ABSTRACT

The complete proton and carbon NMR spectral assignments of a diterpene glycoside isolated from the commercial extract of the leaves of Stevia rebaudiana Bertoni, 13-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy] ent-kaur-16-en-19-oic acid-[(2-O-α-L-rhamnopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl) ester] (1); also known as rebaudioside N, was achieved by the extensive 1D and 2D NMR (¹H and ¹³C, COSY, HMQC, HMBC) as well as mass spectral data. Further, hydrolysis studies were performed on rebaudioside N using acid and enzymatic studies to identify aglycone and sugar residues in its structure.

1. Introduction

Stevia rebaudiana (Bertoni) is a perennial shrub belonging to the family of Asteraceae (Compositae) native to Brazil and Paraguay, but now grown commercially in a number of areas, particularly in Japan, Taiwan, Korea, Mainland China, Thailand and Indonesia [1,2]. Extracts of the leaves of S. rebaudiana have been used for decades to sweeten food and beverages in Japan, South America and China. The major constituents in the leaves of S. rebaudiana are the potently sweet glycosides namely steviolbioside, stevioside, rebaudiosides A and E, dulcoside A and rubusoside; which are glycosides of the diterpene steviol, ent-13-hydroxykaur-16-en-19-oic acid [3,4]. These compounds are also known as Stevia sweeteners.

Recently Ohta et al. have reported several minor steviol glycosides from S. rebaudiana Morita including rebaudioside N [5], however, they have not reported isolation or complete spectral assignment of pure rebaudioside N. As a part of our research related to the discovery of natural sweeteners and sweetener enhancers, we are herewith describing the isolation, characterization and complete ¹H and ¹³C NMR spectral assignments for the diterpene glycoside 13-[(2-O-β-D-glucopyranosyl-3- O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy]-ent-kau r-16-en-19-oic-acid-[(2-O-α-L-rhamnopyranosyl-3-O-βD-glucopyranosyl-β-D-glucopyranosyl) ester] (1) which is also known as rebaudioside N (Figure 1) isolated from the commercial extract of Stevia rebaudiana Bertoni. The complete NMR assignments were achieved on the basis of 1D (¹H and ¹³C) and 2D (COSY, HMQC and HMBC) NMR as well as high resolution mass spectroscopic data. Acid and enzymatic hydrolysis studies on compound 1 were carried out to identify aglycone and sugar residues.

2. Experimental

2.1. General Instrumentation Procedures

HPLC analysis was performed using a Dionex UPLC ultimate 3000 system (Sunnyvale, CA), including a quaternary pump, a temperature controlled column compartment, an auto sampler and a UV absorbance detector. Phenomenex Luna NH₂ with guard column, 150 × 3.0 mm, 3 µm (100A) were used for the characterization of rebaudioside N (1). NMR spectra were acquired on a Varian INOVA 600 MHz instrument with

a 5 mm HCN probe using standard pulse sequences. The NMR spectra were performed in C₅D₅N; chemical shifts are given in d (ppm), and coupling constants are reported in Hz. The spectral data was referenced to the residual solvent signal (d_H 7.19, and d_C 123.5 for pyridine-d₅). IR spectral data was acquired using a Perkin Elmer 400 Fourier Transform Infrared (FT-IR) Spectrometer with Universal attenuated total reflectance (UATR) polarization accessory. MS and MS/MS data were generated with a Thermo LTQ-FTMS mass spectrometer (100,000 resolutions) equipped with a Nano spray ionization source. Samples were diluted with methanol and introduced via infusion using the onboard syringe pump.

2.1.1. Isolation of Compound 1

Compound 1 was purified by repeated isocratic elution (72% acetonitrile in water) of the commercial extract of Stevia rebaudiana Bertoni using Dionex UPLC ultimate 3000 system with Phenomenex Luna NH₂ guard column. Collected the peak eluting at t_R 7.03 min; and dried the corresponding solution under nitrogen yielded 1.

2.1.2. Characterization of 13-[(2-O-β-DGlucopyranosyl-3-O-β-D-Glucopyranosyl- β-D-Glucopyranosyl) oxy] Ent-Kaur-16-en-19 -Oic Acid-[(2-O-α-L-Rhamnopyranosyl-3-O-β- D-Glucopyranosyl-β-D-Glucopyranosyl) Ester] (Rebaudioside N, 1)

White powder; IR ν_max: 3317, 2945, 1725, 1063, 914 cm⁻¹; ¹H-NMR (600 MHz, C₅D₅N, δ ppm) and ¹³C-NMR (150 MHz, C₅D₅N, δ ppm) spectroscopic data see Table 1; HRMS (M + H)⁺ m/z 1275.5493 (calcd. for C₅₆H₉₁O₃₂: 1275.5478).

2.1.3. Acid Hydrolysis of Compound 1

To a solution of compound 1 (5 mg) in MeOH (10 ml) was added 3 ml of 5% H₂SO₄ and the mixture was refluxed for 24 hours. The reaction mixture was then neutralized with saturated sodium carbonate and extracted with ethyl acetate (EtOAc) (2 × 25 ml) to give an aqueous fraction containing sugars and an EtOAc fraction containing the aglycone part. The aqueous phase was concentrated and compared with standard sugars using the TLC systems EtOAc/n-butanol/water (2:7:1) and CH₂Cl₂/MeOH/water (10:6:1) [6-8]; the sugars were identified as D-glucose and L-rhamnose.

2.1.4. Enzymatic Hydrolysis of Compound 1

Compound 1 (1 mg) was dissolved in 10 ml of 0.1 M sodium acetate buffer, pH 4.5 and crude pectinase from Aspergillus niger (50 uL, Sigma-Aldrich, P2736) was added. The mixture was stirred at 50˚C for 96 hr. The product precipitated out during the reaction and was filtered and then crystallized. The resulting product obtained from the hydrolysis of 1 was identified as steviol (4) by comparison of its co-TLC with standard compound and ¹H NMR spectral data [9].

3. Results and Discussion

Compound 1 was isolated as an crystalline material and its molecular formula has been deduced as C₅₆H₉₀O₃₂ on the basis of its positive ESI TOF mass spectrum which showed [M + H]⁺ ion at m/z 1275.5493, and this composition was supported by ¹³C NMR spectral data. The ¹H NMR spectrum of 1 showed the presence of two methyl singlets at δ 1.17 and 1.51, two olefinic protons as singlets at δ 5.05 and 5.69 of an exocyclic double bond, nine methylene and two methine protons between δ 0.72 - 2.60, characteristic for the ent-kaurane diterpenoids isolated earlier from the genus Stevia [10-12]. The basic skeleton of ent-kaurane diterpenoids was supported by COSY (H-1/H-2; H-2/H-3; H-5/H-6; H-6/H-7; H-9/H-11; H-11/H-12) and HMBC (H-1/C-2, C-10; H-3/C-1, C-2, C-4, C-5, C-18, C-19; H-5/C-4, C-6, C-7, C-9, C-10, C-18, C-19, C-20; H-9/C-8, C-10, C-11, C-12, C-14, C-15; H-14/C-8, C-9, C-13, C-15, C-16 and H-17/C-13, C-15, C-16) correlations. The ¹H NMR spectrum of 1 showed the presence of six sugar units in its structure by the presence of the anomeric protons resonating at δ 5.03, 5.06, 5.37, 5.58, 6.22, and 6.32; which was further supported by the MS/MS spectrum of 1 in the positive ESI mode showed the fragment ions at m/z 1113, 951, 805, 643, 481 and 319. Acid hydrolysis of

1 with 5% H₂SO₄ afforded the sugars D-glucose and L-rhamnose, which were identified by direct comparison with authentic samples by TLC [6-8]. Enzymatic hydrolysis of 1 furnished an aglycone which was identified as steviol (4) by comparison of ¹H NMR and co-TLC with standard compound [9]. The large coupling constants observed for the five anomeric protons of the glucose moieties at δ 5.03 (d, J = 7.8 Hz), 5.06 (d, J = 7.6 Hz), 5.37 (d, J = 8.4 Hz), 5.58 (d, J = 7.8 Hz), and 6.22 (d, J = 8.4 Hz), suggested their β-orientation as reported for steviol glycosides [9-12]. The sixth anomeric sugar corresponding to that of L-rhamnosyl unit was identified as a doublet at δ 6.32 (J = 1.4 Hz) suggesting its α-orientation [5]. The ¹H and ¹³C NMR values for all the carbons in 1 were assigned on the basis of COSY, HSQC and HMBC correlations (Table 1).

Based on the results from NMR spectral data and hydrolysis experiments of 1, it was concluded that there are five β-D-glucosyl units and an α-L-rhamnosyl unit in its structure connected to the aglycone steviol. A close comparison of the ¹H and ¹³C NMR values of 1 with rebaudioside M (2) and rebaudioside A (3) [5] suggested the presence of a 2,3-disubstituted β-D-glucosyl unit at C-13 in the form of ether linkage and another 3-substituted β-D-glucosyl unit at C-19 position in the form of an ester linkage, leaving the assignment of the additional α-L-rhamnosyl unit. The downfield shift for both the ¹H and ¹³C chemical shifts at C-2′ of sugar I suggested that the additional α-L-rhamnosyl moiety has been attached at this position. This was confirmed by the key HMBC correlations: H-2′/C-1′, C-3′, C-1′′′′′ and H-1′′′′′/C-2′, C-2′′′′′, C-3′′′′′. Based on the results from chemical and spectral studies, 1 was assigned as 13-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl) oxy] ent-kaur-16-en-19-oic acid-[(2-O-α-L-rhamnopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl) ester]. The structure was further supported by the key COSY and HMBC correlations as shown in Figure 2.

To the best of our knowledge, this is the first report of the isolation of rebaudioside N (1) from S. rebaudiana Bertoni. Though partial NMR spectral data has been reported earlier for rebaudioside N (1) by Ohta et al. [5], this

is the first report of complete ¹H and ¹³C NMR spectral assignments based on 1D (¹H and ¹³C) and 2D (COSY, HMQC and HMBC) NMR as well as high resolution mass spectroscopic data which was supported by enzymatic and acid hydrolysis studies.

4. Conclusion

We are herewith reporting the isolation, complete ¹H and ¹³C NMR spectral assignments for 13-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy]-ent-kaur-16-en-19-oic-acid-[(2-O-α-L-rhamnopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)

ester], also known as rebaudioside N (1) on the basis of extensive 1D and 2D NMR as well as high resolution mass spectral data. Further, acid hydrolysis furnished D-glucose suggesting the presence of two sugar units that were identified as β-D-glucose and α-L-rhamnose; and enzymatic hydrolysis furnished steviol.

5. Acknowledgements

We wish to thank Dr. Shaoxiong Wu, and Dr. Bing Wang of Emory University, Atlanta, USA for obtaining some selected spectral data and other chemistry related help.

REFERENCES

NOTES

^*Corresponding author.