Synthesis of ent-Kaurane Diterpene Monoglycosides

Synthesis of two ent-kaurane diterpene glycosides, steviol 19-O-β-D-glucopyranosiduronic acid (steviol glucuronide, 5 ), and 13-hydroxy ent-kaur-16-en-19-oic acid-β-D-glucopyranosyl ester (7) has been achieved from a common starting material, steviol, using phase transfer catalyst. Also, synthesis of an additional 17-nor-ent-kaurane glycoside, namely 13-methyl-16-oxo-17-nor-ent-kauran-19-oic acid-β-D-glucopyranosyl ester (10) was performed using the starting material isosteviol and similar synthetic methodology. Synthesis of all three steviol glycosides was performed using straightforward chemistry and their structures were characterized on the basis of 1D and 2D NMR as well as mass spectral (MS) data.


Introduction
The major constituents isolated from the leaves of Stevia rebaudiana Bertoni (family: Asteraceae) are the potently sweet diterpenoid glycosides stevioside, and rebaudioside A. These compounds which are known as Stevia sweeteners are glycosides of the diterpene steviol, ent-13-hydroxykaur-16-en-19oic acid [1]. Stevioside tastes about 150-250 times sweeter than sucrose, whereas rebaudioside A tastes about 200-300 times sweeter than sucrose; both are non-caloric. In some parts of the world, including Japan, South Korea, Israel, Mexico, Paraguay, Brazil, Argentina, and Switzerland, these steviol glycosides are used to sweeten food products and beverages. As a part of our continuing research to OPEN ACCESS discover natural sweeteners, we have reported the isolation of several glycosides from the commercial extract of S. rebaudiana [2][3][4][5][6][7][8]. Apart from isolating novel compounds from S. rebaudiana and utilizing them as possible natural sweeteners or sweetness enhancers, we are also engaged in understanding the physicochemical profiles of steviol glycosides in various systems of interest and their metabolites, as well as their characterization [9]. Though many steviol glycosides have been reported in the literature, focused synthetic studies were not carried out. In this article, we present the synthesis of three ent-kaurane diterpene glycosides possessing steviol and isosteviol skeletons, and the characterization of their structures based on extensive NMR and mass spectroscopic data.

Spectroscopy
The structural characterization of 5, 7 and 10 was performed on the basis of one dimensional ( 1 H, 13 C), two-dimensional ( 1 H-1 H COSY, 1 H-13 C HMQC, 1 H-13 C HMBC) NMR and mass spectral data, as well as in comparison with literature values [3,10]. The attachment of sugars at the C-19 position of the steviol skeleton in 5 and 7, and the isosteviol skeleton in 10 was characterized by the key HMBC correlation of the anomeric protons of the respective sugar moieties with the C-19 carbonyl group. The 1 H-and 13 C-NMR values for all the protons and carbons were assigned on the basis of COSY, HMQC and HMBC correlations and are given in Tables 1 and 2. The anomeric protons in all three glycosides 5, 7 and 10 were observed as doublets at δ 5.44 (d, 7.8 Hz), 5.41 (d, 8.2 Hz) and 5.38 (d, 8.2 Hz) respectively, suggesting their β-configuration similar to that of the steviol glycosides isolated from S. rebaudiana [2][3][4][5][6][7][8]. This suggested that even though esterification was performed using their corresponding α-derivatives; the configuration of the final products 5, 7, and 10 was observed to correspond to their β-derivatives; an identical phenomenon was reported earlier for glycosylation of triterpene acids under phase transfer catalytic conditions [11].

General
Melting points were measured using a SRS Optimelt MPA 100 instrument and are uncorrected. Optical rotations were recorded using a Rudolph Autopol V at 25 °C and IR spectral data was acquired using a Perkin Elmer 400 Fourier Transform Infrared (FT-IR) spectrometer equipped with a universal attenuated total reflectance (UATR) polarization accessory. NMR spectra were acquired on Bruker Avance DRX 500 MHz using standard pulse sequences. Chemical shifts are given in δ (ppm), and coupling constants are reported in Hz. HRMS and MS/MS data were generated with a Waters Premier Quadrupole Time-of-Flight (Q-TOF) mass spectrometer equipped with an electrospray ionization source operated in the positive-ion mode and ThermoFisher Discovery OrbiTrap in the positive mode of electrospray. Samples were diluted with water: acetonitrile (1:1) containing 0.1% formic acid and introduced via infusion using the onboard syringe pump. HPLC was performed on an Agilent 1100 system using a Phenomenex Prodigy ODS (3) column (250 × 21.2 mm, 5 μm) or a Beckman Gold system with a Zorbax amino (150 × 4.6 mm, 5 μm) column.

Isolation
Purification of compound 5 was carried out using the Beckman HPLC system using an isocratic solvent method; UV Detection: 210 nm; Mobile Phase A: 75% CH 3  Steviol acetate (2): Steviol acetate (2) was prepared from steviol (1) as reported in the literature and characterized based on the 1 H-NMR and mass spectral data and comparison with the spectral data reported in the literature [12]. 1  Steviol glucuronide (5): Compound 2 (0.633 g, 1.76 mmol) was dissolved in CH 2 Cl 2 (25 mL) and distilled water (3 mL). TBAB (10 mg), K 2 CO 3 (0.62 g, 4.50 mmol), and methyl acetobromo-α-Dglucuronate (0.80 g, 2.02 mmol) were added at room temperature. The reaction mixture was refluxed for 48 h, cooled to room temperature and the layers were separated. The aqueous layer was extracted with additional amounts of CH 2 Cl 2 (2 × 20 mL) and the combined organic layer was washed with water (2 × 30 mL) and brine (40 mL). Concentration of the CH 2 Cl 2 layer under vacuum furnished a residue (1.05 g), which showed a peak at m/z 677 in its mass spectrum corresponding to the (M+H) + ion of 3. Compound 3 (1.00 g, 14.7 mmol) was dissolved in 10% solution of Et 3 N in MeOH-H 2 Ohexane (10:2:1, 23 mL) and stirred at room temperature for 48 h. The MeOH and hexane were evaporated under vacuum and the resulting product (0.73 g) was identified as 4 on the basis of its mass spectrum, which showed a peak at m/z 509 corresponding to its (M+H) + ion. Compound 4 (0.70 g) was dissolved in H 2 O (20 mL) and KOH (0.16 g, 0.28 mmol) was added. The mixture was stirred at room temperature for 48 h and the mixture was subjected to evaporation under reduced pressure. Purification of the residue obtained after evaporation was performed using HPLC furnished 420 mg of 5. 13-Hydroxy-ent-kaur-16-en-19-oic acid-β-D-glucopyranosyl ester (7): To a solution of 2 (2.00 g, 5.56 mmol) in CH 2 Cl 2 (35 mL) and distilled water (4 mL); TBAB (30 mg), K 2 CO 3 (1.94 g, 14.06 mmol), and acetobromo-α-D-glucose (2.88 g, 7 mmol) were added at room temperature. The reaction mixture was refluxed for 48 h, cooled to room temperature and the layers were separated. The aqueous layer was extracted with additional CH 2 Cl 2 (2 × 50 mL) and the combined organic layer was washed with water (2 × 60 mL) and brine (100 mL). Concentration of the CH 2 Cl 2 layer under vacuum furnished a residue (2.23 g) which was identified as 6 on the basis of its EIMS spectrum which showed a peak at m/z 691 corresponding to its (M+H) + ion.  (10): To a solution of 8 (0.70 g, 2.20 mmol) in CH 2 Cl 2 (15 mL) and distilled water (4 mL) was added TBAB (15 mg), K 2 CO 3 (1.00 g, 7.25 mmol), and acetobromo-α-D-glucose (1.25 g, 3.04 mmol). The reaction mixture was refluxed for 48 h, cooled to room temperature and worked-up as described above to furnish a residue (1.20 g) which showed a peak at m/z 649 in its EIMS spectrum corresponding to the (M+H) + ion of 9. Compound 9 (1.00 g, 1.54 mmol) was deacetylated as described above and upon usual work-up afforded a residue which was purified by HPLC to yield 500 mg of 10. White powder, mp 172. 5

Conclusions
In conclusion, three ent-kaurane diterpene glycosides 5, 7 and 10 were synthesized from the natural products steviol and isosteviol through simplified procedures. To the best of our knowledge, this is the first report of the synthesis of C-19 glycosidic linkages on the steviol and istosteviol skeletons. The structures of all the synthesized compounds were characterized on the basis of NMR (1D and 2D) and mass spectral data, as well as in comparison with the data reported in the literature.