Isolation from Stevia rebaudiana of DMDP acetic acid, a novel iminosugar amino acid: synthesis and glycosidase inhibition profile of glycine and β-alanine pyrrolidine amino acids
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DMDP acetic acid [N-carboxymethyl-2,5-dideoxy-2,5-imino-d-mannitol] 5 from Stevia rebaudiana is the first isolated natural amino acid derived from iminosugars bearing an N-alkyl acid side chain; it is clear from GCMS studies that such derivatives with acetic and propionic acids are common in a broad range of plants including mulberry, Baphia, and English bluebells, but that they are very difficult to purify. Reaction of unprotected pyrrolidine iminosugars with aqueous glyoxal gives the corresponding N-acetic acids in very high yield; Michael addition of both pyrrolidine and piperidine iminosugars and that of polyhydroxylated prolines to tert-butyl acrylate give the corresponding N-propionic acids in which the amino group of β-alanine is incorporated into the heterocyclic ring. These easy syntheses allow the identification of this new class of amino acid in plant extracts and provide pure samples for biological evaluation. DMDP N-acetic and propionic acids are potent α-galactosidase inhibitors in contrast to potent β-galactosidase inhibition by DMDP.
KeywordsIminosugars Amino acid Stevia β-Galactosidase α-Galactosidase Hydroxy proline Iminosugar acid Sugar amino acids
Results and discussion
Isolation of DMDP acetic acid 5 from Stevia rebaudiana
Steviamine 21, [with the same relative pyrrolidine ring stereochemistry as galacto-DMDP 10] was the first alkaloid isolated from Stevia (Michalik et al. 2010; Thompson et al. 2009) and has shown potential anti-metastatic activity by inhibiting the hexosaminidase released by a breast cancer cell line (Ramessur et al. 2010). A number of intensely sweet glycosides of steviol are widely available commercially; yet, the iminosugars of Stevia are hitherto unreported despite their strong biological activity. The iminosugar content can be as high as 0.5% of stevia leaves and comprises mainly DMDP 1; some 2 g of DMDP along with 50 mg of DMDP acetic acid 5 were isolated from 2.2 kg of leaves from Stevia rebaudiana Betoni (Asteraceae), a herb native to South America. Many plants containing iminosugars also contain their N-acetic and N-propionic acids which have so far evaded identification and isolation due to the complex free amino acid profile of most plants (Bell 1976).
Synthesis of pyrrolidine N-acetic acids from unprotected iminosugars
Synthesis of pyrrolidine and piperidine N-propionic acids from unprotected iminosugars and polyhydroxylated amino acids
Concentration of iminosugar pyrrolidine carboxylic acids giving 50% inhibition of various glycosidases
All commercial reagents were used as supplied. Methanol was purchased dry from Alfa Aesar in sure-seal bottles. All other solvents were used as supplied (Analytical or HPLC grade), without prior purification. Melting points were recorded on a Kofler hot block and are uncorrected. Optical rotations were recorded on a Perkin-Elmer 241 polarimeter with a path length of 1 dm. Concentrations are quoted in g 100 mL−1. Infrared spectra were recorded on a Perkin-Elmer 1750 IR Fourier Transform spectrophotometer using thin films on a diamond ATR surface (thin film). Only the characteristic peaks are quoted. Low-resolution mass spectra (m/z) were recorded on an Agilent 6120 spectrometer and high-resolution mass spectra (HRMS m/z) on a Bruker microTOF mass analyzer using electrospray ionization (ESI). Nuclear magnetic resonance (NMR) spectra were recorded on Bruker AVIII 400 HD nanobay and Bruker DQX 400 spectrometers (1H: 400 MHz and 13C: 100.6 MHz) in the deuterated solvent stated. All chemical shifts (δ) are quoted in ppm and coupling constants (J) in Hz. Residual signals from the solvents were used as an internal reference, except in the case of deuterium oxide, where acetonitrile was used as the reference. The syntheses of all N-alkyl derivatives were carried out directly from their unprotected parent iminosugars and amino acids as previously prepared: 2,5-dideoxy-2,5-imino-d-mannitol (DMDP) 1 (Best et al. 2010), 2,5-dideoxy-2,5-imino-d-mannonic acid [(3S)-3-hydroxy-bulgecinine] 13 (Best et al. 2010), and deoxynojirimycin (DNJ) 2 (Best et al. 2010) and 2,5-dideoxy-2,5-iminogalactitol (galacto-DMDP) 10 from glucuronolactone (Ayers et al. 2012), 1,4-dideoxy-1,4-imino-d-arabinitol (DAB) 7 and trans,trans-dihydroxyproline 16 from diacetone glucose (Fleet and Witty 1990).
Isolation of DMDP acetic acid 5 and DMDP 1 from Stevia
Leaves of commercially supplied S. rebaudiana Betoni (Asteraceae), a herb native to South America, were used for the isolation of DMDP acetic acid 5. Stevia leaf (2.2 kg) was obtained from Cotswold Health Products UK (batch 060621) and extracted in water for 15 h. The filtrate was applied to a cation exchange column (Amberlite IR120) in the H+ form and washed with water, and then the amino acids and SAA displaced with 2 M ammonia solution. After removal of the ammonia under reduced pressure at 40 °C, the material was applied to anion exchange resin (Amberlite CG400 OH– form) and washed with water to give an unretained fraction containing DMDP 1 and the neutral and acidic compounds displaced with 1 M acetic acid. Both fractions were reduced in volume at reduced pressure and the DMDP purified using Amberlite CG50 resin in the ammonia form with 20 water fractions collected. The DMDP acetic acid 5 was purified from other amino acids by applying the CG400 retained fraction back on the CG400 in the acetate form and 20 fractions collected with water. Analysis of fractions was by GCMS of the trimethylsilylation of aliquots of the fractions. DMDP 1 gives a distinctive EI fragmentation with major ions 217 (100%), 258 (50%), and 348 (80%) 436 M+ − 15 (methyl) (2%) amu (retention time 8.7 min) and the DMDP acetic acid 5 with retention time 10.8 min has ions 147 (100%), 298 (50%), and 478 (80%), 566 M+ − 15 (methyl) (2%) amu. Mannitol as reference on the same mid-polarity column has a retention time of 10.5 min. Yields were: DMDP 1 (2 g) and DMDP acetic acid 5 (50 mg). The GCMS and NMR spectra of DMDP 1 and DMDP acetic acid 5 were identical to those of the synthetic compounds in the paper.
General procedure for pyrrolidine N-acetic acids
Glyoxal solution (40% w/w) in water (1.1 eq) was added to a stirred solution of the corresponding iminosugar (1.0 eq) in water (15 mg/mL). The mixture was stirred at 70 °C for 14–17 h, after which the reaction mixture was concentrated in vacuo. The remaining residue was loaded onto a Dowex ion-exchange resin (50WX8, H+). The column was flushed with water and then eluted with 2 M aqueous ammonia. The ammoniacal fraction was concentrated in vacuo to afford the corresponding compounds.
DMDP 1 (20 mg, 0.12 mmol) gave DMDP acetic acid 5 (27 mg, 100%), [α] D 25 + 5.9 (c 0.15, H2O); νmax (thin film) 3235 (OH), 1614, 1393 (COO); δH (D2O, 400 MHz) 3.77 (2H, m, H-2 and H-5), 4.00 (6H, m, CH2N, H1 and H6), 4.23 (2H, d, J3,2/J4,5 5.6, H3 and H4); δC (D2O, 100.6 MHz) 52.8 (CH2N), 57.2 (C1 and C6), 70.5 (C2 and C5), 74.8 (C3 and C4), 171.3 (C=O); m/z (ESI+): 222 ([M+H]+, 21%), 244 ([M+Na]+, 100%); HRMS (ESI+): found 244.0793 ([M+Na]+); C8H15NNaO6 requires 244.0792.
DAB 7 (11 mg, 0.08 mmol) gave 8 (13 mg, 82%) [α] D 25 +20.7 (c 0.47, H2O); νmax (thin film) 3234 (OH), 1614, 1396 (COO); δH (D2O, 400 MHz) 3.63 (1H, m, H2), 3.66 (1H, dd, J5,4 5.4, J5,5′ 7.0, H5), 3.83 (1H, m, H5′), 3.87 (1H, d, J1,1′ 16.0, H1), 4.01 (2H, m, CH2N), 4.07 (1H, d, J1′,1 16.0, H1′), 4.15 (1H, t, J3,4/J3,2 3.6, H3), 4.38 (1H, m, H4); δC (D2O, 100.6 MHz) 58.2, 58.4 (CH2N and C1), 60.4 (C5), 74.2, 74.4 (C2 and C4), 76.6 (C3), 170.8 (C=O); m/z (ESI+): 192 ([M+H]+, 15%), 214 ([M+Na]+, 100%); HRMS (ESI+): found 214.0687 ([M+Na]+); C7H13NNaO5 requires 214.0686.
galacto-DMDP 10 (15 mg, 0.09 mmol) gave the N-acetic acid 11 (20 mg, 99%) [α] D 25 0.0 (c 0.30, H2O); νmax (thin film): 3360 (OH), 3250 (OH), 1741 (COO); δH (D2O, 400 MHz): 3.06 (2H, ddd, J2,1 4.3, J2,3 5.3, J2,1′ 6.1, H2), 3.24 (2H, s, CH2N), 3.54 (2H, dd, J1,2 4.3, J1,1′ 11.3, H1), 3.61 (2H, dd, J1′,2 6.1, J1′,1 11.3, H1′), 4.25 (2H, d, J3,2 5.3, H3); δC (D2O, 100.6 MHz): 57.0 (CH2N), 60.2 (C1), 66.4 (C2), 72.3 (C3), 180.4 (C=O); m/z (ESI+): 222 ([M+H]+, 100%), 244 ([M+Na]+, 35%); (ESI−): 220 ([M−H]−, 100%). HRMS (ESI+): found 220.0830 ([M−H]−); C8H14NO6 requires 220.0827.
General procedure for N-propionic acids
tert-Butylacrylate (10 eq) was added to a stirred solution of the corresponding iminosugar or hydroxylated proline (1 eq) in methanol (10 mg/mL). The mixture was stirred at 50 °C for 48 h, after which the reaction was shown to be complete by mass spectrometry. The mixture was concentrated in vacuo to afford the crude ester, which was dissolved in a mixture of trifluoroacetic acid (0.5 mL) and water (0.5 mL) and stirred at room temperature. After 48 h, the solvents were removed in vacuo. The remaining residue was loaded onto a Dowex ion-exchange resin (50WX8, H+). The column was flushed with water and then eluted with 2 M aqueous ammonia. The ammoniacal fraction was concentrated in vacuo to afford the title compounds.
DMDP 1 (33 mg, 0.20 mmol) gave DMDP propionic acid 6 (48 mg, 100%) [α] D 25 − 25.2 (c 0.1, methanol); νmax (thin film) 3229 (OH), 1577, 1390 (COO); δH (D2O, 400 MHz) 2.56 (2H, m, CH2COO), 3.45 (2H, t, J 6.4, CH2N), 3.59 (2H, bs, H2 and H5), 3.93 (4H, d, J1,2/J6,5 3.2, H1 and H6), 4.13 (2H, d, J3,2/J4,5 3.2, H3 and H4); δC (D2O, 100.6 MHz) 31.7 (CH2COO), 46.7 (CH2N), 57.5 (C1 and C6), 70.4 (C2 and C5), 76.3 (C3 and C4), 179.1 (C=O); m/z (ESI+): 236 ([M+H]+, 100%), 258 ([M+Na]+, 89%), 493 ([2M+Na]+, 100%); HRMS (ESI+): found 236.1131 ([M+H]+); C9H18NO6 requires 236.1129.
DAB 1 (9 mg, 0.07 mmol) gave DAB propionic acid 9 (14 mg, 100%) [α] D 25 + 12.7 (c 0.43, water); νmax (thin film) 3239 (OH), 1579, 1388 (COO); δH (D2O, 400 MHz) 2.70 (2H, m, CH2COO), 3.40 (1H, m, CH2N), 3.54 (2H, m, H2 and H5), 3.72 (2H, m, H5′ and CH2N), 3.99 (2H, m, H1 and H1′), 4.16 (1H, m, H3), 4.37 (1H, m, H4); δC (D2O, 100.6 MHz) 31.9 (CH2COO), 53.3 (CH2N), 58.7 (C1 and C5), 74.2, 75.5, 76.6 (C2, C3, and C4), 178.0 (C=O); m/z (ESI+): 206 ([M+H]+, 92%), 228 ([M+Na]+, 100%), 433 ([2M+Na]+, 46%); HRMS (ESI+): found 228.0847 ([M+Na]+); C8H15NNaO5 requires 228.0842.
galacto-DMDP 10 (21 mg, 0.13 mmol) gave the propionic acid 12 (25 mg, 83%) [α] D 25 0.0 (c 1.1, H2O); νmax (thin film): 3451 (OH), 3343 (OH), 1738 (COO); δH (D2O, 400 MHz): 2.51–2.63 (2H, m, CH2COO), 3.00 (2H, a–q, J2,1/J2,1′/J2,3 5.1, H2), 3.25 (2H, a–t, J 6.0, CH2N), 3.48 (2H, dd, J1,2 4.5, J1,1′ 11.2, H1), 3.55 (2H, dd, J1′,2 5.8, J1′,1 11.2, H1′), 4.20 (2H, d, J3,2 5.0, H3); δC (D2O, 100.6 MHz): 33.2 (CH2COO), 48.5 (CH2N), 60.1 (C1), 66.3 (C2), 72.2 (C5), 180.4 (C=O); m/z (ESI+): 236 ([M+H]+, 100%), 258 ([M+Na+], 85%); (ESI−): 234 ([M−H]−, 100%). HRMS (ESI−): found 234.0990 [M−H]−; C9H16NO6 requires 234.0983.
N-(2-Carboxyethyl)-2,5-dideoxy-2,5-imino-d-mannonic acid 15
tert-Butylacrylate (0.25 mL, 1.7 mmol) and triethylamine (0.04 mL, 0.29 mmol) were added to a stirred solution of the iminomannonic acid 13 (30 mg, 0.17 mmol) in methanol (1.5 mL). The mixture was stirred at 50 °C for 48 h and then concentrated in vacuo. The crude was re-dissolved in a mixture of trifluoroacetic acid (1.5 mL) and water (1.5 mL), and the resulting solution was stirred at room temperature. After 48 h, the reaction was judged to be complete by mass spectrometry. The solvents were removed in vacuo and the remaining residue was loaded onto an Amberlite CG-400 strongly basic ion-exchange resin. The column was flushed with water and then eluted with 2 M acetic acid. The acidic fraction was concentrated in vacuo to afford the propionic acid 15 (42 mg, 100%). [α] D 25 − 6.7 (c 0.75, water); νmax (thin film) 3262 (OH), 1711, 1621 (COO); δH (D2O, 400 MHz) 2.90 (2H, m, CH2COO), 3.64 (1H, m, CH2N), 3.86 (1H, m, CH2N), 4.02 (1H, m, H5), 4.09 (2H, m, H6), 4.13 (1H, d, J2,3 2.8, H2), 4.28 (1H, t, J4,5/J4,3 2.8, H4), 4.33 (1H, d, J3,2/J3,4 2.8, H3); δC (D2O, 100.6 MHz) 30.3 (CH2COO), 47.3 (CH2N), 57.2 (C6), 72.3 (C5), 74.2 (C2), 76.8 (C4), 79.2 (C3), 170.8, 174.9 (C=O); m/z (ESI−): 248 ([M−H]−, 100%); HRMS (ESI+): found 272.0738 ([M+Na]+); C9H15NNaO7 requires 272.0741.
N-(2-Carboxyethyl)-2,5-dideoxy-2,5-imino-d-lyxonic acid 18
The trans,trans-dihydroxyproline 16 (33 mg, 0.22 mmol) gave the propionic acid 18 (47 mg, 100%) by the same procedure used for the synthesis of 15. [α] D 25 − 2.3 (c 0.48, water); νmax (thin film): 3367 (OH), 1623, 1707 (COO); δH (D2O, 400 MHz, HCl Salt): 2.77–2.85 (2H, m, CH2COO), 3.51–3.54 (3H, m, CH2N and H5), 3.77 (1H, d, H5′, J5′,5 12.1), 4.04 (1H, s, H2), 4.24 (1H, s, H4), 4.43 (1H, s, H3); δC (D2O, 100.6 MHz, HCl salt): 30.3 (CH2COO), 52.7 (CH2N), 60.4 (C5), 74.4 (C4), 76.2 (C2), 79.1 (C3), 174.7, 177.3 (C=O); m/z (ESI+): 220 ([M+H]+, 100%); HRMS (ESI+): found 220.0806 [M+H]+; C8H14NO6 requires 220.0816.
N-(2-carboxyethyl)-1,5-dideoxy-1,5-imino-d-glucitol (DNJ-propionic acid) 20
tert-Butyl acrylate (0.15 mL, 1.04 mmol) was added to a stirred solution of DNJ 2 (17 mg, 0.1 mmol) in methanol (1 mL). The mixture was stirred at 50 °C for 41 h after which more tert-butyl acrylate (0.15 mL) was added. After a further 24 h, the reaction mixture was concentrated in vacuo. The crude residue was re-dissolved in a mixture of trifluoroacetic acid (1 mL) and water (1 mL), and the resulting solution was stirred at rt. After 48 h, the solvents were removed in vacuo and the remaining residue was loaded onto a Dowex ion-exchange resin (50WX8 H+). The column was flushed with water and then eluted with 2 M aqueous ammonia. The ammoniacal fraction was reduced in vacuo to afford the N-propionic acid 20 as white solid (25 mg, 99%). [α] D 20 − 8.0 (c, 0.95 in water); νmax (thin film) 3206 (OH), 1563 (COO); m.p. 196–198 °C; δH (D2O, 400 MHz) 2.52 (m, 2H, CH2COO), 2.65 (t, Jgem = J1,2 = 11.6, 1H, H-1), 2.71 (m, 1H, H-5), 3.12 (m, 1H, CH2N), 3.27 (d, Jgem = 11.6, J1′,2 = 4.8, 1H, H-1′), 3.33 (m, 1H, CH2N), 3.38 (t, J3,2 = J3,4 = 9.4, 1H, H-3), 3.53 (t, J4,5 = J4,3 = 9.4, 1H, H-4), 3.66 (ddd, J2,1′ = 4.8, J2,3 = 9.4, J2,1 = 11.6, 1H, H-2), 3.95 (dd, J6,5 = 2.4, J6,6′ = 13.5, 1H, H-6), 4.00 (dd, J6′,5 = 2.7, J6′,6 = 13.5, 1H, H-6′); δC (D2O, 100.6 MHz) 31.9 (CH2COO), 49.6 (CH2N), 54.6 (C-1), 56.2 (C-6), 65.6 (C-5), 68.1 (C-2), 69.3 (C-4), 77.7 (C-3), 180.2 (C=O); m/z (ESI+): 236 ([M+H]+, 100%), 258 ([M+Na]+, 42%), 493 ([2M+Na]+, 25%); HRMS (ESI+): found 236.1137 ([M+H]+); C9H18NO6+ requires 236.1129.
Glycosidase inhibition (Kato et al. 2015)
The enzymes α-glucosidase (from rice, yeast, and A. niger), β-glucosidase (from almond, bovine liver, and A. niger), α-galactosidase (from coffee beans), β-galactosidase (from bovine liver), α-mannosidase (from jack bean), β-mannosidase (from snail), α-l-rhamnosidase (from Penicillium decumbens), α-l-fucosidase (from bovine kidney), β-glucuronidases (from E. coli and bovine liver), amyloglucosidase (A. niger, and Rhizopus sp.), p-nitrophenyl glycosides, and various disaccharides were purchased from Sigma-Aldrich Co. Brush border membranes were prepared from the rat small intestine (Kessler et al. 1978), and were assayed at pH 6.8 for rat intestinal maltase using maltose. For rat intestinal maltase activities, the reaction mixture contained 25 mM maltose and the appropriate amount of enzyme, and the incubations were performed for 10 min at 37 °C. The reaction was stopped by heating at 100 °C for 3 min. After centrifugation (600g; 10 min), the resulting reaction mixture were added to the Glucose CII-test Wako (Wako Pure Chemical Ind., Osaka, Japan). The absorbance at 505 nm was measured to determine the amount of the released d-glucose. Other glycosidase activities were determined using an appropriate p-nitrophenyl glycoside as substrate at the optimum pH of each enzyme. The reaction mixture contained 2 mM of the substrate and the appropriate amount of enzyme. The reaction was stopped by adding 400 mM Na2CO3. The released p-nitrophenol was measured spectrometrically at 400 nm.
Glycosides from leaves of S. rebaudiana are widely used as low calorie sweeteners (Samuel et al. 2018). Other beneficial properties from Stevia extracts have been noted (Chatsudthipong and Muanprasat 2009; Zangeneh et al. 2017); DMDP 1 was shown to be the major iminosugar in Stevia together with smaller quantities of the DMDP acetic acid 5. Although such polyhydroxylated amino acids are difficult to isolate, they may readily be obtained from unprotected pyrrolidine iminosugars with aqueous glyoxal as the corresponding N-acetic acids and from both pyrrolidine and piperidine iminosugars and of polyhydroxylated prolines with tert-butyl acrylate as the N-propionic acids. The syntheses will allow the easy identification of this new class of amino acid in plant extracts and provide pure sample for biological evaluation. The potent β-galactosidase inhibition by DMDP 1 was replaced by potent α-galactosidase inhibition by DMDP N-acetic 5 and propionic 6 acids; in contrast, galacto-DMDP 10 and both its acetic 11 and propionic 12 acids are α-galactosidase inhibitors.
This work was supported by Fundación Ramón Areces (RFM), a Grant-in-Aid for Scientific Research (C) from the Japanese Society for the Promotion of Science (JSPS KAKENHI Grant number JP17K08362) (AK).
Compliance with ethical standards
Conflict of interest
The authors declare that there are no conflicts of interest.
Research involving human participants and/or animals
This article does not contain any studies with human participants or animals performed by any of the authors.
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