A Novel Diterpene Glycoside with Nine Glucose Units from Stevia rebaudiana Bertoni

Transcription

1 biomolecules Article A Novel Diterpene Glycoside with Nine Glucose Units from Stevia rebaudiana Bertoni Indra Prakash 1, *, Gil Ma 1, Cynthia Bunders 1, Romila D. Charan 2, Carine Ramirez 2, Krishna P. Devkota 2 Tara M. Snyder 2 1 The Coca-Cola Company, One Coca-Cola Plaza, Atlanta, GA 30313, USA; gilma@coca-cola.com (G.M.); cynbun@gmail.com (C.B.) 2 AMRI-Albany, Analytical Development, Albany, NY 12212, USA; Romila.Charan@amriglobal.com (R.D.C.); Carine.Ramirez@amriglobal.com (C.R.); Krishna.Devkota@amriglobal.com (K.P.D.); Tara.Snyder@amriglobal.com (T.M.S.) * Correspondence: iprakash@coca-cola.com; Tel.: ; Fax: Academic Editor: Hans Vliegenthart Received: 18 November 2016; Accepted: 23 January 2017; Published: 31 January 2017 Abstract: Following our interest in new diterpene glycosides with better taste priles than that Rebaudioside M, we have recently isolated characterized Rebaudioside IX a novel steviol glycoside from a commercially-supplied extract Stevia rebaudiana Bertoni. This molecule contains a hexasaccharide group attached at C-13 central diterpene core, contains three additional glucose units when compared with Rebaudioside M. Here we report complete structure elucidation based on extensive Nuclear Magnetic Resonance () analysis ( 1 H, 13 C, Spectroscopy (COSY), Single Quantum Coherence-Distortionless Enhancement Polarization Transfer (HSQC-DEPT), Multiple Bond (HMBC), Total Spectroscopy (TOCSY), Nuclear Overhauser Effect Spectroscopy (NOESY)) spectral data this novel diterpene glycoside with nine sugar moieties containing a relatively rare 1 6 α-linked glycoside. A steviol glycoside bearing nine glucose units is unprecedented in literature, could have an impact on natural sweetener catalog. Keywords: Diterpene glycosides; nine sugar steviol glycoside; structure elucidation; ; Stevia rebaudiana Bertoni; 1 6 glycoside linkage 1. Introduction Humans are born with a predisposition to like sweet tastes. Sweet taste has always been associated with pleasure, consumers enjoy eating or drinking sweet-tasting items. Recently, health-conscious consumers have placed an increasing priority on reducing ir caloric intake without compromising sweetness. This has led food industry to search for novel natural high-potency sweeteners. In past ten years, we have looked to leaves Stevia rebaudiana Bertoni for sweet-tasting steviol glycosides [1 3]. This family natural products which possesses a diterpene core has been used to sweeten food products for hundreds years in South America [4]. However, by early 21st century, chemical structures only a limited number steviol glycosides had been characterized. These included stevioside, Rebaudioside A F, dulcoside A, steviolbioside [5]. In recent years, a greater number minor steviol glycosides have been identified that are present in trace quantities in dried extracts S. rebaudiana leaves [6 11]. However, taste prile physico-chemical properties most isolated purified steviol glycosides have not been reported. Recently, we have focused our efforts on finding new steviol glycosides with a superior taste prile similar structure to Rebaudioside M. Rebaudioside M one minor components in S. rebaudiana leaf extract shows an improved sweet taste prile, reduced perception bitterness, Biomolecules 2017, 7, 10; doi: /biom

2 Biomolecules 2017, 7, Biomolecules 2017, 7, S. rebaudiana leaf extract shows an improved sweet taste prile, reduced perception bitterness, reduced astringency, reduced lingering bitterness compared with that or known steviol glycosides, reduced astringency, including Rebaudioside reduced lingering A [12,13]. bitterness Our compared most recent withwork that uncovered or known a unique steviol Rebaudioside glycosides, including M derivative Rebaudioside with a hydroxyl A [12,13]. group Our most at recent C 15 work position uncovered aditerpene unique Rebaudioside core [14]. In addition, M derivative we with have a hydroxyl developed group biotransformation at C-15 positionreactions diterpene to produce core [14]. Rebaudioside In addition, we M have Rebaudioside developed biotransformation M2 [15]. reactions to produce Rebaudioside M Rebaudioside M2 [15]. In our continued search to find to find potentially potentially significant significant sweet sweet diterpene diterpene glycosides glycosides that are that present are present only in only minor inabundance minor abundance in S. rebaudiana, in S. rebaudiana, we have weisolated have isolated a novel a novel diterpene diterpene glycoside glycoside from commercial from commercial leaf extracts leaf extracts provided provided by PureCircle by PureCircle (Negeri Sembilan, (Negeri Sembilan, Malaysia). Malaysia). The novel compound, The novel Rebaudioside compound, Rebaudioside IX, has nine IX, has glucose nine glucose units, units, in which in which (2-O-β-D-glucopyranosyl-3-O-β-Dglucopyranosyl)-D-glucopyranosylmoiety is isattached to Glc III Rebaudioside M as a sugar linkage in an α-configuration. α configuration. To best our knowledge, a steviol glycoside with nine glucose (2 O β D glucopyranosyl 3 O β Dglucopyranosyl) D glucopyranosyl units is unprecedented in scientific literature, α linkage α-linkage present in Rebaudioside IX is relatively rare in steviol glycoside family. This paper describes isolation structure elucidation novel nine sugar nine-sugar diterpene glycoside (1) (Figure 1) on basis extensive spectroscopic studies. Figure 1. Structures Rebaudioside IX (1) Rebaudioside M (2) Results Results Discussion Discussion Compound Compound 1 was was isolated isolated as as a white white powder. powder. Accurate Accurate measurement measurement using using high high resolution resolution spectrometry spectrometry (HRMS) (HRMS) provided provided an an exact exact at at m/z m/z in in negative negative electrospray electrospray ionization time flight ionization-time--flight (ESI-TOF) (ESI TOF) spectrum spectrum corresponding corresponding to a molecular to a molecular formula formula C 74 H 120 O 48 C74H120O48 (calculated (calculated for C for C74H119O48: ). HRMS Nuclear Magnetic Resonance () data 74 H 119 O 48 : ). HRMS Nuclear Magnetic Resonance () data indicated indicated that (1) had that an (1) additional had an additional glucotriosyl glucotriosyl unit when unit compared when compared with Rebaudioside with Rebaudioside M (2). M (2). A series series experiments experiments including including 1 H,, 13 C,, 1 H- H 1 H correlation correlation spectroscopy spectroscopy (COSY), (COSY), 1 H H- 13 C Single Single Quantum Quantum Coherence Distortionless Coherence-Distortionless Enhancement Enhancement Polarization Polarization Transfer Transfer (HSQC-DEPT), (HSQC DEPT), 1 H- H 13 C Multiple Multiple Bond Bond (HMBC), (HMBC), 1 H- H 1 H Nuclear Nuclear Overhauser Overhauser Effect Effect Spectroscopy Spectroscopy (NOESY), (NOESY), Total Total Spectroscopy Spectroscopy (TOCSY) (TOCSY) were were performed performed to to allow allow elucidation elucidation structure structure (1) (1) (see (see Supplementary Supplementary Materials Materials for for representative representative 2D 2D spectra). spectra). The The 2D 2D data data indicated indicated that that central central core core glycoside glycoside was was a diterpene. a diterpene. The The 1 H 1 H spectrum spectrum (Figure (Figure 2) 2) HSQC DEPT HSQC-DEPT data data (1) (1) indicated indicated presence presence two two methyl methyl singlets singlets at at δ , 1.37, two two proton proton singlets singlets corresponding corresponding to an exocyclic double bond at δh to an exocyclic double bond at δ , nine methylene two methine protons between H , nine methylene two methine protons between

3 Biomolecules 2017, 7, δ H characteristic for ent-kaurene diterpene central core, similar to data reported for Rebaudioside M or steviol glycosides [4,7]. The presence an ent-kaurene diterpenoid aglycone central core was furr confirmed by 1 H- 1 H COSY correlations H-1/H-2; H-2/H-3; H-5/H-6; H-6/H-7; H-9/H-11; H-11/H-12 1 H- 13 C HMBC correlations H-3/C-2, C-4, C-18; H-5/C-4, C-6, C-10, C-19; H-9/C-8, C-10, C-11, C-12, C-14, C-15, C-20; H-12/C-13; H-14/C-15, C-16; H-15/C-13, C-16, H-17/C-13, C15; H-18/C-3, C-4, C-5, C-19; H-20/C-1, C-5, C-9, C-10. The complete 1 H 13 C assignments central diterpene core (1) are provided in Table 1. The relative stereochemistry in central diterpene core was assigned based on Nuclear Overhauser Effect (NOE) correlations. In NOESY spectrum (1), NOE correlations between H-14/H-20 indicated that H-14 H-20 were on same face rings. Similarly, NOE correlations between H-9/H-5 as well as H-5/H-18, but an absence NOE correlations between H-9/H-14 indicated that H-5, H-9, H-18 were on opposite face rings to that H-14 H-20, as presented in Figure 1. These data thus indicated that relative stereochemistry in central diterpene core (1) was retained during glycosylation, is same as that for Rebaudioside M (Figure 1). Table 1. 1 H 13 C Nuclear Magnetic Resonance () ( MHz, 300 K) assignments aglycone compound (1) in pyridine-d 5. Atom No. 13 C 1 H m, 1.86 m m, 2.29 m m, 2.30 m d (13.1) m, 2.39 m m, 1.80 m d (7.9) m, 1.88 m m, 2.69 m m, 2.74 d (10.6) m, 2.02 m bs, 5.71 bs s s The 1 H 1 H- 13 C HSQC-DEPT data for (1) showed signals for nine anomeric protons at δ H 6.40 (δ C 95.3), δ H 5.80 (δ C 104.6), 5.76 (δ C 100.6), 5.48 (δ C 104.8), 5.42 (δ C 96.6), 5.40 (δ C 104.3), 5.29 (δ C 104.6), 5.10 (δ C 106.5), 5.06 (δ C 105.4), indicating presence nine glucose units. This was supported by fragment ions in negative Electrospray Ionization Tem Mass Spectrometry (ESI MS/MS) spectrum (1), which showed loss one hexose unit at m/z , followed by loss two hexose units at m/z sequential loss six hexose moieties at m/z , , , , , The complete assembly glycoside structure was determined on basis COSY, HSQC-DEPT, HMBC correlations, as well as TOCSY data. The TOCSY experiments were used extensively to assign protons nine glucose units, while HSQC-DEPT HMBC data allowed carbon assignments established connectivity glucose units. Thus, long-range 1 H- 13 C correlations observed in HMBC experiment from anomeric proton at δ H 6.40 (δ C 95.3) to a carbonyl carbon at δ C (C-19) allowed its assignment as anomeric proton

4 Biomolecules 2017, 7, Biomolecules 2017, 7, ) to a carbonyl carbon at δc (C 19) allowed its assignment as anomeric proton Glc I (Figure Glc I (Figure 2). The 1 2). H coupling The 1 H coupling sequence sequence from Glc from I anomeric Glc I anomeric proton (δh proton 6.40) (δto H H ) (δh to4.51) H-2 (δ through H 4.51) through H 3 (δh 5.11), H-3 (δh 4 H 5.11), (δh 4.20), H-4 (δ H 5 H 4.20), (δh 4.14), H-5 (δ H 4.14), oxymethylene oxymethylene protons, H 6 protons, (δh 4.20 H-6 (δ H 4.32), 4.20 was 4.32), established was established using a combination using a combination COSY COSY TOCSY data. TOCSY The data. carbons The carbons C 2 (δc 77.2), C-2 (δc 3 C 77.2), (δc C ), (δc 4 C 89.0), (δc 70.5 C-4 or (δ C or or70.7), 70.6 C 5 or 70.7), (δc 78.9), C-5 (δ C 78.9), C 6 (δc 62.2) C-6 were (δ C 62.2) n were assigned nbased assigned on HSQC based on DEPT HSQC-DEPT data confirmed data confirmed by HMBC bycorrelations HMBC correlations H 1/C 3, H-1/C-3, C 5; H 2/C 1, C-5; H-2/C-1, C 3; H 3/C 2; C-3; H-3/C-2; H 4/C 5. H-4/C-5. The higher The frequency higher frequency 1 H 13 C 1 Hchemical 13 Cshifts chemical shifts C 2 C 3 C-2positions C-3 positions suggested suggested possible possible placement placement glycosyl glycosyl units at units se at positions, se positions, which was which confirmed was confirmed by HMBC by HMBC correlations. correlations. Thus, Thus, long range long-range 1 H 13 C 1 H- correlations 13 C correlations observed observed in inhmbc HMBC experiment experiment from from anomeric anomeric protons protons at δh at δ5.80 H 5.80 (Glc (Glc V, V, H 1) H-1) to to carbon at at δc δ C (Glc (GlcI, I, C 2) C-2) δh δ H (Glc VI, H 1) H-1) to carbon at δδc C 89.0 (Glc I, C-3) C 3) established 2,3-O-branched-D-glucodiosyl 2,3 O branched D glucodiosyl substituent in in Glc Glc I to I to be be same same as as that that in in Rebaudioside M. M. data data did did not not indicate any any furr sugar linkages to Glc I, Glc V, Glc VI. The large coupling constants for anomeric protons Glc I I (δ(δh H 6.40, d, d, J 3 = 8.2 Hz), Glc V (δ (δh H 5.80, d, J 3 = Hz), Glc GlcVI VI (δh (δ H 5.29, 5.29, d, d, J 3 J= = 7.9 Hz) Hz) indicated indicated ir ir β configuration. β-configuration. The The complete complete 1 H 1 H 13 C assignments 13 C assignments Glc V Glc VGlc VI Glc was VI based was based on extensive on extensive analysis analysis 2D 2D data, data, are provided are provided in Table in Table 2, while 2, while key key COSY COSY HMBC HMBC correlations correlations are provided are provided in Figure in Figure ppm Figure 2. 1 H spectrum RebaudiosideIX IX (1) (1) (500 (500 MHz, K) K) in in pyridine d5. pyridine-dppm: 5. ppm: parts parts per per million. million.

5 Biomolecules 2017, 7, Biomolecules 2017, 7, Figure Figure Summary Summary key key Multiple Multiple Bond Bond (HMBC) (HMBC) Spectroscopy Spectroscopy (COSY) (COSY) correlations correlations used used to to assign assign C-13 C 13 C-19 C 19 glycoside glycoside region region compound compound (1). (1). Table 2. 1 H 13 C ( MHz, 300 K) assignments C-19 glycosides compound (1) Table in pyridine-d 2. 1 H 13 C ( MHz, 300 K) assignments C 19 glycosides compound 5. (1) in pyridine d5. Sugar Sugar Atom No. 13 C C 1 H H Glc Glc I I d (8.2) d (8.2) m m m m or 70.6 or m or or m m m, m 4.32 m m, 4.32 m Glc V d (7.4) Glc V d 4.19 (7.4) m m m or 71.6 or m m or or m m m, m 4.63 m Glc VI m, d (7.9) m Glc VI d 3.95 (7.9) m m m or m m m or m 4.09 or 4.36 m m, 4.30 m m Chemical shifts could not be unequivocally assigned due to very close chemical shifts or overlapping resonances m or 4.36 m, 4.30 m Furr Chemical analysis shifts could not be unequivocally 2D assigned data due allowed to very close assignment chemical shifts or overlapping remaining six sugars resonances. in (1). The HMBC correlation from anomeric proton at δ H 5.42 (δ C 96.6 via HSQC-DEPT) to a quaternary carbon at δ C 88.0 (C-13) allowed its assignment as anomeric proton Glc II (Figure Furr 2). The analysis 1 H coupling sequence 2D from data allowed Glc II anomeric assignment proton (δ remaining six sugars H 5.42) to H-2 (δ H 4.19) in (1). The HMBC correlation from anomeric proton at δh 5.42 (δc through H-3 (δ 96.6 via HSQC DEPT) to a H 4.93), H-4 (δ H 4.09), H-5 (δ H 3.88), oxymethylene protons, H-6 (δ H 4.19 or quaternary carbon at δc ), was established 88.0 (C 13) using allowed a combination its assignment COSY as anomeric TOCSY proton data. Glc The II carbons (Figure 2). The 1 H coupling sequence from Glc II anomeric proton (δh 5.42) to H 2 (δh C-2 (δ 4.19) through H 3 C 81.2), C-3 (δ C 88.6), C-4 (δ C 70.5 or 70.6 or 70.7), C-5 (δ C ), C-6 (δ C ) (δh 4.93), H 4 (δh 4.09), H 5 (δh 3.88), oxymethylene protons, H 6 (δh were n assigned based on HSQC-DEPT data confirmed by HMBC correlations 4.19 or 4.22 H-1/C-3; 4.33), was established using a combination COSY TOCSY data. The carbons C 2 (δc 81.2), C 3 (δc 88.6), C 4 (δc 70.5 or 70.6 or 70.7), C 5 (δc ), C 6 (δc ) were n assigned based

6 Biomolecules 2017, 7, H-2/C-1, C-3; H-3/C-2, C-4; H-4/C-3, C-5, C-6. As in Glc I, higher-frequency 1 H 13 C chemical shifts C-2 C-3 positions suggested glycosyl substituents at se positions in Glc II, this was confirmed by HMBC correlations. Thus, in Glc II, long-range 1 H- 13 C correlations from anomeric proton at δ H 5.48 (Glc III, H-1) to carbon at δ C 81.2 (Glc II, C-2) anomeric proton at δ H 5.40 (Glc IV, H-1) to carbon at δ C 88.6 (Glc II, C-3) in HMBC experiment established 2,3-O-branched-D-glucodiosyl substituent in Glc II to be same as that present in Rebaudioside M. The large 3-bond coupling constants for anomeric protons Glc II (δ H 5.42, d, J 3 = 8.0 Hz), Glc III (δ H 5.48, d, J 3 = 7.4 Hz), Glc IV (δ H 5.40, d, J 3 = 8.0 Hz) indicated ir β-configuration. The key COSY HMBC correlations are provided in Figure 2, complete 1 H 13 C assignments sugars are provided in Table 3. While data did not indicate any furr sugar linkages to Glc II or Glc IV, relatively higher frequency C-6 (δ c 72.1) in Glc III HMBC correlations from Glc III H-6 (δ H ) to a carbon at δ C suggested a sugar linkage at this position, this was subsequently confirmed by HMBC correlations. Table 3. 1 H 13 C ( MHz, 300K) assignments C-13 glycosides compound 1 in pyridine-d 5. Sugar Atom No. 13 C 1 H Glc II d (8.0) m m # or 70.6 or m m m or 4.22 m, 4.33 m Glc III d (7.4) or m m m m m, 4.50 m Glc IV d (8.0) or m m or m or 4.15 m m m or 4.22 m, 4.32 m Glc VII d (3.5) m m or 70.6 or m m m, ~4.5 m Glc VIII d (7.7) m m or m or 4.16 m m or 3.85 m ~4.2 m to ~4.5 m Glc IX d (7.8) m m or m or 4.16 m m or 3.85 m ~4.2 m to ~4.5 m Chemical shifts could not be unequivocally assigned due to very close chemical shifts or overlapping resonances. # Proton resonance obscured by water resonance, assignment based on Total Spectroscopy (TOCSY) 2D data.

7 Biomolecules 2017, 7, One three remaining sugars had an anomeric proton with a smaller coupling (δ H 5.76, d, J 3 = 3.5 Hz) suggesting an α-linked sugar in structure, while or two had large couplings (δ H 5.10, d, J 3 = 7.7 Hz δ H 5.06, d, J 3 = 7.8 Hz) suggesting a β-configuration. The anomeric proton at δ H 5.76 showed a long-range 1 H- 13 C correlation to carbon at δ C 72.1 (Glc III, C-6), thus establishing a 1 6 α-linkage between Glc VII Glc III (Figure 2). The 1 H coupling sequence from Glc VII anomeric proton (δ H 5.76) to H-2 (δ H 4.13) through H-3 (δ H 4.60), H-4 (δ H 4.16), H-5 (δ H 4.41), oxymethylene protons, H-6 (δ H 4.36 ~4.5), was established using a combination COSY TOCSY data. The Glc VII carbons C-2 (δ C 81.4), C-3 (δ C 84.7), C-4 (δ C 70.5 or 70.6 or 70.7), C-5 (δ C 74.0), C-6 (δ C ) were n assigned based on HSQC-DEPT data confirmed by HMBC correlations H-1/C-2, C-3, C-5; H-2/C-1, C-3; H-3/C-2, C-4; H-5/C-4. The placement two remaining glucose moieties with anomeric protons at δ H 5.10 (δ C 106.5) δ H 5.06 (δ C 105.4) was established by HMBC correlations from anomeric protons at δ H 5.10 (Glc VIII, H-1) to Glc VII C-2 (δ C 81.4) δ H 5.06 (Glc IX, H-1) to Glc VII C-3 (δ C 84.7), thus indicating attachment se two sugars at C-2 C-3, respectively, Glc VII. Reciprocal HMBC correlations from Glc VII, H-2 to δ C (Glc VIII, C-1), H-3 to δ C (Glc IX, C-1) furr confirmed this assignment. In Glc VIII, COSY correlations between anomeric proton (δ H 5.10) to H-2 (δ H 4.06) through H-3 (δ H 4.17) established assignment Glc VIII H-2 H-3, while H-4 (δ H 4.10 or 4.16), H-5 (δ H 3.81 or 3.85), oxymethylene protons, H-6 (δ H ~4.2 to ~4.5) were assigned based on TOCSY data. The carbons at C-2 (δ C 75.7), C-3 (δ C ), C-4 (δ C 72.1 or 74.0), C-5 (δ C ), C-6 (δ C ) were assigned based on HSQC-DEPT HMBC data. Glc IX was similarly assigned. COSY correlations between Glc IX anomeric proton (δ H 5.06) proton at δ H 3.98 established assignment Glc IX, H-2 resonance Glc IX H-3 (δ H 4.10), H-4 (δ H 4.10 or 4.16), H-5 (δ H 3.81 or 3.85), oxymethylene protons, H-6 (δ H ~4.2 to ~4.5), were assigned on basis TOCSY data. Carbons C-2 (δ C 76.1), C-3 (δ C ), C-4 (δ C 72.1 or 74.0), C-5 (δ C ), C-6 (δ C ) were assigned based on HSQC-DEPT HMBC data. Some 1 H 13 C chemical shifts could not be unequivocally assigned due to peak overlap, for se, eir an alternate chemical shift value or a specific range values is provided. Thus, extensive analysis 2D data established connectivity complete assignments all nine sugars in this novel glycoside. The key COSY HMBC correlations 1 are provided in Figure 3, complete 1 H 13 C assignments are provided in Tables Experimental Section 3.1. General Experimental Procedures for Rebaudioside IX (1) Isolation Purification Isolation (1) by Preparative High-Performance Liquid Chromatography (HPLC): The purification 18.3 g stevia extract was performed in three chromatographic steps (see Supplementary Materials for representative HPLC traces from se steps). All processes used a Waters Symmetry Shield RP18 ( mm, 7 µm, p/n WAT248000; Water Corporation, Milford, MA, USA) column at ambient temperature; flow rate at 40 ml/min; detection by UV (210 nm). All gradient steps were linear. All mobile phases were premixed by volume, contained 0.1% acetic acid (HOAc) (v/v). The primary process used following method: Mobile Phase A: 20% acetonitrile (MeCN) in water; Mobile Phase B: 30% MeCN in water; Gradient: 0 10 min (100A:0B), min (0A:100B), 32 min (100A:0B); Injection load: 3 g stevia extract dissolved in 30 ml dimethylsulfoxide (DMSO), n diluted with 90 ml water with 0.1% HOAc. The fraction interest (containing (1) based on chromatographic MS priles, see below for methods) was concentrated from eluent via rotary evaporation (R-114 Rotary Evaporator; Buchi Corporation, New Castle, DE, USA). The concentrated aqueous solution was loaded as is in next purification step. The second chromatographic step used following method: Mobile Phase A: 15% MeCN in water; Mobile Phase B: 30% MeCN in water; Gradient: 0 5 min (100A:0B), min (0A:100B),

8 Biomolecules 2017, 7, min (100A:0B); Injection load: 75 ml aqueous sample concentrate. Fractions interest were pooled, concentrated in vacuo, lyophilized (FTS System benchtop lyophilizer; Labconco Corporation, Kansas City, MO, USA), yielding 62 mg a solid powder. The third chromatographic step used an isocratic mobile phase consisting 18% MeCN in water; Injection load: 62 mg sample dissolved in 10 ml water. The isolated compound was partially concentrated from eluent via rotary evaporation, n furr concentrated on a solid phase extraction (SPE) cartridge, followed by rotary evaporation lyophilization. The purity final product was 90.3% as confirmed by Liquid Chromatography-Charged Aerosol Detection (LC-CAD). Approximately 4.5 mg (1) was available for spectroscopic spectrometric analyses. Fraction analysis: analysis preparative purification fractions were performed using following method. Synergi Hydro-RP column ( mm, 4 µm, p/n 00B-4375-A0; Phenomenex, Torrance, CA, USA); Column Temp: 55 C; Mobile Phase A: % NH 4 OAc % HOAc in water; Mobile Phase B: MeCN; Flow Rate: 1.0 ml/min; Injection volume: 10 µl. Detection was by UV (210 nm) CAD. Primary fractions were analyzed using following method: Gradient: min (75A:25B), 10 min (71A:29B), 16.5 min (70A:30B), min (66A:34B), min (30A:70B), 27.5 min (75A:25B). Fractions from secondary tertiary purification steps were analyzed using following method: Gradient: 0 5 min (90A:10B), 20 min (70A:30B), min (50A:50B), min (90A:10B). Gradient segments were linear. The peak for (1) was observed at a retention time (tr) approximately 19.7 min. Liquid Chromatography-Mass Spectrometry(LC-MS) Analyses: LC-MS analysis enhanced fractions was carried out on a AB Sciex API 150EX spectrometer (Applied Biosystems SCIEX, Waltham, MA, USA) using Turbo Spray with a window Da using following method. Synergi Hydro-RP (Phenomenex); Column Temp: Ambient; Mobile Phase A: 15% MeCN in water; Mobile Phase B:30% MeCN in water; Gradient: 0 5 min (100A:0B), 5 25 min (0A:100B), min (0A:100B), 32 min (100A:0B), Flow Rate: 1.0 ml/min; Injection volume: 10 µl Mass Spectrometry The ElectroSpray Ionization-Time-Off-Flight (ESI-TOF) spectra MS/MS data were generated with a Waters Q-T Premier spectrometer (Waters Coporation) equipped with an electrospray ionization source. The sample was diluted with H 2 O-MeCN (1:1) by 50-fold introduced via infusion using onboard syringe pump analyzed by negative ESI Nuclear Magnetic Resonance Spectroscopy The samples Rebaudioside IX (1) (~5 mg in 700 µl pyridine-d 5 or ~2 mg in 180 µl pyridine-d 5 ) were prepared, data were acquired on Bruker Avance 500 MHz (Bruker BioSpin, Billerica, Massachusetts, USA) instrument with a 5 mm broad b probe 2.5 mm inverse detection probe. The 1 H 13 C spectra were referenced to pyridine signal at δ H 8.72 δ C ppm, respectively. All data were acquired at 300 K (see Supplementary Materials for examples representative 2D spectra not presented in main text) Material Sources The material used for isolation compound (1) was a commercial Stevia rebaudiana Bertoni extract, VSPC , received from PureCircle, Malaysia. 4. Conclusions Based on extensive spectroscopic studies, structure novel diterpene glycoside (1) with nine glucose units was established as 13-[(2-O-(6-O-α-D-glucopyranosyl-(2-O-β-D-glucopyranosyl-3-Oβ-D-glucopyranosyl)-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl)-β-D-glucopyranosyl)oxy] entkaur-16-en-19-oic acid-[(2-o-β-d-glucopyranosyl-3-o-β-d-glucopyranosyl-β-d-glucopyranosyl) ester]. Evaluation data led to conclusion that this compound has an additional glucotriosyl

9 Biomolecules 2017, 7, moiety (2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl)-β-D-glucopyranosyl attached at Glc III Rebaudioside M as a 1 6 sugar linkage in an α-configuration. To best our knowledge, this is first report a steviol glycoside with nine sugars. In addition to this novel feature, α 1 6 sugar linkage present in Rebaudioside IX is also rare for this class compounds. Rebaudioside IX is an important finding, exps our understing diversity diterpene glycosides in leaves S. rebaudiana Bertoni. The finding demonstrates that new structurally unique Rebaudiosides can still be isolated identified from S. rebaudiana, that approach exemplified here fers opportunities to find new sweeteners for health-conscious consumer. Supplementary Materials: The following are available online at Figure S1: Preparative HPLC trace from primary chromatographic process, Figure S2: Preparative HPLC trace from second chromatographic process, Figure S3: Preparative HPLC trace from third chromatographic process, Figure S4: 1 H Nuclear Magnetic Resonance () spectra, full scale expansions Rebaudioside IX (1), Figure S5: 13 C spectrum Rebaudioside IX (1), Figure S6: 1 H- 1 H Spectroscopy (COSY) spectrum Rebaudioside IX (1), Figure S7: 1 H- 13 C Single Quantum Coherence-Distortionless Enhancement Polarization Transfer (HSQC-DEPT) spectrum Rebaudioside IX (1), Figure S8: 1 H- 13 C Multiple Bond (HMBC) spectra, full scale expansion Rebaudioside IX (1), Figure S9: 1 H- 1 H Nuclear Overhauser Effect Spectroscopy (NOESY) spectrum Rebaudioside IX (1). Acknowledgments: We wish to thank PureCircle (Malaysia) for providing a commercial supply Stevia extract. Author Contributions: Indra Prakash, Gil Ma, Cynthia Bunders, Romila D. Charan Krishna P. Devkota wrote manuscript did interpretation data. Romila D. Charan performed experiments did interpretation data. Carine Ramirez Tara Snyder isolated Rebaudioside IX. Conflicts Interest: The authors declare no conflict interest. References 1. Prakash, I.; DuBois, G.E.; King, G.A.; Upreti, M. Rebaudioside A Composition Method for Purifying Rebaudioside A. U.S. Patent 2007/029582, 20 December Prakash, I.; DuBois, G.E.; Clos, J.F.; Wilkens, K.L.; Fosdick, L.E. Development rebiana, a natural, non-caloric sweetener. Food Chem. Toxicol. 2008, 46, S75 S82. [CrossRef] [PubMed] 3. Prakash, I.; Chaturvedula, V.S.P.; Markosyan, A. Isolation, Characterization Sensor Evaluation a Hexa β-d-glucopyranosyl Diterpene from Stevia rebaudiana. Nat. Prod. Commun. 2013, 8, [PubMed] 4. Soejarto, D.D.; Compadre, C.M.; Medon, P.J.; Kamath, S.K.; Kinghorn, A.D. Potential sweetening agents plant origin. II. Field search for sweet-tasting Stevia species. Econ. Bot. 1983, 37, [CrossRef] 5. Ceunen, S.; Geuns, J.M.C. Steviol Glycosides: Chemical Diversity, Metabolism, Function. J. Nat. Prod. 2013, 76, [CrossRef] [PubMed] 6. Ohta, M.; Sasa, S.; Inoue, A.; Tamai, T.; Fujita, I.; Morita, K.; Matsuura, F. Characterization Novel Steviol Glycosides from Leaves Stevia rebaudiana Morita. J. Appl. Glycosci. 2010, 57, [CrossRef] 7. Chaturvedula, V.S.P.; Rhea, J.; Milanowski, D.; Mocek, U.; Prakash, I. Two Minor Diterpene Glycosides from Leaves Stevia rebaudiana. Nat. Prod. Commun. 2011, 6, [PubMed] 8. Chaturvedula, V.S.P.; Prakash, I. Additional Minor Diterpene Glycosides from Stevia rebaudiana. Nat. Prod. Commun. 2011, 6, [PubMed] 9. Chaturvedula, V.S.P.; Prakash, I. Structures Novel Diterpene Glycosides from Stevia rebaudiana. Carbohydr. Res. 2011, 346, [CrossRef] [PubMed] 10. Chaturvedula, V.S.P.; Upreti, M.; Prakash, I. Structures Novel α-glucosyl Linked Diterpene Glycosides from Stevia rebaudiana. Carbohydr. Res. 2011, 346, [CrossRef] [PubMed] 11. Chaturvedula, V.S.P.; Upreti, M.; Prakash, I. Diterpene Glycosides from Stevia rebaudiana. Molecules 2011, 16, [CrossRef] [PubMed] 12. Prakash, I.; Markosyan, A.; Chaturvedula, V.S.P.; Campbell, M.; San Miguel, R.I.; Purkayastha, S.; Johnson, M. Methods for Purifying Steviol Glycosides. PCT Patent WO 2013/096420, 27 June Prakash, I.; Markosyan, A.; Bunders, C. Development Next Generation Stevia Sweetener: Rebaudioside M. Foods 2014, 3, [CrossRef]

10 Biomolecules 2017, 7, Prakash, I.; Ma, G.; Bunders, C.; Devkota, K.P.; Charan, R.D.; Ramirez, C.; Snyder, T.M.; Priedemann, C. A New Diterpene Glycoside: 15α-Hydroxy-Rebaudioside M Isolated from Stevia rebaudiana. Nat. Prod. Commun. 2015, 10, [PubMed] 15. Prakash, I.; Bunders, C.; Devkota, K.P.; Charan, R.D.; Ramirez, C.; Priedemann, C.; Markosyan, A. Isolation Characterization a Novel Rebaudioside M Isomer from a Bioconversion Reaction Rebaudioside A comparison Studies Rebaudioside M Isolated from Stevia rebaudiana Bertoni Stevia rebaudiana Morita. Biomolecules 2014, 4, [CrossRef] [PubMed] 2017 by authors; licensee MDPI, Basel, Switzerl. This article is an open access article distributed under terms conditions Creative Commons Attribution (CC BY) license (