Columns
CHIMIA 2020, 74, No. 10 829
OAc
solvents DMSO, dichloromethane, ethylacetate, acetic acid and
methanol were of analysis grade and from SigmaAldrich (Buchs,
Switzerland).NMRspectraweredoneinD O(99.8%D),obtained
from ARMAR (Döttingen/Switzerland). 25,6-Isopropylidene-l-
ascorbic acid (>99%) was bought from Fluorochem (Hadfield,
United Kingdom) and the cation-exchange resin, IR-120 (H+)
from Merck (Darmstadt, Germany). Benzyl bromide (98%),
tetra-O-acetyl-a-d-glucopyranosyl bromide (>93%), Pd/C
(10%), sodium methanolate (25 wt.% in methanol) were obtained
from Sigma Aldrich (Buchs, Switzerland). Deionized water was
used from Milli-Q purification system (Millipore AG, Zug,
Switzerland).
O
O
AcO
AcO
O
Br
H
O
O
H
H
O
BnBr (1 eq.)
K2CO3 (0.85 eq.)
O
OAc
O
O
O
(1.5 eq.)
O
O
OAc
BnO
O
NaHCO3/1M KCl
CH2Cl2, 40¡C, 48h
DMSO, 50¡C, 4h
OH
OH
HO
O
AcO
AcO
O
O
36%
Ph
59%
OAc
1
2
3
HO
HO
H
O
HO
1.) MeONa (0.4 eq.)
H
HO
1.) Pd-C (10%), H2 /EtOAc, rt, patm, 2h
2.) AcOH aq. / 60¡C, 1.5h
2.) IR-120(H+
)
O
OH
OAc
HO
O
18%
HO
78%
O
HO
HO
O
AcO
AcO
O
O
O
OH
4
5
Oxalic acid solution: 10 g/L were solubilized in water. Oxalic
acid protects the AA from oxidation but impedes 2-β-gAA
detection.
Scheme 1. Schematic presentation of the four synthesis steps.
Percentage under the reaction arrow indicates the synthesis yield of the
individual step.
2.3 Sample Homogenization
First, 1 g of rhizomes and 5 g of stems and leaves, respectively,
were weighted. Rhizomes and stems were mixed (Ultraturax
CA, USA) equipped with a precolumn (30×4.6 mm, cation H
cartridge for amino column). The mobile phase was composed of
T25, IKA, Staufen, Germany) for 2 min at 13’500 rounds/min
with 10 mL of the oxalic acid solution for AA analysis or with constant flow rate of 0.5 mL/min. The column temperature was set
to 40 °C. AA and 2-β-gAA were detected at 254 nm. The retention
5 mmol/L sulfuric acid and was delivered in an isocratic mode at a
water for 2-β-gAA analysis. To prepare the leaves, the Ultraturax
was used for 2 min at 9’500 rounds/min with 20 mL of the oxalic
acid solution or water.
time was 8.6 min for AA and 11.3 min for 2-β-gAA with a total
run time of 30 min. Chromatographic peaks were identified by
comparison of retention times and UV spectra with those obtained
for the standard AA and the synthesis product 2-β-gAA. AA was
2.4 Extraction Procedure
The method described by Toyoda-Ono et al.[12]
with
modifications proposed by Kosi ska-Cagnazzo et al.[9] was used
to extract 2-β-gAA and AA. Briefly, the homogenized sample was
ultrasonicated for 10 min at 35 kHz (VWR, Dietikon, Switzerland).
quantified by external calibration. Quantification of 2-β-gAA was
done traditionally with the AA calibration curve and a conversion
factor reported by Tai and Godha.[19]
After centrifugation for 15 min at 2800g (NUVE,Ankara, Turkey),
the supernatant was collected. To the deposit, 5 mL of oxalic acid Dry weight (dw) of the raw material was determined with
2.7 Dry Weight
solution (10 g/L oxalic acid in water) for AA extraction, or only
water (for 2-β-gAA extraction) were added and blended with a
Vortex Genius 3 (IKA, Chavannes-de-Bois, Switzerland). The
a halogen moisture analyzer (Mettler Toledo, Greifensee,
Switzerland). The temperature was set at 110 °C. All samples
were analyzed in triplicate.
plant material was extracted twice, the extracts were combined and
adjusted to 20 mL. For stem analysis, 10 mL of extract were used.
Again, the plant material was extracted twice, the extracts were
combined and adjusted to 50 mL. After filtration (0.45 µm nylon
syringe filter CHROMAFIL®, Machery-Nagel, Düren, Germany),
the extracts were directly analyzed by HPLC.
2.8 Presentation of Results
Results of dry weight,AA and 2-β-gAA are indicated as mean
± standard deviation from triplicate analysis.
3. Results and Discussion
The structure of the synthesized reference compound for the
vitamin C analog has been confirmed by NMR in D2O (4.67 ppm).
2.5 Synthesis of 2-O-β-d-Glucopyranosyl-l-ascorbic
acid
1H, 13C, COSY, HSQC and HMBC were recorded. The 1H-NMR
The total synthesis of 2-β-gAA was realized over four steps
spectrum of the 2-β-gAA exhibited two doublets on the anomeric
regions (4.88 and 4.79 ppm), one for the ascorbic acid moiety and
one for the glucose unit. All the other signals consist of typical
carbohydrate signals with the five exocyclic protons resonating
(Scheme 1). Commercially available 5,6-isopropylidene-l-
ascorbic acid was selectively benzyl protected at the 3-hydroxyl
position[12] (yield: 36%), followed by the glycosylation with tetra-
O-acetyl-a-d-glucopyranosyl bromide at the 2-hydroxyl position
between 3.55 and 4.00 ppm. The four remaining endocyclic
(yield:59%).[16] Thegenerated2-O-glucosidewasdebenzylatedby
protons are between 3.45 and 3.30 ppm. The 13C-NMR spectrum
hydrogenolysis in the presence of Pd/C and deisopropylidenated
under acidic conditions (yield: 78%).[12] After that, the acetyl
groups were removed under MeONa treatment, neutralized with
a cation-exchange resin, IR-120 (H+) and recrystallized to obtain
displayed 12 resonances for three sp2 hybridized carbons (173.7,
164.7 and 119.4), three oxymethine carbons (77.7, 70.3 and
63.3), and a glucose unit (103.5, 77.6, 76.7, 74.2, 70.5, and 61.7).
The obtained 1H and 13C-NMR spectra were like those published
some 10 mg of 2-β-gAA (yield: 18%).[17] To confirm identity of
by Toyoda-Ono et al.[12]
1
the synthesis product, H and 13C NMR in D2O were recorded
The contents of AA and 2-β-gAA of the rhizomes, stems and
leaves are summarized in the Table 1. For an easier comparison,
this table gives also the analogue’s content of goji berries. The
2-β-gAA was found in all three plant tissues of the Lycium
barbarum plant analyzed. The analogue’s content ranged from
3.34 mg/100 g dw for the leaves up to 12.6 mg/100 g dw for
with a Bruker 400 MHZ UltraShild (broad band indirect, BBI).
2.6 HPLC Analysis of Ascorbic Acid and 2-O-β-d-
Glucopyranosyl-l-ascorbic acid
AnAgilent 1220 Infinity series liquid chromatograph (Agilent
Technologies, CA, USA) comprised of an auto-sampler, a binary
pumpandaG4294BUV-DADdetector(AgilentTechnologies110 a previous study, the analogue was observed exclusively in the
Series, Agilent Technologies, CA, USA) was employed for the
berries,[12] a possible consequence of the more efficient analytical
chromatographic separation as previously described.[18] Briefly, 5
equipment used in the present study. Nevertheless, the analogue
µL were analyzed with an amino column (Aminex HPX-87H Ion
exclusion, 300×7.8 mm i.d., particle size 5 µm, Bio-Rad, Hercules,
the rhizomes with the stems containing 4.05 mg/100 g dw. In
content is much higher in goji berries ranging from 40 to 280
mg/100 g dw.[18] The highest relative standard deviation is
Amini-Rentsch, Lara
