Identification, Chemical Synthesis, and Sweetness Evaluation of Rhamnose or Xylose Containing Steviol Glycosides of Stevia (Stevia rebaudiana) Leaves

Abstract

Steviol glycosides obtained from Stevia rebaudiana leaves are increasingly used in the food industry as natural low-calorie sweeteners. Among them, the sweetness of major glycosides composed of glucose residues (e.g., stevioside and rebaudioside A) has been widely studied. However, the properties of minor natural products containing rhamnose or xylose residues are poorly investigated. In this study, five unreported steviol glycosides containing rhamnose or xylose were extracted from our developing stevia leaves, and their sweetness was evaluated. The highly glycosylated steviol glycosides were identified, and their structures were examined by fragmentation analysis using mass spectrometry. Chemical synthesis of these glycosides confirmed their structures and allowed sensory evaluation of minor steviol glycosides. Our study revealed that a xylose-containing glycoside, rebaudioside FX1, exhibits a well-balanced sweetness, and thus, it is a promising candidate for natural sweeteners used in the food industry.

1. Introduction

Stevia rebaudiana (Bertoni) Bertoni leaves contain a secondary metabolite called steviol glycoside, in which steviol, a type of diterpenoid, is modified with diverse sugar chains (Figure 1). Some steviol glycosides exhibit sweetness that is nearly 300 times higher than that of sucrose and they are therefore utilized as a low-calorie sweetener in the food industry.¹ Currently, artificial sweeteners such as aspartame and acesulfame potassium are widely utilized. However, natural low-calorie sweeteners like steviol glycosides are more promising for gaining public acceptance.

Figure 1.

Structure of a steviol glycoside.

The major steviol glycoside in stevia leaves used in the food industry is rebaudioside A, containing four glucose (Glc) residues. Stevioside, a tri-glucosylated steviol, is a precursor of rebaudioside A and is the most abundant glycoside in the leaves of the plant. These two glycosides are the main substances responsible for the sweetness of stevia. In addition, some steviol derivatives containing different types of constituent sugars are reported. For example, rebaudioside C, also known as dulcoside B, has a glycan containing a rhamnose (Rha) at the C-13 position of steviol, in which one Glc of rebaudioside A is replaced by a Rha. Rebaudioside F, in which one Glc is replaced by a xylose (Xyl), has been detected in stevia leaves.^2,3 As minor components, steviol glycosides with a highly glycosylated C-19 position of steviol have been also identified. Ohta et al. identified 21 steviol glycosides, including 10 novel compounds, from hybridization breeding of S. rebaudiana Bertoni (Morita Kagaku Kogyo Co., Ltd.) using electrospray ionization mass spectrometry (ESI-MS) and ¹H- and ¹³C-nuclear magnetic resonance (NMR).⁴ Furthermore, many groups have reported steviol glycosides extracted from stevia leaves and stevia tablets.⁵⁻¹³

The relationship between the structure and the sweetness of steviol glycosides has been actively studied. It has been reported that the glycan sequence in both the C-19 (R¹) and C-13 (R²) positions of the steviol core affects sweetness.¹ Stevioside is known to provide sweetness that is about 250 times higher than that of sucrose. Rebaudioside A is about 300 times sweeter than sucrose and has a better taste quality than stevioside. Extension of the C-19-ester-linked glycosyl unit has been reported to increase sweetness.¹⁴ Rebaudioside D exhibits an increased sweetness and weaker bitter taste compared to other steviol glycosides. Rebaudioside M, a minor steviol glycoside, has been reported to exhibit high sweetness intensity with a very slight licorice or bitter aftertaste.¹⁵ Furthermore, differences in the constituent sugar also affected the taste quality. Replacement of the Glcβ (1–2) unit in rebaudioside A by a Rhaα (1–2) unit, yielding rebaudioside C, led to a large decrease in sweetness.¹⁶ Furthermore, replacement of the Glcβ (1–2) unit in rebaudioside A by a Xylβ (1–2) unit, yielding rebaudioside F, did not influence sweetness.¹⁷

Some stevia varieties resulting from breeding may contain trace amounts of steviol glycosides whose structures are not yet identified. The presence of such steviol glycosides in trace amounts may contribute to the flavor characteristics of the stevia extracts. Although highly glycosylated steviol is the subject of many reports, very few studies report on highly glycosylated steviol containing Rha or Xyl, and no information on their flavor characteristics has been obtained.

Based on the above, we focused on the exploration and sweetness evaluation of minor steviol glycosides using liquid chromatography (LC)-MS/MS. In this study, we prove the presence of unreported components containing Rha or Xyl in our developing stevia leaves. We determine the structures of five newly identified steviol glycosides, named “dulcoside” series for Rha-containing ones and “rebaudioside FX” series for Xyl-containing ones. Furthermore, these compounds are chemically synthesized to verify their individual taste quality.

2. Materials and Methods

2.1. Plant Material and Screening of Stevia Leaves

The plant material was prepared from two novel stevia plant lines, Cultivar A (EM3–4) and Cultivar B (EM2-27-15) developed by Suntory Global Innovation Center Limited (SIC), which contain relatively high rebaudioside C and D.¹⁸

The samples were prepared as follows: 10.0 mg of lyophilized dried stevia leaves was weighed into a glass vial and 1.0 mL of water/methanol (1/1 vol/vol) was added as an extracting solvent. The mixture was subjected to ultrasonic irradiation in an ultrasonic cleaner (AS ONE, AS52GTU, 35 kHz) at 25 °C for 20 min. The resulting extract was 10-fold diluted with water/methanol and filtrated through a filter with a pore size of 0.45 μm (Cosmonice filter S for organic solvents, Nacalai Tesque, Inc., Kyoto, Japan). Extracts were subjected to LC–MS and were screened based on the estimated molecular weights of steviol glycosides.

2.2. Chemicals

Acetonitrile and methanol (HPLC grade) were purchased from Nacalai Tesque, Inc. The suppliers of steviol glycosides were as follows: Nagara science co., ltd. (Gifu, Japan) for rebaudioside A and rebaudioside C, Wako pure chemical industries ltd. (Osaka, Japan) for rebaudioside F and rebaudioside D, ChromaDex, Inc. (Irvine, CA, USA) for rebaudioside N, and Carbosynth ltd. (Berkshire, UK) for rebaudioside M.

2.3. Liquid Chromatography–Mass Spectroscopy Measurements

A prominence LC-20 AD system (Shimadzu Corporation, Kyoto, Japan) was used together with an SM-C18 (4.6 × 250 mm) column (Imtakt Corporation, Kyoto, Japan). The mobile phase consisted of 0.2% acetic acid containing Milli-Q water as solvent A and methanol as solvent B. The binary gradient was 0–5 min, 10% B; 5–20 min, 10–70% B; 20–25 min, 70–100% B; 25–30 min, 100% B. The flow rate of the mobile phase was 0.4 mL/min. The stevia leaf extract for LC–MS analysis was diluted 10 times, and 5 μL was injected after filtration. An Orbitrap Elite mass spectrometer (Thermo Fisher Scientific Inc., San Jose, CA, USA) equipped with an ESI ion source was used for the detection. The MS measurement was conducted in a negative ion mode at an m/z range of 150–2000 with the resolution set at 60,000. The MS/MS collision-induced dissociation (CID) measurements were carried out by selecting the targeted m/z of the estimated molecular formula comprising various sugar compositions. Regarding fragmentation, the energy required was produced via irradiation at a standard collision energy of 35.

2.4. Evaluation of the Sweetness and Quality of Steviol Glycosides

The sweetness levels of the authentic steviol glycosides (rebaudioside A, D, and M) and the identified steviol glycosides (dulcoside C, D, F, rebaudioside FX1, and FX2) were evaluated by comparison with the sweetness of sucrose. Each glycoside was dissolved in pure water to prepare a test beverage sample. Sucrose standard solutions were prepared from 0.5 to 5.0 g/100 mL in 0.5 g increments. The evaluation was performed by seven (rebaudioside FX1) or four (rebaudioside FX2) panelists trained in the sweetener sensory perception. Sweetness points were scored by correlating the test beverages with sucrose standards of the same level of sweetness intensity. The sweetness rate values were obtained by the normalization of the sweetness points as follows: sweetness level to sucrose = sweetness point/concentration (g/100 mL).

The taste quality was evaluated in terms of sweetness on-set, sweetness linger, bitter taste, and bitter aftertaste. Each steviol glycoside solution was adjusted to the equivalent sweetness of 5 degree Brix according to the sweetness rate obtained above. Sucrose standard was given 3 points in each category and the samples were scored in 0.5-point increments. The higher the value, the faster the sweetness on-set, the longer the sweetness linger, the higher the bitter taste, and the longer the bitter aftertaste.

The sensory evaluation was performed by exhaling (not swallowing) after tasting.

3. Results and Discussion

To explore and characterize minor steviol glycosides, we applied the following procedure: (i) detection and structural estimation of new compounds from the LC–MS/MS analysis of stevia leaf extracts, (ii) chemical synthesis of the estimated structures and matching with the ingredients in stevia leaf extracts, and (iii) sensory evaluation.

3.1. Detection and Structural Estimation of the Unreported Steviol Glycosides from LC–MS/MS Measurements of Stevia Leaf Extracts

3.1.1. Fragmentation Pattern Analysis of Authentic Steviol Glycosides

LC–MS/MS is a useful method for screening steviol glycosides. Some steviol derivatives in stevia leaves or stevia tablets can be easily identified by corelating their retention time (R_t) and m/z values with those of commercially available authentic samples. If no matching occurs, structural information of the compound can be obtained by detailed analysis of the MS/MS fragmentation pattern.^19,20 First, we measured the R_t, molecular ion mass number, and fragmentation of the authentic samples: rebaudiosides A, C, F, D, M, and N (Table 1). We revealed that the highest ion intensity in the MS/MS (the first-step fragmentation) of a steviol glycoside corresponds to the fragment whose ester-linked glycans at position 19 (C-19) are wholly eliminated. Therefore, even for the unidentified compounds, the difference of the m/z values between the observed fragment ion and the molecular ion would provide the formula of the glycan attached to C-19. Then, the MS³ (the second-step fragmentation) pattern from the highest product ion in the MS/MS as a precursor ion, in which the ester-linked sugars were eliminated, gave the information on the glycan at C-13. In contrast to the glycan at C-19, the sugars in the glycan at C-13 were sequentially eliminated. Regarding rebaudioside C that has Rhaα (1–2)[Glcβ (1–3)]Glc at C-13, the priority for cleavage is Glc (loss of 162 u), Rha (loss of 146 u), and finally the sugar moiety directly linked at C-13. On the contrary, rebaudioside F with Xylβ (1–2)[Glcβ (1–3)]Glc at C-13 gave almost similar intensities for the cleavage ions for Glc or Xyl.

Table 1. LC–MS/MS Data of the Commercially Available Authentic Steviol Glycosides.

^aThe precursor ion for MS³ is underlined.

^bThe number in parentheses is the relative intensity of the peak.

3.1.2. LC–MS/MS Analysis of Steviol Glycosides in Stevia Leaves

LC–MS analysis of the stevia leaf extract gave 18 peaks corresponding to steviol glycosides (Figure S1). Among them, rebaudioside A (m/z 965.4, R_t 29.9 min), rebaudioside C (m/z 949.4, R_t 30.2 min), rebaudioside F (m/z 935.4, R_t 30.1 min), rebaudioside D (m/z 1127.4, R_t 28.4 min), rebaudioside I (m/z 1127.4, R_t 30.1 min), rebaudioside N (m/z 1273.5, R_t 28.6 min), and rebaudioside M (m/z 1289.5, R_t 29.0 min) were identified by corelation with the authentic samples. Other peaks were subjected to MS/MS and MS³ data analyses based on the reports of Ohta et al. or Perera et al. (Table 2).^3,19 Four peaks were identified as Rha-containing, of which three peaks observed at m/z 1111.4 were estimated to have 3 Glc and 1 Rha. Considering the major product ion in each MS/MS spectrum, the peaks were assigned as rebaudioside K possessing 2 Glc residues at C-19 (R_t 28.2 min, the MS/MS major product ion [M – 324 – H]⁻), rebaudioside J possessing 1 Glc and 1 Rha at C-19 (R_t 28.8 min, the MS/MS major product ion [M – 308 – H]⁻), and rebaudioside H possessing 1 Glc (R_t 29.5 min, the MS/MS major product ion [M – 162 – H]⁻). The peak observed at m/z 1435.6 was estimated as rebaudioside O possessing 3 Glc and 1 Rha at C-19 (R_t 28.4 min, the MS/MS major product ion [M – 632 – H]⁻). Similarly, rebaudioside T (m/z 1259.5, R_t 29.6 min, the MS/MS major product ion [M – 456 – H]⁻) and Unknown 3 reported by Perera et al. (m/z 1097.4, R_t 28.8 min, the MS/MS major product ion [M – 324 – H]⁻) were categorized as the Xyl-containing family.¹⁹ Finally, we discovered five peaks corresponding to new steviol glycosides (Table 3) and determined their structures, as shown in Figure 2. The details of the structural determination are described below. We named them dulcoside C (m/z 1273.5, R_t 28.1 min), dulcoside D (m/z 1257.5, R_t 28.8 min), dulcoside F (m/z 1095.4, R_t 29.0 min), rebaudioside FX1 (m/z 1097.4, R_t 29.2 min), and rebaudioside FX2 (m/z 1259.5, R_t 29.4 min). The Rha-containing steviol glycosides were named after dulcoside B, while the Xyl-containing ones were named after rebaudioside F.

Table 2. LC–MS/MS Data of Steviol Glycosides Identified Based on Previous reports^3,19.

^aThe precursor ion for MS³ is underlined.

^bThe number in parentheses is the relative intensity of the peak.

Table 3. LC–MS/MS Data of the Newly Identified Steviol Glycosides.

^aThe precursor ion for MS³ is underlined.

^bThe number in parentheses is the relative intensity of the peak.

Figure 2.

Structures of newly identified steviol glycosides.

3.1.3. Structural Estimation of the Steviol Family Containing Rha (Dulcoside C, D, and F)

Figure 3a shows the mass chromatogram (m/z range 1273.40–1273.60) corresponding to the deprotonation negative ion [M – H]⁻ of the chemical formula C₅₆H₉₀O₃₂, containing 5 Glc and 1 Rha. Two peaks were detected at 28.2 and 28.6 min. In the MS/MS spectrum, the peak at 28.6 min gave a major product ion peak at m/z 803.4, corresponding to the elimination of 2 Glc and 1 Rha (Figure 3c), which was assigned to rebaudioside N. In contrast, the peak at 28.2 gave a major peak at m/z 787.4 corresponding to the elimination of 3 Glc moieties (Figure 3b). The eliminated unit of this compound was the same as that of rebaudioside M. It could be assumed that this compound follows similar biosynthetic pathways with rebaudioside M since they were both detected in the same leaf. The MS³ spectrum was obtained from the major peak at m/z 787.4 in the MS/MS spectrum as the precursor ion. Product ions were detected at m/z 641.3, 625.3, and 479.3 with relative intensities of 30, 100, and 15%, respectively. This spectrum is consistent with the MS³ fragmentation pattern of rebaudioside C (949.4 → 787.4 →) and rebaudioside K, which have the same C-13 oligosaccharide arrangement [Rhaα (1–2)[Glcβ (1–3)]Glcβ1-]. According to the MS/MS and MS³ fragmentation analyses, the structure of the new compound was estimated to be Rhaα (1–2)[Glcβ (1–3)]Glcβ1- at C-13 and Glcβ (1–2)[Glcβ (1–3)]Glcβ1- at C-19. It was therefore named dulcoside C.

Figure 3.

LC–MS/MS spectra of steviol glycosides containing 5 Glc and 1 Rha. (a) Mass chromatogram (m/z range 1273.40–1273.60) corresponding to the deprotonation negative ion [M – H]⁻ of the chemical formula C₅₆H₉₀O₃₂, (b) MS/MS and MS³ spectra of the peak observed at 28.2 min. (c) MS/MS and MS³ spectra of the peak observed at 28.6 min.

In addition, the LC peaks affording the fragment peak of m/z 787.4 in the MS/MS spectrum were investigated since they would indicate the same oligosaccharide arrangement as dulcoside C at C-13. Two new compounds were observed, named dulcoside D (m/z 1257.5, R_t 28.8 min) and dulcoside F (m/z 1095.4, R_t 29.0 min). Considering the potential of rhamnose transferase, Rha might be contained in both C-13 and C-19 instead of Glc. In the MS/MS spectra, the eliminated fragment of dulcoside D corresponded to 2 Glc and 1 Rha while that of dulcoside F corresponded to 1 Glc and 1 Rha (Table 3). Furthermore, the MS³ spectra of both dulcoside D and dulcoside F were consistent with that of dulcoside C. Their structures are presented in Figure 2 and dulcoside F is a biosynthetic precursor of dulcoside D.

3.1.4. Structural Estimation of the Unreported Steviol Family Containing Xyl (Rebaudioside FX1 and FX2)

Figure 4a shows the mass chromatogram (m/z range 1097.40–1097.60) corresponding to the deprotonated negative ion [M – H]⁻ from the chemical formula C₄₉H₇₈O₂₇, which contains 4 Glc and 1 Xyl. Three peaks were detected at 28.8, 29.2, and 29.6 min. The peak observed at 28.8 min (Figure 4b) was estimated to be unknown 3 reported by Perera et al. (Table 2).¹⁹ The peak observed at 29.6 min was ascribed to an in-source fragment ion of rebaudioside T. Figure 4c shows the MS/MS and MS³ spectra of the peak observed at 29.2 min. In the MS/MS spectrum, the main peak at m/z 803.4, corresponding to the elimination of 1 Glc and 1 Xyl (−294 Da), was detected. Since this compound contains Xyl, it was named rebaudioside FX1. In the MS³ spectrum from the precursor ion at m/z 803.4, the product ions were detected at m/z 641.3, 623.3, 479.3, and 413.2 with relative intensities of 100, 10, 10, and 10%, respectively. This MS³ fragmentation pattern is consistent with that of rebaudioside A. Thus, the structure was estimated as Glcβ (1–2)[Glcβ (1–3)]Glcβ1- at position C-13 and Xylβ(1–2)Glcβ1- at C-19. Similarly, rebaudioside FX2 (m/z 1259.5, R_t 29.4 min) was estimated to have Xylβ (1–2)[Glcβ (1–3)]Glcβ1- at C-13 and Glcβ (1–2)[Glcβ (1–3)]Glcβ1- at C-19, by LC–MS/MS analysis (Table 3).

Figure 4.

LC–MS/MS spectra of steviol glycosides containing 4 Glc and 1 Xyl. (a) Mass chromatogram (m/z range 1097.40–1097.60) corresponding to the deprotonation negative ion [M – H]⁻ of the chemical formula C₄₉H₇₈O₂₇, (b) MS/MS and MS³ spectra of the peak observed at R_t 28.8 min, and (c) MS/MS and MS³ spectra of the peak observed at R_t 29.2 min.

3.2. Chemical Syntheses of Newly Identified Steviol Glycosides

The sugar compositions of the new glycosides were estimated; however, the position and the stereochemistry of the attached sugars could not be determined by MS analysis. In addition, the possibility of the presence of 6-deoxy-Glc instead of Rha²¹ or aglycon isomer²² could not be excluded since they have been detected in stevia extract. Therefore, the putative steviol glycosides (Figure 2) were synthesized to confirm their structures and conduct sensory evaluations.

3.2.1. Design of Synthesis for Steviol Glycosides

Several groups have studied the chemical synthesis of steviol glycosides,²³⁻²⁸ reporting that the exo-methylene group in steviol causes rearrangement of the double bond and addition of water under acidic conditions. Glycosylation of the tertiary hydroxyl group at C-13 of steviol requires careful handling because of side reactions. Schmidt, Zhang, and Sun group successfully overcame this problem by subjecting Schmidt’s donors and Yu’s donors at low temperatures for glycosylation at C-13 and a phase-transfer catalyst for esterification at C-19, to synthesize several glycosides.^23,27,28 However, the glycosylation reaction at C-13 is still considered a step that requires skilled synthetic techniques and careful attention. To obtain the stevioside moieties more easily, some commercially available analogs highly glycosylated at C-13 were purchased. We considered that steviol containing a glycan at C-13 can derive from natural products and the unfavorable substituent at C-19 can be removed by alkali-hydrolysis. Then, the chemically synthesized glycans were attached at the 19-carboxyl group.

Various glycans are connected to the carboxy group at C-19 of steviol via a β linkage. β selective glycosylation is required without participation of the neighboring group since the desired glycan has an additional sugar at C-2 and not an acyl substituent. The presence of a double bond in steviol limits the hydrogenation conditions for the final deprotection. Meanwhile, although the glycan at C-19 is connected via an ester bond, it was reported that the hindered ester is resistant to the removal of acyl-type protective groups.²⁶ Taking the above points into consideration, the retrosynthetic route was designed, as shown in Scheme 1 (using dulcoside C as an example).

Scheme 1. Retrosynthetic Strategy for Dulcoside C.

3.2.2. Synthesis of Steviol Acceptors

The synthesis of steviol acceptors possessing a glycan at C-13 and a free carboxylic acid at C-19 was conducted according to Scheme 2. The steviol acceptor containing a Rha (2) was obtained from the commercially available rebaudioside C through alkali hydrolysis and acetylation of the hydroxyl groups of the glycan of 1 in high yield. Similarly, the steviol acceptor consisting of a Glc (4) was synthesized from rebaudioside A via compound 3, and the one containing a Xyl (6) was obtained from rebaudioside F via compound 5 in an efficient manner.

Scheme 2. Synthesis of Steviol Acceptors.

NaOH, MeOH, reflux.

Ac₂O, Pyr., RT, 92% (2 steps).

NaOH, MeOH, reflux.

Ac₂O, Pyr., RT, 80% (2 steps).

NaOH, MeOH, reflux.

Ac₂O, Pyr., RT, 90% (2 steps).

3.2.3. Synthesis of the Glycan Donor

The glycan donor was synthesized according to Scheme 3 to bond to the carboxy group at position 19 of steviol. In the case of a trisaccharide donor consisting of only Glc, the methoxyphenyl (MP) glucoside (7) was protected with a benzylidene acetal to afford compound 8, and glycosylation with the Glc donor (9) at both C-2 and C-3 produced the trisaccharide 10. Then, the benzylidene group was converted into Ac groups to afford the compound 11. The trisaccharide hemiacetal (12) was produced by deprotecting the MP group of the compound 11.

Scheme 3. Synthesis of the Glycan Donor Consisting of Only Glc.

PhCH(OMe)₂, 10-camphorsulfonic acid, MeCN, RT, 77%.

9 (2.2 equiv), TMSOTf, CH₂Cl₂, molecular sieves 4A, RT.

p-toluenesulfonic acid, EtOH/H₂O, RT.

Ac₂O, Pyr. RT, 54% (3 steps).

cerium ammonium nitrate, MeCN/H₂O = 4/1, 0 °C, 29%.

During synthesis of the glycan donor containing a Rha, coupling of the Glc derivative (13) and the Rha donor (14) afforded disaccharide (15), and deprotection of the benzylidene group afforded compound 16 (Scheme 4). Acetylation of 16 followed by the deprotection of the MP group produced the disaccharide hemiacetal (17). In addition, the trisaccharide 19 was obtained by benzylidenation and glycosylation of 18 with the Glc donor (9). Compound 21 was produced by converting the benzylidene group into the Ac group and the cleavage of the MP group.

Scheme 4. Synthesis of the Glycan Donors Containing Rhamnose.

14 (1.1 equiv), TfOH, CH₂Cl₂, molecular sieves 4A, RT, 81%.

H₂, Pd(OH)₂, EtOH/THF, RT.

Ac₂O, Pyr. RT.

cerium ammonium nitrate, MeCN/H₂O = 2/1, RT, 66% (3 steps).

PhCH(OMe)₂, 10-camphorsulfonic acid, MeCN, RT.

9 (1.1 equiv), TfOH, CH₂Cl₂, molecular sieves 4A, 0 °C 22% (3 steps).

H₂, Pd(OH)₂, EtOH/THF, RT.

Ac₂O, Pyr. RT.

cerium ammonium nitrate, MeCN/H₂O = 2/1, RT, 84% (3 steps).

In the synthesis of the glycan donor containing a Xyl, the reaction of the Glc derivative (22) and the Xyl donor (23) afforded disaccharide (24) and deprotection of the allyl group produced compound 25 in a good yield (Scheme 5).

Scheme 5. Synthesis of the Glycan Donor Containing Xylose.

23 (1.1 equiv), BF₃·Et₂O, CH₂Cl₂, molecular sieves 4A, −20 °C 88%.

PdCl₂, AcOH/H₂O, RT, 75%.

3.2.4. Condensation of the Glycan Donor and Steviol Acceptor

The condensation reaction between the glycan donor and the steviol acceptor was examined using model sugars. Glycosylation through an oxonium cation intermediate was unfavorable because of the acid-sensitivity of the acceptor and poor β-selectivity. For example, the coupling of an imidate donor and a steviol acceptor in the presence of TMSOTf and MS4A at −20 °C mainly afforded α glycosides even in acetonitrile. Thus, S_N2-type reactions were examined. Among them, a Mitsunobu reaction between a hemiacetal donor and a steviol acceptor in the presence of highly reactive N,N,N′,N′-tetramethylazodicarboxamide (TMAD)²⁹ and PBu₃ in 1,4-dioxane at 60 °C provided the desired β-steviol glycoside in good yield.

The Mitsunobu reaction was also applied to the target glycan donors and the steviol acceptors (Scheme 6). In entry 1, the hemiacetal donor 12 and the steviol acceptor 2 were glycosylated to give the desired steviol glycoside 26 in a 47% yield with complete β selectivity. The α glycoside was produced in very low quantities when the glycan donors (21 or 17) containing a Rha at 2-position (R⁴) of Glc of the reducing end were used (Entries 2 and 3). On the contrary, when glycan donors or the steviol acceptors containing a Xyl were used, the desired glycoside was obtained in good yields exhibiting complete β selectivity (Entries 4 and 5).

Scheme 6. Coupling Reaction of the Glycan Donors and Steviol Acceptors.

α/β ratio was estimated by NMR analysis in CDCl₃.

The yield and stereoselectivity would depend on the anomeric ratio of the donor 1-OH group and the rate of interconversion during the reaction process. High β-selectivity may be attributed to the bulky steviol acceptors attacking the activated α-phosphonium intermediate from the β-face, exhibiting less steric hindrance (Figure 5). Contrarily, attacking the β-phosphonium intermediate from the α-face would be more difficult due to the 1,3-diaxial repulsion and the steric hindrance of 2-OR⁴. As an exception, when R⁴ was α-L-Rha, the β-selectivity slightly decreased. This might be because α-L-Rha, which is connected via an axial bond, changes the intermediate conformation. When the stable conformations of disaccharides were calculated in which Rha, Xyl, or Glc is attached to the 2O-position (R⁴) of Glc, only the Glc residue in Rhaα1-2Glc (β) could take the ¹C₄ conformation (Figure S3). Although the calculation was performed for simple disaccharide models without protective groups in the gas phase, the result suggested that the Rha in 17 or 21 may allow the Glc residue to take the ¹C₄ conformation and the steviol acceptor to attack from the equatorial side with less steric hindrance, resulting in the α linkage.

Figure 5.

Possible reaction mechanisms.

The protected steviol glycosides were treated with NaOMe in MeOH, leading to the desired steviol glycosides in high yields (Scheme 7).

Scheme 7. Deprotection of Protected Steviol Glycosides.

All synthetic samples were matched with newly discovered steviol glycosides from stevia leaves, according to their LC retention times, LC–MS/MS fragmentation patterns, and MS³ fragmentations (Table 3 and Figure S2). Thus, the structures of these steviol glycosides were unambiguously confirmed, as shown in Figure 2.

3.3. Sensory Evaluation of Newly Identified Steviol Glycosides

3.3.1. Evaluation of the Sweetness Level of Steviol Glycosides

The sweetness of authentic steviol glycosides (rebaudioside A, D, and M) and five newly identified ones (dulcoside C, D, F, and rebaudioside FX1, FX2) was evaluated by comparison with sucrose standard solutions. Since the Rha-containing dulcoside series (dulcosides C, D, and F) exhibited significantly lower sweetness compared to rebaudioside A, they were excluded from evaluation. Rebaudioside FX1 and FX2 showed potent sweetness. First, the sweetness of rebaudioside FX1 was therefore compared with that of the authentic samples. Each solution (0.012 g/100 mL) was tasted by seven trained panelists. Rebaudioside FX1 scored the highest sweetness level, with an average of 4.25 points (the equal level to 4.25 g/100 mL of sucrose solution, Table 4). The sweetness points of rebaudioside A, D, and M were 3.79, 3.87, and 4.06, respectively. Comparing the obtained sweetness levels to sucrose, rebaudioside FX1 was 354 times higher, and rebaudioside A, D, and M were 312, 318, and 341 times higher, respectively (Table 4). Similarly, the sweetness level of rebaudioside FX2 was evaluated by four panelists and was scored slightly lower than that of rebaudioside A (Table S1).

Table 4. Sweetness Level and the Rating Point of Rebaudioside FX1.

		sucrose	rebaudioside A	rebaudioside D	rebaudioside M	rebaudioside FX1
sweetness level to sucrose		1	312	318	341	354
sweetness point (n = 7)	maximum		4.85	4.85	4.19	5.00
	average		3.79	3.87	4.06	4.26
	minimum		2.95	3.09	3.40	3.84

3.3.2. Evaluation of the Sweetness Quality of Rebaudioside FX1

Since rebaudioside FX1 exhibited the highest sweetness, it was further compared with sucrose, rebaudioside A, D, and M in terms of sweetness on-set, sweetness linger, bitter taste, and bitter aftertaste by seven panelists. Sucrose (5 degree Brix) was given 3 points as a standard, and each steviol glycoside was scored in 0.5-point increments. In each criterion, the higher the value, the faster the sweetness on-set, the longer the sweetness linger, the higher the bitter taste, and the longer the bitterness aftertaste. The results are presented in Table 5. All tested steviol glycosides exhibited a slightly slower sweetness on-set compared to sucrose. Generally, the lingering sweetness and bitterness affect the taste in a negative way. The stevia extract exhibits slight bitterness and longer aftertaste, features that are problematic for replacing sugar. Rebaudioside FX1 showed shorter sweetness linger than sucrose, and its bitter taste and aftertaste were improved compared with those of rebaudioside A, which is abundantly used in the food industry. Among the previously reported steviol glycosides, rebaudioside D has been evaluated as a well-balanced natural sweetener; however, it exhibits problems in solubility. The results of the sensory evaluation of rebaudioside FX1 were comparable to those of rebaudioside D, whereas rebaudioside FX1 exhibited good solubility at the tested concentrations.

Table 5. Sensory Evaluation for Rating Attributes.

	sucrose	rebaudisoide A	rebaudioside D	rebaudioside M	rebaudioside FX1
sweetness on-set	3	2.50 ± 0.93	2.38 ± 0.88	2.50 ± 0.76	2.38 ± 0.51
sweetness liger	3	3.31 ± 0.92	2.75 ± 0.60	2.81 ± 0.84	2.75 ± 0.80
bitter taste	3	3.44 ± 0.62	3.06 ± 0.50	3.13 ± 0.44	3.06 ± 0.32
bitter aftertaste	3	3.25 ± 0.65	3.19 ± 0.37	3.31 ± 0.46	3.13 ± 0.23

In summary, we discovered five new steviol glycosides containing Rha or Xyl. Highly glycosylated derivatives such as rebaudioside M have attracted attention owing to their improved sweetness and solubility. Although steviol glycosides containing sugars other than Glc have received less attention, Purkayastha et al. reported the detection of rebaudioside FX1 from stevia extract³⁰ shortly after we filed our patent application.³¹ Our sweetness evaluation demonstrated that rebaudioside FX1 is a promising candidate for next-generation sweeteners. Our analytical strategy is a useful tool for searching minor steviol glycosides containing sugars other than Glc in stevia leaf extracts or tablets.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jafc.3c01753.

• Sweetness level and rating point of steviol glycosides; LC–MS chromatogram of the leaf extract; comparison of the MS chromatogram and MS spectra of the newly discovered steviol glycosides with those of synthetic samples; conformational analyses of model disaccharides; and synthetic method, compound data for new compounds (PDF)

Author Contributions

^⊥ T.W. and K.F. contributed equally to this work.

The authors declare no competing financial interest.