1H ΝΜR chemical shift assignment, structure and conformational elucidation of hypericin with the use of DFT calculations – The challenge of accurate positions of labile hydrogens
Graphical abstract
1H ΝΜR chemical shift assignment, determination of accurate labile hydrogen positions, beyond the limits of X-ray diffraction methods, and structure and conformational elucidation of hypericin with the use of DFT calculations has been achieved.
Introduction
Hypericin (phenanthro[1,10,9,8,o,p,q,r,a]perylene-7,14-dione]), Fig. 1, is a red-colored phytochemical which, together with hyperforin, is one of the principal active constituents of Hypericium (Saint John's wort).1, 2 Hypericin has been reported to act as an antibiotic antiviral3 and non-specific kinase inhibitor,4 to inhibit the growth of a variety of neoplastic cell types5 and to modify levels of neurotransmitters in brain regions involved in the pathophysiology of depression.6 Furthermore, hypericin has been intensively investigated as an agent in photodynamic therapy due to its extensive chromophore system.7
Theoretical calculations indicated that the 7,14 dioxo tautomer of hypericin Q,7, 14 with the carbonyl groups in positions 7 and 14 (Fig. 1) is the most stable by at least 45 kJ/mol. This tautomer is present in solution,1 as well as in the crystalline state,8 which indicated a propeller conformation with dihedral angles C(3)–C(3a)–C(3b)–C(4) and C(10)–C(10a)–C(10b)–C(11) of 21.8° and 28.5°, respectively.
Hypericin is very acidic with pKa = 1.7–2.09, 10 due to the proton on the hydroxy group on C-4 (C-3) (Fig. 2). The X-ray structure of the hypericinate, Hy− (ionic form of hypericin), demonstrates a pronounced helical twist.9 The steric interaction between the two methyl groups of C-10 and C-11 results in a dihedral angle C(10)–C(10a)–C(10b)–C(11) of 32.4°, and between the two hydroxy groups on C-3 and C-4 in a dihedral angle C(3)–C(3a)–C(3b)–C(4) of 19.2°. The anion is stabilized by two major factors: a strong hydrogen bonding with the adjacent hydroxy group on C-39 and the negative charge on the oxygen which is delocalized by resonance to the carbonyl group on C-7.11 Because of the symmetry of hypericin, the same effects result from the loss of the proton from the hydroxy group on C-3. The anion exists as the 7,14-dioxo tautomer (1a) and its conformation is almost identical to the propeller conformation of the parent hypericin. The energy barrier of interconversion of the two propeller conformations has been calculated to be ≥ 80 kJ/mol.10 Kinetic investigation of the propeller enantiomerization barrier of hypericin also resulted in a value of about 80 kJ/mol.12
Investigations using temperature dependent 1H NMR and 2D ROESY spectroscopy in DMSO-d6 and in acetone-d6 indicated that hypericin exists in both solvents as a single tautomer of the neutral structure 1 (Fig. 1).13 Furthermore, the assignment of the bay hydroxy protons OH-3,4 was attributed to a broad resonance at ∼8.1–8.3 ppm. On the contrary, Dax et al.14 suggested that in DMSO-d6 hypericin exists in the ionic structure of the Q7, 14 on the basis of a strong deshielded broad resonance of the bay OH group at δ = 17.3–17.5 ppm which corresponds to an integral of one proton. The assignment of the bay OH-3,4 signal in the neutral state of hypericin in acetone-d613 was questioned by Skalkos et al.15 with the use of variable temperature 1H NMR spectroscopy. At low temperatures (∼215 K) the exchange process of labile OH protons reduces significantly, which allowed the assignment of a signal at δ = 11.6–11.8 ppm, with an integral of two protons, to the bay OH groups. The 3 ppm shift to low frequency of the bay OH-3,4 signal as compared to the signals of the hydroxy OH-8,13 protons (δ = 14.2 ppm) and the OH-1,6 protons (δ = 14.8 ppm) was attributed to the weaker hydrogen bonding interaction of the two bay hydroxy groups due to dihedral angle constraints between the two phenyl moieties groups.
Recent developments in quantum chemical methods for calculating NMR chemical shifts and advances in computer power16 have led to an increasing number of studies which support the assignment of individual protons and carbons17 with emphasis in the structure of complex natural products18 including solvent effects.19, 20, 21, 22, 23 It is our goal to both critically evaluate conflicting literature 1H NMR signal assignment, and to highlight the capabilities of DFT calculations that can be used to address resonance assignments, ionization state, accurate labile hydrogen positions beyond the limits of X-ray diffraction methods, and structural and conformational properties of natural products.
Section snippets
Computational methods
Calculations were performed with the DFT method, with the B3LYP hybrid functional24, 25 as implemented in the Gaussian 09W package.26 NMR chemical shifts were calculated with the GIAO (Gauge Independent Atomic Orbital) method at the B3LYP/6-311 + G(2d,p) level using the CPCM (Conductor-like Polarizable Continuum Model) model.27 The molecular structures of hypericin, hypericinate and hypericin + solvent discrete hydrogen bonded complexes were minimized at the B3LYP/6-31 + G(d) basis set and
Structural properties of hypericin + solvent (1:1) and (1:2) discrete hydrogen bonded complexes
Table S1 illustrates selected inter- and intramolecular structural data of hypericin + acetone 1:1 complexes which were energy minimized at the B3LYP/6-31 + G(d), TPSSh/TZVP and CAM-B3LYP level of theory in the gas phase or using the CPCM/IEF-PCM models. In all cases the distorted skeleton of the hypericin molecule shows a pronounced propeller conformation (Fig. 3) with dihedral angles C(3)–C(3a)–C(3b)–C(4) and C(10)–C(10a)–C(10b)–C(11) ranging from 24.6° to 26.3° and 33.2° to 33.5°,
Conclusions
We have demonstrated that DFT calculations of 1H NMR chemical shifts can provide correct resonance assignments, accurate determination of labile hydrogen positions beyond the limits of X-ray diffraction methods, and structural and conformational properties of natural products. More specific:
(i) Excellent linear correlation between the computed and experimental 1H NMR chemical shifts can be obtained for moderate basis sets. However, accurate structural data of hydrogen bonding distances and
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