RQ3, A natural rebaudioside D isomer, was obtained from

Article Cite This: J. Agric. Food Chem. 2019, 67, 8020−8028

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RQ3, A Natural Rebaudioside D Isomer, Was Obtained from Glucosylation of Rebaudioside A Catalyzed by the CGTase Toruzyme 3.0 L Qingbin Guo,†,§,# Tongtong Zhang,†,‡,# Nifei Wang,§ Yongmei Xia,*,†,‡ Zhuoyu Zhou,†,‡ Jian-rong Wang,¶ and Xuefeng Mei¶ †

State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, Jiangsu 214122, China School of Chemical and Materials Engineering, Jiangnan University, Wuxi, Jiangsu 214122, China § State Key Laboratory of Food Nutrition and Safety, Tianjin University of Science and Technology, Ministry of Education, Tianjin 300457, China ¶ Pharmaceutical Analytical & Solid-State Chemistry Research Center, Shanghai Institute of Materia Medical, Chinese Academy of Sciences, Shanghai 201203, China

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‡

S Supporting Information *

ABSTRACT: In this study, a monoglucosyl rebaudioside A product was isolated from the mixture of glucosylated rebaudioside A obtained from the most reported and industrial used cyclodextrin glycosyl transferase, Toruzyme 3.0 L (CGTase, Toruzyme 3.0 L). The molecular structure of the monoglucosyl rebaudioside A was characterized using LC-MS/MS and methylation analysis combined with 1D and 2D NMR, indicating that it is 13-[(2-O-(3-α-O-D-glucopyranosyl)-β-D-glucopyranosyl-3-O-βD-glucopyranosyl-β-D-glucopyranosyl)oxy] ent-kaur-16-en-19-oic acid β-D-glucopyranosyl ester (also known as RQ3, which naturally exists in Stevia extract as an isomer of rebaudioside D). This study may help to further understand the reaction mechanism of glucosylation of steviol glycoside assisted by Toruzyme 3.0 L in the aspect of molecule linkage pattern, and also benefit the application of the glucosylated rebaudiosides. KEYWORDS: rebaudioside, steviol glycoside, glycosylation, transglucosylation, NMR

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INTRODUCTION Steviol glycosides (SGs) might be the most attractive natural noncaloric sweetener.1,2 To date, 64 steviol glycosides have been identified from the leaves of Stevia rebaudiana (FAO JECFA Monographs 20, 2017, ISSN 1817-7077), among which stevioside and rebaudioside A (Supporting Information, Figure 1s) are the two major components. For decades, efforts have been made to explore the structure−sweetness relationship of steviol glycosides.3−7 It has been found that extending the C-19-ester-linked glycosyl unit led to an improvement of the edulcorant quality.3 For example, rebaudioside D (Figure 1s) and rebaudioside M, which carry two and three C-19 linked glycosyl units, respectively, both demonstrate improved edulcorant quality over that of stevioside or rebaudioside A. Hence, enzymatic glucosylation of stevioside and rebaudioside A have been extensively studied8−17 to improve the edulcorant quality;9,15−23 the resultant products are so-called glucosyl steviol glycosides (GSGs). Some enzymatic-produced GSGs have been reported to have structures that are the same as the natural, identical SGs, such as rebaudioside A from stevioside10,24,25 and rebaudioside D,26,27 rebaudioside I,28 and rebaudioside M2 from rebaudioside A;26,29 most of them were produced by using UDPglucosyltransferases (UGTs) and sucrose synthase with UDPglucose as the glucosyl donor. The UDP-glucose pathway normally provides high product specificity and subsequent © 2019 American Chemical Society

coupling of a few glucosyl groups onto the glycoside acceptors, with monoglucosylated product as the major product.24−31 Unlike the UDP-glucose pathway, glucosylation of steviol glycosides catalyzed by CGTs (cyclodextrin glycosyl transferase) produces many more glucosyl groups onto the acceptors16,17,32−34 because CGTs act as multifunctional enzymes and most CGTs take oligosaccharides as glucosyl donor. In addition, CGTs would generate α-glucosides; subconsciously and logically, it is easy to think that CGTs do not induce naturally occurring steviol glycosides because most glucose residues in the natural steviol glycosides exist as β-configuration. As a matter of fact, the isomeric rebaudiosides (such as the set of rebaudioside I, D, and D2 and the set of rebaudioside M and M2) differ only in the linkage patterns, but their edulcorant quality as like sweetness are quite different. For example, the sweetness of Reb A is 250-fold that of sucrose, while its isomer Reb E has a sweetness of 150-fold that of sucrose, respectively. There are no sweetness data of the isomeric rebaudioside D or M sets yet. Prakash et al. (used UGTs with UDP-glucose)27,28,32 and Gerwig, te Poele, Devlamynck, and Yang et al.8,35−37 (used Received: Revised: Accepted: Published: 8020

April 23, 2019 June 14, 2019 June 18, 2019 June 18, 2019 DOI: 10.1021/acs.jafc.9b02545 J. Agric. Food Chem. 2019, 67, 8020−8028

Article

Journal of Agricultural and Food Chemistry

Standards of rebaudioside I and rebaudioside D were purchased from Shanghai ZZBio Co., Ltd., China. All other reagents were of analytical grade and used as received. Glucosyl rebaudioside A was prepared as follows: 10 mL of rebaudioside A aqueous solution (20 g/L), 10 mL of β-cyclodextrin aqueous solution (20 g/L), and 10 U CGTase/g rebaudioside A were mixed in a 100 mL flask with shaking at 70 °C for 24 h. The composition of the raw products was analyzed by HPLC as presented in the Supporting Information (Figure 2s). The component possessing the same HPLC retention time as rebaudioside I/D (standards) was designated as compound I for subsequent structure determination (in Figure 2s, Supporting Information). The compound I was isolated from the glucosyl rebaudioside A mixture with a preparative chromatography system (Kromasil C18; DAC50 column from Jiangsu Hanbang Science and Technology Co. Ltd. of China, ⌀ 50 mm × 500 mm, 7−8 MPa) and identified under the running conditions as follows. Using Waters 2695 HPLC system (Waters, United States) equipped with a Kromasil C18 column and a UV detector (detected at 210 nm), the samples were eluted using a mixture of sodium phosphate buffer (10 mmol/L, pH 2.6) and acetonitrile (68:32, v/v) and the flow rate was 1.0 mL/min. The sample obtained was stored for subsequent analysis. LC/MS Analysis. LC/MS analysis was carried out by using a Thermo Scientific Q-Exactive Benchtop Orbitrap Mass Spectrometer connected with a Vanquish Flex Binary UPLC System (Massachusetts, USA). The UPLC system consisted of a binary pump with a vacuum degasser, an autosampler, a UV/vis photodiode array detector, and a column compartment. A Thermo Scientific (Waltham, MA) GlycanPac 135 AXH-1 column (150 mm × 21 mm, 3 μm) was used for separation. Two mobile phases, solvent A (5 mM ammonium formate 99.9% H2O + 0.1% formic acid) and solvent B (99.9% acetonitrile + 0.1% formic acid), were used with 70% B and 30% A as the elution buffer. The column compartment was controlled at 25 °C, the flow rate was fixed at 0.2 mL/min, and the injection amount was 2 μL. The positive heated-electrospray ionization (HESI) mode was used for data collection. The optimized HESI conditions were as follows: sheath gas, 45 arbitrary units; auxiliary gas, 10 arbitrary units; sweep gas, 2 arbitrary units; S-lens RF level, 50; capillary temperature, 250 °C; and auxiliary heater temperature, 400 °C. The spray voltage was 3.5 kV. The automatic gain control target and maximum injection time for full mass data collection were 3e6 and 240 ms, respectively. Methylation Analysis. Methylation analysis of compound I was conducted using the method of Ciucanu and Kerek.42 The samples after reduction were dissolved in dimethyl sulfoxide, sonicated at 40 °C for 2 h, and transferred to a 70 °C water bath for 2 h with constant stirring. After that, dry sodium hydroxide powder was added to the solution under constant stirring at room temperature followed by 2.5 h of constant stirring after adding 0.3 mL of methyl iodide. The mixture was extracted with 1 mL of methylene chloride, passed through a sodium sulfate column, and then dried with nitrogen gas. The methylated oligosaccharide was hydrolyzed by adding 0.5 mL of 4 M trifluoroacetic acid to the sample in a test tube, sealing the tube, heating it at 100 °C for 6 h, cooling, and then drying with N2. The sample was then dissolved with distilled water (0.3 mL), and the hydrolysate was reduced using 5 mg of sodium borodeuteride and acetylated with acetic anhydride (0.5 mL) for 2 h. Aliquots of the resultant partially methylated alditol acetates (PMAA) were injected into a GC-MS system (ThermoQuest Finnigan, San Diego) fitted with a SP-2330 column (Supelco, Bellefonte, PA, USA; ⌀ 0.25 mm × 30 m, 0.2 μm) and an ion trap MS detector. The running conditions were as follows: 160−210 °C at 2 °C/min and then 210−240 °C at 5 °C/min. 1D and 2D NMR Analyses. The sample was dissolved in 4 mL of D2O at 30 °C with stirring for 2 h and then freeze-dried. This procedure was repeated three times. Then samples were redissolved in 3 mL of D2O and transferred into an NMR testing tube before analysis. High-resolution 1H and 13C NMR spectra were recorded at 500.13 and 125.78 MHz, respectively, on a Bruker ARX500 NMR spectrometer operating at 30 °C. A 5 mm inverse geometry 1 H/13C/15N probe was used. Chemical shifts are reported relative

glucansucrase enzyme with sucrose as glucosyl donor) determined structures of the synthetic products using a full set of NMR and MS analyses. However, although most industrial manufacturers produce glucosyl steviol glycosides with CGTase Toruzyme 3.0 L (a commercial cyclodextrin glycosyl transferase), the linkage pattern of GSGs obtained from CGTase still remain unclear. Furthermore, there are still more reports identifying the molecular structure of enzymatic GSG using only LC-MS and HPLC analysis with known rebaudiosides as reference. JECFA (Joint FAO/WHO Expert Committee on Food Additives) provided a standard HPLC protocol to identify 13 steviol glycosides in 2016 tentatively (FAO JECFA Monographs 19, 2016), and then formally in 2017 with dozens of steviol glycosides (FAO JECFA Monographs 20, 2017). However, using the HPLC profile only might mislead the identification of enzymatic GSGs (Figure 2s, Supporting Information). Yet there are still a few enzymatic GSGs products in research articles that have been identified only according to HPLC protocol and LC-MS. In addition, because the GSG obtained with Toruzyme 3.0 L has been in the market for decades, and there are many claimed enzymatic or fermented rebaudioside D and rebaudioside M that are going to blow up the market, it is necessary to unravel the molecular structure of the main component and share a detailed stepwise protocol for the structural identification of steviol glycosides with a clear linkage pattern. In industry, the most popular glucosyltransferases, such as CGTase and other α-amylase, have been extensively applied to catalyze the transglucosylation of stevioside or rebaudioside A with dextrins as glucosyl donors. 11,12,14−17,33,34,38 The CGTase-intermediated reaction has multiple pathways that could yield many products via hydrolysis, coupling, and disproportionation.16 The obtained GSGs that possess a high percentage of glucosyl units are used as sweet flavoring agents in both perfumes and drug formulas.39 The GSGs with less than four glucosyl units at the C19 position are supposed to be intense sweeteners;40 their molecular weights and rough structures have been elucidated, but the linkage patterns at present still need to be clearly unraveled.41 Furthermore, although the function of CGTs and proposed reaction mechanism in the literature16 do not support the production of rebaudioside D from rebaudioside A, the HPLC profile of the glucosyl rebaudioside A generated by Toruzyme 3.0 L could provide some peaks at exactly the same retention times as that of rebaudioside D or rebaudioside I (Supporting Information, Figure 2s), which may lead to a misinterpretation. In this study, using the commercial CGTase (Toruzyme 3.0 L) as glucosyltransferase and rebaudioside A with β-cyclodextrin as substrates, a relative massive monoglucosyl rebaudioside A (Supporting Information, Figure 2s) was obtained from the glucosyl rebaudioside A mixture, which possessed the same HPLC retention time as rebaudioside D or I (Supporting Information, Figure 2s). The detailed molecular structure was herein characterized using LC-MS/MS, methylation analysis and 1D and 2D NMR in a stepwise manner.

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MATERIALS AND METHODS

Materials. Rebaudioside A (RA, 97%, HPLC) was provided by Zhucheng Haitian Pharm Co., Ltd., China. β-Cyclodextrin (β-CD, 98.0%, HPLC) was purchased from Jiangsu Fengyuan Biotechnology Company, China. CGTase Toruzyme 3.0 L from Thermoanaerobacter sp. (coupling activity, 114 U/mL) was gifted by Novozymes, China. 8021

DOI: 10.1021/acs.jafc.9b02545 J. Agric. Food Chem. 2019, 67, 8020−8028

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H]+, [M − 2G + H]+, [M − 3G + H]+, [M − 4G + H]+, and [M − 5G + H]+ with relatively lower intensities were also observed in the MS/MS spectra. However, the linkage patterns were still not clear. Methylation Analysis. Methylation analysis was used to help identify the glucosyl groups by their number and position. Three sugar residues from compound I were identified with methylation analysis, which were T-GlcP, 4-GlcP, and 2,3GlcP, respectively, as shown in Figure 3s (Supporting Information). According to the peak area, the molar ratios of T-GlcP, 4-GlcP, and 2, 3-GlcP were 7:1:2. Sugar units in rebaudioside A include only T-GlcP and 2,3-GlcP (Figure 1s, Supporting Information), but when a new glucosyl residue was added because of the designated reaction, the T-GlcP to which the added sugar connected in rebaudioside A was then converted to 4-GlcP, and the added sugar existed as T-GlcP in the new compound I. The methylation analysis also confirmed that the new compound I was not rebaudioside D or rebaudioside I. Rebaudioside I contains residues of T-GlcP, 3-GlcP, and 2, 3-GlcP, and rebaudioside D comprises T-GlcP, 2-GlcP, and 2, 3-GlcP. Through enzymatic glucosylation reaction, an extra T-GlcP units was inserted to rebaudioside A molecule; however, the location and the configuration (α- or β-form) of the added sugar had not been determined thus far. Therefore, 1D and 2D NMR spectroscopy was employed to solve these problems. 1D NMR Analysis. The full 1H NMR spectrum in Figure 2a can be simply divided into two zones, peaks with chemical shifts ranging from 3 to 5.5 ppm, which represent the sugar units and peaks with chemical shifts less than 2.3 ppm, which are derived from the aglycone. According to the spectrum in Figure 2b, seven peaks, including δ 5.36 (d, J = 8.1 Hz, 1H), 5.33 (d, J = 4.0 Hz, 1H), 5.12 (s, 1H), 4.87 (s, 1H), 4.79 (d, J = 7.7 Hz, 1H), 4.73 (d, J = 7.7 Hz, 1H), and 4.66 (d, J = 7.6 Hz, 1 H) were detected in the anomeric region (4.6−5.5 ppm), while only five peaks showed duplicate peak splitting, which were labeled as A, B, C, D, and E, from low to high field, respectively (Figure 2). Peaks at 5.12 and 4.87 ppm, which did not show any splitting, were likely derived from the aglycone group of compound I. This result was confirmed by the 2D NMR spectra. The high J coupling distances for peaks A (8.1 Hz), C (7.7 Hz), D (7.7 Hz), and E (7.6 Hz) suggested that residues A, C, D, and E adopted a β-configuration, while residue B, with a relatively low J coupling distance (4 Hz), showed an α-configuration. Peak integration in the proton spectrum could reflect the abundance of the sugar residues. Peak areas of A, B, C, D, and E were at the ratio of 1.00:0.75:1.03:0.95:0.81, indicating that all the sugar residues existed in a similar ratio in compound I. Peaks at 1.20 and 0.86 ppm were assigned to the −CH3 group of the 18 and 20 positions of aglycone (Figure 1s, Supporting Information), and both peaks showed relatively high intensity (Figure 2c). The proton chemical shifts of the aglycone group (from positions 1 to 20, as shown in Figure 1s, Supporting Information) were all successfully assigned using COSY (Figure 3) and TOCSY (Figure 4) experiments and all the peaks are labeled in Figure 2c. Figure 2d shows the 13C spectrum of compound I. The chemical shifts of the sugar units were mostly distributed in the range of 60−103 ppm, while the spectrum of the aglycone group was mostly located in the range of 10−60 ppm. Three peaks were also observed downfield in the spectrum (19, 178.5 ppm; 16, 152.7 ppm; and 17, 104.9 ppm) because of the

to trimethylsilyl propionate in D2O for 1H (0.0 ppm, external standard) and 1,4-dioxane in D2O for 13C (66.5 ppm, external standard). Homonuclear 1H/1H correlation experiments (COSY and TOCSY) and heteronuclear 1H/13C correlation experiments (HSQC and HMBC) were run using the standard Bruker pulse sequences.

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RESULTS AND DISCUSSION LC-MS/MS Analysis. Compound I had the same HPLC retention time as standard rebaudioside I, and it also presented another HPLC retention time that was the same as that of standard rebaudioside D in another slightly modified HPLC protocol according to the JECFA method (Figure 2s, Supporting Information), respectively. The results from LCMS/MS analysis of compound I are presented in Figure 1. Its

Figure 1. Total ionic chromatogram (a), mass spectrum (b), and tandem MS (MS/MS) spectrum of compound I (c).

molecular formula was deduced as C50H80O28 based on the LC-MS/MS data, which showed the presence of an [M + H]+ ion at m/z 1129 together with [M + NH4]+ at m/z 1146 (Figure 1a,b). This result suggested that compound I is a monoglucosyl rebaudioside A, which is as expected. This result was also confirmed by the MS/MS spectra (Figure 1c), from which five sugar units, including [2G]+, [3G]+, [4G]+, and [5G]+, were all detected. Meanwhile, fragments of [M − G + 8022


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Figure 2. 1D NMR spectra of compound I. (a) Full proton NMR spectrum, (b) enlarged proton NMR spectrum for the anomeric region of the sugar units, (c) enlarged proton NMR spectrum for the aglycone group, and (d) 13C NMR spectrum.

the full correlations of protons within one sugar ring. The assignments of the COSY spectra are normally started from the anomeric proton (H-1). Taking the pyranose sugar ring as an example, the coupling network follows the order of H-1 couples with H-2, H-2 couples with H-1 and H-3, H-3 couples with H-2 and H-4, H-4 couples with H-3 and H-5, and H-5 couples with H-4 and H-6/H-6′. Following this rule, the full chemical shifts of the above-mentioned five sugar residues were all identified. In this study, the cross peaks of H-1/H-2 of those residues were all labeled in the enlarged section for sugar units (Figure 3b). For the aglycone group, as shown in Figure 3c, most of the cross peaks demonstrated the correlations of two protons under the same carbon atoms. For example, cross peaks between 1−1′, 2−2′, 3−3′, 7−7′, 11−11′, 12−12′, 14− 14′, and 15−15′ could be detected (Figure 3c). Similarly, the TOCSY spectrum (Figure 4a) could also be simply divided into two zones, the sugar unit zone (Figure 4b) and the aglycone zone (Figure 4c). As shown in Figure 4b, the full connectivity of each sugar residue was labeled, and the data obtained were helpful as Supporting Information to confirm or

carboxyl group (19) and CC double bonds (16 and 17), as shown in Figure 1s (Supporting Information). The anomeric 13 C of all the sugar units as well as signals derived from the aglycone were well-resolved, which were therefore easily identified, while the nonanomeric 13C of sugar units, which were mostly located in the spectrum range of 68−77 pm, were enlarged and labeled in Figure 3b. Notably, some peaks with relatively low intensity (e.g., peaks at 65.51 and 73.38 ppm) were also observed, indicating that compound I might also contain some other structural isomers at a relatively smaller percentage. 2D NMR Analysis. The full proton chemical shifts of compound I were all identified according to both the COSY (Figure 3) and TOCSY spectra (Figure 4), and all the proton chemical shifts are summarized in Table 1s (Supporting Information). COSY and TOCSY spectra both demonstrate the interactions between protons of one sugar ring, while the COSY spectrum provides only the correlations between neighboring protons and the TOCSY spectrum demonstrates 8023


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Figure 3. COSY spectra of compound I. (a) Full spectrum, (b) enlarged spectrum for the anomeric region for the sugar units, and (c) enlarged proton spectrum for the aglycone group.

support data obtained by the COSY spectrum. In addition, the total correlation of protons within the aglycone zone was also marked, as shown in Figure 4c. As the positions of 4, 9, 8, 13, and 16 in the aglycone have no protons, the proton connectivities in the aglycone ring can therefore be divided into three groups: 1−1′−2−2′−3−3′, 5−6−6′−7−7′, and 9− 11−11′−12−12′ (Figure. 4c). In addition, the H and H′ correlation at position 14 was also detected.

Figure 5 shows the HSQC (Figure 5a,b) and HMBC (Figure 5c) spectra of compound I and demonstrates the 1H and 13C correlations, from which the HSQC shows the correlation peaks of the directly connected 1H and 13C atoms, while the HMBC exhibits 1H and 13C correlations within a three-bond distance, which can therefore be used to determine the sequences of different sugar residues. The directly linked 1 H−13C correlations detected by HSQC have been excluded in 8024


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Figure 4. TOCSY spectra of compound I. (a) Full spectrum, (b) an enlarged anomeric spectrum region for the sugar units, and (c) an enlarged proton spectrum for the aglycone group.

for the five sugar residues as well as the 1H−13C correlations derived from the aglycone zone were all assigned (Table 1s). For the HMBC spectrum, both the inter- and intra-

the HMBC spectrum. In this study, peaks of compound I were assembled into three main zones in the full HSQC spectrum, as shown in Figure 5a, and the complete chemical shifts of 13C 8025


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Figure 5. HSQC spectrum (a) and enlarged HSQC (b) and HMBC (c) spectra of the aglycone zone for compound I.

connectivities of 1H−13C among different residues were determined, while only intercorrelation peaks that are helpful in determining the sequence of the sugar and aglycone residues are labeled in Figure 5c. To summarize, the following intercorrelations were detected: H-1 of residue B and C-3 of

residue C; H-1 of residue C and C-2 of residue E; H-1 of residue D and C-3 of residue E; H-1 of residue E and C-13 of aglycone residue; and H-1 of residue A and C-19 of the aglycone residue. Sequences, including B1-3C1-2E1-13aglycone, D1-3E, and A1-19aglycone were established. The detailed 8026



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structure of compound I was therefore established as shown in Figure 6. In summary, the structure of compound I was

Article

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (Y.X.). ORCID

Qingbin Guo: 0000-0002-2049-5771 Tongtong Zhang: 0000-0001-7130-2442 Nifei Wang: 0000-0002-3389-6769 Yongmei Xia: 0000-0002-3045-6151 Zhuoyu Zhou: 0000-0002-6865-062X Jian-rong Wang: 0000-0002-0853-7537 Xuefeng Mei: 0000-0002-8945-5794 Author Contributions #

Q.G. and T.Z. contributed equally to this work.

Funding

Financial support from the National Natural Science Foundation of China (31772017, 31371837) and the National First-Class Discipline Program of the Light Industry Technology and Engineering (LITE2018-03) are appreciated. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful to Yue Wenyan for her help with sample preparation. A special gratitude to the reviewers and the Editor for their valuable advice, patience, and assistance.

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proposed, as shown in Figure 6, namely, as 13-[(2-O-(3-α-O-Dglucopyranosyl)-β-D-glucopyranosyl-3-O-β-D-glucopyranosylβ-D-glucopyranosyl)oxy] ent-kaur-16-en-19-oic acid β-D-glucopyranosyl ester. This compound was previously termed as RQ3,7 which was found in Stevia rebaudiana leaves and is an isomer of rebaudioside I and rebaudioside D;43 this structure was also found in a sample of glucosylated steviol glycosides that was obtained from a cyclodextrin glycosyltransferase (produced by Bacillus stearothermophilus) of the stevia extract containing stevioside, rebaudioside A, rebaudioside C, and dulcoside.32 Therefore, with cyclodextrin as glucosyl donor, the transglucosylation of rebaudioside A induced by CGTase Toruzyme 3.0 L could yield steviol glycoside RQ3, and the structure of this monoglucosylated rebaudioside A was fully elucidated. This result could also help understand the reaction mechanism of glucosylation of steviol glycoside assisted by Toruzyme 3.0 L regarding the molecular linkage pattern.

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REFERENCES

(1) Carrera-Lanestosa, A.; Moguel-Ordóñez, Y.; Segura-Campos, M. Stevia rebaudiana Bertoni: A Natural Alternative for Treating Diseases Associated with Metabolic Syndrome. J. Med. Food 2017, 20 (10), 933−943. (2) Gantait, S.; Das, A.; Mandal, N. Stevia: A Comprehensive Review on Ethnopharmacological Properties and In Vitro Regeneration. Sugar Tech 2015, 17 (2), 95−106. (3) Kasai, R.; Kaneda, N.; Tanaka, O.; Yamasaki, K.; Sakamoto, I.; Morimoto, K.; Okada, S.; Kitahata, S.; Furukawa, H. Sweet DiterpeneGlycosides of Leaves of Stevia rebaudiana Bertoni Synthesis and Structure-Sweetness Relationship of Rebaudiosides-A, -D, -E and Their Related Glycosides. Nippon Kagaku Kaishi 1981, 1981 (5), 726−735. (4) Darise, M.; Mizutani, K.; Kasai, R.; Tanaka, O.; Kitahata, S.; Okada, S.; Ogawa, S.; Murakami, F.; Chen, F. H. Enzymic Transglucosylation of Rubusoside and the Structure-Sweetness Relationship of Steviol-Bisglycosides. Agric. Biol. Chem. 1984, 48 (10), 2483−2488. (5) Mizutani, K.; Miyata, T.; Kasai, R.; Tanaka, O.; Ogawa, S.; Doi, S. Study on improvement of sweetness of steviol bisglycosides: Selective enzymic transglucosylation of the 13-o-glycosyl moiety. Agric. Biol. Chem. 1989, 53 (2), 395−398. (6) Chéron, J. B.; Casciuc, I.; Golebiowski, J.; Antonczak, S.; Fiorucci, S. Sweetness prediction of natural compounds. Food Chem. 2017, 221, 1421−1425. (7) Gerwig, G. J.; te Poele, E. M.; Dijkhuizen, L.; Kamerling, J. P. Stevia Glycosides: Chemical and Enzymatic Modifications of Their Carbohydrate Moieties to Improve the Sweet-Tasting Quality. Adv. Carbohydr. Chem. Biochem. 2016, 73, 1−72. (8) Gerwig, G. J.; te Poele, E. M.; Dijkhuizen, L.; Kamerling, J. P. Structural analysis of rebaudioside A derivatives obtained by Lactobacillus reuteri 180 glucansucrase-catalyzed trans-α-glucosylation. Carbohydr. Res. 2017, 440, 51−62. (9) Ko, J. A.; Nam, S. H.; Park, J. Y.; Wee, Y.; Kim, D.; Lee, W. S.; Ryu, Y. B.; Kim, Y. M. Synthesis and characterization of glucosyl stevioside using Leuconostoc dextransucrase. Food Chem. 2016, 211, 577−582.

Figure 6. Proposed structure of the compound I, i.e., RQ3.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.9b02545. Molecular structures of rebaudioside A, rebaudioside D, and rebaudioside I; HPLC spectrum of rebaudioside I, glucosyl rebaudioside A, rebaudioside D, and compound I; LC-MS spectrum of compound I after 0.5 M HCl hydrolysis and 0.5 M NaOH hydrolysis; TIC and MS spectrum of the PMAA for compound I derived from the methylation analysis; NMR chemical shifts of compound I (PDF) 8027


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RQ3, A natural rebaudioside D isomer, was obtained from

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