Synthesis and Biological Evaluation of Cyanogenic Glycosides

Article abstract of DOI:10.1080/07328303.2015.1105249

An efficient procedure for the synthesis of cyanogenic glycosides with different carbohydrate units was developed. Amygdalin (3), prunasin (1), sambunigrin (2), and neoamygdalin (21) were prepared according to the elaborated method, and biological tests, including antifungal, antibacterial, and cytotoxic activities, were performed.

Full text of DOI:10.1080/07328303.2015.1105249

Journal of Carbohydrate Chemistry
ISSN: 0732-8303 (Print) 1532-2327 (Online) Journal homepage: http://www.tandfonline.com/loi/lcar20
Synthesis and Biological Evaluation of Cyanogenic
Glycosides
Dmitry V. Yashunsky, Ekaterina V. Kulakovskaya, Tatiana V. Kulakovskaya,
Olga S. Zhukova, Mikhail V. Kiselevskiy & Nikolay E. Nifantiev
To cite this article: Dmitry V. Yashunsky, Ekaterina V. Kulakovskaya, Tatiana V. Kulakovskaya,
Olga S. Zhukova, Mikhail V. Kiselevskiy & Nikolay E. Nifantiev (2015): Synthesis and
Biological Evaluation of Cyanogenic Glycosides, Journal of Carbohydrate Chemistry, DOI:
10.1080/07328303.2015.1105249
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Date: 30 November 2015, At: 20:54

Journal of Carbohydrate Chemistry, 00:1–15, 2015
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ISSN: 0732-8303 print / 1532-2327 online
DOI: 10.1080/07328303.2015.1105249
Synthesis and Biological
Evaluation of Cyanogenic
Glycosides
Dmitry V. Yashunsky,1 Ekaterina V. Kulakovskaya,2 Tatiana V.
Kulakovskaya,2 Olga S. Zhukova,2 Mikhail V. Kiselevskiy,3 and
Nikolay E. Nifantiev1
1Laboratory of Glycoconjugate Chemistry, N.D. Zelinsky Institute of Organic
Chemistry, Russian Academy of Sciences, Leninsky Prospect 47 119991, Moscow,
Russia
2Skryabin Institute of Biochemistry and Physiology of Microorganisms, Russian
Academy of Sciences, Pushchino, Moscow Region 142290, Russia
3Laboratory of Cell Immunity, N.N. Blokhin Russian Cancer Research Center,
Kashirskoe shosse, 24 115478, Moscow, Russia
GRAPHICAL ABSTRACT
An efficient procedure for the synthesis of cyanogenic glycosides with different carbohy-
drate units was developed. Amygdalin (3), prunasin (1), sambunigrin (2), and neoamyg-
dalin (21) were prepared according to the elaborated method, and biological tests, in-
cluding antifungal, antibacterial, and cytotoxic activities, were performed.
Keywords Cyanogenic glycosides; Glycosylation; Nitrile formation; Cytotoxic activity;
Cancer cells
Received September 1, 2015; accepted October 5, 2015.
Address correspondence to Professor Nikolay E. Nifantiev, Laboratory of Glycoconju-
gate Chemistry, N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sci-
ences, Leninsky prospect, 47, Moscow B-334 119991, Russia. E-mail: nen@ioc.ac.ru
1

D.V. Yashunsky et al.
2
INTRODUCTION
Cyanogenic glycosides or cyanoglycosides account for approximately 90%
of the wider group of plant toxins known as cyanogens. The key char-
acteristic of these toxins is cyanogenesis (the formation of free hydrogen
cyanide) and it is associated with cyanohydrins that have been stabilized by
glycosylation (attachment of sugar residues) to form the cyanogenic glyco-
sides. Cyanoglycosides are found in numerous structural variations through-
out the plant kingdom.[1] The biological and chemical interest in cyanogenesis
has stimulated the investigation of biochemical properties of cyanoglycosides
such as (R)-prunasin (1), (S)-sambunigrin (2), (R)-amygdalin (3), and others
(Fig. 1). In particular, amygdalin is the largely used component of the tra-
ditional Chinese medicine (produced from bitter almond) and therefore has
been investigated for nearly 200 years (for review see ref. [2]). Nevertheless,
these studies produced quite contradictory data about the anticancer activity
of amygdalin and its analogs.[3,4] Despite the failure of clinical tests to demon-
strate the anticancer effects of amygdalin in the United States and Europe,[5]
a few studies have examined other pharmacological activities of amygdalin.
It was reported recently that this agent could also exert anti-inflammatory
and antinociceptive effects as it led to in vitro suppression of cyclooxygenase-2
(COX-2) and inducible nitric oxide synthase (iNOS) expression in mouse BV2
microglial cells.[6–9]
Figure 1: Structures of the natural cyanogenic glycosides 1–3.

Cyanogenic Glycosides
3
RESULTS AND DISCUSSION
The goal of the present investigation was to elaborate a universal approach
for chemical preparation of a set of different cyanogenic glycosides and to sys-
tematically evaluate the biological properties of the synthesised derivatives,
including anticancer, antifungal, and antibacterial activities, by using known
experimental in vitro models.
Selectively protected functionalized benzyl glucosides, such as 4 or 5
(Fig. 2), are the most convenient intermediate in the synthesis of amygdalin,
prunasin, and their analogs and isomers due to simple (a) conversion of an
amide group into a nitrile group and (b) elongation of the sugar chain at the
6-position of the glucose unit with different glycosyl donors. Alternatively, it
is possible to use mandelonitrile directly as an acceptor in glycosylation with
different glucosyl donors. It is known[10] that glucose pentaacetate could react
with racemic mandelonitrile in the presence of TMSOTf to give a mixture of
acetylated derivative of prunasin and sambunigrin. It could be a convenient
way toward obtaining a mono- and disaccharide derivative of mandelonitrile,
but fast epimerization at the mandelonitrile chiral center under basic condi-
tions makes a deacylation step very problematic. The intermediates 4 and 5
could easily be obtained from the known diastereomerically pure derivative
6.[11]
In the original work,[11] glucoside 6 was obtained by glycosylation of man-
delamide (R-7) with trichloroacetimidate 8 in the presence of boron trifluoride
Figure 2: Intermediates in the synthesis of cyanogenic glycosides.

D.V. Yashunsky et al.
4
etherate in ether with a yield of 70%. It should be noted that such a trans-
formation sometimes could be inefficient. For instance, it is known[12] that the
reaction of galactose pentaacetate with racemic mandelic amide in the pres-
.
ence of BF3 Et2O only gives a rather poor yield (33%). An attempt to reproduce
the same glycosylation with trichloroacetimidate (8) and racemic mandelamide
(7) in the presence of the same activator in ether resulted in the formation of
only traces of the desired product. Replacement of ether with dichloromethane
as the solvent afforded a mixture of target glucosides 9 with 65% yield; how-
ever, stereoselectivity of glycosylation was moderate (α:β ratio = 1:5; Sch. 1).
It is known[13,14] that benzoylated imidate donors are much more effective in
comparison to the acetylated imidate donors in terms of yield and stereoselec-
tivity of glycosylation of different acceptors. Indeed, using benzoylated glucose
trichloroacetimidate 10 as a donor allowed an improvement in the stereoselec-
tivity of glycosylation, and as a result, a mixture of glucosides 11 was obtained
with 57% yield and an α:β ratio of 1:25.
A disadvantage of this reaction was the formation of a considerable amount
(40%) of α-glucosyl fluoride 12 as a side product. Although the formation of glu-
cosyl fluoride is an uncommon fact, there is one example[15] where glycosylation
with trichloroacetimidate derivative of per-acetylated galactose in the presence
.
of BF3 Et2O gave a 21% yield of 2,3,4,6-tetra-O-acetyl-α-D-galactopyranosyl
fluoride as a by-product.
.
Scheme 1: Reagents and conditions: (a) BF3 Et2O (ex), CH2Cl2, rt; (b) HCl-MeOH, CH2Cl2;
(c) MeONa-CH2Cl2-MeOH; (d) Ac2O-Py.

Cyanogenic Glycosides
5
Surprisingly, when 6-O-acetylated imidate 13 was used in the glycosylation
of 7, no formation of the corresponding glucosyl fluoride was observed, and
a mixture of 14 (β-isomer) and 15 (α-isomer) in a ratio of 10:1 was obtained
with a total yield of 80%. Selective removal of the acetyl group in 14 with
hydrogen chloride in a mixture of dichloromethane and methanol resulted in
the formation of diastereomeric 6-OH derivatives 16 (30%) and 17 (55%), which
could easily be separated by conventional silica gel column chromatography.
The stereochemistry of the chiral center in the mandelic acid residue in 16
and 17 was determined by their transformation to fully acetylated derivatives
18 and 6; physical and spectral properties of the latter were identical to those
described in the literature.[11]
The transformation of pure diastereomers 16 and 17 into (S)-sambunigrin
(2) and (R)-prunasin (1), respectively, should include the steps of dehydration
of the amide group into a nitrile group and the removal of the benzoyl protect-
ing groups. The conversion of amides into nitriles is a well-known procedure in
synthetic organic chemistry.[16] The most efficient methods include the treat-
ment with trifluoroacetic anhydride (TFAA)-Py[17] or using “activated” DMSO
species.[18] However, the presence of the free hydroxyl group in derivatives 16
and 17 prevents the application of oxidative conditions of activated DMSO
species. Furthermore, final deacylation of mandelonitriles under basic condi-
tions can lead to fast epimerization at the chiral center of the mandelonitrile
moiety.[11] Therefore, the deacylation step should precede dehydration of the
amide group into a nitrile group. Taking this into account, we have elaborated
an efficient two-step procedure to convert derivatives 16 and 17 into the target
compounds (Sch. 2).
Thus, complete deacylation with
a catalytic amount of NaOMe
in dichloromethane–methanol followed by treatment with an excess
of TFAA in Pyr, subsequent de-O-trifluoroacetylation of the reaction
Scheme 2: Reagents and conditions: (a) MeONa-CH2Cl2-MeOH; (b) TFAA-Py, 1,4-dioxane.

D.V. Yashunsky et al.
6
products with methanol and anion-exchange resin (HCO3−), and pu-
rification on
a silica gel using the known 5-component (20:5:15:6:4
EtOAc:MeOH:acetone:CH2Cl2:H2O) solvent system[19] produced the targets 2
and 1 in almost quantitative yields.
To obtain amygdalin (3), 6-OH derivative 17 was subjected to BF3·Et2O-
promoted glycosylation with trichloroacetimidate 13 in dichloromethane. In
this case, the reaction proceeded smoothly at rt and gave disaccharide 19 stere-
ospecifically in 75% yield (Sch. 3).
Employing the two-step procedure used for the preparation of 1 and 2, we
converted derivative 19 into amygdalin 3 in almost quantitative yield. Pure
intermediate compound 20 was also isolated for use in biological tests. Simi-
larly, we prepared neoamygdalin 21 from 6-OH derivative 16 and trichloroace-
timidate 13. Stereospecific glycosylation led to disaccharide 22 in 80% yield;
further transformation of 22 provided the desired neoamygdalin 21 almost
quantitatively.
The set of five compounds 1–3, 20, and 21 was subjected to antifun-
gal, antibacterial, and cytotoxic activities tests. The yeasts Saccharomyces
cerevisiae VKM Y-1173 (model culture) and Filobasidiella neoformans IGC
3957 (cryptococcosis pathogen) and Escherichia coli K-12 were used as test
cultures for antifungal and antibacterial activities, and the results are pre-
sented in Table 1. None of the tested compounds possessed antibiotic activity
against the tested microorganisms at rather high concentration of chemical
agent.
Cytotoxic activities of the above set of target compounds 1–3, 20, and
21 are summarized in Table 2. The most active among tested compounds
.
Scheme 3: Reagents and conditions: (a) BF3 Et2O (ex), CH2Cl2, rt; (b) MeONa-CH2Cl2-
MeOH; (c) TFAA-Py, 1,4-dioxane.

Cyanogenic Glycosides
7
Table 1: The growth of test cultures in the presence of amygdalin (3) and related
compounds at a concentration 1.7 mg/mL
Culture densitya (at 495 nM)
Filobasidiella
neoformans IGC
3957
Saccharomyces
cerevisiae VKM
Y-1173
Escherichia coli
Studied agent
K-12
Control
0.325 0.017
0.353 0.047
0.366 0.019
0.335 0.027
0.332 0.019
0.318 0.034
0.537 0.047
0.522 0.074
0.503 0.058
0.533 0.022
0.522 0.058
0.515 0.053
0.376 0.009
0.322 0.019
0.346 0.023
0.381 0.032
0.389 0.008
0.343 0.027
1
2
3
21
20
a Average optical density values of three experiments.
were glucosides 1 and 2, which in fact demonstrated some cytotoxic ef-
fect (cell viabilities were 37.5% and 28.5%, respectively) only at the concen-
tration of 10−2 M. All tested compounds at concentrations of 10−3 to 10−4
M had no effect on cell growth. These findings are consistent with Park
et al., who established similar levels of cytotoxicity for amygdalin deriva-
tives against human colon cancer cells at comparable levels of exposure
concentrations.[20]
It is interesting to note that compound 20, which has no CN group (and
thus is not cyanogenic), possesses comparable cytotoxic activity (cell viability:
48.2%) as compounds 1 and 2. This finding suggests that the activity of amyg-
dalin and related compounds may be connected to properties other than their
cyanogenic ability.
Table 2: Cytotoxicity of amygdalin (3) and other compoundsa
Viability of HCT-116 cells, %
Studied agent
10−2
M
10−3
M
10−4
M
1
37.5 2.7
28.5 2.3
87.5 2.5
71.4 3.2
48.2 1.8
91.1 3.7
85.7 3.9
87.6 3.1
98.2 2.2
92.9 4.1
97.0 2.2
100 2.8
100 2.1
98.0 1.3
99.4 1.7
2
3
21
20
a Independent experiments were repeated four times. Results are presented as mean SE (P
< 0.05).

D.V. Yashunsky et al.
8
CONCLUSION
In conclusion, an efficient procedure for the synthesis of cyanogenic glycosides
with different carbohydrate units was elaborated. Compounds 1–3, 20, and 21
demonstrated weak or absent biological activity under the experimental condi-
tions used. A further increase in the activity of the studied compounds may be
achieved by modification of their carbohydrate moieties, which can now be per-
formed by using the developed chemical approaches. Studies in this direction
are in progress in our group and will be reported elsewhere.
EXPERIMENTAL SECTION
Chemical Synthesis
General methods
NMR spectra were recorded on a Bruker Avance 600 instrument. Shifts
are given in ppm with respect to the TMS signal and coupling constants (J)
are given in Hertz. Signal assignment was made using COSY and HSQC ex-
periments. HRMS (ESI) were obtained on a MicrOTOF II (Bruker Daltonics)
instrument. Optical rotations were measured using a JASCO P-2000 polarime-
ter at 22 to 24◦C in the specified solvents. TLC was performed on silica gel 60
F254 plates (E. Merck), and visualisation was accomplished using UV light or
by charring at ∼150◦C with 10% (v/v) H3PO4 in ethanol. Column chromatogra-
phy was carried out on silica gel 60 (40–63 μm, E. Merck). All air- or moisture-
sensitive reactions were carried out using dry solvents under dry argon. Chem-
icals were purchased from Acros, Fluka, or Aldrich and used without further
purification.
General procedure A: Glycosylation with trichloroacetimidates in the presence
.
of BF3 Et2O
To a mixture of trichloroacetimidate (1 eq.) and monohydroxyl acceptor
.
(1.1–1.3 eq.) in CH2Cl2 (5 mL/mmol), BF3 Et2O (1.7 eq. relative to donor plus
acceptor) was added and the resulting mixture was stirred for an additional 10
to 20 min at rt, quenched with satd. Aq. NaHCO3, and extracted with CH2Cl2.
The organic layer was separated, dried, and concentrated. The residue was pu-
rified by silica gel column chromatography to provide a glycosylation product.
(R,S)-(2,3,4,6-tetra-O-acetyl-α,β-D-glucopyranosyloxy)(phenyl)acet
amide (9). A mixture of isomers 9 (528 mg, 65%) was obtained from donor 8
(834 mg, 1.69 mmol) and acceptor 7 (280 mg, 1.85 mmol, 1.1 eq.) according to
the general procedure A.

Cyanogenic Glycosides
9
(R,S)-(2,3,4,6-tetra-O-benzoyl-α,β-D-glucopyranosyloxy)(phenyl)ace
tamide (11) and 2,3,4,6-tetra-O-benzoyl-α-D-glucopyranosyl fluo-
ride (12). A mixture of isomers 11 (644 mg, 57%) and glucosyl fluoride
12 (370 mg, 40%) was obtained from donor 10 (1.15 g, 1.55 mmol) and
acceptor 7 (300 mg, 2 mmol, 1.29 eq.) according to the general procedure
A.
(R,S)-(6-O-acetyl-2,3,4-tri-O-benzoyl-β-D-glucopyranosyloxy)
(phenyl)acetamide (14) and (R,S)-(6-O-acetyl-2,3,4-tri-O-benzoyl-α-
D-glucopyranosyloxy)(phenyl)acetamide (15). A mixture of isomers 14
+ 15 (235 mg, 80%) was obtained from donor 13 (300 mg, 0.442 mmol and
acceptor 7 (87 mg, 0.575 mmol, 1.3 eq.) according to the general procedure A.
(R)-[6-O-(6-O-Acetyl-2,3,4-tri-O-benzoyl-β-D-glucopyranosyl)-
2,3,4-tri-O-benzoyl-β-D-glucopyranosyloxy](phenyl)aceta
mide (19) (66 mg) was obtained in a yield of 75% from donor 13 (68 mg,
0.1 mmol, 1.3 eq.) and acceptor 17 (48 mg, 0.077 mmol) according to the gen-
eral procedure A: amorphous powder; [α]D –24◦ (c 0.7, CHCl3); δH (600 MHz,
CDCl3): 1.90 (3 H, s, Ac), 3.84 (1 H, dd, J5,6a = 6.8, J6a,6b = 10.9, H-6a’), 3.90 (1
Н, m, H-5”), 3.99 (1 H, m, H-5’), 4.08 (1 H, dd, J5,6a = 1.7, H-6b’), 4.17 (1 H, dd,
J5,6a = 2.9, J6a,6b = 12.2, H-6a”), 4.24 (1 H, dd, J5,6a = 5.3, H-6b”), 4.53 (1 H, d,
J1,2 = 7.9, H-1’), 4.90 (1 H, d, J1,2 = 7.8, H-1”), 5.15 (1 H, s, H-7), 5.37 (1 H, t,
J3,4 = J4,5 = 9.7, H-4’), 5.47 (2 H, m, H-2’and CONHH), 5.53 (2 H, m, H-2” and
H-4”), 5.72 (1 H, dd, J2,3 = 9.7, H-3’), 5.90 (1 H, t, J2,3 = J3,4 = 9.5, H-3”), 6.80
(1 H, br.s, CONHH), 7.12–8.03 (35 Н, m, 7xPh); δC (150 MHz, CDCl3): 20.5
(Ac), 62.5 (C-6”), 67.7 (C-6’), 69.4 (C-4”), 69.7 (C-4’), 71.7 (C-3’), 72.0 (C-2”), 72.2
(C-2’), 72.25 (C-5”), 72.7 (C-3”), 73.8 (C-5’), 79.3 (C-7), 97.5 (C-1’), 100.9 (C-1”),
128.0–135.0 (7×Ph), 165.1–165.7 (6×Bz), 170.5 (Ac), 171.8 (C-8); HMRS-ESI
(positive mode): m/z 1164.3297 [M+Na]+; calcd. for C64H55NNaO19 1164.3260.
(S)-[6-O-(6-O-Acetyl-2,3,4-tri-O-benzoyl-β-D-glucopyranosyl)-2,3,4-
tri-O-benzoyl-β-D-glucopyranosyloxy](phenyl)acetamide (22) (56 mg)
was obtained in a yield of 80% from donor 13 (54 mg, 0.079 mmol, 1.3 eq.)
and acceptor 16 (38 mg, 0.061 mmol) according to the general procedure A:
amorphous powder; [α]D –7◦ (c 1, CHCl3); δH (600 MHz, CDCl3): 2.00 (3 H, s,
Ac), 3.55 (1 Н, m, H-5”), 3.82–3.93 (3 H, m, H-5’ and H-6’), 4.17 (1 H, dd, J5,6a
= 3.0, J6a,6b = 8.9, H-6a”), 4.21 (1 H, dd, J5,6a = 5.0, H-6b”), 4.56 (1 H, d, J1,2
=
7.9, H-1”), 4.85 (1 H, d, J1,2 = 7.8, H-1’), 5.20 (1 H, s, H-7), 5.34–5.41 (3 H, m,
H-2”, H-4’ and CONHH), 5.43 (1 H, t, J3,4 = J4,5 = 9.7, H-4”), 5.57 (1 H, dd, J2,3
= 9.5, H-2’), 5.70 (1 H, t, J2,3 = 9.7, H-3”), 5.80 (1 H, t, J3,4 = 9.6, H-3’), 6.49 (1
H, br.s, CONHH), 7.22–8.05 (35 Н, m, 7×Ph); δC (150 MHz, CDCl3): 20.6 (Ac),
62.3 (C-6”), 67.2 (C-6’), 69.2 (C-4”), 69.3 (C-4’), 71.9 (C-5’), 72.0 (C-2”), 72.2
(C-2’), 72.4 (C-3”), 72.6(C-3’), 75.0 (C-5”), 81.4 (C-7), 100.7 (C-1’), 100.8 (C-1”),
126.8–136.6 (7×Ph), 165.1–165.7 (6×Bz), 170.5 (Ac), 172.1 (C-8); HMRS-ESI
(positive mode): m/z 1164.3275 [M+Na]+; calcd. for C64H55NNaO19 1164.3260.

D.V. Yashunsky et al.
10
(S)-2,3,4-tri-O-benzoyl-β-D-glucopyranosyloxy(phenyl)acetamide
(16) and (R)-2,3,4-tri-O-benzoyl-β-D-glucopyranosyloxy(phenyl)acetam
ide (17). To a solution of the mixture of isomers 14 and 15 (350 mg,
0.526 mmol) in methylene chloride (3 mL), a solution of HCl in methanol
(6 mL, prepared by addition of 0.6 mL of AcCl to 14 mL of MeOH at 0◦C) was
added at rt. The reaction mixture was kept at rt for 17 h and coevaporated
with toluene to dryness, and the residue was separated by silica gel column
chromatography (1:1 toluene–EtOAc → 1:2 toluene–EtOAc) to provide 16
(98 mg, 30%): amorphous powder; [α]D –31◦ (c 1, CHCl3); δH (600 MHz, CDCl3):
1.80 (1 H, br.s, OH), 3.53 (1 H, dd, J5,6a = 5.7, J6a,6b = 12.7, H-6a’), 3.58 (1 H,
dd, J5,6a = 2.3, H-6b’), 3.67 (1 Н, m, H-5’), 4.98 (1 H, d, J1,2 = 7.8, H-1’), 5.12
(1 H, s, H-7), 5.41 (1 H, t, J3,4 = J4,5 = 9.7, H-4’), 5.51 and 6.61 (2 H, both br.s,
CONH2), 5.59 (1 H, dd, J2,3 = 9.3, H-2’), 5.93 (1 H, t, H-3’), 7.10–8.00 (20 H,
m, 4×Ph); δC (150 MHz, CDCl3): 61.4 (C-6’), 69.3, 72.4(2), and 75.0 (C-2’, 3’,
4’, 5’), 82.8 (C-7), 101.1 (C-1’), 127.3–136.8 (4×Ph), 165.6 and 165.7(2) (3×Bz),
172.0 (C-8); HMRS-ESI (positive mode): m/z 648.1836 [M+Na]+; calcd. for
C35H31NNaO10 648.1840; and 17 (180 mg, 55%): amorphous powder; [α]D –17◦
(c 1, CHCl3); δH (600 MHz, CDCl3): 2.20 (1 H, br.s, OH), 3.67 (1 H, m, H-5’),
3.73 (1 H, dd, J5,6a = 4.4, J6a,6b = 13.0, H-6a’), 3.86 (1 H, dd, J5,6a = 1.6, H-6b’),
4.66 (1 H, d, J1,2 = 7.9, H-1’), 5.31 (1 H, s, H-7), 5.52 (1 H, t, J3,4 = J4,5 = 9.7,
H-4’), 5.63 (1 H, dd, J2,3 = 9.9, H-2’), 5.80 and 6.90 (2 H, both br.s, CONH2),
5.85 (1 H, t, H-3’), 7.12–7.95 (20 H, m, 4×Ph); δC (150 MHz, CDCl3): 61.1 (C-6’),
69.4, 72.0, 72.1 and 75.0 (C-2’, 3’, 4’, 5’), 79.2 (C-7), 97.7 (C-1’), 127.8–135.0
(4×Ph), 165.7(2) and 166.0 (3×Bz), 172.1 (C-8); HMRS-ESI (positive mode):
m/z 648.1834 [M+Na]+; calcd. for C35H31NNaO10 648.1840.
(S)-2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyloxy(phenyl)acetamide
(18). Glucoside 16 (34 mg, 0.054 mmol) was dissolved in CH2Cl2 (0.5 mL) and
MeOH (1 mL), and 1 M sodium methoxide (0.02 mL) was added. The mixture
was kept at rt for 2 h. The mixture was made neutral with Amberlite IR-120
(H+). The resin was filtered off and thoroughly washed with MeOH, and the
combined filtrates were concentrated. The residue was dissolved in Py (1 mL),
and acetic anhydride (0.2 mL) was added. The reaction mixture was stirred at
rt for 3 h and coevaporated with toluene. The residue was purified by silica gel
column chromatography (2:1 toluene–acetone) to provide 18 (26 mg, quant.):
amorphous powder; [α]D +14◦ (c 1.1, CHCl3); δH (600 MHz, CDCl3): 2.00, 2.01,
2.02, and 2.09 (12 H, all s, 4×Ac), 3.61 (1 Н, m, H-5’), 3.95 (1 H, dd, J5,6a
2.1, J6a,6b = 12.3, H-6a’), 4.12 (1 H, dd, J5,6a = 5.3, H-6b’), 4.69 (1 H, d, J1,2
=
=
7.9, H-1’), 5.05 (1 H, t, J3,4 = J4,5 = 9.7, H-4’), 5.12 (1 H, dd, J2,3 = 9.5, H-2’),
5.13 (1 H, s, H-7), 5.23 (1 H, t, H-3’), 5.82 and 6.52 (2 H, both br.s, CONH2),
7.31–7.49 (5 Н, m, Ph); δC (150 MHz, CDCl3): 20.5, 20.6, 20.65, and 20.7
(4×Ac), 61.7 (C-6’), 68.1, 71.7, 72.0, and 72.5 (C-2’, 3’, 4’, 5’), 82.0 (C-7), 100.1
(C-1’), 127.1–135.9 (Ph), 169.3, 169.6, 170.2 and 170.5 (4×Ac), 173.0 (C-8);

Cyanogenic Glycosides
11
HMRS-ESI (positive mode): m/z 504.1474 [M+Na]+; calcd. for C22H27NNaO11
504.1476.
(R)-2,3,4,6-tetra-O-acetyl-β-D-glucopyranosyloxy(phenyl)acetamide
(6). Glucoside 6 (32 mg) was synthesized from glucoside 17 (42 mg,
0.067 mmol) as was described for preparation of 18 in a quantitative
yield: crystals; mp 145–146◦C (hexane-EtOAc); [α]D –39◦ (c 1, CHCl3); δH
(600 MHz, CDCl3): 2.01(2), 2.02, and 2.13 (12 H, all s, 4×Ac), 3.60 (1 Н, m,
H-5’), 4.12 (1 H, br.d, J6a,6b = 12.5, H-6a’), 4.28 (1 H, dd, J5,6a = 5.0, H-6b’),
4.35 (1 H, d, J1,2 = 7.5, H-1’), 5.05–5.15 (3 H, m, H-2’, 3’, 4’), 5.17 (1 H, s, H-7),
5.85 and 6.79 (2 H, both br.s, CONH2), 7.29–7.40 (5 Н, m, Ph); δC (150 MHz,
CDCl3): 20.5, 20.6, 20.65, and 20.7 (4×Ac), 61.7 (C-6’), 68.2, 71.2, and 72.0(2)
(C-2’, 3’, 4’, 5’), 79.4 (C-7), 97.5 (C-1’), 127.0–135.0 (Ph), 169.4, 169.8, 170.0, and
170.6 (4xAc), 172.0 (C-8); HMRS-ESI (positive mode): m/z 504.1478 [M+Na]+;
calcd. for C22H27NNaO11 504.1476.
General procedure B: Synthesis of unprotected derivatives of mandelic nitrile
The acyl-protected derivative of mandelic amide (1 eq.) was dissolved in
CH2Cl2 (2 mL/mmol) and MeOH (4 mL/mmol), and 1 M sodium methoxide
(0.05 mL/mmol) was added. The mixture was kept at rt for 2 h. The mixture
was made neutral with Amberlite IR-120 (H+). The resin was filtered off and
thoroughly washed with MeOH, and the combined filtrates were concentrated.
The residue was dissolved in dioxane (4 mL/mmol) and Py (2 mL/mmol), and
trifluoroacetic anhydride (TFAA) (1.5 eq. per free hydroxyl group) was added.
The reaction mixture was stirred at rt for 2 h, quenched with water, diluted
with methanol, and treated with an excess of Amberlyst A-26 (HCO3−). The
resin was filtered off and thoroughly washed with 50% aq. MeOH, and the
combined filtrates were concentrated. The residue was purified by silica gel col-
umn chromatography (20:5:15:6:4 EtOAc:MeOH:acetone:CH2Cl2:H2O) to pro-
vide unprotected derivatives of mandelic nitrile.
(S)-β-D-Glucopyranosyloxy(phenyl)acetonitrile [(S)-Sambunigrin]
(2) (23 mg) was obtained in near-quantitative yield from 16 (35 mg, 0.08 mmol)
according to the general procedure B: amorphous powder; [α]D –83◦ (c 1.0,
H2O); δH (600 MHz, CD3OD): 3.25–3.47 (4 H, m, H-2’, 3’, 4’, 5’), 3.70 (1 H,
dd, J5,6a = 6.3, J6a,6b = 12.0, H-6a’), 3.94 (1 H, dd, J5,6a = 1.7, H-6b’), 4.69 (1
H, d, J1,2 = 7.7, H-1’), 6.04 (1 H, s, H-7), 7.40–7.62 (5 Н, m, Ph); δC (150 MHz,
CD3OD): 62.9 (C-6’), 68.7 (C-7), 71.7, 74.8, 78.1, and 78.7 (C-2’, 3’, 4’, 5’), 102.2
(C-1’), 118.6 (C-8), 128.8, 130.0, 130.9, and 135.3 (Ph); HMRS-ESI (positive
mode): m/z 318.0938 [M+Na]+; calcd. for C14H17NNaO6 318.0948.
(R)-β-D-Glucopyranosyloxy(phenyl)acetonitrile [(R)-Prunasin] (1)
(14 mg) was obtained in near-quantitative yield from 17 (21 mg, 0.048 mmol)
according to the general procedure B: amorphous powder; [α]D –39◦ (c 1.0,
H2O); δH (600 MHz, CD3OD): 3.21–3.45 (4 H, m, H-2’, 3’, 4’, 5’), 3.71 (1 H,

D.V. Yashunsky et al.
12
dd, J5,6a = 6.2, J6a,6b = 12.0, H-6a’), 3.94 (1 H, dd, J5,6a = 2.3, H-6b’), 4.26 (1
H, d, J1,2 = 7.1, H-1’), 5.90 (1 H, s, H-7), 7.42–7.62 (5 Н, m, Ph); δC (150 MHz,
CD3OD): 63.0 (C-6’), 68.7 (C-7), 71.7, 75.0, 78.1, and 78.5 (C-2’, 3’, 4’, 5’), 102.2
(C-1’), 119.6 (C-8), 129.1, 130.3, 131.1, and 135.0 (Ph); HMRS-ESI (positive
mode): m/z 318.0941 [M+Na]+; calcd. for C14H17NNaO6 318.0948.
(R)-[(6-O-β-D-Glucopyranosyl-β-D-glucopyranosyl)oxy](phenyl)ace
tonitrile [(R)-Amygdalin] (3) (20 mg) was obtained in near-quantitative
yield from 19 (43 mg, 0.045 mmol) according to the general procedure B:
crystals; mp 210–211◦C (EtOH); [α]D +29◦ (c 1.0, H2O); δH (600 MHz, D2O):
3.32–3.57 (7 H, m, H-2’, 2”, 3’, 3”, 4’, 4”, 5”), 3.62 (1 H, m, H-5’), 3.74 (1 H, dd,
J5,6a = 5.9, J6a,6b = 12.5, H-6a”), 3.92 (2 H, m, H-6b” and H-6a’), 4.22 (1 H,
dd, J5,6a = 1.7, J6a,6b = 11.9, H-6b’), 4.59 (1 H, d, J1,2 = 7.9, H-1”), 4.62 (1 H,
d, J1,2 = 7.9, H-1’), 5.90 (1 H, s, H-7), 7.52–7.65 (5 Н, m, Ph); δC (150 MHz,
D2O): 62.0 (C-6”), 69.6 (C-6’), 70.3 (C-7), 70.4, 70.9, 74.0, 74.4, 76.6, 75.7, 76.9,
and 77.1 (C-2’, 2”, 3’, 3”, 4’, 4” 5’, 5”), 102.7 (C-1’), 104.1 (C-1”), 119.8 (C-8),
129.0, 130.6, 131.7, and 133.7 (Ph); HMRS-ESI (positive mode): m/z 480.1473
[M+Na]+; calcd. for C20H27NNaO11 480.1476.
(S)-(6-O-β-D-Glucopyranosyl-β-D-glucopyranosyloxy)(phenyl)aceto
nitrile [(S)-Neoamygdalin] (21) (19 mg) was obtained in near-quantitative
yield from 22 (40 mg, 0.042 mmol) according to the general procedure B:
amorphous powder; [α]D –15◦ (c 1.4, H2O); δH (600 MHz, CD3OD): 3.20–4.20
(12 H, m, H-2’, 2”, 3’, 3”, 4’, 4”, 5’, 5”, 6a,b’, 6a,b”), 4.35 (1 H, d, J1,2 = 7.7,
H-1”), 4.61 (1 H, d, J1,2 = 7.7, H-1’), 5.89 (1 H, s, H-7), 7.36–7.56 (5 Н, m, Ph);
δC (150 MHz, CD3OD): 60.9 (C-6”), 67.2 (C-7), 68.5 (C-6’), 69.5, 69.6, 72.7, 73.1,
75.3, 75.7, 75.8, and 75.9 (C-2’, 2”, 3’, 3”, 4’, 4” 5’, 5”), 100.0 and 103.1 (C-1’, 1”),
116.8 (C-8), 127.2, 128.5, 129.4, and 132.5 (Ph); HMRS-ESI (positive mode):
m/z 480.1459 [M+Na]+; calcd. for C20H27NNaO11 480.1476.
(R)-(6-O-β-D-Glucopyranosyl-β-D-glucopyranosyloxy)(phenyl)aceta
mide [(R)-Amygdalin amide] (20). The acyl-protected derivative of man-
delic amide 19 (40 mg, 0.035 mmol) was dissolved in CH2Cl2 (0.5 mL) and
MeOH (1 mL), 1 M sodium methoxide (0.02 mL) was added, and the mixture
was kept at rt for 2 h. The mixture was made neutral with Amberlite IR-120
(H+). The resin was filtered off and thoroughly washed with MeOH, and the
combined filtrates were concentrated. The residue was purified by silica gel
column chromatography (20:5:15:6:4 EtOAc:MeOH:acetone:CH2Cl2:H2O) to
22
provide (R)-amygdalin amide 20 (16 mg, quant.): amorphous powder; [α]D
–97◦ (c 1, H2O); δH (600 MHz, CD3OD): 3.22–3.43 (8 H, m, H-2’, 2”, 3’, 3”, 4’,
4”, 5’, 5”), 3.67 (1 H, dd, J5,6a = 5.3, J6a,6b = 11.9, H-6a”), 3.79 (1 H, dd, J5,6a
=
5.8, J6a,6b = 11.5, H-6a’), 3.88 (1 H, dd, J5,6a = 1.9, H-6b”), 4.13 (1 H, d, J1,2
= 7.7, H-1”), 4.15 (1 H, dd, J5,6a = 1.9, H-6b’), 4.42 (1 H, d, J1,2 = 7.8, H-1’),
5.31 (1 H, s, H-7), 7.32–7.54 (5 Н, m, Ph); δC (150 MHz, CD3OD): 63.0 (C-6”),
70.0 (C-7), 71.8 (C-6’), 71.9, 75.1, 75.4, 77.5, 77.7, 78.2, 78.3, and 80.0 (C-2’,
2”, 3’, 3”, 4’, 4” 5’, 5”), 101.0 and 105.1 (C-1’, 1”), 129.5, 129.7, 129.9, and 133.5

Cyanogenic Glycosides
13
(Ph), 174.0 (C-8); HMRS-ESI (positive mode): m/z 498.1574 [M+Na]+; calcd.
for C20H29NNaO12 498.1582.
Antifungal and Antibacterial Tests
The yeasts Saccharomyces cerevisiae VKM Y-1173 (model culture) and
Filobasidiella neoformans IGC 3957 (cryptococcosis pathogen) and Escherichia
coli K-12 were used as test cultures. F. neoformans was grown in a glucose-
peptone medium containing (g/L): glucose, 10; peptone (enzymatic, United
States), 5; yeast extract (Fluka, Germany), 4. The cultivation was carried out
in flasks with 200 mL of the medium on a shaker (145 rpm) at 28◦C for 2 days.
S. cerevisiae was grown in the YPD medium containing (g/L): glucose, 20; pep-
tone (enzymatic, USA, 20; yeast extract (Fluka, Germany), 10. The cultivation
was carried out in flasks with 200 mL of the medium on a shaker (145 rpm)
at 28◦C for 24 h. The bacterial culture was grown in a 5.5 liquid medium con-
taining (g/L): yeast extract, 1; soya extract, 30; amino peptide, 60; Tryptone, 5;
pH 7.2. The cultivation was carried out in tubes on a shaker (145 rpm) at 28◦C
for 24 h. The cultures were used to assess the ability to grow in the presence
of compounds 1–3, 20, and 21 in immunological plates. The tested compounds
were dissolved in deionized water to a concentration of 10 mg/mL. The cultures
were incubated in plate wells (the incubation mixture contained 0.035 mL of
0.01 M citrate buffer, pH 7.0 [for F. neoformans] or water [for the other two
cultures], 0.015 mL of cell suspension, and 0.01 mL of the aqueous solution
of tested compound) for 1 h at rt. The concentration of tested compounds in
the mixture was 1.7 mg/mL. The cells incubated in the presence of equivalent
amounts of 0.01 M citrate buffer, pH 7.0, or water were used as a control. Next,
0.25 mL of the media described above was added to each well, the cultivation
was performed in a thermal shaker at 30◦C for 1 day for S. cerevisiae and E.
coli and 2 days for F. neoformans, and the optical density of the culture was
measured at 495 nM (plate photometer Saphir, Russia).
Cell Culture
The human colon cancer cell line HCT-116 was obtained from the Russian
Cancer Research Center. Cells were cultured in RPMI-1640 medium (Gibco,
NY, USA) supplemented with 10% heat-inactivated FBS (Gibco, NY, USA).
Cultures were maintained in a humidified incubator at 37◦C in an atmosphere
of 5% CO2. The medium was changed every 2 days.
Cytotoxicity Assay
Cell viability was determined using the 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assay. Cells were seeded in quadrupli-
cate at a concentration of 2×104 cells/well on a 96-well plate (Nunc, Thermo
Scientific, San Jose, CA, USA). The final volume was adjusted to 100 μL/well.

D.V. Yashunsky et al.
14
HCT-116 cells were incubated with amygdalin and amygdalin’s derivatives at
concentrations of 10−2, 10−3, and 10−4 М for 72 h. After MTT (Sigma, MO,
USA) was added to each group, the cells were incubated for 4 h. Then, 30 μL
of 1 mg/mL MTT stain was added to each well and the plate was incubated
at 37◦C for 4 h. Next, 100 μL of dimethyl sulfoxide (Sigma) stop solution was
added to each well. The plate was shaken at rt for 10 to 20 min. The viabil-
ity was measured with a microtiter plate reader (Multiscan MS, Labsystem,
Finland) at a test wavelength of 540 nm. The optical density was calculated
as the difference between the reference wavelength and the test wavelength.
Percentage viability was calculated as: (treated sample/control sample)×100.
FUNDING
This work was supported by the Russian Science Foundation (RSF grant 14-
50-00126).
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