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. 2026 Sep 10;11(37):55994–56000. doi: 10.1021/acsomega.6c05861

Structural Revision of a Nornicotine Glycoside: Identification of N‑[(β-D-xylopyranosyl)methyl]nornicotine from Cherry-Red Tobacco

Yong Li 1, Tao Pang 1,*, Junli Shi 1, Zhongbang Song 1, He Xie 1, Ge Bai 1
PMCID: PMC13613952  PMID: 42799094

Abstract

A nornicotine glycosidepreviously reported with an incorrect structurewas isolated from cherry-red tobacco and structurally re-elucidated. Its molecular formula (C15H22N2O5) was determined by high-resolution electrospray ionization mass spectrometry. Acid hydrolysis combined with gas chromatography–mass spectrometry (GC-MS) preliminarily identified the aglycone as nornicotine. Comprehensive analysis of one-dimensional (1H, 13C, DEPT) and two-dimensional (COSY, HSQC, HMBC) nuclear magnetic resonance (NMR) data unambiguously established the planar structure, confirming the sugar moiety is a six-membered pyranose linked to the pyrrolidine nitrogen of nornicotine via an N–CH2–C bond. Density functional theory (DFT)-based NMR chemical shift calculations were performed for four possible stereoisomers (β-D, β-L, α-D, α-L). Linear regression analysis showed the β-D stereoisomer had the highest fitting correlation coefficient (R 2 = 0.9952 for 13C and R 2 = 0.9801 for 1H), significantly outperforming the others. The mean absolute error (MAE) was 2.41 ppm for 13C NMR chemical shifts and 0.28 ppm for 1H NMR chemical shifts, indicating excellent agreement between theoretical and experimental data. Combined with the prevalent distribution of β-D configuration in natural glycosides, the sugar moiety’s stereo configuration was determined as β-D, confirming it as β-D-xylopyranose. This work corrects the long-standing misassignment of the nornicotine glycoside’s sugar moiety.

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Introduction

Tobacco (Nicotiana tabacum L.) is an economically important cash crop and a rich source of bioactive secondary metabolites, among which alkaloids and their derivatives are the most characteristic components. These compounds not only play crucial roles in the chemical defense and ecological adaptation of tobacco plants but also serve as important chemotaxonomic markers for the genus Nicotiana. Nicotine is the predominant alkaloid in normal cultivated tobacco, accounting for more than 95% of the total alkaloid content; , importantly, it is the core component responsible for the addictive properties of tobacco as a smokable product, primarily by regulating the secretion of dopamine in the central nervous system. , However, some tobacco variants exhibit unique alkaloid metabolic profiles due to genetic variations, leading to significant changes in the types and contents of alkaloids. ,

Cherry-red tobacco, a special flue-cured tobacco variant derived from normal cultivated tobacco, is distinguished by its genetic characteristic of converting a large proportion of nicotine to nornicotine (the demethylation product of nicotine) during growth and curing. , This unique metabolic shift results in a distinct chemical composition compared to normal tobacco, making cherry-red tobacco an ideal material for exploring novel nornicotine-derived metabolites. − As a key secondary metabolite in cherry-red tobacco, nornicotine has a hydrogen substituent on its pyrrolidine nitrogen atom; this N–H structure renders the nitrogen atom relatively active, allowing it to undergo a series of substitution reactions. Specifically, the active nitrogen atom can be acylated to generate acylated metabolites, nitrosated to tobacco-specific nitrosamine, glycosylated to form glycoside conjugates-the focus of this study-or undergo other substitution reactions. Glycosylation is a common plant modification process that often modulates the solubility, stability, and biological properties of parent compounds, and the nornicotine glycosides derived from this reaction are the core research object of the present work.

Phytochemical investigations on tobacco nornicotine glycosides have been extremely limited, with only one relevant study formally reported to date. This study, published by Koiwai et al. in 1979, describes the isolation and structural characterization of a nornicotine glycoside (1-(1′-2′S-Nornicotino)-1-deoxy-β-d-fructofuranose), which was assigned a fructose moiety and a C-glycosidic bond. However, limited by the technical conditions at that time, the nuclear magnetic resonance (NMR) data provided for structural analysis only included low-resolution 1H NMR and 13C NMR spectra, which are insufficient to fully verify the correctness of the proposed structure. Consequently, the sugar moiety in the reported compound was likely misassigned.

In this study, a nornicotine glycoside (Figure ) was isolated from cherry-red tobacco, and its structure was comprehensively elucidated using a combination of acid hydrolysis-GC-MS, high-resolution NMR spectroscopy (1D and 2D), HRESIMS, and density functional theory (DFT)-based NMR calculations. Our results confirmed that the isolated compound is identical to the one reported by Koiwai et al. (1979), but with a corrected sugar moiety: the previously proposed five-membered fructofuranose was revised to a six-membered β-D-xylopyranose, which contains a nonprotonated anomeric carbon and is linked to the pyrrolidine nitrogen of nornicotine via an N–CH2–C bond. Furthermore, we determined the stereo configurration of the sugar moiety as β-D through DFT-NMR calculations of the four possible stereoisomers (β-D, β-L, α-D, α-L), which was supported by the prevalent distribution of β-D configuration in natural glycosides. Thus, this study not only corrects the long-standing structural misassignment of the nornicotine glycoside reported by Koiwai et al. but also clarifies the accurate structure and stereochemistry of this compound, providing reliable structural information for further phytochemical, chemotaxonomic, and biological studies on tobacco variants and their alkaloid metabolites.

1.

1

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Structure of the title compound.

Results and Discussion

Structural Identification of the Title Compound

HRESIMS Analysis

The HRESIMS spectrum (Figure a) of the title compound showed a quasi-molecular ion peak at m/z [311.1606]+, consistent with the theoretical exact mass of [C15H22N2O5 + H]+ (calcd for 311.1607), confirming its molecular formula as C15H22N2O5 with 5 degrees of unsaturation. MS2 analysis of [311.1606]+ revealed four consecutive mass spectral peaks with a mass loss of 18 (corresponding to H2O), indicating the presence of four hydroxyl groups (Figure b). Fragment ions included a peak at m/z 149.1074, consistent with the characteristic ions of nornicotine, indicating the presence of a nornicotine unit (consistent with the two nitrogen atoms in the molecular formula). The mass difference (311 – 149 = 162) suggested a glycosyl moiety, which aligns with the four hydroxyl groups and five oxygen at oms in the molecular formula. These results initially indicated the title compound is a nornicotine glycoside, consistent with the biological characteristics of cherry-red tobacco (high nicotine-to-nornicotine conversion).

2.

2

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Primary (a) and secondary (b) mass spectra of the title compound. In the secondary mass spectrum, four consecutive dehydrated ion peaks and the fragment ion peaks of nornicotine are labeled.

Acid Hydrolysis Products Analysis

Acid hydrolysis of the purified title compound, followed by GC-MS analysis, revealed significant chromatographic peaks of nornicotine and its silylated products (identified via NIST library) (Figure ). Two obvious hexose peaks were also detected; NIST library search showed ∼80% similarity to common hexoses, but no match was verified by literature retention indices, suggesting the sugar moiety has a relatively special structure. These results directly confirmed that the title compound consists of a nornicotine moiety and a glycosyl moiety, laying a foundation for further structural elucidation.

3.

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Total ions chromatograms of the title compound before and after hydrolysis. After oximation and silylation, the target compound showed two chromatographic peaks with very close retention times (41.03 and 41.11 min) on the gas chromatogram, whose retention indices relative to n-alkanes were 2678.6 and 2684.1, respectively. Peaks of 25.85 and 26.67 min were initially identified as hexose.

NMR Spectral Analysis

The 1H NMR spectrum (800 MHz, MeOD) of the title compound showed four aromatic proton signals (δ 8.52, 8.43, 7.94, 7.42, see Figure S1), characteristic of the pyridine ring in nornicotine. Aliphatic proton signals (δ 4.03–1.73) were assigned to the aliphatic moiety of nornicotine and the glycosyl moiety. The 13C NMR spectrum (201 MHz, MeOD, see Figure S2) showed 15 carbon signals, consistent with C15H22N2O5; combined with DEPT (Figure S2) and HSQC (Figures and S3) spectra, all carbon and proton signals were unambiguously assigned, with no conflicting information.

4.

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Observed HSQC, HMBC and COSY. For HMBC, the arrows indicate correlations from the corresponding carbon atoms to the attached hydrogen atoms.

Based on HRESIMS and hydrolysis-GC-MS results (confirming a nornicotine glycoside), HSQC-derived carbon–proton combinations were divided into three parts: pyridine ring, tetrahydropyrrole ring, and glycosyl moiety. Chemical shifts confirmed δ 125.60­(CH), 137.60­(CH), 140.89­(C), 149.07­(CH), 149.96­(CH) belong to the pyridine; δ 24.2­(CH2), 35.38­(CH2), 57.04­(CH2), 69.02­(CH) belong to the tetrahydropyrrole ring (confirmed by COSY coupling, see Figures and S4). The remaining signals (δ 60.06­(CH2), 70.99­(CH), 71.51­(CH), 71.99­(CH), 98.26­(C), 64.14­(CH2)) were assigned to the glycosyl moiety.

The pyridine ring connection mode was determined as 140.89­(C)-137.60­(CH)-125.60­(CH)-149.07­(CH)-N-149.96­(CH)-140.89­(C) by COSY and HMBC correlations (Figures , S4, and S5). Both COSY and HMBC signal are clear and complete.

For the tetrahydropyrrole ring, HMBC coupling between δ 69.02 (CH) proton and pyridine ring signals (δ 137.6, 140.89, 149.96) confirmed δ 69.02 as the direct link between the two rings. COSY and HMBC correlations established the connection sequence as 69.02­(CH) −35.38­(CH2) −24.2­(CH2) −57.04­(CH2) -N-69.02­(CH).

The glycosyl moiety connection to the tetrahydropyrrole ring was also determined by HMBC: δ 60.06 (CH2) protons showed HMBC correlations with tetrahydropyrrole signals (δ 57.04, 69.02), confirming it as the carbon directly connected to the nitrogen atom. Given its chemical shift (60.06), δ 60.06 cannot link directly to an oxygen atom, so its other end connects to a glycosyl carbon.

The remaining five glycosyl carbons (one degree of unsaturation, δ 64.14–98.26) each connect to an oxygen atom (no carbonyl groups, δ < 160). MS2 (four hydroxyl groups) and HSQC (one nonprotonated carbon) excluded carbon–carbon double bonds (which would require δ > 100 and more nonprotonated carbons), so the degree of unsaturation arises from a ring possibly formed by an ether oxygen.

The nearly identical chemical shifts of the three glycosyl carbons (δ 70.99, 71.51, 71.99) suggest that their chemical environments are similar. Carbon chemical shifts around 70 suggest that each of these carbon atoms likely bears a hydroxyl group. δ 98.26 (downfield) suggests a hydroxyl group and an ether oxygen, while δ 64.14 (upfield) suggests an ether oxygen (weakening deshielding). COSY confirmed direct connections of δ 71.51–71.99 and δ 64.14–70.99. HMBC correlations between δ 64.14 (CH2) protons and δ 98.26 (C) support a C–O–C connection. In summary, the glycosyl carbon connection sequence was determined as 60.06­(CH2)-98.26­(C)-71.99­(CHOH)-71.51­(CHOH)-70.99­(CHOH)-64.14­(CH2OH)-O-98.26­(C). The nornicotine moiety connects to the sugar ring by the N–CH2 (δ 60.06) group and the nonprotonated anomeric carbon (δ 98.26).

ROESY spectroscopy (800 MHz, MeOD) detected NOE signals only between geminal protons of methylene groups (δ 35.38, 60.06, 57.04, 64.14, Figure S6). No cross NOE correlations between nongeminal protons were observed, attributed to spatial distances greater than 3.5 Å (NOE detection threshold), consistent with the six-membered pyranosyl ring’s chair conformation and exoface orientation of the nornicotine moiety.

DFT-NMR Calculations for Stereo Configuration

To clarify the title compound’s stereo configuration, four possible stereoisomers (β-D, β-L, α-D, α-L) were constructed. The constructed stereoisomers were preoptimized with molecular mechanics force field (MM2), then subjected to DFT geometric optimization (B3LYP/def2-SVP) and 1H/13C NMR chemical shift prediction. Linear regression analysis of the experimental chemical shifts and the predicted chemical shifts showed the β-D configuration had the highest correlation coefficient (Table ), significantly outperforming the others. The β-D isomer’s mean absolute error (MAE) values (1H: 0.28 ppm, 13C: 2.41 ppm) meet reliability criteria for stereo configuration identification. − Combined with the prevalence of β-D configurations in natural glycosides the title compound’s stereo configuration was determined as β-D, confirming the sugar moiety as β-D-xylopyranose (the only six-membered pyranose pentose consistent with the planar structure and β-D configuration).

1. Linear Fitting Result of the Calculated and Experimental 1H and 13C Chemical Shifts for the Four Possible Stereo Structures (β-D, β-L, α-D, α-L) of the Title Compound .

    β-D β-L α-D α-L
1H NMR curve y = 0.9881x + 0.1807 y = 0.9258x + 0.6262 y = 0.9799x + 0.1764 y = 0.993x + 8.5818
R 2 0.9801 0.9437 0.9714 0.9429
MAE 0.28 0.39 0.33 0.44
13C NMR curve y = 1.0069x + 7.1452 y = 1.0044x + 7.06 y = 0.993x + 8.5818 y = 0.9996x + 6.022
R 2 0.9952 0.9911 0.9926 0.9823
MAE 2.41 2.98 2.97 4.58
Energy Total (Eh) –1069.04007 –1069.04169 –1069.02446 –1069.01477
  ΔE (kcal/mol) 1.02 0 10.81 16.91
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a

MAE stands for mean absolute error in the table. ΔE values are relative to the lowest-energy isomer (β-L).

The relative energies of the four stereoisomers after geometry optimization were compared (Table ). The α-d and α-L isomers lie 10.81 and 16.91 kcal/mol above the global minimum (β-L), respectively, which is consistent with the well-established predominance of β-configurations among natural glycosides and rules out any significant contribution from the α-anomers under the experimental conditions. Notably, the β-D isomerwhich shows the best agreement with experimental NMR datais only 1.02 kcal/mol higher in energy than β-L. This small energy difference is within the typical uncertainty of DFT methods for diastereomeric pairs and cannot by itself serve as a reliable criterion for configurational assignment. Instead, the unambiguous discrimination between β-D and β-L relies on the DFT-NMR chemical shift comparison, as described below.

Conformational analysis. To evaluate the potential impact of conformational averaging on the DFT-NMR predictions, a systematic conformational analysis was performed for the best-fitting β-d isomer. The four hydroxyl groups of the sugar moiety (C13–OH, C14–OH, C26–OH, C27–OH) were rotated by ±120° about the C–O bond, generating eight unique starting conformers. Each conformer was fully optimized at the B3LYP-D3­(BJ)/6–31G­(d) level, and the NMR chemical shifts were calculated at the GIAO-mPW1PW91/6–311+G­(2d,p) level. After optimization, six of the eight conformers converged to geometries with energies within 0.01 kcal/mol of the original global minimum, indicating a flat potential energy surface with respect to OH rotation. The remaining two conformers (C14–OH and C27–OH rotated variants) converged to higher-energy structures (ΔE = 3.1 and 7.5 kcal/mol, respectively), with Boltzmann populations below 0.5% at 298 K. Boltzmann-weighted 13C chemical shifts were computed and compared with the single-conformer prediction. The maximum change in any individual 13C chemical shift upon Boltzmann averaging was only 0.38 ppm, and the 13C MAE remained essentially unchanged (2.41 ppm for single-conformer vs 2.39 ppm for Boltzmann-weighted). For 1H NMR, the maximum change was 0.05 ppm. These results demonstrate that conformational averaging does not significantly affect the calculated NMR chemical shifts for this compound, validating the single-conformer approach employed herein. The complete conformational analysis data are provided in the Supporting Tables (Tables S1, S2, and S3).

Structural Re-Evaluation vs the Early Report (Koiwai et al., 1979)

Koiwai et al. (1979) first isolated a nornicotine–sugar conjugate from cherry-red tobacco. The molecular formula of the present compound, C15H22N2O5, matches that reported by Koiwai et al. (1979), as supported by HRESIMS data showing a protonated molecular ion at m/z 311.1607 ([M + H]+). Combined with the identical plant source and shared nornicotine–sugar conjugate nature, the compound characterized herein is most likely the same metabolite reported in the earlier study. However, a critical difference exists in the structural assignment of the glycosyl moiety: Koiwai et al. proposed a five-membered fructofuranose ring, whereas the present study unambiguously establishes that the sugar unit exists as a six-membered pyranosyl ring, based on the following combined spectroscopic and computational evidence.

The δ 64.14 (CH2) protons show strong HMBC correlations with the adjacent nonprotonated carbon, indicating a two- or three-bond through-bond coupling within a rigid cyclic structure. For a furanose exocyclic CH2OH group, this would require undetectable four-bond or longer coupling (4J+), which is inconsistent with the intense cross-peak observed experimentally. This key HMBC evidence directly demonstrates that the methylene group is embedded within the sugar ring rather than existing as a flexible terminal hydroxymethyl side chain, thereby ruling out the five-membered fructofuranose structure proposed previously.

To further corroborate this conclusion, DFT-NMR calculations were performed on the literature-assigned five-membered furanose form and the four possible six-membered pyranose structures (β-D, β-L, α-D, and α-L) proposed herein (Table ). Linear regression analysis revealed that the five-membered furanose structure exhibited significantly poorer agreement with experimental chemical shifts (1H NMR: R 2 = 0.933, MAE = 0.45 ppm; 13C NMR: R 2 = 0.976, MAE = 5.18 ppm) with larger systematic deviations, compared not only to the best-fitting β-D pyranose isomer but also to all four six-membered pyranose configurations, thereby independently validating the spectroscopic assignment, ruling out the five-membered furanose form, and confirming the glycosyl unit as a six-membered pyranosyl ring (with β-D being the most consistent with experimental data) instead of the originally reported five-membered fructofuranose form.

The structural misassignment of the glycosyl moiety as a five-membered fructofuranose ring by Koiwai et al. primarily stems from the limitations of analytical techniques available at that time, with only 1D 1H and 13C NMR (100 MHz) employed and no 2D NMR spectroscopy (e.g., HMBC) to provide direct through-bond connectivity evidence for distinguishing ring-embedded methylene from exocyclic terminal CH2OH. Additionally, the spectroscopic characteristics of exocyclic CH2OH and ring methylene are highly similartheir 13C chemical shifts and proton coupling constants show negligible differences in 1D NMR, making unambiguous differentiation impossible by this means alone. Furthermore, the assignment relied heavily on comparison with reported spectral data of known fructofuranosides rather than direct structural evidence, with no consideration of the unique electronic and steric features of this nornicotine N-glycoside that deviates from common O-glycosides, leading to an inference based on spectral similarity rather than definitive structural proof.

Possible Biosynthetic Origin

The title compound consists of (S)-nornicotine and β-D-xylopyranose linked through a C–N–C bridge. In Nicotiana species, nornicotine is formed by demethylation of nicotine, a reaction catalyzed by the cytochrome P450 enzyme CYP82E4 (nicotine N-demethylase). This enzyme has been shown to be responsible for nornicotine accumulation in cherry-red tobacco. The sugar moiety is proposed to be derived from UDP-α-D-xylose, a common glycosyl donor in plant secondary metabolism, and the formation of the N–CH2 linkage is tentatively attributed to a UDP-glycosyltransferase (UGT) capable of N-glycosylation. To date, however, no specific UGT catalyzing nornicotine N-glycosylation has been characterized, and the proposed biosynthetic pathway remains speculative.

Experimental Section

General Experimental Procedures

Nuclear magnetic resonance (NMR) spectra (1H, 13C, DEPT, COSY, HSQC, HMBC, ROESY) were recorded on a Bruker AVANCE III 800 MHz spectrometer (Bruker BioSpin GmbH, Karlsruhe, Germany) with tetramethylsilane (TMS) as the internal standard; chemical shifts (δ) were expressed in ppm, and coupling constants (J) were given in Hz. High-resolution electrospray ionization mass spectrometry (HRESIMS) data were acquired on a Bruker Impact II mass spectrometer (Bruker BioSpin GmbH, Karlsruhe, Germany). LC-QTOF analysis was performed on a Waters Acquity UPLC system coupled to a Bruker Impact II Q-TOF mass spectrometer. Chromatographic separation was achieved using a Waters UPLC HSS T3 column (1.8 μm, 2.1 mm × 10 cm) with a mobile phase consisting of water (A) and acetonitrile (B), both containing 0.1% formic acid, at a flow rate of 0.3 mL/min. The gradient elution program was adjusted to ensure target compound separation: initial 40% A/60% B, linearly changed to 5% A/95% B within 1.00 min and held until 3.00 min, then reverted to 40% A/60% B at 3.01 min and maintained until 5.0 min for re-equilibration. The mass spectrometer was operated in positive electrospray ionization (ESI) mode, with an end plate offset of 500 V, capillary voltage of 4500 V, nebulizer gas pressure of 1.8 bar, dry gas flow of 8.0 L/min, and dry temperature of 220 °C, acquiring full scan data over 50–1300 m/z. GC-MS analysis was performed on an Agilent 8890 GC system coupled to a single quadrupole mass spectrometer. Separation was achieved using an HP-5MS UI column (30 m × 250 μm × 0.25 μm) with helium as the carrier gas at a constant flow of 1 mL/min. The oven temperature program was optimized for better separation: initial temperature 60 °C (held for 1 min), ramped at 5 °C/min to 280 °C (held for 15 min), followed by a postrun at 290 °C for 3 min. The inlet was operated at 250 °C with a split ratio of 20:1. The mass spectrometer was operated in electron ionization (EI) mode at 70 eV, with the ion source and quadrupole temperatures set at 230 and 150 °C, respectively. Full scan data were acquired over a mass range of 33–500 m/z, with a solvent delay of 7.00 min. A BUCHI Pure C-850 semipreparative chromatography system (BUCHI Labortechnik AG, Flawil, Switzerland) was used for separation, and a manually packed glass crude separation column (particle size 50 μm, size 49 mm × 460 mm) was employed. Secondary separation was performed using an Agilent semipreparative column (C18, 10 μm × 10 mm × 25 cm). All solvents used were of analytical grade (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China), and water was double distilled.

Plant Material and Sample Preparation

The leaves of cherry-red tobacco (N. tabacum L.) were collected from the Yuxi Yanhe Experimental Base of Yunnan Academy of Tobacco Agricultural Sciences in August 2024. For sample preparation, mature middle and upper tobacco leaves were baked according to the standard three-stage flue-curing procedure. After baking, tobacco leaves with obvious cherry-red spots were selected, the main veins were removed, and the leaves were crushed and sieved through a 40-mesh sieve for later use.

Extraction, Isolation and Purification

Sample Extraction: 100 g of crushed tobacco leaf powder was weighed into a 2000 mL beaker, 1000 mL of water was added to the beaker, and the mixture was subjected to ultrasonic extraction for 20 min. The extraction solution was then filtered, and the supernatant was transferred to a new beaker. Approximately 800 mL of supernatant was collected.

Crude Separation with a Glass Column: The upper cover plate of the manually packed glass crude separation column (particle size 50 μm, size 49 mm × 460 mm) was opened for sample loading, with a single sample loading volume of 100 mL. After sample loading, chromatographic separation was performed. The mobile phase consisted of methanol and water. The flow rate was 20 mL/min, and the detection wavelength of the UV detector was 200 nm. The gradient program was set as follows: 40% methanol from 0 to 15 min; methanol increased from 40% to 80% from 15 to 35 min; finally, 80% methanol was maintained for 40 min to end the separation. LC-QTOF confirmed that the target compound appeared as an obviously large peak between 55 and 64 min, and this fraction was collected. The crude separation with the glass column was performed 8 times in total, and the collected fractions were combined.

Nitrogen Blowing Concentration: All fractions obtained from the 8 runs were transferred to 50 mL centrifuge tubes, and the fractions in the centrifuge tubes were placed on a nitrogen blowing instrument to remove most of the methanol solvent by nitrogen blowing. The remaining part was diluted with water to 10 mL.

Secondary Separation with Semi-Preparative Chromatographic Column: The sample loading volume was 1 mL, the flow rate was 5 mL/min, and the mobile phase was composed of methanol and water. The gradient program was set as follows: 30% methanol from 0 to 2 min; methanol increased from 30% to 60% from 2 to 8 min; finally, 60% methanol was maintained for 17 min. This process was performed 10 times in total. LC-QTOF confirmed that the target peak appeared between 14 and 16 min, and the peak was collected and transferred to a lyophilizer for lyophilization to obtain the crude product of the title compound.

Purity Determination

Approximately 2 mg of the lyophilized sample was taken and added to 100 μL of methoxylamine pyridine solution (20 mg/mL) for oximation reaction at 37 °C for 90 min, then 80 μL of MSTFA was added for silylation reaction at 37 °C for 30 min. After the reaction, the reaction solution was transferred to a gas chromatography sampling vial for GC-MS analysis. Blank control experiments were performed simultaneously; peak areas corresponding to blank impurities were subtracted, and the purity was calculated as the ratio of the target peak area to the total peak area.

Acid Hydrolysis for Preliminary Structure Identification

The title compound (5.0 mg) was dissolved in 2 M HCl (2 mL) and refluxed at 95 °C for 2 h. The reaction mixture was neutralized with 2 M NaOH to pH 7, then the solution was lyophilized. The lyophilized hydrolysate was treated and detected using the same two-step derivatization (oximation-silylation) and GC-MS analysis method as in purity determination. The retention indices (RI) of the hydrolyzed products were calculated, and the mass spectra were compared with the NIST library for preliminary identification of the aglycone and sugar moiety.

DFT-NMR Calculations

To verify the structural correctness of the title compound, DFT-NMR calculations were performed using ORCA_6.1.1 software. The correlation coefficient (R 2) and mean absolute error (MAE) between the calculated and experimental chemical shifts were calculated using a reference method to evaluate the consistency of the structure. Four stereoisomers (β-D, β-L, α-D, α-L) of the title compound were constructed using Chem3D and preoptimized with the MM2 force field. Briefly, the initial 3D structure of each stereoisomer was constructed, and geometry optimization was carried out at the B3LYP-D3­(BJ)/6–31G­(d) − level. The NMR chemical shifts were calculated by the GIAO , -mPW1PW91/6–311+G­(2d,p)method with MeOD as the solvent, and TMS was used as the internal standard for calibration.

Spectral Data of the Title Compound

New nornicotine glycoside (the title compound): amorphous powder; 1H NMR (800 MHz, MeOD) δ 8.52 (t, J = 3.0 Hz, 1H), 8.43 (ddd, J = 17.6, 4.9, 1.6 Hz, 1H), 7.94 (tt, J = 6.4, 1.9 Hz, 1H), 7.42 (ddd, J = 16.3, 7.9, 4.8 Hz, 1H), 3.99 (dd, J = 12.5, 1.4 Hz, 1H), 3.81 (tt, J = 3.5, 1.9 Hz, 1H), 3.73 (dd, J = 9.7, 3.5 Hz, 1H), 3.69–3.63 (m, 1H), 3.63–3.57 (m, 1H), 3.55 (dd, J = 12.5, 1.9 Hz, 1H), 3.38 (d, J = 9.8 Hz, 1H), 2.98 (d, J = 12.8 Hz, 1H), 2.52–2.45 (m, 1H), 2.37 (d, J = 12.8 Hz, 1H), 2.24 (dddd, J = 12.6, 9.2, 7.6, 5.1 Hz, 1H), 2.07–1.97 (m, 1H), 1.93 (dtd, J = 12.8, 6.3, 3.4 Hz, 1H), 1.73 (dddd, J = 12.6, 10.2, 8.9, 6.3 Hz, 1H); 13C NMR (201 MHz, MeOD) δ 149.96, 149.07, 140.89, 137.60, 125.60, 98.26, 71.99, 71.51, 70.99, 69.02, 64.14, 60.06, 57.04, 35.38, 24.20; HRESIMS m/z [311.1606]+ (calcd for C15H22N2O5 + H, [311.1607]).

Supplementary Material

ao6c05861_si_001.pdf (785.5KB, pdf)
ao6c05861_si_002.pdf (255.7KB, pdf)

Acknowledgments

This research was supported by the foundations from China National Tobacco Company Yunnan branch (2023530000241028, 2025530000241023 and 2023530000241023) and Yunnan Daguan Laboratory (YNDG202302ZY03).

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.6c05861.

  • 1H NMR spectrum of the title compound (Figure S1); DEPT spectrum of the title compound (Figure S2); HSQC spectrum of the title compound (Figure S3); HMBC spectrum of the title compound (Figure S4); COSY spectrum of the title compound (Figure S5); ROESY spectrum of the title compound (Figure S6) (PDF)

  • Total energies and optimized Cartesian coordinates (Å) for the four stereoisomers of the title compound calculated at the B3LYP-D3­(BJ)/6–31G­(d) level (Table S1); per-atom deviations between experimental and calculated NMR chemical shifts for the best-fitting β-d isomer (Table S2); conformational analysis results: and energies and Boltzmann weighting of OH-rotated conformers of the β-d isomer at 298 K (Table S3) (PDF)

The authors declare no competing financial interest.

References

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