Efficient chemoenzymatic synthesis of 4-nitrophenyl β-d-apiofuranoside and its use in screening of β-d-apiofuranosidases

Article abstract of DOI:10.1016/j.carres.2016.04.030

4-Nitrophenyl β-d-apiofuranoside as a chromogenic probe for detection of β-d-apiofuranosidase activity was prepared in 61% yield from 2,3-isopropylidene-α,β-d-apiofuranose through a sequence of five reactions. The synthesis involves one regioselective enz

Full text of DOI:10.1016/j.carres.2016.04.030

Carbohydrate Research 430 (2016) 48–53
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Efficient chemoenzymatic synthesis of 4-nitrophenyl β-d-
apiofuranoside and its use in screening of β-d-apiofuranosidases
Peter Kis, Elena Potocká, Vladimír Mastihuba, Mária Mastihubová *
Laboratory of Biocatalysis and Organic Synthesis, Institute of Chemistry, Slovak Academy of Sciences, SK-845 38 Bratislava, Slovakia
A R T I C L E
I N F O
A B S T R A C T
Article history:
Received 9 March 2016
Received in revised form 29 April 2016
Accepted 30 April 2016
Available online 3 May 2016
4-Nitrophenyl β-d-apiofuranoside as a chromogenic probe for detection of β-d-apiofuranosidase activ-
ity was prepared in 61% yield from 2,3-isopropylidene-α,β-d-apiofuranose through a sequence of five
reactions. The synthesis involves one regioselective enzymatic step—benzoylation of primary hydroxyl
of 2,3-isopropylidene-α,β-d-apiofuranose catalysed by Lipolase 100T and stereoselective β-d-
apiofuranosylation of p-nitrophenol using BF3·OEt2/Et3N. The product was used for screening of β-d-
apiofuranosidase activity in 61 samples of crude commercial enzymes and plant materials. Fifteen enzyme
preparations originating from different strains of genera Aspergillus display β-d-apiofuranosidase activ-
ity. The highest activity was found in Rapidase AR 2000 (78.27 U/g) and lyophilized Viscozyme L (64,36 U/g).
© 2016 Elsevier Ltd. All rights reserved.
Keywords:
4-Nitrophenyl β-d-apiofuranoside
β-d-Apiofuranosidase
Aspergillus
1. Introduction
d-Apiofuranose (2-C-(hydroxymethyl)-d-erythrofuranose) is a
branched monosaccharide relatively abundant in the plant kingdom.
It is a natural component of rhamnogalacturonan II as a binding
centre for borate ester bridge between two rhamnogalacturonan II
chains.1–4 d-Apiose also occurs as a structural constituent of plant
defence substances,5 and of different phenylpropanoid glycosides
in medicinal plants such as isoliquiritin apioside,6 luteosides,7
kelampayosides8 or cucurbitosides.9,10
β-d-Apiofuranosidases (apiosidases) are glycosidases cleaving β-d-
apiofuranose residues from the side-chains of rhamnogalacturonan
II, or from low molecular plant metabolites. They find practical use
for example in wine technology as side-activity of industrial
pectinases releasing volatile aroma substances from their glyco-
sidic precursors.11,12 Due to their relatively marginal role in food and
beverage technologies, only a limited number of studies of
apiosidases appeared, mostly from the laboratory of Günata,11,13,14
who used 4-nitrophenyl β-d-apiofuranoside (1, Fig. 1) as a chro-
mogenic probe for their assaying. They tested occurrence of this
specific glycosidase in only a limited number of commercial enzyme
preparations. Production of several of these enzyme preparations
was abandoned or they have been renamed. For example, Günata
et al. isolated this enzyme from Klerzyme 200, which is now pro-
duced under the label Rapidase AR-2000 (DSM, USA).
Fig. 1. Structure of 4-nitrophenyl β-d-apiofuranoside.
Our interest in straightforward synthesis of biologically active
phenylpropanoid substances harbouring β-apiosyl moiety in the
glycon part led us to an idea of enzymatic apiosylation of precur-
sors of the target molecules. Since no pure apiosidase is in the
market, extensive search for an inexpensive, easily available source
of this enzyme has been started to provide it in reasonable amounts
and costs without the necessity of microbial screenings, cultiva-
tions and enzyme preconcentration or purification. Our laboratory
has a long-lasting experience in screening of interesting enzyme side-
activities in present-day crude industrial enzymes or various plant
materials and their use in synthetic procedures.15–22 The purpose
of this report is therefore to present our experience with screen-
ing of these crude enzyme materials for presence of β-apiosidase,
including a new way of synthesis of its chromogenic substrate and
modification of the current spectrophotometric method.
2. Results and discussion
2.1. Synthesis of chromogenic substrate
*
Corresponding author. Institute of Chemistry, Slovak Academy of Sciences,
Dúbravská cesta 9, SK-845 38 Bratislava, Slovakia. Tel.: +4212 59410 246; fax: +4212
59410 222.
Synthesis of 4-nitrophenyl β-d-apiofuranoside 1 has been re-
E-mail address: chemjama@savba.sk (M. Mastihubová).
ported as early as in 1992, but no preparative details were given
http://dx.doi.org/10.1016/j.carres.2016.04.030
0008-6215/© 2016 Elsevier Ltd. All rights reserved.

P. Kis et al. / Carbohydrate Research 430 (2016) 48–53
49
Scheme 1. Synthesis of β-d-apioside 1 from isopropylidenated apiose 2.
other than a very general methodology.14 The authors glycosylated
4-nitrophenol by per-O-acetylated d-apiose23,24 under catalysis with
p-toluenesulfonic acid. A mixture of α- and β-product (12% and
59%, respectively) was obtained and separated by column
chromatography.
We have developed an effective, simple and selective multigram
synthesis of 4-nitrophenyl β-d-apiofuranoside 1 from stable and crys-
talline 2,3-O-isopropylidene-α,β-d-apiofuranose (2, Scheme 1).
Obviously, 2,3-O-isopropylidene 2 has been already synthe-
sized from d-mannose,23–25 d-xylose,26 L-arabinose27 as starting
compounds and its benzylidene analogue has been prepared from
L-ribose.10 We chose synthesis of 2 from d-mannose as the most
convenient and verified way for preparative purposes, following Ho23
and Hammerschmidt et al.25
The necessity of strongly acidic conditions for removal of stable
2,3-O-isopropylidene group of the apiofuranose ring limits usabil-
ity of 2 for synthesis of apiofuranosides.4,28 The transformation of
2 to peracylated apiofuranose derivative was therefore required.
Compound 2 was firstly acetylated, but acetyl function at primary
hydroxyl was rather unstable during removal of 2,3-O-isopropylidene
moiety. Previous works24,29 also reported moderate yields of this re-
action (42% and 30%, respectively). We have therefore decided to
introduce more stable benzoyl group to preserve the erythro-
configuration of apiose after removal of isopropylidene (Scheme 1).
Authors in the previous work25 benzoylated 2 at low temperature
from −78 °C to −65 °C for 16 hours using benzoyl chloride and
pyridine in dichloromethane and obtained 3 in 71% yield.
Deisopropylinated product 4 was acetylated without previous pu-
rification and 5 was obtained in 79% yield.
Regioselective enzymatic acylation by commercial lipases is be-
coming a common method in protection strategies of sugars.30,31 After
short screening of available lipases and reaction conditions, the
benzoylation of 2 by Lipolase 100T19 with vinyl benzoate in toluene
was found as an appropriate method for preparative purposes. Com-
pound 3 was isolated in 95% yield. After removal of 2,3-O-
isopropylidene group from 3 by 80% formic acid, product 4 was
obtained in 93% yield and conventionally acetylated in pyridine
catalysed with 4-N,N-dimethylaminopyridine. We obtained
benzoylated triacetate 5 as a promising donor for glycosylation of
p-nitrophenol in almost quantitative yield.
Acetoxy group has been a very convenient anomeric leaving group
in glycosylation reactions. Furthermore, the neighbouring group effect
of the 2-O-acyl moiety could be used for prevailing formation of
β-anomer in 1,2-trans glycosylation. Generally, conventional
glycosylation methods applied to furanosides showed low
stereoselectivity. In our previous experiments, the donor 5 was di-
rectly used in glycosylation of p-nitrophenol catalysed by Lewis acids
SnCl4 and BF3·OEt2. Mixtures of anomers were however obtained
when only reaction temperatures and times were varied to opti-
mize the reaction. The anomerization caused by acidic conditions
was therefore suppressed by addition of Et3N to the reaction.32 Use
of BF3·OEt2 (10 equivs) and Et3N (1 equiv) was found to be optimal
and the reaction afforded stereoselectively the kinetically fa-
voured β-glycoside 6 in 78% yield after isolation. Final deacylation
under Zemplén conditions (MeONa/MeOH) afforded the target chro-
mogenic substrate 1 in 91% yield.
2.2. Assay of β-apiosidase
The apiosidase assay introduced by Günata14 is a simple, reli-
able method suitable for fast analysis of multiplex sets of samples.
The principle of the method is notoriously known from routine assays
of other glycosidases: 4-nitrophenol after its enzymatic release from
1 (Scheme 2) forms yellow coloration after alkalinization of the assay
solution. We have slightly modified the method for our screening
purposes, the modification being in different working wavelength
(410 nm instead of 400 nm) and different stopping solution (satu-
rated solution of borax instead of sodium carbonate). The main
change lies in use of two working buffers to increase the chance for
detection of the activity since glycosidases of various origins may
have quite different pH range of activity.
2.3. Screening of β-D-apiosidase
Besides pectinases known for apiosidase content from previ-
ous studies (Rapidase AR-2000)13,14 or from declaration of producer
Scheme 2. Principle of β-apiosidase assay.

50
P. Kis et al. / Carbohydrate Research 430 (2016) 48–53
Table 1
β-d-Apiosidase activity in liquid enzyme preparations
Declared activity
Commercial name
Source
Activity [U/mL]
pH 5.0
pH 6.5
Pectinases and polygalacturonases
Pectinex UF
Ultrazym AFP
TP714L
Aspergillus aculeatus
Aspergillus sp.
Aspergillus sp.
Aspergillus niger
not available
1.34 0.08
0.58 0.05
0.55 0.03
0.03 0.01
n. d.
0.31 0.03
0.34 0.01
0.38 0.02
n. d.
n. d.
n. d.
Peclyve LVG
Pectinex Yield Mash
Pectinex Ultra Mash
Rohavin LX
A. aculeatus
not available
n. d.
n. d.
n. d.
Rohavin MX
not available
n. d.
n. d.
β-Glucanases and cellulases
Glucoamylases
Viscozyme L
Novozym 188
Depol 692L
A. aculeatus
A. niger
Aspergillus sp. & Trichoderma
Trichoderma reesei
A. niger (Bacillus subtilis)
A. niger
A. niger
A. niger
A. niger
5.43 0.29
2.04 0.05
0.25 0.02
n. d.
n. d.
n. d.
n. d.
n. d.
n. d.
3.79 0.20
0.73 0.08
0.06 0.01
n. d.
n. d.
n. d.
n. d.
n. d.
n. d.
Celluclast
Dextrozyme DX 1,5X
Dextrozyme GA 1,5X
Spirizyme Ultra
Amylyve AG 400 L
AMG 300 L
β-Galactosidase
Earthworm hydrolases
Lactozym 3000 L
Vermistimul
Protamyl Bio V
Protamyl Bio D
Drep A
Kluyveromyces lactis
Eisenia foetida
Eisenia foetida
Eisenia foetida
Eisenia foetida
n. d.
n. d.
n. d.
n. d.
n. d.
n. d.
n. d.
n. d.
n. d.
n. d.
n. d. – not detected (detection limit = 0.03 U/mL).
(Lallzyme BETA), scale of various liquid and powdered commer-
cial enzyme preparations as well as a small number of plant materials
have been tested. Based on the mentioned literary data, enzymes
originating from genus Aspergillus were preferentially selected for
the screenings. For better comparison of results, we organized data
from the screening to two sets – liquid and solid enzymes. Table 1
summarizes apiosidase activities displayed by liquid samples. The
highest activity was found in crude β-glucanases Viscozyme L and
Novozym 188. Less significant or minor levels of apiosidases were
found in several pectinases and cellulases. In general, all products
displaying β-apiosidase were products originating exclusively from
aspergilli, mostly A. niger or A. aculeatus, but several aspergilli prod-
ucts, especially glucoamylases, did not possess this activity. This may
be in accordance with the prior statement of Dupin et al.14 about
inducible character of this enzyme. The enzyme cocktails from fungi
other than aspergilli, from yeasts or from earthworms Eisenia foetida
did not display the apiosidase activity.
Similar pattern of β-apiosidase occurrence was observed among
the powdered enzymes (Table 2) – all enzymes positive for this
activity originate from genus Aspergillus, the highest level being
found in Rapidase AR 2000, a pectinase from A. niger. Significant
levels were found also in β-glucosidase for winemaking Lallzyme
BETA, Amano α-galactosidase DS and pectinase Rapidase Expres-
sion. It is worth to point that three tested lipases from A. niger
also possess levels of apiosidase, which are comparable with
previously reported apiosidase source Ultrazym 10013,14 (Table 2).
Typically for fungal glycosidases, activity of the enzymes was
higher at pH 5.
ties of apiosidase on levels comparable to its content in Rapidase
AR 2000, or Lallzyme BETA, respectively. This makes β-apiosidase
available in high amounts and low cost for both scientific and in-
dustrial purposes.
3. Conclusion
We have developed an efficient chemoenzymatic synthesis of
4-nitrophenyl β-d-apiofuranoside 1 from readily available 2. The
preparation includes two important selective steps: enzymatic
regioselective benzoylation of primary hydroxyl and optimized
stereoselective 1,2-trans-glycosylation using BF3·OEt2 as promotor
and Et3N for suppression of anomerization. The chromogenic
apiosidase probe 1 was produced in 61% overall yield from 2 (40%
in ten steps from d-mannose).
The probe was used to screen 61 crude commercial enzymes and
other biological materials for presence of β-apiosidase. We have
found that the enzyme is surprisingly abundant among enzymes
available in the market. Only the fungal enzymes produced by as-
pergilli possessed the β-apiosidase activity, significantly high levels
being found in Rapidase AR 2000, Lallzyme BETA, Amano
α-galactosidase DS and ultrafiltered and lyophilized glycanases
Viscozyme L and Novozym 188.
4. Experimental
4.1. General methods
β-Apiosidase activity was not identified in plant materials rich
for other glycosidases (including β-glucosidase and diglycosidases)
such as lyophilized crude fruit juice and defatted seed meals or grape
pressing cake. These results confirm the statement of Sarry et al.33
that β-apiosidase is absent in whole grape berry pulp or berry skin,
although the berry skin is a rich source of exoglycosidases in-
volved in aroma development in must and wine.
The liquid glycanases Viscozyme L and Novozym 188 are inex-
pensive, widely used industrial enzymes. After their ultrafiltration
and lyophilization, powdered catalysts were obtained, with activi-
The reactions were performed with commercial reagents and sol-
vents purchased in satisfactory purity (min. 95%) from local
subsidiaries of Sigma-Aldrich, Merck, Acro¯ s Organics, VWR,
Mikrochem, Centralchem, Slavus. d-Mannose was obtained as a gift
from Technikum, Institute of Chemistry, Slovak Academy of Sci-
ences in purity above 98%. Solvents were refluxed for 2 h over an
appropriate drying agent (dichloromethane—P2O5, methanol–Mg/
I2) under an argon atmosphere and subsequently distilled off and
used in reaction. All reactions were monitored on TLC plates (Silicagel
60, F254, 0.25 mm, E. Merck, Darmstadt, Germany). The spots of

P. Kis et al. / Carbohydrate Research 430 (2016) 48–53
51
Table 2
β-d-Apiosidase activity in powdered materials
Declared activity
Commercial name
Source
Activity [U/g]
pH 5.0
pH 6.5
Pectinases, polygalacturonases
and β-glucanases
Rapidase AR 2000
Lallzyme BETA
Rapidase Expression
Ultrazym 100
Pentopan 500 BG
Ultraflo (lyof)
Rohavine Color
VERON 191
Rohalase GMP
Fungal pectinase (Fluka)
Fungal pectinase (Calbiochem)
Pectin esterase
Rohament pectinase
α-galactosidase DS
Lallzyme Cuvée Blanc
Fungal lactase
Lactase F
Aspergillus niger
A. niger
A. niger
78.27 3.98
11.48 0.63
6.93 0.08
2.74 0.16
n. d.
n. d.
n. d.
n. d.
n. d.
n. d.
n. d.
n. d.
n. d.
11.19 0.39
4.85 0.27
n. d.
46.85 4.29
6.77 0.27
3.79 0.40
1.33 0.08
n. d.
n. d.
n. d.
n. d.
n. d.
n. d.
n. d.
n. d.
n. d.
7.78 0.42
2.20 0.19
n. d.
A. niger
Thermomyces lanuginosus
Humicola insolens
Not available
Not available
Not available
Fungal
Fungal
Lycopersicon esculentum
Not available
A. niger
A. niger
Aspergillus oryzae
A. oryzae
α-Galactosidase
β-Glucosidase
β-Galactosidase
n. d.
n. d.
Lipases
Lipolyve AN
Enzeco Lipase
Lipase A
Prolyve PAC
Viscozyme L
Novozym 188
campus tree
rowan
buckwheat
tartaric buckwheat
apricot
A. niger
A. niger
A. niger
A. niger
A. aculeatus
A. niger
3.10 0.11
2.77 0.14
2.63 0.07
n. d.
64.36 4.76
15.81 0.95
n. d.
n. d.
n. d.
n. d.
n. d.
2.34 0.07
1.73 0.08
2.22 0.15
n. d.
30.86 0.91
7.34 0.22
n. d.
n. d.
n. d.
n. d.
n. d.
Protease
Lyoph. glycanasesa
Lyoph. crude fruit juices
Defatted seed meals
Sophora japonica
Sorbus aucuparia
Fagopyrum esculentum
F. tataricum
Prunus armeniaca
P. persica
peach
grape
grape
n. d.
n. d.
n. d.
n. d.
n. d.
n. d.
Vitis viniferab
Vitis viniferab
Seed meal press cake
n. d. – not detected (detection limit = 1.2 U/g).
a
Ultrafiltered and lyophilised glycanases positive for β-apiosidase in liquid form.
Mix of varieties Furmint and Lipovina.
b
compounds were detected with UV lamp (λmax = 254 nm) or by char-
ring the plates with 5% orcinol in 10% (v/v) ethanolic solution of
H2SO4 by drying at ca. 200 °C. Column chromatography was per-
formed on Silica gel 60 (0.035–0.070 mm, pore diameter ca. 6 nm,
Acro¯s Organics). Melting points were determined with a Kofler hot-
stage and are uncorrected. Optical rotations were measured with
a Jasco P-2000 polarimeter at 20 °C in chloroform or methanol. NMR
spectra were recorded at 25 °C on either Varian 400 MR or Bruker
AVANCE III HD 400 MHz equipped with Prodigy CryoProbe. 1H NMR
spectra were recorded at 400 MHz and shifts are referenced to in-
ternal solvents or Me4Si as internal standard, respectively. 13C NMR
spectra were recorded at 101 MHz and shifts are referenced to in-
ternal solvents. Chemical shifts are expressed in parts per million
(d scale) downfield from tetramethylsilane and are referenced to
the residual proton in the NMR solvent (CDCl3: δ 7.26 ppm, δ
77.23 ppm and CD3OD: δ 3.31 ppm, δ 49.15 ppm). For correct com-
plete assignment of signals a two-dimensional homonuclear
correlation technique (H-H COSY) and heteronuclear correlation tech-
nique (H-C HSQC) were performed. Mass spectra were measured
on Orbitrap Velos PRO, Thermo Scientific with parameters HESI
(heated electrospray): 50 °C, capillary temperature 275 °C, spray
voltage +3.5 kV, auxilliary gas 0, sheath gas flow rate 15. Spectro-
photometric assays were performed on UV-VIS Spectrophotometer
UV–1800 (Shimadzu, Kyoto, Japan). Incubations in small volumes
were performed in Eppendorf tubes on Thermomixer comfort
(Eppendorf AG, Hamburg, Germany). Ultrafiltration was per-
formed on filter VIVAFLOW 200 (10,000 MWCO PES) purchased from
Sartorius Stedim Biotech (GmbH, Goettingen, Germany) with peri-
staltic pump Masterflex L/S (Cole-Parmer International, Vernon Hills,
IL, USA). Lyophilization of the ultrafiltrated enzymes was per-
formed on freeze dry system LABCONCO Freezone 18 overnight.
4.2. Biocatalysts
Crude commercial preparation Rapidase AR-2000 was a kind gift
from Patrice Pellerin (DSM, USA), Rapidase Expression from O.K.
SERVIS BioPro, s.r.o. (Czech Republic), local supplier of DSM, Lallzyme
BETA and Lallzyme Cuvée Blanc were purchased from Lallemand
(Canada). Enzyme preparations from Novozymes: Lipolase 100T,
Viscozyme L, Novozym 188, Pectinex UF, Ultrazym AFP, Pectinex Yield
Mesh, Pectinex Ultra Mash, Celluclast, Dextrozyme DX 1,5X,
Dextrozyme GA 1,5X, Spirizyme Ultra, Lactozym 3000 L, AMG 300 L,
Pentopan 500 BG and Ultraflo were gift from Marián Illáš (Biotech
s.r.o., Slovakia), α-galactosidase DS, Lactase F and Lipase A were pur-
chased from Amano Enzyme (USA). Peclyve LVG, Amylyve AG 400 L,
Lipolyve AN and Prolyve PAC were gift from Lyven (France). Sample
of Fungal lactase was a gift from Shin Nihon (Japan) and Enzeco Mi-
crobial Lipase Concentrated was a gift from Enzyme Development
Corporation (USA). Rohavin LX, Rohavin MX, Rohavine Color, VERON
191, Rohament and Rohalase GMP were gift from Barentz Slovakia
(local supplier of AB Enzymes GmbH, Germany), TP714L and Depol
692L from Biocatalysts (UK) and few preparations for wastewater
treatment were purchased from Karel Pecl-EKOVERMES (Pusteˇjov,
Czech republic). Tomato pectin esterase was from Sigma and prepa-
rations of three fungal pectinases were from Sigma, Fluka and
Calbiochem.
Plant materials as fruits of Sophora japonica and Sorbus aucuparia
were harvested in local sources. Seeds of Fagopyrum tataricum var.
Madawaska were kind gift from Iveta Cicˇová (Research Institute of
Plant Production, Slovakia). Seeds of Fagopyrum esculentum, Prunus
armeniaca and Prunus dulcis were purchased from the local sup-
plier. Seed and pressing cake obtained after grape oil production
were kind gift from Alžbeta Zlatnická (J. & J. Ostrožovicˇ, Slovakia).
ˇ

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P. Kis et al. / Carbohydrate Research 430 (2016) 48–53
4.3. 5-O-Benzoyl-2,3-O-isopropylidene-D-apiofuranose (3)
0.113 mol, 7.5 equiv). The stirring was continued at r.t. for further
96 h. Reaction mixture was then poured on the mixture of crushed
ice (60 g) and aqueous 1M hydrochloric acid (60 mL) under vigor-
ous stirring. The resulting mixture after 1 h stirring was extracted
with chloroform (3 × 25 mL). Combined organic extracts were washed
with aqueous 1M hydrochloric acid (3 × 25 mL), saturated NaHCO3
(3 × 25 mL) and the chloroform fraction was dried over anhydrous
sodium sulphate. After filtration and evaporation to dryness, the
residue was purified by column chromatography (toluene/ethyl
acetate = 4/1) to give 5 as yellow oil (5.59 g, 98 %, α:β = 1:4); Rf = 0.53
(CHCl3/MeOH = 9:1), Rf = 0.85 (CH2Cl2/MeOH = 18:1), Rf = 0.69
(toluene/ethyl acetate = 1:1), 5β Rf = 0.43 (toluene/ethyl acetate = 2:1),
To a solution of 2 (12 g, 0.08 mol) in toluene (1.2 L) was added
vinyl benzoate (23.71 g, 0.16 mol) and Lipolase 100T (180 g). The
reaction mixture was shaken at 40 °C and 200 rpm for 10 days. The
reaction was stopped, the enzyme filtered off, washed with acetone,
washings were combined with the filtrate and the solvents evapo-
rated. The concentrated crude mixture was diluted with toluene and
washed with saturated NaHCO3. Organic fractions were dried over
anhydrous sodium sulphate and concentrated in vacuo. The residue
was purified by column chromatography (toluene/ethyl acetate = 5/
1) to give 3 as yellowish-brown oil (22.84 g, 95%, α:β = 1:4), which
upon crystallization gave 3 as white needles; Mp 71–72 °C (diethyl
ether/hexane), lit.25 mp = 64–67 °C; Rf = 0.52 (toluene/ethyl acetate,
1/1, v/v); [α]D20 = −28.0 (c 1.0, CHCl3), lit.25 [α]D20 = −31.2 (c 1.3, CH2Cl2);
1H NMR (400 MHz, CDCl3) 3α: δ 1H NMR (400 MHz, CDCl3) δ 1.47
(s, 3H), 1.59 (s, 3H), 3.68 (d, J = 10.6 Hz, 1H, H-4b), 4.01 (d, J = 10.6 Hz,
1H, H-4a), 4.02 (d, J = 11.8 Hz, 1H, OH), 4.47 (d, J = 3.3 Hz, 1H, H-2),
4.48 (d, J = 11.8 Hz, 1H, H-5b), 4.56 (d, J = 11.7 Hz, 1H, H-5a), 5.09
(dd, J = 11.7, 3.2 Hz, 1H, H-1), 7.44–7.48 (m, 2H, H-Ph), 7.57–7.62 (m,
1H, H-Ph), 8.02–8.04 (m, 2H, H-Ph); 13C NMR (100 MHz, CDCl3), 3α:
δ 27.6 (CH3), 27.8 (CH3), 65.2 (C-5), 70.6 (C-4), 81.8 (C-2), 89.7 (C-
3), 98.1 (C-1), 115.3 (C-Me2), 128.7 (2xCH-Ph), 129.8 (C-Ph), 129.9
(2xCH-Ph), 133.7 (CH-Ph), 166.2 (COPh); 1H NMR (400 MHz, CDCl3)
3β: δ 1.41 (s, 3H, CH3), 1.50 (s, 3H, CH3), 3.18 (d, J = 2.7 Hz, 1H, OH),
4.09 (d, J = 10.0 Hz, 1H, H-4b), 4.17 (d, J = 10.0 Hz, 1H, H-4a), 4.53 (s,
1H, H-2), 4.52 (d, J = 11.8 Hz, 2H, H-5b), 4.64 (d, J = 11.7 Hz, 1H, H-5a),
5.49 (d, JH,OH = 2.7 Hz, 1H, H-1), 7.42–7.46 (m, 2H, H-Ph), 7.54–7.59
(m, 1H, H-Ph), 8.07–8.09 (m, 2H, H-Ph); 13C NMR (100 MHz CDCl3)
3β: δ 27.6 (CH3), 27.7 (CH3), 66.1 (C-5), 74.6 (C-4), 87.3 (C-2), 90.4
(C-3), 102.3 (C-1), 114.0 (C-Me2), 128.6 (2xCH-Ph), 129.6 (C-Ph),
129.9 (2xCH-Ph), 133.4 (CH-Ph), 166.4 (COPh). ESI-HRMS C15H18O6Na
m/z [M + Na]+ Anal. Calcd. 317.09956; Found 317.09941.
5α Rf = 0.36 (toluene/ethyl acetate = 2:1), lit.25 5β Rf = 0.22 (hexane/
20
ethyl acetate = 3:1), 5α Rf = 0.16 (hexane/ethyl acetate = 3:1); [α]D
=
– 21.3 (c 1.0, CHCl3), lit.25 5β [α]D20 = – 31.5 (c 0.7, CH2Cl2), 5α
[α]D20 = 48.5 (c 0.9, CH2Cl2); 1H NMR (400 MHz, CDCl3), 5α: δ 2.05
(s, 3H, CH3), 2.11 (s, 3H, CH3), 2.12 (s, 3H, CH3), 4.34 (d, J = 10.7 Hz,
1H, H-4b), 4.42 (d, J = 10.7 Hz, 1H, H-4a), 4.65 (d, J = 11.9 Hz, 1H,
H-5b), 4.90 (d, J = 11.9 Hz, 1H, H-5a), 5.48 (d, J = 4.7 Hz, 1H, H-2),
6.42 (d, J = 4.7 Hz, 1H, H-1), 7.44–7.50 (m, 2H, H-Ph overlapping with
5β), 7.56–7.62 (m, 1H, H-Ph, overlapping with 5β), 8.01–8.04 (m,
2H, H-Ph); 13C NMR (101 MHz, CDCl3) 5α: δ 20.5 (CH3), 21.1 (CH3),
21.4 (CH3), 63.8 (C-5), 72.3 (C-2), 72.8 (C-4), 81.2 (C-3), 94.4 (C-1),
128.7 (CH-Ph overlapping with 5β), 129.6 (C-Ph), 130.0 (CH-Ph over-
lapping with 5β), 133.6 (CH-Ph overlapping with 5β), 165.9 (COPh),
1
169.4 (COCH3), 169.6 (COCH3), 169.9 (COCH3); H NMR (400 MHz,
CDCl3), 5β: δ 2.07 (s, 3H, CH3), 2.10 (s, 3H, CH3), 2.13 (s, 3H, CH3),
4.28 (d, J = 10.5 Hz, 1H, H-4b), 4.48 (d, J = 10.5 Hz, 1H, H-4a), 4.82
(d, J = 12.4 Hz, 1H, H-5b), 4.99 (d, J = 12.4 Hz, 1H, H-5a), 5.60 (s, 1H,
H-2), 6.17 (s, 1H, H-1), 7.44–7.50 (m, 2H, H-Ph), 7.56–7.62 (m, 1H,
H-Ph), 8.08–8.10 (m, 2H, H-Ph); 13C NMR (101 MHz, CDCl3), 5β: δ
20.7 (CH3), 21.2 (CH3), 21.2 (CH3), 63.2 (C-5), 73.6 (C-4), 76.2 (C-2),
83.3 (C-3), 99.2 (C-1), 128.7 (2xCH-Ph), 129.9 (C-Ph), 130.0 (2xCH-
Ph), 133.6 (CH-Ph), 166.1 (COPh), 169.2 (COCH3), 169.3 (COCH3), 169.8
(COCH3); ESI–HRMS C18H20O9Na m/z [M + Na]+ Anal. Calcd. 403.09995;
Found 403.09975.
4.4. 5-O-Benzoyl-D-apiofuranose (4)
Aqueous formic acid (30 mL, 80 wt %) was added to a solution
of 3 (6.0 g, 0.02 mol) in dichloromethane (10 mL) and the reaction
mixture was refluxed for 5.5 h at 60 °C. The reaction mixture was
then concentrated under reduced pressure to about a half of a
volume and several times co-evaporated with toluene until all formic
acid was removed. After evaporation to dryness, the residue was
purified by column chromatography (toluene/ethyl acetate = 1/4) to
afford white amorphous solid 4 (4.73 g, 93%, α:β = 0.48:1); Mp = 138–
143 °C (methanol); Rf = 0.31 (ethyl acetate), [α]D20 = – 5.4 (c 1.0,
methanol); 1H NMR (400 MHz, CD3OD) 4α: δ 3.98 (d, J = 5.9 Hz, 1H,
H-2), 3.99 (t, J = 10.0 Hz, 2H, CH2-4), 4.32 (d, J = 11.4 Hz, 1H, H-5a),
4.38 (d, J = 11.4 Hz, 1H, H-5b), 5.26 (d, J = 4.7 Hz, 1H, H-1), 7.51–
7.45 (m, 2H, H-Ph), 7.64–7.58 (m, 1H, H-Ph), 8.10–8.03 (m, 2H, H–Ph);
13C NMR (101 MHz, CD3OD) 4α: δ 68.3 (C-5), 73.8 (C-2), 74.5 (C-
4), 77.6 (C-3), 98.3 (C-1), 129.8 (2xCH-Ph), 130.9 (2xCH-Ph), 131.3
(C-Ph), 134.6 (1xCH-Ph), 168.0 (COPh); 1H NMR (400 MHz, CD3OD)
4β: δ 3.86 (d, J = 9.8 Hz, 1H, H-4b), 3.89 (d, J = 3.8 Hz, 1H, H-2), 4.17
(d, J = 9.8 Hz, 1H, H-4a), 4.38 (t, J = 11.8 Hz, CH2-5), 5.22 (d, J = 3.8 Hz,
1H, H-1), 7.51–7.45 (m, 2H, 2xCH-Ph), 7.64–7.58 (m, 1H, 1xCH-
Ph), 8.10–8.03 (m, 2H, 2xCH-Ph); 13C NMR (101 MHz, CD3OD), 4β:
δ 67.8 (C-5), 74.5 (C-4), 78.7 (C-3), 79.3 (C-2), 104.3 (C-1), 129.8
(2xCH-Ph), 130.9 (2xCH-Ph), 131.3 (C-Ph), 134.6 (1xCH-Ph), 168.0
(COPh); ESI–HRMS C12H14O6Na m/z [M + Na]+ Anal. Calcd. 277.06826;
Found 277.06737.
4.6. 4-Nitrophenyl 2,3-di-O-acetyl-5-O-benzoyl-β-D-apiofuranoside
(6)
To a mixture of 5 (5.0 g, 0.013 mol) and 4-nitrophenol (2.7 g,
0.0195 mol, 1.5 equiv), a solution of triethylamine (1.32 g, 0.013 mol,
1 equiv) in dry dichloromethane (50 mL) was added under an at-
mosphere of argon. Then
a solution of boron trifluoride
diethyletherate (18.91 g, 0.13 mol, 10 equiv) in dry dichloromethane
(30 mL) was added at room temperature over a period of 30 min.
and then the solution was stirred for additional 1 h. The reaction
mixture was then diluted with dichloromethane (70 mL) and satu-
rated NaHCO3 (100 mL) was added under stirring to the reaction
mixture. After separation of phases, the organic fraction was dried
over magnesium sulphate and the filtrate was concentrated under
reduced pressure. The residue was chromatographed on silica gel
(toluene/ethyl acetate = 5:1) to give the title compound 6 (4.66 g,
78 %), which upon crystallization from 2-propanol gave pale yellow
solid; Mp = 127–129 °C (2-propanol); Rf = 0.61 (toluene/ethyl acetate,
1/1, v/v); [α]D20 −117.3 (c 1.0, CHCl3), 1H NMR (400 MHz, CDCl3), 1H
NMR (400 MHz, CDCl3): δ 2.10 (s, 3H, CH3), 2.16 (s, 3H, CH3), 4.37
(d, J = 10.6 Hz, 1H, H-5a), 4.51 (d, J = 10.6 Hz, 1H, H-5b), 4.93 (d,
J = 12.4 Hz, 1H, H-4a), 5.02 (d, J = 12.4 Hz, 1H, H-4b), 5.76 (s, 1H, H-1),
5.79 (s, 1H, H-2), 7.11 (d, J = 9.2 Hz, 2H, 2xCH-NPh), 7.46–7.50 (m,
2H, 2xCH-Ph), 7.58–7.63 (m, 1H, CH-Ph), 8.09–8.11 (m, 2H, 2xCH-
Ph), 8.20 (d, J = 9.2 Hz, 2H, 2xCH-NPh); 13C NMR (101 MHz, CD3OD):
δ 20.7 (CH3), 21.3 (CH3), 63.5 (C-5), 73.8 (C-4), 76.9 (C-2), 83.5 (C-
3), 104.1 (C-1), 116.6 (2xCH-NPh), 126.0 (2xCH-NPh), 128.7 (2xCH-
Ph), 129.6 (C-Ph), 130.0 (2xCH-Ph), 133.6 (CH-Ph), 143.0 (C-NPh),
160.9 (C-NPh), 166.1 (COPh), 169.3 (COCH3), 169.9 (COCH3);
4.5. 5-O-Benzoyl-1,2,3-tri-O-acetyl-D-apiofuranose (5)
Acetic acid anhydride (9.19 g, 0.09 mol, 6 equiv) and 4-N,N-
dimethylaminopyridine (0.185 g, 0.0015 mol, 10 mol %) were added
at 0 °C to a stirred solution of 4 (3.81 g, 0.015 mol) in pyridine (8.90 g,

P. Kis et al. / Carbohydrate Research 430 (2016) 48–53
53
ESI–HRMS m/z C22H21NO10Na [M + Na]+ Anal. Calcd 482.10577; Found
482.10518.
Contract No. 26240120001 as well as the “Amplification of the Centre
of Excellence on Green Chemistry Methods and Processes”, CEGreenII,
Contract No. 26240120025). The authors thank I. Uhliariková for NMR
measurements and S. Vlcˇková for mass measurements.
4.7. 4-Nitrophenyl β-D-apiofuranoside (1)
Compound 6 (4.66 g, 0.0099 mol) was dissolved in dry metha-
nol (90 mL). Then 0.5 M sodium methanolate was added (21 mL,
0.0105 mol) and the reaction was stirred for 20 min at laboratory
temperature. After this time the reaction mixture was neutralized
with Dowex 50W–X8, filtered and evaporated to dryness. Crude
product was dissolved in warm 2-propanol and placed in refriger-
ator for one day to afford pale yellow solid (2.46 g, 91 %); Mp = 109–
110 °C (2-propanol), lit.14 mp = 104–105 °C (Et2O/MeOH/n–pentane);
Supplementary material
Supplementary data to this article can be found online at
doi:10.1016/j.carres.2016.04.030.
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This work was supported by the Slovak Research and Develop-
ment Agency under the contract No. APVV-0846-12, the Scientific
Grant Agency of the Ministry of Education of Slovak Republic and
Slovak Academy of Sciences (VEGA) under the projects No. 2/0138/
12 and No. 2/0157/16 and also the Research & Development
Operational Programmes funded by the ERDF (“Centre of Excel-
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