Structural analysis of rebaudioside A derivatives obtained by Lactobacillus reuteri 180 glucansucrase-catalyzed trans-a - glucosylation

The wild-type Gtf180-D N glucansucrase enzyme from Lactobacillus reuteri 180 was found to catalyze the a -glucosylation of the steviol glycoside rebaudioside A, using sucrose as glucosyl donor in a trans-glucosylation process. Structural analysis of the formed products by MALDI-TOF mass spectrometry, methylation analysis and NMR spectroscopy showed that rebaudioside A is speci ﬁ cally a - D -glucosylated at the steviol C-19 b - D -glucosyl moiety (55% conversion). The main product is a mono-( a 1 / 6)-glu- cosylated derivative (RebA-G1). A series of minor products, up to the incorporation of eight glucose residues, comprise elongations of RebA-G1 with mainly alternating ( a 1 / 3)- and ( a 1 / 6)-linked glucopyranose residues. These studies were carried out in the context of a program directed to the improvement of the taste of steviol glycosides via enzymatic modi ﬁ cation

Due to the growing awareness and concerns for human health related to excessive consumption of sugar (sucrose), the application of steviol glycosides as non-caloric bio-alternatives for sucrose and as substitutes for artificial (synthetic) sweeteners is strongly promoted nowadays [4e8].Since a couple of years, steviol glycosides have been permitted for use as food additive and sweetener in the USA [9] and in Europe (E960) [10,11].However, despite their intense sweetness and diverse beneficial pharmacological properties [12e15], the main drawback for successful commercialization of Stevia sweeteners is their slight bitterness and unpleasant (metallic) aftertaste, experienced by more than half of the human population.
For natural steviol glycosides with b-D-glucopyranosyl units as constituents, it has been reported that the ratio of the number of glucose units at the C-13 site to that at the C-19 site of the steviol core has a relationship with the sweetness as well as with the quality of taste of the steviol glycosides [16,17].To improve the Abbreviations: FID, free induction decay; FWHM, full width at half maximum; GLC-EI-MS, gas-liquid chromatography electron impact mass spectrometry; HPAEC, high-pH anion-exchange chromatography; HSQC, 1 H-detected hetero-nuclear single-quantum coherence spectroscopy; MALDI-TOF-MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; MLEV, composite pulse devised by M. Levitt; NMR, nuclear magnetic resonance; RebA, rebaudioside A; RebB, rebaudioside B; ROESY, rotating-frame nuclear Overhauser enhancement spectroscopy; Ste, stevioside; TLC, thin-layer chromatography; TOCSY, total correlation spectroscopy; UVeVIS, ultravioletevisible; WEFT, water eliminated Fourier transform.
* Corresponding author.E-mail address: L.Dijkhuizen@rug.nl(L.Dijkhuizen). 1 These authors contributed equally to this work.taste, especially for food applications, chemical and enzymatic modifications of the carbohydrate moieties of specific steviol glycosides have been investigated, and showed promising results [3,16,18e25].
With a focus on enzymatic modifications of steviol glycosides to produce derivatives with improved organoleptic properties, we have incubated rebaudioside A with sucrose and the wild-type Gtf180-DN glucansucrase enzyme from Lb. reuteri 180.Here, we present detailed structural analyses by MALDI-TOF mass spectrometry, methylation analysis and 1D/2D 1 H/ 13 C NMR spectroscopy of obtained a-D-glucopyranosylated products.1) shows four Glc residues (Glc1, Glc2, Glc3 and Glc4) with a total of fourteen free hydroxyl groups, which can act as acceptors for transglucosylation reactions.In view of the reported enzymatic activity of the wild-type Gtf180-DN glucansucrase enzyme from Lb. reuteri 180 (Section 1) [28e31], at first instance, elongations at HO-3, HO-4 and HO-6 are expected.

Incubation of rebaudioside
RebA (50 mM) was incubated at 37 C with 10 U/mL wild-type Gtf180-DN enzyme in sodium acetate buffer, pH 4.7, containing 1.0 M sucrose.After 3 h, a second batch of 1.0 M sucrose was added to the reaction mixture, and the incubation was prolonged for 21 h.After removal of glucose, fructose, gluco-oligo/polysaccharide (products of the "natural activity" of the enzyme), protein material, and residual sucrose from the reaction mixture via solid-phase extraction, HPLC analysis of the (a-glucosylated) RebA mixture showed a complex pattern of peaks, as visualized in Fig. 3. Fraction F1 had the same retention time as the acceptor substrate RebA.For further analysis, fractions F1-F9 were isolated.
MALDI-TOF-MS analysis of the fractions F1-F9 showed a series of quasi-molecular ions [MþNa] þ , in accordance with an extension of RebA (F1) with one (F2) to eight (F9) glucose residues, respectively (Supplementary Information Fig. S1).However, in view of the HPLC peak clusters within some fractions, groups of isomeric components with the same molecular mass can be expected.Integration of the HPLC peaks in Fig. 3 revealed that 55% of RebA  1 and 2. For an explanation of the code system used, see Fig. 4.

Structural analysis of HPLC fraction F1
NMR analysis of fraction F1 confirmed the presence of acceptor substrate RebA (Fig. 5, 1 H NMR spectrum of F1, identical to Supplementary Information Fig. S3; Tables 1 and 2).Methylation analysis (Table 3) revealed the presence of terminal Glcp and 2,3-di-O-substituted Glcp in the molar ratio of 3:1, as expected for RebA.

Structural analysis of HPLC fraction F2
Methylation analysis of F2 (RebAþ1Glc) (Table 3) showed a 3:1 molar ratio for terminal Glcp and 2,3-di-O-substituted Glcp, indicating that the additional Glcp residue is attached to a terminal Glcp residue of RebA.The detection of a 6-mono-O-substituted Glcp residue suggested a product with a Glcp(1 / 6) extension.
The 1 H NMR spectrum of F2 (Fig. 5; enlarged anomeric region in Supplementary Information Fig. S6) exhibited the typical steviol core signal pattern as seen for RebA (F1).Besides the four b- The latter signal correlated with a 13 C resonance at d C 99.5 in the HSQC spectrum (Fig. 6).
In a similar way as described for RebA in the Supplementary Information, using 2D NMR spectroscopy (TOCSY with different mixing times, ROESY and HSQC, Fig. 6), the 1 H/ 13 C chemical shifts of the steviol core (Table 1) and the five Glc residues (Table 2) of F2 were assigned.The 1 H and 13 C chemical shift sets of Glc2, Glc3 and Glc4 are nearly identical to those of F1 (RebA), suggesting that no modifications had occurred in the carbohydrate moiety at the   .96;T ¼ 300 K) [40].The absence of / 3)Glcp(1 / or / 4) Glcp(1 / elements in the methylation analysis of F2 (Table 3) together with the absence of an a-anomeric 1 H signal at d H ~5.27, typical for a terminal Glc(a1 / 3) or Glc(a1 / 4) residue (see fraction F3P2, Section 2.4), supports the absence of such extensions at Glc1.
In summary, the mono-transglucosylated product from fraction F2 is RebA with a Glc(a1 Glc1] using UDP-Glc as glucosyl donor and the b-glucosyltransferase UGTSL2 enzyme as biocatalyst [41].Furthermore, a Glc(a1 / 6) extension of Glc1 of stevioside was demonstrated in a transglucosylation reaction with maltose as donor substrate in the presence of Biozyme L [20].

Structural analysis of HPLC fraction F4
Methylation analysis of HPLC fraction F4 (RebAþ3Glc) showed the presence of 6-and 3-mono-O-substituted Glcp in the molar ratio 2.6:1.0;furthermore, the molar ratio of terminal and 2,3-di-Osubstituted Glcp is 3:1 (Table 3).Although the integration of the anomeric protons in the 1 H NMR spectrum of F4 (Fig. 5) suggest the presence of one major component, in view of the mentioned structures present in fraction F3, it would be expected that also fraction F4 contains a mixture of different compounds with the same molecular mass.As already discussed for F2 and F3, also in F4 the extra a-linked Glc residues are only located at the steviol C-19 site [alkaline treatment of F4 yielded only RebB (F1-4S; Supplementary Information Fig. S7)].The 1 H/ 13 C NMR assignments of the F4 steviol aglycone and carbohydrate moieties, derived from TOCSY, HSQC and ROESY measurements, are presented in Tables 1  and 2, respectively (spectra not shown).

Structural analysis of HPLC fractions F5-F9
As is evident from the HPLC profile in Fig. 3, fractions F5 (RebAþ4Glc) to F9 (RebAþ8Glc) represent complex mixtures of RebA derivatives.Inspection of the 1D 1 H NMR spectra of F5 to F9 (spectra not shown) learned that the steviol core regions (0.80e2.20 ppm) are identical and the carbohydrate bulk regions (3.20e4.20 ppm) similar to those in the 1D 1 H NMR spectra of F3 and F4.When treated with alkali, each fraction was converted into RebB (F1-4S; Supplementary Information Fig. S7), meaning that also in the case of F5 to F9 a-glucosylation had only occurred at the steviol C-19 glucosyl moiety of RebA.The patterns of anomeric signals in the 1 H NMR spectra of F5 to F9 are comparable with those of F3 and F4, indicating that the elongated carbohydrate chains at the steviol C-19 site of F5 to F9 are built up from (a1 / 6)-and (a1 / 3)-linked Glc residues.More specifically, the spectra showed relative increase in intensities of the H-1 resonance at d H 5.25 and the H-5 resonance at d H 4.10, derived from internal / 6) Glc(a1 / 3) residues, and the H-1 resonance at d H 4.87, derived from terminal Glc(a1 / 6) and internal / 6)Glc(a1 / 6) residues.Methylation analysis of these high-molecular-mass fractions showed an increase of (a1 / 6) and (a1 / 3) linkages towards almost equal molar amounts (Table 3; only traces of 4-linked Glcp were observed), suggesting the preference of the wild-type Gtf180-DN enzyme for the synthesis of alternating (a1 / 6)/(a1 / 3) linkages in the major formed RebA derivatives.Furthermore, the finding of low amounts of 3,6-di-O-substituted Glcp residues in the methylation analysis of these fractions reflects also the possibilities of branching.

Conclusions
Over the years, several types of carbohydrate-active enzymes have been used in the glycosylation reactions of steviol glycosides (see review Ref. [3]).With respect to trans-a-glucosylations, cyclodextrin glycosyltransferase systems, introducing elongations with a-D-Glcp-(1 / 4) units at both the steviol C-13 and C-19 sites of stevioside, rubusoside and rebaudioside A (RebA), gained great attention.In these bioconversions, glucose donors such as cyclodextrins, maltodextrins and starches are used.But also other aglucosidase transglycosylation systems were investigated, i.e. glucansucrase enzymes combined with sucrose as glucose donor.
Recent studies in our research group have shown that (mutant) Gtf180-DN glucansucrase of Lb. reuteri strain 180 introduces glucose residues from sucrose into RebA, specifically at the C-19 site.In this report the structural analysis of the formed products with wild-type Gtf180-DN glucansucrase as biocatalyst has been described in detail.Structural biological aspects, including comparison of transglycosylation activities and differences in conversion percentages of wild-type and 82 mutant Gtf180-DN glucansucrase enzymes, molecular docking studies of the Gtf180-DN e RebA complex, and sweetness/bitterness tests will be described elsewhere [te Poele et al., manuscript in preparation].As a first step, the Glc(b1 / C-19 residue of RebA was found to be elongated with a Glc(a1 / 6) residue (RebA-G1).Further extensions, up to eight Glc units, comprised mainly Glc(a1 / 6) and Glc(a1 / 3) residues; in the trisaccharide, elongation evidence for a termination with a Glc(a1 / 4) residue was found.The major higher a-glucosylated RebA products are built up by elongation of the Glc(a1 / 6)Glc(b1 / C-19 moiety with alternating (a1 / 3)/ (a1 / 6)-linked Glc units, accompanied by products with only (a1 / 6)-linked sequences in lesser amounts.In the highermolecular-mass components also 3,6-branching is indicated.

Preparation of a-D-glucosylated rebaudioside A products
Incubations of RebA (50 mM) were performed in 5 mL 25 mM sodium acetate (pH 4.7), containing 1 mM CaCl 2 , in the presence of 10 U/mL Gtf180-DN enzyme at 37 C and 24 h.Two batches of 1.0 M sucrose donor substrate were added at t ¼ 0 and t ¼ 3 h, respectively.One unit (U) of enzyme is defined as the amount of enzyme required for producing 1 mmol fructose from sucrose per min in reaction buffer, containing 1.0 M sucrose at 37 C.In this case, 1 U corresponded to 0.038 mg Gtf180-DN.The full rationale for these incubation conditions will be described in detail elsewhere [te Poele et al., manuscript in preparation].The pool of glucosylated RebA products was isolated by solid-phase extraction (SPE) using a Strata-X 33m Polymeric Reversed Phase column (Phenomenex, Utrecht, The Netherlands).Briefly, the SPE column was conditioned with 6 bed volumes methanol and subsequently equilibrated with 6 bed volumes de-ionized water.After loading of the sample, the column was washed with 6 bed volumes de-ionized water to remove enzyme, glucose, fructose, gluco-oligo/polysaccharides and residual sucrose.Then, the mixture of RebA products was eluted with 6 bed volumes 50% acetonitrile.Subsequently, the mixture was fractionated on a Luna 10 mm NH 2 semi-preparative chromatography column (250 mm Â 10 mm, Phenomenex), using an Ul-tiMate 3000 HPLC system (ThermoFisher Scientific, Amsterdam, The Netherlands), equipped with a VWD-3000 UV-VIS detector (monitoring at 210 nm).Separations were obtained at a flow-rate of 4.6 mL/min under gradient elution conditions (solvent A ¼ acetonitrile; solvent B ¼ 0.025% aqueous acetic acid), starting with a 2-min isocratic step with 80% solvent A in B followed by a linear gradient of 80 to 50% solvent A in B over 38 min.The manually collected fractions were evaporated to dryness under a stream of nitrogen, and the residues were re-dissolved in deionized water and directly lyophilized.Fresh newly dissolved samples were used for analysis.

Alkaline hydrolysis
To release the carbohydrate moiety linked to the C-19 carboxyl group, 4 mg RebA and 4 mg of each transglucosylated product were individually dissolved in 1 mL 1.0 M NaOH and the solutions were heated at 80 C for 2.5 h, then cooled down, and neutralized with 6 M HCl.The modified product fractions were isolated using Strata-X 33m Polymeric Reversed Phase columns (Phenomenex), as described in Section 4.2.

Methylation analysis
Steviol glycoside samples were permethylated using CH 3 I and solid NaOH in (CH 3 ) 2 SO, as described previously [49], then hydrolyzed with 2 M trifluoroacetic acid (2 h, 120 C) to give the mixture of partially methylated monosaccharides.After evaporation to dryness, the mixture, dissolved in H 2 O, was reduced with NaBD 4 (2 h, room temperature).Subsequently, the solution was neutralized with 4 M acetic acid and boric acid was removed by repeated co-evaporation with methanol.The obtained partially methylated alditol samples were acetylated with 1:1 acetic anhydride-pyridine (30 min, 120 C).After evaporation to dryness, the mixtures of partially methylated alditol acetates, dissolved in dichloromethane, were analyzed by GLC-EI-MS on an EC-1 column (30 m Â 0.25 mm; Alltech), using a GCMS-QP2010 Plus instrument (Shimadzu Kratos Inc., Manchester, UK) and a temperature gradient (140e250 C at 8 C/min) [50].

Mass spectrometry
Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was performed on an Axima™ mass spectrometer (Shimadzu Kratos Inc.), equipped with a nitrogen laser (337 nm, 3 ns pulse width).Positive-ion mode spectra were recorded using the reflector mode at a resolution of 5000 FWHM and delayed extraction (450 ns).Accelerating voltage was 19 kV with a grid voltage of 75.2%.The mirror voltage ratio was 1.12 and the acquisition mass range was 200e6000 Da.Samples were prepared by mixing on the target 1-mL sample solutions with 1 mL aqueous 10% 2,5-dihydroxybenzoic acid in 70% acetonitrile as matrix solution.

NMR spectroscopy
Resolution-enhanced 1D/2D 500-MHz 1 H/ 13 C NMR spectra were recorded in D 2 O on a Bruker DRX-500 spectrometer (Bijvoet Center, Department of NMR Spectroscopy, Utrecht University).To avoid overlap of anomeric signals with the HOD signal, the spectra were run at 334 K. Data acquisition was done with Bruker Topspin 2.1.Before analysis, samples were exchanged twice in D 2 O (99.9 atom% D, Cambridge Isotope Laboratories, Inc., Andover, MA) with intermediate lyophilization, and then dissolved in 0.6 mL D 2 O.It should be noted that during longer stay in aqueous solution, partial degradation of steviol glycosides can occur due to loss of the carbohydrate moiety from the steviol C-19 carboxyl group and possible formation of the D 15À16 isomer and the D 16À17 hydration products [51,52].Therefore, fresh solutions of ~4 mg/mL (~4 mM) were used for all NMR measurements.Suppression of the HOD signal was achieved by applying a WEFT (water eliminated Fourier transform) pulse sequence for 1D NMR experiments and by a presaturation of 1 s during the relaxation delay in 2D experiments.The 2D TOCSY spectra were recorded using an MLEV-17 (composite pulse devised by Levitt et al.) [53] mixing sequence with spin-lock times of 20, 50, 100 and 200 ms.The 2D 1 H-1 H ROESY spectra were recorded using standard Bruker XWINNMR software with a mixing time of 200 ms.The carrier frequency was set at the downfield edge of the spectrum in order to minimize TOCSY transfer during spinlocking.Natural abundance 2D 13 C-1 H HSQC experiments ( 1 H frequency 500.0821MHz, 13 A with the Gtf180-DN glucansucrase enzyme and sucrose Inspection of the molecular structure of rebaudioside A {RebA, 13-[(2-O-b-D-glucopyranosyl-3-O-b-D-glucopyranosyl-b-D-glucopyranosyl)oxy]ent-kaur-16-en-19-oic acid b-D-glucopyranosyl ester} (Fig.

Fig. 6 .
Fig.6.HSQC, TOCSY (mixing time 200 ms) and ROESY spectra of the carbohydrate part of HPLC fraction F2 (RebA-G1), recorded in D 2 O at 334 K.In the HSQC spectrum, means cross-peak H-3/C-3 of residue Glc2, etc.; assignments in red reflect the substituted positions of the residues.In the ROESY spectrum, the inter-residual cross-peaks confirming the Glc4(b1 / 3)Glc2, Glc3(b1 / 2)Glc2 and Glc5(a1 / 6)Glc1 linkages are indicated with red boxes.(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) C signals of the carbohydrate moiety at the steviol C-13 site (for the anomeric regions: Glc2, d H-1 4.715, d C-1 97.6; Glc3, d H-1 4.810, d C-1 103.6;Glc4, d H-1 4.702; d C-1 103.9).These results, together with the alkaline-treatment data of F3, indicated that also in F3P2 the carbohydrate moiety at the steviol C-13 site was not modified.The remaining carbohydrate signals in the anomeric region of the 1 H NMR spectrum of F3P2 (Fig. 7; enlarged anomeric region in Supplementary Information Fig. S8) represent a heterogeneous b-anomeric signal of Glc1 (steviol C-19 site) at d H ~5.42, a heterogeneous a-anomeric signal at d H ~4.88 (Glc5), overlapping with one of the C-17 steviol protons, and a heterogeneous a-anomeric signal at d H ~5.28 (Glc6).The Glc1 H-1 signal is clearly built up from two doublets at d H 5.425 and 5.410 (J 1,2 8.3 Hz; peak ratio 2.7:1.0), the Glc5 H-1 signal from two doublets at d H 4.873 and 4.857 (J 1,2 4.1 Hz; peak ratio 2.8:1.0), and the Glc6 H-1 signal from two doublets at d H 5.275 and 5.265 (J 1,2 4.3 Hz; peak ratio 2.8:1.0),suggesting the presence of two compounds in

Fig. 8 .
Fig. 8. HSQC spectrum (200 ms) of the carbohydrate part of HPAEC fraction F3P2, plotted at high level (resonances from RebA-G2b).means cross-peak H-3/C-3 of residue etc.; assignments in red reflect the substituted positions of the residues.Note Glc6 H-5/C-5 (d 3.93/73.4),stemming from terminal Glc(a1 / 3).(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) C frequency 125.7552MHz) were recorded without decoupling during acquisition of the 1 H FID. The NMR data were processed using the MestReNova 9 program (Mestrelab Research SL, Santiago de Compostella, Spain).Chemical shifts (d) are expressed in ppm by reference to internal acetone (d H 2.225 for 1 H and d C 31.07 for 13 C).

Table 1
a In ppm relative to internal acetone (d 2.225 for 1 H and d 31.07 for 13 C).
a c Substituted carbon positions are indicated in italics.

Table 3
Methylation analysis of the carbohydrate moieties in RebA and a-glucosylated RebA products (fractions F1-F5 and F9).
a R t