Class of sucrose esters and a method for their preparation
11377463 · 2022-07-05
Assignee
Inventors
Cpc classification
C12P19/18
CHEMISTRY; METALLURGY
C07H1/00
CHEMISTRY; METALLURGY
International classification
Abstract
A new class of sucrose esters and a method for their preparation is described herein.
Claims
1. A method for the preparation of a β-D-fructofuranosyl-(2,1)-α-D-uronic acid or an ester thereof which comprises the step of fructosylating a D-uronic acid salt or ester thereof in the presence of B. megaterium levansucrase (Bm-Ls).
2. The method of claim 1, wherein the D-uronic acid is D-galacturonic acid or D-glucuronic acid.
3. The method of claim 1, wherein the D-uronic acid salt is an alkali metal salt.
4. The method of claim 1, wherein the ester is a residue of the formula—CO—O—R, wherein R is a hydrocarbon residue having 1 to 30 carbon atoms which may be interrupted by one or more heteroatoms and which may be substituted by one or more functional groups.
5. The method of claim 1, wherein the method is for the preparation of a β-D-fructofuranosyl-(2,1)-α-D-uronic acid ester, further wherein an esterification step is conducted prior or after the fructosylation step.
6. The method of claim 5, wherein the esterification step is conducted in the presence of tetrabutylammonium fluoride.
7. The method of claim 3, wherein the alkali metal salt is sodium salt.
Description
BRIEF DESCRIPTION OF THE FIGURES
(1) The invention is further illustrated by the figures, wherein
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EXAMPLES
(27) Chemicals were purchased from commercial sources (Sigma Aldrich, VWR Chemicals, Carbosynth) and applied without further purification. Solvents were distilled prior to use. Deuterated solvents for NMR measurements were obtained from Deutero and used as received. NMR spectra were measured on a BRUKER AVANCE 400 FT-NMR at 25° C. Proton chemical shifts (δ scale) are expressed as parts per million (ppm) and were determined relative to a residual protic solvent as an internal reference (D.sub.2O: δ=4.79 ppm, MeOD: δ=3.31 ppm). Data for .sup.1H-NMR spectra are listed as follows: chemical shift (δ ppm) (multiplicity, integration, coupling constants (Hz), assigned proton). Couplings are indicated as: d=doublet, dd=doublet of doublet, ddd=doublet of doublet of doublet, m=multiplet. .sup.13C-NMR spectra were recorded with the same BRUKER spectrometer at 100.9 MHz. Carbon chemical shifts (δ scale) are indicated in in parts per million (ppm) as well and calibrated to the carbon resonance of the respective solvent (MeOD: δ=49.00 ppm). Mass spectrometry (MS) measurements were performed on a BRUKER Daltonics autoflex//(electronspray ionization, ESI) instrument.
(28) Experimental Section
(29) Expression of B. megaterium levansucrase SacB
(30) One single colony of freshly transformed E. coli bearing the selected plasmids was used to inoculate 10 mL LB-medium containing 10 μg/mL kanamycin. Precultures were incubated over night at 37° C. and used to inoculate 250 mL LB-medium with the appropriate antibiotic. Expression of the levansucrase SacB was induced when cells reached an OD.sub.600 of around 0.6 by adding IPTG (isopropyl-β-D-thiogalactoside) at a final concentration of 0.5 mM. Cultures were incubated over night at 20° C. Cells were harvested by centrifugation and resuspended in 7 mL of 50 mM phosphate buffer pH 6.6. After sonication, the extracts were cleared by centrifugation at 13000×g.
(31) Activity Assays of SacB
(32) For the DNS assay, FTs containing 0.5 M sucrose solutions were incubated in 50 mm phosphate buffer pH 6.6 in a total volume of 500 μL at 650 rpm (Thermomixer compact, Eppendorf, Germany). Five samples of 70 μL each were taken at different time points after the reaction was started (0, 2, 4, 6 and 8) and mixed with 70 μL DNS. Samples were heated at 95° C. for 5 min and cooled down for 2 min at 4° C. For quantification, samples were diluted 1:6 with water and the absorbance measured at 540 nm. Data was processed using a calibration curve of absorbance versus glucose concentration and the change in absorbance (slope) was calculated.
(33) Synthesis of D-glucuronic acid benzyl ester (5b)
(34) ##STR00004##
(35) D-glucuronic acid (4.00 g, 20.6 mmol) was dissolved in DMF (20 mL) and 1 M TBAF added (22 mL of 1 M solution in THF) at 0° C. Benzylbromide (3.52 g, 2.44 mL, 20.6 mmol) was dropped slowly to the reaction mixture at 0° C. and stirred at room temperature for a further 18 h. The solvent was removed under reduced pressure. The residue was purified via column chromatography over silica gel (CH.sub.2Cl.sub.2/MeOH 4:1) to yield ester 5b as colorless solid (4.21 g, 14.8 mmol, 72%). R.sub.f: 0.50 (CH.sub.2Cl.sub.2/MeOH 4:1). .sup.1H-NMR (400 MHz, D.sub.2O/MeOD): δ=7.48-7.37 (m, 10 H, H—Ar, α/β), 5.28-5.25 (m, 4 H, H-7, α/ϵ), 5.24 (d, 1 H, .sup.3J=3.9 Hz, H-1-α), 4.67 (d, 1 H, .sup.3J=8.1 Hz, H-1β), 4.39 (d, 1 H, .sup.3J=10.1 Hz, H-5-α), 4.11 (d, 1 H, .sup.3J=9.8 Hz, H-5-β), 3.71 (dd, 1 H, .sup.3J=9.4 Hz, .sup.3J=9.4 Hz, H-3-α), 3.57 (dd, 1 H, .sup.3J=10.1 Hz, .sup.3J=9.4 Hz, H-4-α), 3.55 (dd, 1 H, .sup.3J=9.4 Hz, .sup.3J=3.9 Hz, H-2-α), 3.54 (dd, 1 H, .sup.3J=9.8 Hz, .sup.3J=9.2 Hz, H-4-β), 3.49 (dd, 1 H, .sup.3J=9.2 Hz, .sup.3J=9.2 Hz, H-3-β), 3.28 (dd, 1 H, .sup.3J=8.1 Hz, .sup.3J=9.2 Hz, H-2-β) ppm. .sup.13C-NMR (101 MHz, D.sub.2O/MeOD): δ=171.2 (COO-β), 170.3 (COO-α), 134.9 (2×Cq-Ar), 128.8 (3 C, 3×CH—Ar), 128.7 (2 C, 2×CH—Ar), 128.3 (4 C, 4×CH—Ar), 127.5 (CH—Ar), 96.2 (C-1-1-β), 92.4 (C-1-α), 75.2 (C-3-β), 74.7 (C-5-β), 73.6 (C-2-β), 72.3 (C-3-α), 71.5 (C-4-α), 71.3 (C-4-β), 70.9 (C-2-α), 70.7 (C-5-α), 67.9 (2 C, OCH.sub.2, α/β) ppm. HRMS-ESI (+), m/z): 307.07882 [M+Na].sup.+, calcd. for C.sub.13H.sub.16O.sub.7Na.sup.+307.07866.
(36) Synthesis of D-glucuronic acid iso-propylic ester (5c)
(37) ##STR00005##
(38) D-glucuronic acid (4.00 g, 20.6 mmol) and Ag.sub.2CO.sub.3 (2.84 g, 10.3 mmol) were dissolved in 40 mL H.sub.2O and 40 mL MeOH and the mixture was stirred at room temperature for 1 h. The solvents were evaporated under reduced pressure and the resulting grey solid was dissolved in 100 mL DMF at 40° C. 2-lodopropane (17.5 g, 10.3 mL, 103 mmol) was added and the reaction mixture was stirred for 4 h at 40° C. The resulting suspension was filtrated and the solvent of the filtrate was removed under reduced pressure. The residue was purified via column chromatography (SiO.sub.2, CH.sub.2Cl.sub.2/MeOH 4:1) to achieve the ester 5c 2.39 g, 10.1 mmol, 49%) as a colorless solid. R.sub.f: 0.32 (CH.sub.2Cl.sub.2/MeOH 4:1). .sup.1H-NMR (400 MHz, D.sub.2O/MeOD): δ=5.25 (d, 1 H, .sup.3J=3.7 Hz, H-1-α), 5.14-5.04 (m, 2 H, H-7, α/β), 4.67 (d, 1 H, .sup.3J=8.0 Hz, H-113), 4.30 (d, 1 H, .sup.3J=9.6 Hz, H-5-α), 3.98 (d, 1 H, .sup.3J=9.6 Hz, H-5β), 3.71 (dd, 1 H, .sup.3J=9.4 Hz, .sup.3J=9.4 Hz, H-3-α), 3.56 (dd, 1 H, .sup.3J=9.4 Hz, .sup.3J=3.7 Hz, H-2-α), 3.55 (dd, 1 H, .sup.3J=9.6 Hz, .sup.3J=9.4 Hz, H-4-α), 3.52 (dd, 1 H, .sup.3J=9.6 Hz, .sup.3J=9.2 Hz, H-4β), 3.49 (dd, 1 H, .sup.3J=9.2 Hz, .sup.3J=9.2 Hz, H-3β), 3.28 (dd, 1 H, .sup.3J=9.2 Hz, .sup.3J=8.0 Hz, H-2β), 1.30-1.24 (m, 12 H, 4×CH.sub.3, α/β) ppm. H-3-C-NMR (101 MHz, D.sub.2O/MeOD): δ=171.0 (COO-β), 170.1 (COO-α), 96.2 (C-1-β), 92.4 (C-1-α), 75.2 (C-3-β), 74.8 (C-5-β), 73.6 (C-2β), 72.3 (C-3-α), 71.5 (C-4-β), 71.3 (2 C, C-7, α/β), 71.2 (C-4-α), 71.0 (C-2-α), 70.8 (C-5-α), 20.7 (4 C, 4×CCH.sub.3, α/β) ppm.
(39) Synthesis of β-D-fructofuranosyl-α-D-glucuronic acid benzyl ester (2b)
(40) ##STR00006##
(41) To a solution of D-glucuronic benzyl ester (D-GIcABn) (3.64 g, 12.8 mmol) in Sorensen buffer pH 6.6 (128 μL, 1 M) and sucrose (2.19 g, 6.40 mmol in 2.56 mL H.sub.2O Bm-Ls (final activity 4 U/mL) was added and reacted at 37° C. for 1.5 h. The solvents were evaporated under reduced pressure. The residue was purified via column chromatography (silica-gel, EtOAc/iPrOH/H.sub.2O 6:3:1) to obtain the benzyl ester 2b as a colorless solid (1.29 g, 2.88 mmol, 45%). R.sub.f: 0.45 (EtOAc/iPrOH/H.sub.2O 6:3:1). .sup.1H-NMR (400 MHz, D.sub.2O/MeOD): δ=7.48-7.37 (m, 5 H, H—Ar), 5.40 (d, 1H, .sup.3J=3.9 Hz, H-1), 5.29 (m, 2 H, H-8), 4.40 (d, 1 H, .sup.3J=9.9 Hz, H-5), 4.18 (d, 1 H, .sup.3J=8.7 Hz, H-3′), 3.91 (dd, 1 H, .sup.3J=8.7 Hz, .sup.3J=8.7 Hz, H-4′), 3.80 (m, 1 H, H-5′), 3.75 (dd, 1 H, .sup.3J=9.5 Hz, .sup.3J=9.5 Hz, H-3), 3.62-3.57 (m, 4 H, H-2, H-4, 2×H-1′), 3.46 (m, 2 H, H-6′) ppm. .sup.13C-NMR (101 MHz, D.sub.2O/MeOD): δ=170.7 (COO), 134.9 (1-C, Cq-Ar), 128.8 (3 C, CH—Ar), 126.5 (2 C, CH—Ar), 103.8 (C-2′), 92.2 (C-1), 81.5 (C-5′), 76.0 (C-3′), 73.8 (C-4′), 72.2 (C-3), 71.5 (C-5), 71.2 (C-4), 70.6 (C-2), 68.0 (C-7), 62.2 (C-6′), 60.9 (C-1′) ppm. HRMS-ESI (+), m/z): 469.13165 [M+Na].sup.+ calcd. for C.sub.19H.sub.26O.sub.12Na.sup.+469.13099.
(42) Synthesis of β-D-fructofuranosyl-α-D-glucuronic acid iso-propyl ester (2c)
(43) ##STR00007##
(44) To a solution of D-glucuronic iso-propyl ester (D-GIcAiPr) (0.52 g, 2.20 mmol) in Sörensen buffer pH 6.6 (22 μL, 1 M) and sucrose (0.38 g, 1.10 mmol in 433 μL H.sub.2O) Bm-Ls (final activity 4 U/mL) was added and reacted at 37° C. for 1.5 h. The solvents were evaporated under reduced pressure. The residue was purified via column chromatography (silica-gel, EtOAc/iPrOH/H.sub.2O 6:3:1) to obtain the iso-propyl ester 2c as a colorless solid (0.32 g, 577 μmol, 52%). R.sub.f: 0.40 (EtOAc/iPrOH/H.sub.2O 6:3:1). .sup.1H-NMR (400 MHz, D.sub.2O/MeOD): δ=5.42 (d, 1 H, .sup.3J=3.8 Hz, H-1), 5.12 (m, 1 H, H-7,), 4.31 (d, 1 H, .sup.3J=9.9 Hz, H-5), 4.21 (d, 1 H, .sup.3J=8.7 Hz, H-3′), 3.99 (dd, 1 H, .sup.3J=8.7 Hz, .sup.3J=8.7 Hz, H-4′), 3.87 (ddd, 1 H, .sup.3J=8.7 Hz, .sup.3J=7.5 Hz, .sup.3J=2.7 Hz, H-5′), 3.77-3.72 (m, 1 H, H-1′), 3.74 (dd, 1 H, .sup.3J=9.9 Hz, .sup.3J=9.3 Hz, H-3), 3.63 (m, 3 H, H-1′, 2×H-6′), 3.59 (dd, 1 H, .sup.3J=9.9 Hz, .sup.3J=3.8 Hz, H-2), 3.56 (dd, 1 H, .sup.3J=9.9 Hz, .sup.3J=9.3 Hz, H-4), 1.30-1.27 (m, 6 H, 2×CH.sub.3) ppm. .sup.13C-NMR (101 MHz, D.sub.2O/MeOD): 5=170.5 (COO), 103.9 (C-2′), 92.3 (C-1), 81.5 (C-5′), 76.0 (C-3′), 74.0 (C-4′), 72.3 (C-3), 71.7 (C-5), 71.4 (CCH.sub.3), 71.2 (C-4), 70.5 (C-2), 62.3 (C-1′), 60.8 (C-6′), 20.5 (2 C, CCH.sub.3) ppm. HRMS-ESI (+), m/z): 421.13165 [M+Na].sup.+ calcd. for C.sub.15H.sub.26O.sub.12Na.sup.+ 421.13160.
(45) Synthesis of β-D-fructofuranosyl-α-D-glucuronic acid (2a)
(46) ##STR00008##
(47) A mixture of β-D-fructofuranosyl-α-D-glucuronic iso-propyl ester (100 mg, 251 μmol) and 10 mL of a 0.5 M NaOH solution was stirred at room temperature for 1 h. DOWEX cation exchanger was added to the solution and stirred until pH 7 was reached. The solvents were evaporated under reduced pressure to obtain the sucrose acid 2a as a colorless solid (85.0 mg, 239 μmol, 95%). .sup.1H-NMR (400 MHz, D.sub.2O/MeOD): 5=5.40 (d, 1 H, .sup.3J=3.9 Hz, H-1) 4.21 (d, 1 H, .sup.3J=9.9 Hz, H-5), 4.19 (d, 1 H, .sup.3J=8.7 Hz, H-3′), 4.03 (dd, 1 H, .sup.3J=8.7 Hz, .sup.3J=8.7 Hz, H-4′), 3.86 (ddd, 1 H, .sup.3J=8.7 Hz, .sup.3J=7.2 Hz, .sup.3J=2.7 Hz, H-5′), 3.78-3.70 (m, 2 H, H-1′, H-3), 3.68-3.56 (m, 4 H, H-1′, 2×H-6′, H-2), 3.51 (dd, 1 H, .sup.3J=9.9 Hz, .sup.3J=9.6 Hz, H-4) ppm. .sup.13C-NMR (101 MHz, D.sub.2O/MeOD): δ=174.5 (COO), 103.7 (C-2′), 92.1 (C-1), 81.4 (C-5′), 76.0 (C-3′), 73.7 (C-4′), 72.3 (C-3), 72.2 (C-5), 71.6 (C-4), 70.7 (C-2), 62.1 (C-6′), 60.8 (C-1′) ppm.
(48) Synthesis of D-galacturonic acid benzyl ester (6b)
(49) ##STR00009##
(50) D-galacturonic acid monohydrate (3.00 g, 14.1 mmol) was solved 48 mL DMF with molecular sieve 3 Å and 22 mL 1 M TBAF in THF was added at room temperature. Benzylbromide (2.41 g, 1.67 mL, 14.1 mmol) was added slowly at 0° C. and the reaction mixture was stirred for 72 h at room temperature. The solvent was removed under reduced pressure. The residue was purified via column chromatography over silica gel (CH.sub.2Cl.sub.2/MeOH 4:1) to yield ester 6b as colorless solid (2.52 g, 8.88 mmol, 63%). R.sub.f: 0.47 (CH.sub.2Cl.sub.2/MeOH 4:1). .sup.1H-NMR (400 MHz, D.sub.2O/MeOD): δ=7.46-7.40 (m, 10 H, H—Ar α/β), 5.30 (d, 1 H, .sup.3J=3.9 Hz, H-1-α) 5.27-5.24 (m, 4 H, OCH.sub.2 α/β), 4.78 (m, 1 H, H-5-α), 4.58 (d, 1 H, .sup.3J=7.9 Hz, H-1-β), 4.45 (d, 1 H, .sup.3J=1.4 Hz, H-5-β), 4.31 (dd, 1 H, .sup.3J=3.4 Hz, .sup.3J=1.5 Hz, H-4-α), 4.24 (dd, 1 H, .sup.3J=3.5 Hz, .sup.3J=1.4 Hz, H-4-β), 3.89 (dd, 1 H, .sup.3J=10.2 Hz, .sup.3J=3.4 Hz, H-3-α), 3.79 (dd, 1 H, .sup.3J=10.2 Hz, .sup.3J=3.9 Hz, H-2-α), 3.67 (dd, 1 H, .sup.3J=9.9 Hz, .sup.3J=3.5 Hz, H-3-β), 3.49 (dd, 1 H, .sup.3J=9.9 Hz, .sup.3J=7.9 Hz, H-2-β) ppm. .sup.13C-NMR (101 MHz, D.sub.2O/MeOD): δ=170.7 (COO-β), 169.7 (COO-α), 135.0 (2 C, 2×Cq-Ar), 128.8 (4 C, 4×CH—Ar), 128.7 (2 C, 2×CH—Ar), 128.3 (4 C, 4×CH—Ar), 96.2 (C-1-β), 92.3 (C-1-α), 74.1 (C-3-β), 72.2 (C-5-β), 71.1 (C-2-β), 70.4 (C-3-α), 70.1 (C-4-α), 69.6 (C-4-β), 68.5 (C-2-α), 67.7 (C-5-α), 67.6 (2 C, OCH.sub.2, α/β) ppm.
(51) Synthesis of β-D-fructofuranosyl-α-D-galacturonic acid benzyl ester (3b)
(52) ##STR00010##
(53) To a solution of D-galacturonic benzyl ester (D-GalABn) (200 mg, 704 μmol) in Sorensen buffer (400 μL, 1 M) and sucrose (120 mg, 252 μmol in 8.00 mL H.sub.2O) Bm-Ls (final activity 4 U/mL) was added and reacted at 37° C. for 1.5 h. The solvents were evaporated under reduced pressure. The residue was purified via column chromatography (silica-gel, CH.sub.2Cl.sub.2/MeOH 4:1) to obtain the benzyl ester 3b as a colorless solid (46.0 mg, 103 μmol, 41%). R.sub.f: 0.26 (CH.sub.2Cl.sub.2/MeOH 4:1). .sup.1H-NMR (400 MHz, D.sub.2O/MeOD): δ=7.46-7.41 (m, 5 H, H—Ar), 5.46 (d, 1 H, .sup.3J=3.9 Hz, H-1), 5.30-5.26 (m, 2 H, OCH.sub.2), 4.85 (d, 1 H, .sup.3J=1.5 Hz, H-5), 4.35 (dd, 1 Hz, H-4) 4.18 (d, 1 H, .sup.3J=8.7 Hz, H-3′), 3.96 (dd, 1 H, .sup.3J=10.2 Hz, .sup.3J=3.4 Hz, H-3), 3.92 (dd, 1 H, .sup.3J=8.7 Hz, .sup.3J=8.7 Hz, H-4′), 3.86-3.81 (m, 2 H, H-2, H-5′), 3.64 (s, 2 H, 2×H-1′), 3.60-3.56 (m, 2 H, 2×H-6′) ppm. .sup.13C-NMR (101 MHz, D.sub.2O/MeOD): δ=170.2 (COO), 135.1 (1-C, Cq-Ar), 128.8 (2 C, CH—Ar), 128.7 (1 C, CH—Ar) 128.4 (2 C, CH—Ar), 103.6 (C-2′), 92.3 (C-1), 81.3 (C-5′), 76.2 (C-3′), 73.7 (C-4′), 71.3 (C-3), 69.9 (C-5), 68.4 (C-4), 67.7 (C-2), 67.3 (C-7), 61.9 (C-6′), 60.9 (C-1′) ppm.
(54) Synthesis of β-D-fructofuranosyl-α-D-galacturonic acid (3a)
(55) ##STR00011##
(56) A mixture of β-D-fructofuranosyl-α-D-galacturonic benzylic ester (46 mg, 103 μmol) and 5 mL of a 0.5 M NaOH solution was stirred at room temperature for 1 h. DOWEX anion exchanger was added to the solution 3 h at room temperature. The solvents were removed under reduced pressure to obtain the sucrose acid 3a as a colorless solid (35.3 mg, 99 μmol, 96%). .sup.1H-NMR (400 MHz, D.sub.2O/MeOD): δ=5.35 (d, 1 H, .sup.3J=4.0 Hz, H-1), 4.46 (d, 1 H, .sup.3J=1.5 Hz, H-5), 4.18 (dd, 1 H, .sup.3J=3.4 Hz, .sup.3J=1.5 Hz, H-4), 4.09 (d, 1 H, .sup.3J=3.4 Hz, .sup.3J=8.8 Hz, H-3′), 3.90 (dd, 1 H, .sup.3J=8.8 Hz, .sup.3J=8.8 Hz, H-4′), 3.88 (dd, 1 H, .sup.3J=10.4 Hz, .sup.3J=3.4 Hz, H-3), 3.78-3.71 (m, 2 H, H-2, H-6′), 3.65 (dd, 1 H, .sup.2J=12.6 Hz, .sup.3J=2.9 Hz, H-6′), 3.61-3.55 (m, 3 H, H-5′, 2×H-1′) ppm. .sup.13C-NMR (101 MHz, D.sub.2O/MeOD): 5=174.2 (COO), 103.6 (C-2′), 92.4 (C-1), 81.3 (C-5′), 76.3 (C-3′), 73.7 (2 C, C-4′, C-3), 70.4 (C-5), 68.9 (C-4), 67.5 (C-2), 61.8 (C-6′), 60.8 (C-1′) ppm.
(57) 6-O-Toluolsulfonyl-D-glucopyranose (400 MHz, D.sub.2O/MeOD): δ=7.81-7.77 (m, 4 H, H.sub.arom-Ts), 7.44-7.42 (m. 4 H, H.sub.arom-Ts), 5.00 (d, J=3.68 Hz, 1 H, H1α), 4.42 (d, J=7.80 Hz, 1 H, H1β), 4.28 (m, 2 H, H6α, H6β), 4.16 (m, 2 H, H6α, H6β), 4.16 (dd, J=5.42, 10.58 Hz, 1 H, H6α), 4.11 (dd, J=6.16, 10.56 Hz, 1 H, H6β), 3.90 (ddd, J=2.03, 5.37, 10.05 Hz, 1 H, H5α), 3.61 (t, J=9.28 Hz, 1 H, H3α), 3.43 (ddd, J=1.97, 6.19, 9.73 Hz, 1 H, H5β), 3.28-3.25 (m, J=15.97 Hz, 2 H, H3α, H2β), 3.22-3.17 (m, 2 H), 3.08 (dd, J=7.84, 9.16 Hz, 1 H, H2β), 2.45 (s, 1 H, H-Me an Ts) ppm. .sup.13C-NMR (101 MHz, D.sub.2O/MeOD): δ=149.99 (1 C, Carom-Ts), 146.51 (2 C, Carom-Ts), 138.76 (1 C, Carom-Ts), 134.47 (2 C, Carom-Ts), 131.05 (2 C, Carom-Ts), 129.94 (1 C, Carom-Ts), 129.20 (1 C, Carom-Ts), 127.07 (1 C, Carom-Ts), 125.75 (1 C, Carom-Ts), 98.28 (1 C, 1β), 94.02 (1 C, C1α), 77.99 (1 C, C2α), 76.13 (1 C, C2α), 75.20 (1 C, C5α), 74.81 (1 C, C3α), 73.68 (1 C, C3β), 71.52 (2 C, C6α, C6β), 71.35 (2 C, C4α, C4β), 71.18 (1 C, 5α), 21.65 (2 C, C-Me an Ts) ppm.
(58) Synthesis of 6-O-Toluolsulfonyl-D-glucopyranosyl-β-D-fructofuranoside
(59) 6-O-Toluolsulfonyl-D-glucopyranose (100 mg, 0.23 mmol) and sucrose (6.14 g, 4.80 mmol) are solved in 1 M Sorensen-buffer (5 mL, pH 6.6). The enzyme is added (SacB, 2U). The reaction mixture can be stirred for 1 h at 37° C. The reaction will be stopped by adding 5 mL methanol. The product will be isolated over silica gel with an eluent (water/isopropanol/acidic acid ethylester=1:3:6). 1H-NMR (400 MHz, MeOD):
(60) δ=7.81 (d, J=1.24, 7.15 Hz, 2 H, Harom-Ts), 7.45 (J=8.80 Hz, 2 H, Harom-Ts), 5.28 (d, J=3.76 Hz, 1 H, H1), 4.28 (dd, J=1.91, 10.64 Hz, 1 H, H6), 4.14 (dd, J=10.75, 5.17 Hz, 1 H, H6), 4.06 (d, J=8.26 Hz, 1 H, H3′), 3.98 (ddd, J=1.88, 5.07, 10.14 Hz, 1 H, H5), 3.91 (t, J=8.06 Hz, 1 H, H4′),3.80-3.66 (m, 4 H, 5 H, H5′, H6′, H3), 3.56 (m, J=8.8 Hz, 2 H, H1′, H2′), 3.33 (m, 1 H, H2), 3.22 (dd, J=8.93, 10.28 Hz, 1 H, H4), 2.46 (s,3 H, H-Me an Ts) ppm.
(61) 13C NMR (100-MHz, MeOD):
(62) δ=146.62 (1 C, Carom-Ts), 134.30 (1 C, Carom-Ts), 131.13 (2 C, Carom-Ts), 129.30 (2 C, Carom-Ts), 105.28 (1 C, C2′), 93.40 (1 C, C1), 84.14 (1 C, C5′), 79.29 (1 C, C3′), 76.05 (1 C, C4′), 74.60 (1 C, C3), 73.10 (1 C, C2), 71.90 (1 C, C5), 71.19 (1 C, C4), 70.83 (1 C, C6), 64.28-64.16 (2 C, C1′, C6′), 21.69 (1 C, C-Me an Ts) ppm.
(63) Synthesis of 6-Azido-6-desoxy-D-glucopyranosyl-β-D-fructofuranoside 17.5 mg (35.2 μmol) of 6-O-Toluolsulfonyl-D-glucopyranosyl-β-D-fructofuranoside are solved in DMF (2 mL) and 9.02 mg (0.139 mmol) sodium azide added. The mixture is stirred 96 h at 120° C. Isolation of the product can be done with silica gel and an eluent (water/isopropanol/acidic acid ethylester=1:3:6) and 0.5% triethylamine. 1H-NMR (400 MHz, MeOD): δ=5.40 (d, J=3.96 Hz, 1 H, H1), 4.10 (d, J=8.33 Hz, 1 H, H3′), 4.01 (t, J=8.09 Hz, 1 H, H4′), 3.96 (ddd, J=2.47, 4.75, 9.89 Hz, 1 H, H5), 3.81-3.75 (m, 3 H, H5′, H6′), 3.69 (t, J=9.47 Hz, 1 H, H3), 3.45-3.40 (m, 2 H, H2, H6) 3.34 (s, 1 H, H4) ppm.13C-NMR (100 MHz, MeOD):
(64) δ=105.44 (1 C, C2′), 93.67 (1 C, C1), 83.99 (1 C, C5′), 79.26 (1 C, C3′), 75.96 (1 C, C4′), 74.56 (1 C, C3), 73.32 (1 C, C4), 73.20 (1 C, C2), 72.16 (1 C, C5), 64.09-63.89 (2 C, C1, C6′), 52.78 (1 C, C6) ppm.