flavone moieties of swertisin (1) and embinoidin (2) are given in Table 1, while the spectral data for the sugar moieties are given in Table 2.
The structure of swertisin (1) is shown in Figure 1, with carbons and the 5-OH proton giving rise to doubled signals marked with asterisks. The 1 H-NMR spectrum of swertisin (1) in DMSO-d 6 at 289 K showed that the relative proportion of the major and minor rotamers was 1.00:0.82. The duplication of the signals of the sugar unit and of carbons close to the C-glycosidic bond implied that there was an energy barrier about this bond that prevented fast exchange between the two rotamers. VT 1 H-NMR studies were conducted on swertisin (1) to confirm this supposition. The VT 1 H-NMR studies on swertisin (1) were not performed using DMSO-d 6 as the solvent because the 5-OH signal markers were not detected individually just above the freezing temperature of DMSO-d 6 . On lowering the temperature below 292 K, the sample froze. Therefore, swertisin (1) was dissolved in (CD 3 ) 2 CO-d 6 /DMSO-d 6 (1:1) which had a freezing temperature of 258 K, thereby allowing the 5-OH signal markers to be individually detected. At 262 K and using the 5-OH signals as markers, the two rotamers of swertisin (1), with a relative proportion of 1.00:0.82, were independently detected since they were in slow exchange. At 305 K, the signals for the two rotamers moved even closer but were still detected. Eventually, the two signals coalesced to a single peak at ca. 13.54 ppm at a Coalescence Temperature, T c , of 321 K ( Figure 2). The coalescence of signals was observed for the entire 1 H-NMR spectrum, although the other pairs of signals coalesced at lower temperatures, reflecting their smaller chemical shift differences.  13 C-NMR (150 MHz) spectral data of the flavone nucleus of swertisin (1) and embinoidin (2) in DMSO-d 6 (δ in ppm, J in Hz).   (2) in DMSO-d 6 (δ in ppm, J in Hz).   (1) Figure 2. Effect of Temperature on the 5-OH signal markers in the 1 H-NMR spectrum of swertisin (1).
The free energy of activation for the interconversion between the two unequally populated rotamers of swertisin (1) can be calculated using Eyring's equations (a and b) as modified by Shanan-Atidi and Bar-Eli [15]: where X = 2πτ∆v and ∆P = P A − P B . P A and P B represent the population of the conformers A and B (P A > P B , P A + P B = 1), respectively, and τ is the mean lifetime. T c and ∆v are the coalescence temperature and the chemical shift difference between conformers A and B, respectively. X is obtained using equation (c): From the 1 H-NMR spectrum at 262 K, the frequency difference, Δν, between the 5-OH signals was 11.13 Hz (11.13 s −1 The VT 1 H-NMR studies confirmed the hypothesis that the doubling of signals in the 1 H-and 13 C-NMR spectra at 262 K was due to the presence of two rotamers of swertisin (1) separated by a relatively high energy barrier.
The data derived from the T-ROESY spectrum suggested that the two rotamers slowly rotated about the C-glycosidic (C-1''-C-6) bond. Weak cross-peaks were observed between the respective methoxyl protons (7-OMe), and the H-1'' a and H-2'' b protons. This observation indicated that β-D-glucose rotates about the C-1''-C-6 bond. Therefore, in one rotamer, the H-1'' proton is oriented syn to the 7-OMe group whereas, in the other rotamer, the H-2'' proton is oriented syn to the 7-OMe group.

(b)
The structure of embinoidin (2) is shown in Figure 4, with carbons and the 5-OH proton giving rise to doubled signals marked with asterisks. At 305 K, the 1 H-NMR spectrum of embinoidin (2) in DMSO-d 6 indicated a doubling of the signals which was attributed to the presence of two rotamers, with a relative proportion of 1.00:0.97, in solution. The duplicated 1 H-NMR signals suggested that, at 305 K, there were two rotamers separated by an energy barrier about the C-glycosidic bond which hindered rotation between the rotamers. VT 1 H-NMR studies were performed on embinoidin (2) in order to confirm this hypothesis. At 305 K and using the 5-OH signals as markers, the two rotamers of embinoidin (2) in DMSO-d 6 were in slow exchange so that each rotamer was detected independently. At 348 K, the signals for the two rotamers were still individually detected, although, the two 5-OH signals had broadened and moved closer together. Eventually, the two signals coalesced to a single peak at ca. 13.4 ppm at a Coalescence Temperature, T c , of 356 K ( Figure 5). The coalescence of signals was observed for the entire 1 H-NMR spectrum, although the other pairs of signals coalesced at lower temperatures, reflecting their smaller chemical shift differences. The free energy of activation for the interconversion between the two rotamers of embinoidin (2) was also calculated using Eyring's equations (a and b) as modified by Shanan-Atidi and Bar-Eli [15]. Since the relative proportion of the two rotamers was approximately 1:1 at 305 K, the free energy of activation for rotation, ΔG ‡ rot , was also calculated using Eyring equation for equally populated rotamers [16]. H. S. Gutowsky showed that the rate of rotation, k c , at the temperature of coalescence, T c , 356 K, can be calculated using the following equation [16]: The free energy of activation for rotation, ΔG ‡ rot , at 356 K was calculated using the Eyring equation [16]: where k is the rate constant, k B is Boltzmann's constant, h is Planck's constant, K is the transmission coefficient, T is the temperature in K, R is the universal gas constant and ΔG ‡ is the free energy of activation.
Assuming the transmission coefficient, K, to be unity, converting natural log (ln) to log 10 , and substituting k c and T c into the Eyring equation, this equation becomes [16]: The VT 1 H-NMR studies confirmed the hypothesis that the doubling of signals in the 1 H-and 13 C-NMR spectra at 305 K was due to the presence of two rotamers of embinoidin (2) separated by a relatively high energy barrier.
The data derived from the T-ROESY spectrum again suggested that the two rotamers slowly rotated about the C-glycosidic bond. Weak cross-peaks were observed between the respective methoxyl protons (7-OMe), and both signals of H-1'' a and H-2'' b . This observation indicated that β-D-glucose, which is directly attached to the flavone nucleus, rotates about the C-glycosidic bond. Therefore, in one rotamer, the H-1'' proton is oriented syn to the 7-OMe group whereas, in the other rotamer, the H-2'' proton is oriented syn to the 7-OMe group. Stronger cross-peaks were observed between the H-2'' a and H-1''' a signals, and between the H-2'' b and H-1''' b proton signals. Thus, in each rotamer, the rotation about the C-2''-O-C-1''' bond brings the H-1''' proton in close proximity to the H-2'' proton.

General
Mass spectral data was obtained using a Bruker Daltonics micrOTOF-Q ESI mass spectrometer, and the UV-visible spectra were recorded on a Varian CARY 50 Conc UV-visible spectrophotometer. IR spectra were acquired on Perkin Elmer FTIR RX1 spectrophotometer. Column chromatography was carried out on Merck silica gel 60 (70-230 mesh). At room temperature, the 1 H-, 13 C-, 1 H-1 H COSY, HSQC, HMBC and T-ROESY NMR experiments were performed using a Bruker Avance DRX-600 spectrophotometer which was equipped with a 5 mm PATXI indirect detection probe with a Z gradient coil ( 1 H 90 pulse width = 9.90 s, 13 C 90 pulse width = 12.55 s). The VT 1 H-NMR data were acquired on a Bruker-Avance DRX-400 spectrophotometer equipped with a 5 mm 1 H/ 13 C/ 19 F/ 31 P probe and a BVT-3300 VT controller for temperature regulation and measurement. The samples were dissolved in DMSO-d 6 or (CD 3 ) 2 CO-d 6 /DMSO-d 6 (1:1), chemical shifts are reported in parts per million (ppm) downfield from tetramethylsilane (0 ppm) as the internal standard, and coupling constants (J) are recorded in Hertz (Hz). Operating parameters for the 1 H-detected experiments were an F2 ( 1 H) spectral width of 12335.526 Hz with 64 K data points, whereas the parameters for the 13 C-detected experiments were an F1 ( 13 C) spectral width of 36057.691 Hz with 64 K data points, a 30° pulse width and 8K scans. For the high resolution HSQC spectra, F1 was 28673.971 Hz and F2 was 7936.508 Hz, 256 time increments were linear predicted to 1024, with 16 transients per increment, and for HMBC spectra, F1 was 34711.465 Hz and F2 was 7936.708 Hz with 32 transients per increment and 256 time increments linear predicted to 1024. The 1 H-1 H COSY spectra were processed in the absolute value mode. Phase-sensitive T-ROESY spectra used the same 1 H spectral windows and F2 data points with 256 increments linearly predicted to 1024 with a mixing time of 0.2 s and a relaxation delay of 2.0 s.

Plant Material, Extraction and Isolation
The leaves of A. aripoense, growing at an altitude of ca. 800 m, were collected at El Cerro del Aripo, Trinidad, on January 2008. The plant was identified by Mr. Winston Johnson of the National Herbarium of Trinidad and Tobago, where a voucher specimen (TRIN 36517) was deposited.
A portion of the n-BuOH fraction (22.00 g) was subjected to isocratic column chromatography on silica gel using CHCl 3 /MeOH (75:25) to yield five fractions. The fifth fraction (4.18 g) was further fractionated on a silica gel column using CHCl 3 /MeOH (75:25) to afford four fractions. When concentrated under reduced pressure, the third fraction yielded embinoidin (2, 45.4 mg).