*4.3. Determination of the Absolute Configuration of the Monosaccharides.*

A simple and rapid reversed-phase C18 high-performance and ultra-high-performance liquid chromatography methods have been developed to determine the absolute configuration of several monosaccharides linked to different kind aglycones [75,76]. This method is not only appropriate to DGs from *S. rebaudiana*, but it can also be applied for several classes of glycosides: flavonoid glycosides, alkaloid glycosides and triterpene glycosides [77,78]

among others. The method is based on cleavage of all the monosaccharides linked to the aglycone under acid hydrolysis. Further liquid-liquid partitions with adequate organic solvent are needed to separate the monosaccharides from the aglycone. It is well-known in steviol glycosides that steviol is not the main aglycone recovered but rather a mixture of isosteviol, endo-steviol and steviol. Basically, two step reactions are needed to yield monosaccharide derivatives traceable by UV with enough difference in retention times between D and L monosaccharide enantiomers. The first step reaction is between the monosaccharides and L-cysteine methyl ester to yield the thiazolidine derivatives followed with further addition of phenylisothiocyanate to yield the thiocarbamoyl thiazolidine monosaccharide derivatives. Both step reactions are performed at 60–70 ◦C in pyridine. The identity and absolute configuration of the monosaccharide could be identified by comparison of the retention times of the derivatives with appropriate standards [75,76]. Thiohydantoin is a by-product yielded in the reaction which has earlier retention times in RP-C18 HPLC method than the thiocarbamoyl-thiazolidine sugar derivatives [75].

#### **5. NMR Experiments**

Several approaches have been discussed in previous sections, all of them provide important information for the structure elucidation of the DGs but still those approaches do not fully elucidate the structures of several DGs. The oligosaccharides at position C-19 and isomeric aglycones are not easily identified using previous methods. There is no doubt that NMR is the most powerful technique for the structure elucidation of any compound if an appropriate amount and purity are in hand. Steviol is the main aglycone in DGs from *S. rebaudiana*, although other natural and degradation cores have also been reported (Figure 1). As far as we know, only steviol, isosteviol, endo steviol and the aglycone with the hydrated double bond at 16,17 (Figure 1V) have been isolated as the aglycone forms. The 13C-NMR spectra for the aglycones of isosteviol, steviol and endocyclic steviol isomer are shown in Figure 6. The 1H and 13C NMR chemical shifts of the main aglycones found in *S. rebaudiana* or in DGS from leaves of this plant are listed (Table 4).

Herein, we discuss the key NMR chemical shifts to differentiate the aglycone cores from *S. rebaudiana*. Steviol has present an exocyclic double bond at position C-16 and the glycosylation sites at position C-13 and C-19 (Figure 1I). The double bond is characterized by a quaternary (158.3 ppm) and a methylene olefinic carbon resonance (103.5 ppm, 13C resonances); and proton resonances (5.48 and 5.04 ppm) while endo steviol, has an endocyclic double bond at position C-15 (135.0, 13C) and 5.24 ppm 1H) and C-16 (145.9 ppm 13C) with an additional methyl group at C-17 (12.7 13C and 1.59 ppm 1H) (Figure 1IV). Isosteviol is easily differentiated from the other aglycones due to the presence of a ketone group (221.3 ppm 13C) at C-16 and an additional methyl group (29.9 13C and 1.40 ppm 1H) (Figure 1III). Compounds 7 with a molecular weight of (332 Da) (Figure 1VI) and 8 with 334 Da (Figure 1VII) possess an endocyclic double bond like endo steviol. The slight structural differences are found at position C-17 where a remarkable difference in chemical shifts is observed, compound 7 has an -CHO (191.4 13C and 9.61 ppm 1H) and 8 a -CH2OH group (59.2 13C and 4.11; 4.29 1H). Different from the other cores, compound 4 with a molecular weight of 336 Da (Figure 1V), does not present any double bond, the main difference is found in the groups linked at position C-16, -CH3 (22.2 13C; 1.32 ppm 1H) and -OH (77.1 ppm 1H).

**Figure 6.** Comparison of the 13C-NMR of isosteviol (**A**), steviol (**B**) and endocyclic steviol isomer (**C**).


**Table 4.** 1H and 13C chemical shifts of the compounds **1**–**9**.


**Table 4.** *Cont.*

(a) NMR spectra were recorded in Pyr-*d*5. (b) MeOH-*d*4, **1**: steviol; **2:** C-12 aglycone; **3:** endo steviol isomer; **4:** aglycone with a CH3 and OH groups linked at C-16; **5:** isosteviol; **6:** *ent*-atisene core from 13-[(2-O-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-d-glucopyranosyl) oxy] ent-hydroxyatis-16-en-19-oic acid -β-D-glucopyranosy ester; **7:** aglycone with a CHO group linked at C-16; **8:** aglycone with a CH2OH group linked at C-16; **9:** aglycone from 15α-hydroxy-rebaudioside M. Chemical structures of the aglycone cores are presented in Figure 1: steviol (I); *ent*-atisene (II); isosteviol (III); endo steviol (IV); CH3 and OH at C-16 (V); CHO group at C-16 (VI); CH2OH at C-16 (VII); C-12 linkage (VIII); 15-α-hydroxy-rebaudioside M (IX).

> Compounds 2 (Figure 1VII) and 6 (Figure 1II) share very similar chemical shifts, although different structures have been assigned. Compound 6 was unambiguously elucidated using appropriate 1D and 2D NMR experiments and aglycone was defined as *ent*-13(*S*)-hydroxyatisenoic acid core. If compared with steviol, the main differences were found in C-13 (77.8 ppm 13C), C-14 (37.7 ppm 13C), C-16 (146.7 ppm 13C) and C-17 (108.0 ppm 13C). Structure of compound 2 should be reanalyzed. Compound 9 shows similar chemical shifts that steviol aglycone with a main difference in the chemical shifts of H-15 and C-15 with 3.70 ppm and 80.8, respectively. The NMR chemical shifts of selected DGs with similar structures are compiled from Tables 5–8. In Table 5 are shown the NMR data for DGs with three glucose units while in Table 6, DGs with four glucose units.




**Table 5.** *Cont.*

(a) NMR spectra recorded in Pyr-*d*5, (b) MeOH-*d*4. **10:** stevioside (Y = 2) [79]; **11**: rebaudioside KA (X = 2) [22]; **12:** 12-α-[(2-O-β-Dglucopyranosyl-β-D-glucopyranosyl)oxy]ent-kaur-16-en-19-oic acid β-D-glucopyranosyl ester (Y = 2) [22]; **13:** rebaudioside G (Y = 3) [16] NA: not assigned.

> In Table 7 are presented DGs with four sugar units, one xylose and three glucoses with different arrangements whereas in Table 8, a few examples of DGs with four sugar units, one rhamnose or 6-deoxyglucose together with three glucose with different arrangements.




**Table 6.** *Cont.*

kaur-16-en-18-oic

 acid

kaur-16-en-18-oic

 acid

glucopyranosyl-β-D-glucopyranosyl)oxy]

D-fructofuranosyl-β-D-glucopyranosyl)oxy]

β-D-glucopyranosyl

β-D-glucopyranosyl

 ester (**\***Fru, X = 3) [65]. NA: not assigned.

 ester (**\***Glc, X = 6 as follows

Glcβ(1-6)Glcβ(1-2)Glcβ1-

 [65]; **19**:

13-[(2-*O*-β-D-glucopyranosyl-3-*O*-β-


**Table 7.** 1H and 13C chemical shifts of the compounds **20**–**23**.

(a) NMR spectra recorded in Pyr-*d*5, (b) MeOH-*d*4. **20**: rebaudioside F (\*Glc, \*\*Xyl and \*\*\*Glc) [11]; **21**: rebaudioside R (\*Xyl, \*\*Glc and \*\*\*Glc) [23]; **22**: 13-[(2-*O*-β-D-glucopyranosyl-3-*O*-β-D-xylopyranosyl-β-D-glucopyranosyl)oxy] *ent*-kaur-16-en-19-oic acid β-Dglucopyranosyl ester (\*Glc, \*\*Glc and \*\*\* Xyl) [68]; **23**: 13-[(2-O-β-D-glucopyranosyl-β-D-glucopyranosyl) oxy]-kaur-16-en-18-oic acid-(6-Oβ-D-xylopyranosyl-β-D-glucopyranosyl) ester (\*Glc and \*\*Glc) [37].


**Table 8.** 1H and 13C chemical shifts of the compounds **24**–**27**.

(a) NMR spectra recorded in Pyr-*d*5, (b) MeOH-*d*4. **24**: rebaudioside S [23]; **25**: no trivial or systematic name was assigned (\*Glc) [25]; **26**: 13-[(2-O-6-deoxy-β-D-glucopyranosyl-3-O-β-D-glucopyranosyl-β-D-glucopyranosyl)oxy] ent-kaur-16-en19-oic acid β-D-glucopyranosyl ester (\*6deoxyGlc) [71]; **27**: rebaudioside C (\*Rha) [16].
