H-NMR Studies on Three C-glycosylflavones Exhibiting Rotational Isomerism

Rotational isomerism has been observed on some C-glycosyl flavonoids with special chemical strutures. It may bring rotamers arising from restricted rotation about one single bond. C-glycosyl flavonoids showed a wide range of biological activities. This emphasizes the significance to understand the conformational stability of C-glycosyl flavonoids in drug development. Three C-glycosyl flavonoids, vaccarin (1), isovitexin-2”-O-arabinoside (2) and spinosin (3), were found to exist as rotamers in the light of their featured behavior in nuclear magnetic resonance spectroscopy (NMR). Some separated signals were observed in their H-NMR and C NMR spectra. Variable-temperature (VT) H-NMR experiments were then conducted to study the observation and to determine their rotational energy barriers and then evaluate their conformational stability. The temperature varied from 298K to 363K and separated signals merged at high temperature. With the help of Eyring equation, the rotational energy barriers of compounds 1-3 were calculated and found much lower than 24 kcal/mol. When the energy barrier is high enough (> 24 kcal/mol) and the time scale for interconversion (halflife > 1000 s) is long enough for isolation of individual rotamers, the isomers are termed atropisomers, which thus will display axial charity. According to this standard, the compounds 1-3 may not exhibit atropisomerism and isolation of rotamers should be difficult. Their relatively low energy barriers indicate they can be developed as rapidly equilibrating mixture.


Introduction
Rotational isomer or rotamer is one of a set of conformers arising from restricted rotation about one single bond, which is due to the presence of a sufficiently large rotational barrier to make the phenomenon observable on the time scale of the experiment. When the energy barrier is high enough (> 24 kcal/mol) and the time scale for interconversion (halflife > 1000 s) is long enough for isolation of individual rotamers, the isomers are termed atropisomers, which thus will display axial charity [1][2][3].
Rotational isomerism has been observed on a portion of C-glycosyl flavonoids. Nuclear magnetic resonance spectroscopy (NMR) is the technique that has contributed most to the understanding of this phenomenon. The signals in 1 H and 13 C NMR spectra unexpectedly separate in low temperature but duplication collapses in high temperature, which indicates the existence of rotational isomers [4]. C-glycosyl flavonoids showed a wide range of biological activities and had higher antioxidant and anti-diabetes potential than their corresponding O-glycosyl flavonoids and aglycones [5]. It has become clear that in many cases the majority of activity (toward a desired target) belongs to one rotational isomer, while the other contributes very little [6,7]. This emphasizes the significance to understand the conformational stability of C-glycosyl flavonoids in drug development. The stability of rotamers is often evaluated by kinetics measurements that include various physical terms such as the rate constant (k), half-life (t 1/2 ), and energy barrier (∆G ‡ ) to rotation [3].
Our previous phytochemical studies on Vaccaria hispanica (Miller) Rauschert isolated two C-glycosyl flavonoids, vaccarin (1) and isovitexin-2"-O-arabinoside (2) (Figure 1), both of which exhibited rotational isomerism and existed as rotamers [8]. However, it is still unclear whether the rotational barrier is high enough for atropisomerism to be realized or not.
Modern Chemistry 2021; 9(1): 8-12 9 The conformational stability of these molecules needs to be analyzed. In this paper, we are aimed to determine their rotational energy barriers and then evaluate their conformational stability through variable-temperature (VT) 1 H-NMR experiments. In order to research the effect of the C-7 substituents, commercial spinosin (3) was purchased and subjected to the experiments, as well.

Results and Discussion
C-glycosyl flavonoids 1-3 were subjected to 1 H-NMR and 13 C-NMR experiments at room temperature. Duplicated signals of a part of nucleus appeared in both spectra. Variable-temperature (VT) 1 H-NMR experiments were then conducted from 298 K to 363 K. It turned out that the separated signals coalesced at higher temperature ( Figure 2). This feature suggested compounds 1-3 existed as rotational isomers at room temperature, which should be caused by the restricted rotation around the C (sp 3 )-C (sp 2 ) glucosyl-flavone linkage according to [4].
With the 5-OH signals as markers, the 1 H-NMR spectrum of vaccarin (1) in DMSO-d 6 at 298 K showed that the relative proportion of the major and minor rotamers was 1.336: 1. The two rotamers of vaccarin (1) were independently detected because they were in relatively slow interconverting. At higher temperature, the signals for the two rotamers moved even closer but were still detected. At last, the two signals coalesced to a single peak at δ 13.55 ppm at 353 K, namely at coalescence temperature, T c ( Figure 2). Actually, the other separated signals also coalesced, some of which coalesced at lower temperatures, because their smaller chemical shift difference. The free energy of activation for the interconversion between the two unequally populated rotamers of vaccarin (1) can be calculated using Eyring's equations (1 and 2) as modified by Shanan-Atidi and Bar-Eli [9,10]: where X = 2πτ∆v and ∆P = P A -P B P A and P B mean the respective population of the conformers A and B (P A > P B , P A + P B = 1). τ is the mean lifetime. T c means the coalescence temperature and ∆v is the chemical shift difference between conformers A and B. X is obtained using equation (3): From the 1 H-NMR spectrum of vaccarin (1)  Since the relative proportion of the two rotamers was approximately 1:1 at 298 K, the free energy of activation for rotation, ∆G ‡ , was also calculated using Eyring equation for equally populated rotamers. The rate of rotation, k c , at the temperature of coalescence, T c , 303 K, can be calculated using the following equation [10]: k c = π(∆ν)/√2 k c = π*27.96/√2=62.1 s -1 The free energy of activation for rotation, ∆G ‡ , at 303 K was calculated using the Eyring equation: where k is the rate constant at the temperature T, 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 ∆G ‡ is the free energy of activation.
Assuming the transmission coefficient, K, to be unity, converting natural log (ln) to log10, and substituting k c and T c into the Eyring equation, this equation becomes: ∆G ‡ = 4.57 T c (10.32 + log 10 T c -log 10 k c ) in units of cal/mol = 4.57*303*(10.32+log 10 303 -log 10 62.1) The two methods gave similar results. The 1 H-NMR spectrum of spinosin (3) in DMSO-d 6 at 298 K showed that the relative proportion of the major and minor rotamers was 1.091: 1, using the 5-OH signals as markers. The frequency difference, ∆ν, between the 5-OH signals was 47.14 Hz. The coalescence temperature, T c , was 353 K. The VT 1 H-NMR studies of C-glycosyl flavonoids 1-3 confirmed the hypothesis that the doubling of signals in the 1 Hand 13 C-NMR spectra at 298 K was owing to the presence of two rotamers separated by a relatively high energy barrier ( Table 1). C-glycosyl flavonoids 1 with the largest C-7 substituent had the highest energy barriers. This result indicated that the C-7 substituents had an effect to the rotational isomerism of 6-C-glycosyl flavonoids. The bulker C-7 substituents gave rise to the higher rotational energy barriers. However, they may not exhibit atropisomerism and the isolation of rotamers should be difficult because their energy barriers are much lower than 24 kcal/mol. Based on the amount of rotational barriers, suspected atropisomeric compounds were classified into three categories and accompanying drug development strategies were suggested by LaPlante [1]. Class 1 compounds have rotation barriers around the chiral axis of <20 kcal/mol and possess fast axial rotation rates (t 1/2 less than minutes) with no axial chirality. According to [1], Class 1 rotamers should be developed as purified, single compounds (rapidly equilibrating mixture). Obviously, 6-C-glycosyl flavonoids 1-3 belong to the Class 1 category and can be developed as rapidly equilibrating mixture. We can also presume that other common natural 6-C-glycosyl flavonoids should belong to the Class 1 category, judging from their similar structures and other reported data of energy barriers [11][12][13][14][15]. Therefore, common 6-C-glycosyl flavonoids usually can be developed as rapidly equilibrating mixture.

General Experimental Procedure
1 H NMR and 13 C NMR spectra were recorded with a Bruker Avance III HD 500 MHz instrument. VT-1 H NMR was recorded with a Bruker Avance III 400 MHz instrument (Bruker, Breman, Germany).