Dynamic stereochemistry of rutin (vitamin P) in solution: theoretical approaches and experimental validation

Rutin, vitamin P, was extracted from Salvia macrosiphon and identiﬁed by 1 H, 13 C, 1 H– 1 H COSY, HMQC, and HMBC spectroscopy. In parallel, density functional theory (DFT) using B 3 LYP functional and split- valance 6-311G** basis set has been used to optimize the structures and conformers of rutin. Also experimental and theoretical methods have been used to correlate the dependencies of 1 J , 2 J , and 3 J involving 1 H and 13 C on the C5 00 –C6 00 ( x ), C6 00 –O6 00 ( h ), and C1 000 –O6 00 ( u ) torsion angles in the glycosidic moiety. New Karplus equations are proposed to assist in the structural interpretation of these couplings. 3 J HH depends mainly on the C–C ( x ) torsion angle, as expected, and 2 J HH values depend on both C–C ( x ) and C–O ( h ) torsions. 1 J CH values within hydroxymethyl fragments were also examined and found to depend on r CH , which is modulated by speciﬁc bond orientation and stereoelectronic factors. In all calculations solvent effects were considered using a polarized continuum model (PCM). (cid:2) 2010 Elsevier Ltd. All rights reserved.


Introduction
Bioflavonoids are benzo-c-pyrone derivatives of plant origin with a wide range of physiological activities such as antioxidant, antimicrobial, anti-inflammatory, antiallergenic, antiviral, and anti-tumor properties. [1][2][3][4] Rutin (Fig. 1) is a non-toxic bioflavonoid composed of the flavonol quercetin and the disaccharide rutinose that is found in more than 70 plant species. It is used clinically in therapeutic medicine, 5,6 especially in humans for the treatment of lymphoedema following axillary lymph node excision. 7 It has less toxicity in the human body and has the potential to be a novel therapeutic agent.
Some analytical methods, including capillary electrophoresis, 8 cyclic voltammetry, 9 HPLC, 10 chemiluminescence, 11 electrochemical sensor, 12 spectrophotometry, 13 and sequential injection analysis, 14 have been applied to the determination of the structure and properties of rutin, but it is still necessary to develop methods to quantify rutin in pharmaceutical preparations or crude drugs.
Recently the interaction of rutin with some flavonoids with DNA has been studied, but how flavonoid molecules bind to DNA is currently unclear. The function of the glycosidic ring, for example, is not understood. [15][16][17] In this study we focused on the sugar chain of rutin (rutinose) to determine its conformation in solution. Determination of the conformation of biologically active molecules is often based on NMR spectral data in combination with computational methods. In the present work, we are interested in extending the use of J-coupling constants for the structural, stereochemical, and conformational analysis of vitamin P by definition of the dependences of 3 J HH , 2 J HH , and 1 J CH coupling constants on the 1 H/ 13 C atomic dihedral angles in the sugar chain. So we present the results of studies of chemical shifts and a set of J-couplings about the C5 00 -C6 00 (x), C6 00 -O6 00 (h), and C1 000 -O6 00 (u) bonds, 3 J HH , 2 J HH , and 1 J CH , on rutinose, using experimental and theoretical methods to determine the Karplus equation.

NMR measurements
1 H NMR, 13 C NMR, DEPT, 1 H-1 H COSY, HMQC, and HMBC spectra of rutin were taken at 298 K in DMSO (99.99% D) on a Bruker Avance DRX operating at 500.133 MHz for 1 H and 125.770 MHz for 13 C, using a 5-mm broad band inverse probe with sufficient digital resolution to ensure errors 60.1 Hz in the measured J-couplings.
All 2D NMR spectra were acquired by pulsed field gradient-selected methods. 2D correlation spectroscopy (COSY) was used to confirm 1 H assignments. Heteronuclear multiple quantum correlation (HMQC) and heteronuclear multiple bond correlation (HMBC) were used for 13 C assignments.  2-4 show the 1 H-1 H COSY, HMQC, and HMBC spectra of rutin, respectively, and the 13 C NMR of extracted rutin is in good agreement with that reported in the literature. 18 HMQC and HMBC spectra were recorded using 2048 Â 1024 data matrices; the number of scan and dummy scans were 48 and 16, respectively, in all cases.

Ab initio molecular orbital calculation
Ab initio calculations were carried out with the GAUSSIAN program series 2003. 19 The optimization of the geometry was performed employing a hybrid Hartree-Fock density-functional scheme, the adiabatic connection method, that is, the Becke three-parameter with Lee-Yang-Parr (B 3 LYP) functional of density functional theory (DFT) 20 with the standard basis set, 6-311G**. Full optimizations were performed without any symmetry constraints. We computed the harmonic vibrational frequencies to confirm that an optimized geometry correctly corresponds to a local minimum that has only real frequencies. The solvent effects on the conforma-tional equilibrium have been investigated with a PCM method 21 at the B 3 LYP/6-311G** level. Solvation calculations were carried out for DMSO (e = 46.7) with the geometry optimization for this solvent. Conformational energy profiles around the C5 00 -C6 00 and C1 000 -O6 00 bond in rutinose, Figure 5, were calculated by driving the x and h dihedral angles from 0°to 360°in 30°increments, while allowing the remaining geometrical parameters to relax. In this report the orientations about the C5 00 -C6 00 , C6 00 -O6 00 , and C1 000 -O6 00 are described by torsion angles (x = O5 00 -C5 00 -C6 00 -O6 00 ), (h = C5 00 -C6 00 -O6 00 -C1 000 ), and (u = H1 000 -C1 000 -O6 00 -C6 00 ). For the C5 00 -C6 00 and C1 000 -O6 00 rotamers we used the standard nomenclature, Scheme 1. The O5 00 and C4 00 are the reference atoms and staggered conformers are designated as the gt (x % 60), tg (x % 180), and gg (x % À60).

Geometry optimization of rutin
The rutin structure was fully optimized by the B 3 LYP method using the 6-311G** basis set with no initial symmetry restrictions and assuming a C 1 point group. The optimized geometry of rutin in the gas phase was reoptimized by considering the solvent effect (e = 46.7) using the polarized continuum model (PCM). Tomasi's polarized continuum model defines the cavity as the union of a ser- ies of interlocking atomic spheres. The effect of polarization of the solvent continuum is represented numerically. 21 Figure 5 shows the optimized structure of rutin in DMSO solvent.
A selection of calculated bond distances, bond angles, and dihedral angles are compiled in Table 1. Calculation of vibrational frequencies has confirmed a stationary point with no negative eigenvalue observed in the force constant matrix.

Calculation of chemical shifts and NMR spin-spin coupling constants
NMR computations of absolute shieldings were performed using the GIAO method 22 on the DFT-optimized structure in the presence of solvent. The 1 H and 13 C chemical shifts were calculated by using the corresponding absolute shieldings calculated for Me 4 Si at the same level of theory (Table 2). A good agreement between the experimental and theoretical chemical shifts shows the reliability of DFT calculations for this series of molecules.
Recent investigations have shown that the density functional theory (DFT) method provides accurate predictions of structural parameters 23 and nearly quantitative 13 C-13 C and 13 C-1 H spin couplings in a wide range of bonding environments without the need for scaling. 24 Also Serianni and co-workers have shown that the B 3 LYP method is a suitable method for predicting coupling constants in disaccharides. 25 Consequently, the coupling constants in rutinose, Figure 6, were obtained by finite-field (Fermi contact) double perturbation theory 26   couplings using the least-squares procedure. Specific staggered hydroxymethyl rotamers of rutinose generated by systematically rotating the (x = O5 00 -C5 00 -C6 00 -O6 00 ) and (h = C5 00 -C6 00 -O6 00 -H) torsions, from 0°to 360°in 30°increments by holding both torsion angles at fixed values, were constructed in the GAUSSIAN viewer and subsequently geometrically optimized using B 3 LYP/6-311G**. These structures were reoptimized taking solvent effects into account.

Vicinal (three-bond) 1 H-1 H spin-spin coupling constants
The previous study shows two 3 J HH values, 3 J H5,H6R and 3 J H5,H6S , in some aldohexopyranoside derivatives that are sensitive to x. 27 3 J H5 00 ,H6 00 R and 3 J H5 00 ,H6 00 S (Eqs. 1 and 2) were computed for rutinose using the set of staggered and eclipsed geometries (Table 3). Karplus equations were also derived by including the effect of h, but this processing did not improve the quality of Eqs. 1 and 2 significantly. So two 3 J HH values, 3 J H5 00 ,H6 00 R and 3 J H5 00 ,H6 00 S , are sensitive to    θ ω Figure 6. The tube model of rutinose structure to calculate coupling constants that are sensitive to x, h, and u.

Geminal (two-bond) 1 H-1 H spin-spin coupling constants
2 J H6R,H6S is affected by both x and h, but its dependence on h is significantly greater than its dependence on x. The latter conclusion is supported by previous studies of Serianni and co-workers, 28 which show that the computed 2 J H6R,H6S is related to both x and h.
The additional hyper surface dataset obtained in this work yielded an improved equation (Eq. 3) with substantially smaller rms error. According to the data in Table 3 Eq. 3 relates 2 J H6 00 R,H6 00 S for rutinose to x and h:

One-bond 13 C-1 H spin-spin coupling constants
C-H bond length is a key determinant of the 1 J CH value, with shorter bond (greater s-character) yielding larger couplings. 30 Several structural factors influence C-H bond length: axial versus equatorial bond orientation, vicinal lone-pair effects, 30,31 1,3lone-pair effect, 32 and 1,4-lone-pair effects. 28 The effects of 1,3interactions with oxygen lone-pairs are observed on r C5-H5 and 1 J C5 00 -H5 00 ; therefore, the orientation of the vicinal lone-pairs on O5 00 and the orientation of the C5 00 -H5 00 bond remain fixed in all structures. A plot of calculated 1 J C5 00 ,H5 00 versus r C5 00 -H5 00 is linear (Fig. 8) indicating that the C-H bond length is highly correlated with the magnitude of 1 J CH , with shorter bonds yielding larger coupling constants. The shortest C6 00 -H6 00 bond, and the largest 1 J C6 00 -H6 00 values, is expected for a C6 00 -H6 00 bond that does not experience a bond-lengthening vicinal (anti) O6 00 lone-pair interaction and a bond-shortening 1,3-interaction with an O5 00 lone-pair. A plot of calculated 1 J C6 00 ,H6 00 versus r C6 00 -H6 00 for staggered rotamers is almost linear, too. The above-mentioned results relate the 1 J CH to only two torsion angles x and h. Rotation of the C6 00 -O6 00 bond modulates the stereoelectronic effect of the O6 00 lone-pairs on the C6 00 -H6 00 R and C6 00 -H6 00 S bond lengths, but other effects (1,3-lone-pair interactions with O5 00 and bond orientation) also influence these bond lengths, so the Karplus equations for rutinose that relate 1 J C5 00 -H5 00 and 1 J C6 00 -H6 00 to x and h give relatively large rms errors.
One of the major practical advantages of the angular dependences of 1 J C,H values is the possibility of determination of the glycosidic-bond torsional angles. The dependencies of 1 J C1 000 ,H1 000 on u are examined by systematic rotations about u in rutinose by 10 increments. Computed values of 1 J C1 000 ,H1 000 in optimized structures after every increase in u dihedral angles are shown in Table 4.   The relationship between 1 J C1 000 ,H1 000 and u was parameterized using a complete dataset of 36 data points, yielding Eq. 4, which indicates conformational dependence of coupling constant upon the dihedral angle u. Also Table 5  Rotameric distributions around the C5 00 -C6 00 and C6 00 -O6 00 bonds of the aldohexopyranosyl ring of rutinose can be determined from 3 J H5 00 ,H6 00 R and 3 J H5 00 ,H6 00 S . The limiting values of these couplings depend on assumptions made about the torsion angles and the choice of Karplus equation. The limiting couplings in Table 5 were used to estimate the percentages of C 00 5-C 00 6 and C 00 6-O 00 6 rotamers in rutinose. According to Table 5 small percentages of tg rotamer are observed. Percentages of gt, gg, and tg rotamers were calculated by solving the following three equations simultaneously: 3 J H5;H6R ¼ P gt ð 3 J H5;H6RðgtÞ Þ þ P gg ð 3 J H5;H6RðggÞ Þ þ P tg ð 3 J H5;H6RðtgÞ Þ; 3 J H5;H6S ¼ P gt ð 3 J H5;H6SðgtÞ Þ þ P gg ð 3 J H5;H6SðggÞ Þ þ P tg ð 3 J H5;H6SðtgÞ Þ; and P gt þ P gg þ P tg ¼ 1: In these equations, P is the fraction of the respective rotamer, 3 J H5,H6R(gt) is the standard value of 3 J H5,H6R in the gt rotamer, 3 J H5,H6R(gg) is the standard value of 3 J H5,H6R in the gg rotamer, and so forth. Standard couplings used in the calculations were derived from Eqs. 1 and 2. It is important to appreciate that substitution at O6 00 eliminates O6 00 -H, thereby preventing the measurement of 3 J HCOH , 33 which can be used to evaluate the C-O torsion. In this situation, knowledge of the relationships between 3 J HH , 2 J HH , and h can be especially useful.

Conclusions
Conformational studies of the exocyclic hydroxymethyl group in the disaccharide rutinose in rutin have relied heavily on the use of 3 J HH values to estimate rotamer populations in solution. The strategy was to obtain experimental results from extracted rutin, and then these data were used to test the ability of the DFT methods to estimate the chemical shifts and coupling constants. Good agreement between experimental and theoretical data confirms the accuracy of the B 3 LYP/6-311G** method for calculation of chemical shifts and coupling constants of saccharides.
The results show 3 J HH values in hydroxymethyl fragments are evaluated mostly by the C-C torsion angle (x) and less by the C-O torsion angle (h). Notice that the 2 J HH is determined mainly by the C-O torsion angle (h) in the absence of a hydroxyl proton on O6, when the hydroxyl group is substituted (for example in a (1?6)-glycosidic linkage).
In addition, this report generates new theoretical treatments for flavonoids with a sugar moiety, which makes the interpretation of saccharide conformational analysis more feasible. These results are expected to be helpful for understanding the conformational details of rutin in solution and will give a clue into the design of the binding of rutin to DNA molecules and different enzymes. The present findings make a significant contribution not only for the studies of rutin but also for the related studies of bioflavonoid with a saccharide moiety.