Structural Elucidation of Quinovose-Containing Steviol Glycosides from Enzymatic Biotransformation of Stevia rebaudiana

Abstract

Two steviol glycosides containing quinovose were isolated from a biotransformation mixture of stevia extract derived from Stevia rebaudiana Bertoni leaves. These compounds were elucidated using comprehensive spectroscopic techniques, including nuclear magnetic resonance (NMR) and mass spectrometry (MS). These compounds were designated as Rebaudioside QM and the novel Rebaudioside 2QM. Based on structural similarity, we hypothesize that Stevioside E may serve as a biosynthetic precursor for Rebaudioside QM. Comprehensive LC-MS profiling also suggests potential precursors for Rebaudioside 2QM. Tentative biosynthetic pathways were proposed for both compounds. The presence of these unknown compounds further supports the notion that S. rebaudiana harbours a wide array of yet-undiscovered steviol glycosides, potentially driven by the inherent diversity of UDP-dependent glycosyltransferases (UGTs) within the plant itself. The discovery of Rebaudioside QM and Rebaudioside 2QM expands the known diversity of steviol glycosides and provides new insights into glycosylation patterns in S. rebaudiana, which may support the development and production of novel sweeteners with improved sensory and physicochemical properties.

1. Introduction

The global pursuit of healthier sugar alternatives has intensified in recent years, driven by increasing awareness of the adverse health effects associated with excessive sugar consumption. This trend is further reinforced by regulatory interventions such as sugar taxes, which aim to curb the intake of high-calorie sweeteners [1,2]. As metabolic disorders including obesity, type II diabetes, and cardiovascular diseases continue to rise, the demand for non-caloric, non-cariogenic, and nutritionally beneficial sugar substitutes is growing rapidly [3,4].

Among the most promising natural alternatives are steviol glycosides, a group of intensely sweet diterpenoid compounds extracted from the leaves of Stevia rebaudiana Bertoni. This perennial shrub, belonging to the Asteraceae (Compositae) family, is economically valued for its high concentration of natural sweeteners [5]. The sweetness of stevia leaves is attributed to steviol glycosides, which are tetracyclic diterpenoids differentiated primarily by the number and arrangement of sugar residues, predominantly glucose attached at the C13 and C19 positions of the steviol aglycone.

The biosynthesis of steviol glycosides from steviol aglycone is not fully understood, largely due to limited knowledge of the specific UDP-dependent glycosyltransferases (UGTs) responsible for their formation, which hinders a comprehensive understanding of the biosynthetic pathway [6,7]. Nevertheless, several UGTs have been characterized, including UGT76G1, which catalyzes β(1→3)-glycosylation at the C13 and C19 positions of the sugar moieties attached to steviol, as well as UGT91D2, which catalyzes β(1→2) glycosylation of the sugar moieties [8,9,10,11,12,13].

Commonly identified steviol glycosides include stevioside, Rebaudioside A–O, dulcoside A, steviolbioside, and rubusoside [14,15,16,17]. Interestingly, some steviol glycosides contain sugar residues other than glucose, such as rhamnose and xylose, found in dulcoside A, Rebaudioside C, and Rebaudioside F, respectively. A particularly rare subgroup of steviol glycosides incorporates quinovose moieties, also known as C6-deoxyglucose, with only five such compounds reported to date (Figure 1) [18,19,20,21,22]. Expanding the list of rare sugar-containing steviol glycosides, including those with quinovose, is important, as the presence of non-glucose sugars at different positions may influence sweetness intensity and taste profile [23,24].

One of these quinovose-containing compounds, previously referred to as compound 2c, was obtained through acid and heat treatment of Rebaudioside M and was also detected in stevia extract [20,21]. In the present study, we successfully isolated this compound, which we have named Rebaudioside QM, along with a novel steviol glycoside, Rebaudioside 2QM. Both compounds are structural isomers formed during enzymatic biotransformation of stevia extract and share a molecular weight of 1275.3 g/mol, consistent with a steviol glycoside comprising five glucose units and one quinovose unit. Here, we report the isolation and structural elucidation of both compounds and propose tentative biosynthetic pathways for their formation.

2. Results

This study focuses on the discovery of novel steviol glycosides synthesized from the biotransformation of stevia extract (see Supplementary Figure S1 for the HPLC chromatogram of the initial stevia extract). Two UDP-glucosyltransferases, UGT76G1 and UGTSl2, were employed to catalyze β(1→3) glycosylation and β(1→2) glycosylation of steviol glycosides present in the extract, respectively [8,9,10,11,12,13]. The HPLC chromatogram of stevia extract after biotransformation is shown in Figure 2a. Figure 2b shows the LC-MS chromatogram of the same sample, acquired in selective ion monitoring (SIM) mode at m/z 1273.5, revealing two previously unknown peaks. These peaks were isolated and subjected to structural elucidation. Both compounds were identified as steviol glycosides containing quinovose moieties, which we have named Rebaudioside QM (1) and the newly discovered Rebaudioside 2QM (2).

2.1. Rebaudioside QM (1)

LC-MS analysis of the stevia extract indicated that Rebaudioside QM (1) is present at approximately 1.17% in the final biotransformation mixture on a dry weight basis. Rebaudioside QM (1) was isolated using a three-step purification strategy involving preparative HPLC described in Section 4.2. The final purified compound was obtained as a white powder (91.1% chromatographic purity), with a sharp peak observed in the HPLC chromatogram at 7.25 mins (see Supplementary Figure S2a).

The compound was analyzed by LC-MS in negative ion mode (API-ES), revealing a deprotonated molecular ion at m/z 1273.5 [M-H]⁻ (see Supplementary Figure S3a). This corresponds to a neutral molecular weight of 1274.5 g/mol, which is consistent with a steviol glycoside bearing six hexose units (calculated molecular weight = 1275.3 g/mol). Structure elucidation was performed using a comprehensive set of NMR techniques, including ¹H, ¹³C, ¹H–¹H COSY, ¹H–¹H NOESY, One-Dimensional Nuclear Overhauser Effect Spectroscopy (1D-NOESY), One-Dimensional Total Correlation Spectroscopy (1D-TOCSY), Heteronuclear Single Quantum Coherence–Distortionless Enhancement Polarization Transfer (¹H–¹³C HSQC-DEPT), Heteronuclear Multiple Bond Correlation (¹H–¹³C HMBC) and Heteronuclear Single Quantum Coherence–Total Correlated Spectroscopy (¹H–¹³C HSQC-TOCSY).

The presence of six anomeric protons, evident from ¹H and ¹H–¹³C HSQC-DEPT spectra, confirmed the presence of six sugar units in the structure. Four anomeric protons at δ_H 5.38 (J = 8.0 Hz), δ_H 6.40 (J = 8.0 Hz), δ_H 5.79 (J = 8.0 Hz), and 5.31 ppm (J = 8.0 Hz) appeared as clear doublets with large coupling constants, consistent with β-orientation. The anomeric protons at δ_H 5.49 and 5.46 ppm were overlapped but their apparent doublet pattern (J = 8.0 Hz) remained discernible, also suggesting β-configuration, as confirmed by ¹H–¹³C HSQC-DEPT correlations. The compound was identified by comparison of its ¹H and ¹³C NMR data with those reported in the literature [20,21].

Sugar II was identified as a quinovose residue based on the presence of a characteristic C-6 methyl doublet at δ_H 1.64 ppm. Its anomeric proton exhibited a large coupling constant (J = 8.0 Hz), which is typical for β-D-quinovose. In addition, the overall ¹H and ¹³C NMR patterns agree with reported NMR features of quinovose residues in natural products [25].

The observed chemical shifts were largely consistent with published values, with minor differences in the assignment of C-3 and H-3 between sugars V and VI, likely due to signal overlap in this congested region. The assignment for sugar V was supported by the ¹H–¹H NOESY, where two cross-peaks were observed at 4.35 ppm and 3.92 ppm. The ¹H–¹H COSY correlation between H-2^V (4.22 ppm) and the signal at 4.35 ppm indicated that this resonance corresponds to H-3^V, and therefore the signal at 3.92 ppm was assigned to H-5^V. For sugar VI, only one clear NOESY cross-peak was observed at 3.85 ppm from H-1^VI, which was therefore assigned as H-5^VI and the remaining unassigned resonance was attributed to H-3^VI. Although minor ambiguity is possible due to signal congestion, the key correlations and structural features required for accurate assignment and identification of the compound remained consistent. Full assignments are provided in Supplementary Tables S1 and S2. Figure 3 illustrates the chemical structure of Rebaudioside QM (1), which was established as (13-[(2-O-6-deoxy-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy] ent-kaur-16-en-19-oic acid-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)ester]).

Rebaudioside QM (1) has previously been identified in stevia extract and can be produced through acid and heat treatment of Rebaudioside M [20,21]. To our knowledge, this is the first report of Rebaudioside QM (1) being successfully isolated from the biotransformation of stevia extract using UGT76G1 and UGTSl2 enzymes. Unlike the degradation-based approach described in previous publication, our strategy uses enzymatic glycosylation of steviol glycosides, providing a more controlled and selective route and representing a more sustainable alternative to the degradation-driven approach [20].

2.2. Rebaudioside 2QM (2)

LC-MS analysis of the stevia extract indicated that Rebaudioside 2QM (2) is present at approximately 0.46% in the final biotransformation mixture on a dry weight basis. Rebaudioside 2QM (2) was isolated from the final biotransformation reaction mixture using a three-step purification strategy involving preparative HPLC in Section 4.2. The final purified compound was obtained as a white powder (94.5% chromatographic purity), with a sharp peak observed in the HPLC chromatogram at 10.40 mins (see Supplementary Figure S2b).

The compound was analyzed by LC-MS in negative ion mode (API-ES), revealing a deprotonated molecular ion at m/z 1273.5 [M-H]⁻ (see Supplementary Figure S3b). This corresponds to a neutral molecular weight of 1274.5 g/mol, which is consistent with a steviol glycoside bearing six hexose units (calculated molecular weight = 1275.3 g/mol). Its structure was elucidated using a comprehensive set of NMR techniques, including ¹H, ¹³C, ¹H–¹H COSY, ¹H–¹H NOESY, 1D-NOESY, 1D-TOCSY, ¹H–¹³C HSQC-DEPT, ¹H–¹³C HMBC and ¹H–¹³C HSQC-TOCSY, with spectra provided in the Supplementary Material (see Supplementary Figures S4–S11). Figure 4 illustrates the chemical structure of Rebaudioside 2QM (2).

Combined analysis of mass spectrometry and NMR spectra confirmed that the compound is a glycoside bearing a central diterpenoid core. The HSQC-DEPT spectrum revealed three methyl groups, appearing as singlets at δ_H 1.33 and 1.34 ppm in the ¹H NMR spectrum, and a doublet at δ_H 1.65 ppm (J = 6.2 Hz). Further analysis indicated that the methyl doublet at δ_H 1.65 ppm belongs to a sugar moiety rather than the diterpenoid core. Additionally, a signal at δ_C 104.9 ppm demonstrated the presence of an exo-methylene carbon. This assignment was further supported by correlations of two olefinic protons at δ_H 4.94 and 5.70 ppm with the δ_C 104.9 ppm signal, confirming the presence of an exocyclic double bond. Nine methylene and two methine protons were observed between δ_H 0.77–2.70 ppm, characteristic of the ent-kaurane diterpenoid skeleton previously reported in stevia extracts [21]. The presence of the ent-kaurane diterpenoid aglycone was further supported by ¹H–¹H 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) and ¹H–¹³C HMBC correlations (H-18/C-3, C-4, C-5, C-19; H-20/C-5, C-9, C-10) (see Supplementary Table S3 for complete ¹H and ¹³C assignments of the ent-kaurane diterpenoid).

Correlations observed in the ¹H–¹H NOESY spectrum were used to assign the relative stereochemistry of the diterpene core. NOE correlations between H-5 and H-9 indicated that these protons are located on the same face of the molecule while the absence of NOE correlations between H-14 and H-9 suggested H-14 lies on the opposite face relative to H-9 and H-5. The NOESY data confirmed the relative stereochemistry of the aglycone, further supporting its identity as an ent-kaurane diterpenoid consistent with steviol, the known aglycone of steviol glycosides in S. rebaudiana [21].

Following the identification of steviol as the aglycone (molecular weight = 318.46 g/mol), the compound was hypothesized to be a steviol glycoside with six hexose sugar moieties, one of which is deoxysugar. This inference was based on its observed molecular weight, as determined by LC-MS analysis. The presence of six anomeric protons, evident from ¹H and ¹H–¹³C HSQC-DEPT spectra, confirmed the presence of six sugar units in the structure. Doublets with large coupling constants were observed for two anomeric protons at δ_H 6.32 (J = 8.0 Hz) and 5.66 ppm (J = 8.0 Hz), consistent with β-orientation. The anomeric protons at δ_H 5.38, 5.49, 5.46 and 5.41 ppm were partially overlapped with the residual water signal at δ_H 5.5 ppm but their apparent doublet pattern (J = 8.0 Hz) remained discernible, also suggesting β-configuration, as confirmed by ¹H–¹³C HSQC-DEPT correlations.

The structures of the sugar were elucidated using ¹H–¹H COSY correlations, complemented by 1D-TOCSY and 1D-NOESY experiments, which enabled determination of their stereochemistry and structural features. For sugar IV, the β-orientation of the anomeric proton indicated that both H-1^IV and H-2^IV were axial. The 1D-NOESY experiment irradiating at H-3^IV (δ_H 5.03 ppm) showed the correlations to both H-1^IV and H-5^IV, indicating that both H-3^IV and H-5^IV were also axial, consistent with a β-D-glucopyranose configuration. The 1D-TOCSY experiment irradiating the same proton allowed the identification of H-2^IV and H-4^IV. The correlation observed between H-3^IV and H-1^VI in the NOESY spectrum suggested a 1→3 sugar linkage between Sugar IV and Sugar VI. (see Supplementary Figure S12). Similar analyses were performed for Sugar I. For Sugar II, III and VI, partial signal overlap introduced some ambiguity. Nevertheless, key correlations from 1D-NOESY and 1D-TOCSY enabled assignment of critical protons and confirmation of their overall configuration and their connectivity, in combination with the other NMR experiments discussed earlier. Collectively, these data confirmed that Sugar I, II, III, IV, and VI are β-D-glucose.

For Sugar V, 1D-NOESY and 1D-TOCSY experiments irradiating its anomeric proton (δ_H 5.66 ppm) revealed the chemical shift for H-2^V, H-3^V, H-4^V and H-5^V. Irradiation at H-5^V (δ_H 3.62 ppm) further showed a strong correlation to a methyl doublet at δ_H 1.65 ppm, which also exhibited a strong ¹H–¹H COSY correlation with H-5^V. The correlation at δ_H 4.38 ppm (chemical shift in H-2^IV) observed in the 1D-NOESY spectrum upon irradiation at H-1^V, which was absent in 1D-TOCSY (irradiating at H-1^V and H-3^V), suggested a 1→2 sugar linkage between Sugar V and Sugar IV. Sugar V displayed the same characteristic NMR features as observed for Sugar II in Rebaudioside QM (1), and the data are consistent with literature-reported quinovose residues in natural products, further confirming the identity of this sugar [25]. As illustrated in Figure 5, combination of all experiments confirmed Sugar V as quinovose, linked to Sugar IV via a 1→2 sugar linkage. The ¹H and ¹³C chemical shifts for the glycosides at C-13 and C-19 are summarized in Supplementary Table S4. The structure of Rebaudioside 2QM (2), containing a relatively rare quinovose, was established 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-6-deoxy-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)ester]).

3. Discussion

3.1. Identification and Quantitative Analysis of Potential Precursors

To date, only five steviol glycosides containing a quinovose moiety have been identified in stevia extract. Based on their molecular structures, Stevioside E (3) are hypothesized to be biosynthetic precursors of Rebaudioside QM (1) due to their structural similarity, as both compounds possess a β(1→2) linkage with a quinovose unit at C13 position. To evaluate this hypothesis, we analyzed the initial stevia extract to identify potential precursor compounds.

Comprehensive HPLC and LC-MS analyses were conducted on the stevia extract before and after enzymatic biotransformation to investigate the biosynthesis pathway of Rebaudioside QM (1) and Rebaudioside 2QM (2). LC-MS analysis targeting m/z 949.4 was carried out on the initial stevia extract to search for Stevioside E (3) with molecular weight of 950 g/mol. This analysis revealed multiple uncharacterized peaks, three of which disappeared after biotransformation (designated as Unknown 1, 2 and 3) (Figure 6a,b). The disappearance of the three unknowns coincided with an increased in Rebaudioside QM (1) and Rebaudioside 2QM (2) in the final biotransformation mixture under the applied enzymatic conditions (Figure 6d).

Unknown 1, present at approximately 0.81% in the initial stevia extract, is proposed as a potential precursor to Rebaudioside QM (1), which increased from 0.01% to 1.17% after biotransformation. Similarly, Unknown 2 (0.34%) may serve as a precursor to Rebaudioside 2QM (2), which increased from 0.09% to 0.46%. Unknown 3 (0.2%) may be related to other minor compounds formed during the biotransformation but was not characterized in this study (Figure 6c,d).

Based on these observations, we hypothesize that Unknown 1, likely to be Stevioside E (3), is the precursor for Rebaudioside QM (1), while Unknown 2 is the precursor for Rebaudioside 2QM (2). Both Rebaudioside QM (1) and Rebaudioside 2QM (2) occur at low abundance in S. rebaudiana and are synthesized via enzymatic biotransformation, enabling their detection and structural identification.

3.2. Proposed Biosynthetic Pathway of Rebaudioside QM (1)

Stevioside E (3) is hypothesized to be Unknown 1 and a plausible precursor of Rebaudioside QM (1) based on structural similarity. The proposed biosynthetic pathway of Rebaudioside QM (1) is illustrated in Figure 7. Stevioside E (3) present in the stevia extract undergoes sequential in vitro β(1→2) and β(1→3) glycosylation at the C19 position, catalyzed by UGTSl2 and UGT76G1, forming putative intermediate products PC-1Q4G4 (4) and PC-1Q4G5 (5), respectively. These intermediates are further converted into Rebaudioside QM (1), with PC-1Q4G4 (4) being catalyzed by UGT76G1 and PC-1Q4G5 (5) by UGTSl2. To validate this hypothesis, future work will focus on the isolation and structural elucidation of Unknown 1, which may confirm its role as a biosynthetic precursor in this pathway.

3.3. Proposed Biosynthetic Pathway of Rebaudioside 2QM (2)

To date, no steviol glycosides with a quinovose linkage pattern similar to Rebaudioside 2QM (2) have been identified in S. rebaudiana. Based on the HPLC and LC-MS analyses discussed above, Unknown 2, with a molecular weight of 950 g/mol (consistent with a steviol glycoside comprising three glucose units and one quinovose unit), is proposed as the precursor for Rebaudioside 2QM (2).

Considering possible glycosyl moieties of Unknown 2 and the structural features of Rebaudioside 2QM (2), three possible arrangements of glycosyl moieties are proposed as putative intermediates, designated as PC-1Q3G1 (6), PC-1Q3G2 (7), and PC-1Q3G3 (8). Each contains one quinovose and three glucose units, with the quinovose unit positioned at the β(1→2) linkage of the C19 position of the steviol aglycone. The three proposed intermediates differ in the positioning and linkage patterns of the glucose residues, which may influence their susceptibility to enzymatic glycosylation.

It is hypothesized that these putative precursors undergo sequential enzymatic glycosylation mediated by UGTSl2 and UGT76G1, resulting in the biosynthesis of Rebaudioside 2QM (2) via addition of glucose units at the β(1→2) and β(1→3) glycosidic linkages, respectively, as illustrated in Figure 8. Further investigation is required to validate this hypothesis. Upcoming work will focus on the isolation and structural elucidation of Unknown 1 and Unknown 2 to better understand the proposed biosynthetic pathway of both Rebaudioside QM (1) and Rebaudioside 2QM (2).

3.4. Taste Profile of Steviol Glycosides Containing Non-Glucose Sugar Moieties

The sweetness of steviol glycosides is strongly influenced by their structural features, particularly the number and position of glycosyl groups attached to the steviol backbone [26,27,28,29,30,31,32,33]. In addition to glycosylation patterns, variations in the type of constituent sugars can also markedly influence taste properties. For instance, replacing glucose at the β(1→2) glycosidic linkage at the C13 position of stevioside and Rebaudioside A with rhamnose to form dulcoside A and Rebaudioside C, respectively, significantly reduces their respective sweetness level [34]. In contrast, substitution with xylose at the same β(1→2) position in Rebaudioside A yields Rebaudioside F, which does not substantially alter the sweetness level [35]. Watanabe et al. further identified four minor steviol glycosides containing rhamnose or xylose that generally exhibited lower sweetness potency, with one notable exception. Rebaudioside FX1, an analogue to Rebaudioside D, featuring a xylose moiety at the β(1→2) glycosidic linkage at C19 position, was reported to have higher sweetness level than Rebaudioside D. Conversely, Rebaudioside FX2, an analogue to Rebaudioside M, with a xylose moiety at the β(1→2) glycosidic linkage at C13 position, showed reduced sweetness as compared to Rebaudioside M [24]. These observations suggest that the positions and types of sugar units play important roles in determining the sweetness level of steviol glycosides.

The sensory characteristics and sweetness potency of naturally occurring steviol glycosides bearing quinovose remain unexplored, leaving a notable gap in current knowledge. Kamiya et al. chemically synthesized stevioside analogues by replacing the glucose unit at β(1→2) linkage at the C19 position with a quinovose moiety. This substitution enhanced sweetness moderately compared to stevioside [36]. However, direct attachment of quinovose at the carboxyl group at the C19 position reduced sweetness and increased bitterness relative to steviolbioside [37]. These findings suggest that the presence and position of quinovose can influence the taste profile of steviol glycosides.

Based on discoveries by Watanabe et al. and Kamiya et al., replacing glucose at β(1→2) linkage at the C19 position of steviol glycosides may enhance sweetness compared to their analogues. Rebaudioside 2QM (2), an analogue of Rebaudioside M with quinovose replacing glucose at this position, may therefore exhibit improved sweetness and distinct taste profile compared to Rebaudioside M. Evaluating Rebaudioside QM (1) and Rebaudioside 2QM (2) will not only help determine their sensory profiles but also provide insights into the structure–function relationship, particularly how different linkages and sugar types influence the sweetness and sensory properties of steviol glycosides.

4. Materials and Methods

4.1. Biotransformation

Stevia extract provided by PureCircle Sdn. Bhd. (Bandar Enstek, Malaysia) was used as starting material for one-pot biotransformation reaction using wild type glucosyltransferase UGTS12 and UGT76G1, together with wild type sucrose synthase SuSy_At. The enzyme preparations were performed according to the protocol described previously [8]. UGT76G1 from S. rebaudiana (AAR06912.1), UGTSl2 from Solanum lycopersicum (XP_004250485.1) and SuSy_AT from Arabidopsis thaliana ( NP_001031915.1) were individually expressed using expression vector pET-30a(+) in Escherichia coli BL21 (DE3). The cells were then lysed using BugBuster^® Master Mix (Novagen, Damstadt, Germany) to obtain lysate containing the expressed enzymes, which were subsequently used in biotransformation.

The biotransformation was carried out under the following conditions: the initial reaction solution (1 L) contained approximately 521 units of UGTSl2, 2012 units of SuSy_At, 15 units of UGT76G1, 1.24 mM of uridine diphosphate (UDP), 880 mM sucrose, 4 mM MgCl₂, 50 mM potassium phosphate buffer (pH 6.55) and 100 g stevia extract. The reaction mixture was incubated at 45 °C for 48 h with agitation. Additional UDP (1.24 mM) was supplemented at 12 h, 24 h and again at 36 h to maintain substrate availability throughout the reaction.

After 48 h, 1000 mL of the reaction medium was inactivated by adjusting the pH to 5.5 using phosphoric acid, followed by boiling for 10 min. The mixture was then filtered using cardboard. The filtrate was loaded onto a column containing 1 L of YWD03 resin (Cangzhou Yuanwei, China) pre-equilibrated with water. The resin was washed with 5 L of water, and the water effluent was discarded. Steviol glycosides were eluted from the YWD03 resin column using 5 L of 70% v/v ethanol/water. The ethanol-containing effluent was collected and evaporated using a rotary evaporator to remove ethanol, and the sample was further concentrated. The concentrated sample was then subjected to fractionation and separation by preparative HPLC.

4.2. Isolation and Purification

Isolation of Rebaudioside QM (1) and Rebaudiosde 2QM (2) by HPLC was divided into three chromatographic steps. The first step involved semi-preparative purification using Agilent 1200 Series Preparative LC System (Agilent Technologies, Waldbronn, Germany) equipped with two preparative pump, autosampler, Diode Array Detector and fraction collector with the following condition: Column: Agilent Zorbax SB-C18, 9.4 × 250 mm, 5 μm; Mobile Phase A: Water; Mobile Phase B: MeCN; Gradient: 75% A and 75% B for 15 min, 75–30% A and 25–70% B over 0.5 min and hold for 4.5 min; Post Time: 5 min.; Flow Rate: 5 mL/min; Injection load: 100 µL of 100 mg/mL solution. Detection was by UV (210 nm). The fraction of interest between 17.0 min and 18.0 min was collected over multiple runs and concentrated by rotary evaporation under reduced pressure.

The secondary purification was carried out using an Agilent 1100 Series HPLC system (Agilent Technologies, Waldbronn, Germany) equipped with quaternary pump, autosampler, column compartment, Multiple Wavelength Detector and analytical fraction collector with the following conditions: Column: Agilent Poroshell 120 SB-C18 2.7 µm, 4.6 × 150 mm; Column Temp: 40 °C; Mobile Phase A: Water; Mobile Phase B: MeCN; Gradient: 70% A and 30% B for 11 min, 70–40% A and 30–60% B over 0.5 min and hold for 3.5 min; Post time: 5 min; Flow Rate: 0.8 mL/min; Injection load: 5 µL of 165 mg/mL solution. The fraction of 1 eluting between 5.1 min and 5.6 min, fraction of 2 eluting between 7.6 min and 8.2 min was collected over multiple runs and concentrated by rotary evaporation under reduced pressure.

The final purification of 1 was performed using the same column and conditions, but isocratic mobile phase; 32% MeCN in water, but injection load was 5 µL of 50 mg/mL solution. The broad peak observed at t_R 3.8–4.0 min was collected over multiple runs and dried by rotary evaporation under reduced pressure.

The final purification of 2 was performed using the same column and conditions, but isocratic mobile phase; 28% MeCN in water, but injection load was 5 µL of 30 mg/mL solution. The broad peak observed at t_R 13.5–14.5 min was collected over multiple runs and dried by rotary evaporation under reduced pressure.

The initial stevia extract, final biotransformation mixture, purified 1 and 2 fractions were analyzed using an Agilent 1200 Series HPLC system (Agilent Technologies, Waldbronn, Germany) equipped with binary pump, autosampler, thermostat column compartment and an Agilent 6110 Single Quadrupole LC-MS system operating in negative ion mode with the conditions summarized below. Column: Agilent Poroshell 120 SB-C18 2.7 µm, 4.6 × 150 mm; Column Temp: 40 °C; Mobile Phase A: Water (0.1% HCOOH) and Mobile Phase B: MeCN (0.1% HCOOH) in an isocratic elution with 32% mobile phase A in mobile phase B; Flow Rate: 0.5 mL/min; Injection volume: 2 μL. Detection was by UV (210 nm) and MSD (Scan and SIM Mode, API-ES 500–1500 Da, negative polarity). The yield of 1 was 15.0 mg with 91.1% chromatographic purity, and the yield of 2 was 16.3 mg with 94.5% purity.

4.3. Mass Spectroscopy

The API-ES mass spectra and MS data were generated by an Agilent 6110 Single Quadrupole LC-MS spectrometer (Agilent Technologies, Little Falls, DE, USA) equipped with an atmospheric pressure ionization-electrospray ion source. Samples were analyzed by negative API-ES. Samples were diluted with H₂O:MeCN (7:3) by 1000-fold

4.4. Nuclear Magnetic Resonance

Samples of purified Rebaudioside QM (1) and Rebaudioside 2QM (2) were prepared by dissolving 10 mg of each compound in 200 μL of pyridine-d₅. The ¹H, ¹³C, ¹H–¹H COSY, 1D-NOESY, 1D-TOCSY, ¹H–¹³C HSQC-DEPT, ¹H–¹³C HMBC, ¹H–¹³C HSQC-TOCSY and ¹H–¹H NOESY spectra were acquired on a Bruker 400 MHz spectrometer (Bruker BioSpin GmbH, Rheinstetten, Germany). NMR data were processed using MestReNova software (version 16.0.0-39276, Mestrelab Research S.L., Santiago de Compostela, Spain). Chemical shifts were referenced to pyridine-d₅ (δ_H 7.19, 7.55, 8.71 ppm; δ_C 123.5, 135.5, 149.9 ppm).

4.5. Quantification of Rebaudioside QM (1) and Rebaudioside 2QM (2)

Quantification was performed using calibration curves constructed from purified Rebaudioside QM (1) and Rebaudioside 2QM (2) obtained through isolation. Six concentration levels were prepared for each analyte, and quadratic regression yielded correlation coefficients (R² ≥ 0.999). Details of the calibration levels and the calibration plots are provided in Supplementary Data S1. Stevia extract samples before and after enzymatic biotransformation were diluted approximately 400-fold to ensure peak areas fell within the calibration range.

4.6. Material Sources

Commercial S. rebaudiana extract (from stevia cultivar: PCS-13), sourced from PureCircle Sdn. Bhd., Bandar Enstek, Negeri Sembilan, Malaysia, was used as the initial material for enzymatic biotransformation

5. Conclusions

In this study, we successfully isolated two steviol glycosides containing the rare sugar quinovose: Rebaudioside QM (1), which has a quinovose moiety at the C13 position, and a novel steviol glycoside, Rebaudioside 2QM (2), with quinovose at the C19 position.

Their structures were elucidated using a comprehensive suite of 1D and 2D NMR techniques, enabling precise assignment of their unique sugar residues. Based on the molecular structures, we propose plausible biosynthetic pathways for both Rebaudioside QM (1) and Rebaudioside 2QM (2).

Future work will focus on validating the proposed biosynthetic pathways and evaluating the sensory properties of Rebaudioside QM (1) and Rebaudioside 2QM (2). Current efforts on stevia biotransformation have enabled enzymatic synthesis of the steviol glycosides in sufficient quantities, overcoming the limitation of low abundance in nature. Advances in biotransformation, synthetic biology and metabolic engineering offer promising strategies to design microbial platforms capable of expressing specific UGTs and further optimizing glycosylation pathways. These approaches enable targeted and scalable biosynthesis, offering a sustainable alternative to plant extraction.

The identification of quinovose-containing steviol glycosides adds a new dimension to the structural variety of steviol glycosides and underscores the value of investigating glycosylation mechanisms in S. rebaudiana. Pursuing this line of research not only deepens our understanding of how steviol glycosides are synthesized but also paves the way for the development of novel sweeteners with improved flavour and sensory profiles.