Chemistry of Natural Compounds, Vol. 48, No. 5, November, 2012 [Russian original No.

5, September–October, 2012]

A NEW ARYLSULFATASE FROM THE MARINE

MOLLUSK Turbo chrysostomus

M. S. Pesentseva,1* V. V. Sova,1 Al. S. Silcchenko,1 UDC 577.152.316

A. A. Kicha,1 Ar. S. Silcchenko,1 T. Haertle,2
and T. N. Zvyagintseva1

A new arylsulfatase (EC 3.1.6.1) was isolated from the liver of the marine mollusk Turbo chrysostomus. The
enzyme catalyzed hydrolysis of potassium p-nitrophenylsulfate, did not affect natural fucoidan, and catalyzed
cleavage of sulphate in the C4 position of xylose included in carbohydrate chains of holothurian triterpene
glycosides. Halistanol sulphate and glycosides from starfish that contained sulfates in the aglycon part of
the molecule inhibited the activity of the arylsulfatase. Several holothurian glycosides in small concentrations
(3.5–8 u 10–10 M) increased the enzyme activity. The arylsulfatase activity was maximal at pH 7. Its
molecular weight was 35 kDa according to gel-filtration. The Michaelis constant (KM) for hydrolysis of
p-nitrophenylsulfate was 10 mM. The inactivation half-life of the enzyme was 15 min at 55°C.

Keywords: arylsulfatase, marine mollusks, Turbo chrysostomus, triterpene glycosides, sulfated steroidal glycosides.

Sulfatases catalyze hydrolytic cleavage of sulfate from various organic substrates (EC 3.1.6.-). These enzymes have
been observed in most organisms from bacteria to mammals. The principal role of bacterial sulfatases consists of the utilization
of substances containing sulfates. Mammal sulfatases are involved in transformations of such sulfated substrates as
mucopolysaccharides, sulfolipids, and steroidal hormones [1–4]. Arylsulfatases, steroidsulfatases, and glycosulfatases that
act on sulfated mono- and polysaccharides were found in various organs of marine invertebrates [5–7]. The presence of
arylsulfatase activity in lysosomal cells, which are responsible for the lysis of cellular structures and various chemical compounds
allowed a digestive role to be proposed for arylsulfatases in mollusks [8, 9]. This hypothesis was consistent with the presence
of sulfated substances in the food of mollusks poisonous to plants and a higher level of arylsulfatase activity in their digestive
organs compared with the activity in predatory mollusks [10].
A comparison of the amino-acid sequence of known sulfatases showed that they are evolutionally conservative enzymes
[4]. It was found that the active center of sulfatases contained the unusual residue CD-formylglycine, which is formed in
eukaryote enzymes from cysteine during post-translation and from serine, in prokaryote enzymes [11, 12]. A high degree of
amino-acid sequence homology was observed in arylsulfatases and phosphatases [13, 14]. The evolutions of these enzyme
groups are probably related.
Many sulfatases are not highly specific enzymes. For example, steroidsulfatases of mollusks and mammals exhibit
arylsulfatase activity whereas homogeneous arylsulfatases contain traces of steroidsulfatase activity [15]. Natural sulfated
sugars turned out to be substrates for arylsulfatases and not for glycosulfatases [16]. Sulfoesterase from a marine mollusk
acted effectively on both synthetic substrates p-nitrocatecholsulfate and L-fucose sulfated at C-2 in addition to natural
fucoidan [17]. Several researchers thought that the existing nomenclature of sulfatases did not correspond completely to their
specificity [16].
The goal of the present work was to study the specificity of a new sulfatase isolated from the marine mollusk Turbo
chrysostomus inhabiting the littoral zone of the Socialist Republic of Vietnam.

1) G. B. Elyakov Pacific Institute of Bioorganic Chemistry, Far-East Branch, Russian Academy of Sciences, 690022,
Vladivostok, fax: (423) 231 40 50, e-mail: mpesentseva@gmail.com; 2) Laboratoire d’Etude des Interactions des Molecules
Alimentaires, Institut National de la Recherche Agronomique, B. P. 71627, 44316 Nantes, Cedex 3, France. Translated from
Khimiya Prirodnykh Soedinenii, No. 5, September–October, 2012, pp. 759–764. Original article submitted April 16, 2012.
0009-3130/12/4805-0853 ©2012 Springer Science+Business Media New York 853
TABLE 1. Specificity of Sulfatase from T. chrysostomus and Effect of Natural Glycosides on its Activity

Compound MW, Da Desulfating activity Inhibition, I50, M Activation, A50, M

Halistanol sulfate (1) 754 – 3 u 10–8 8 u 10–10

Holothurin A (2) + No inhibition No activation
Pseudostichoposide A (3) 1158 + No inhibition 3.5 u 10–10
Pseudostichoposide B (4) 1260 + No inhibition 7.9 u 10–10
Cucumarioside H (5) 1330 + No inhibition 7.5 u 10–10
Cucumarioside H2 (6) 1348 + No inhibition 7.4 u 10–10
Cucumarioside H5 (7) 1330 + No inhibition 7.5 u 10–10
Okhotoside B2 (8) 1348 + No inhibition 7.4 u 10–10
Okhotoside B3 (9) 1348 – No inhibition 7.4 u 10–10
Koreoside A (10) 1412 + No inhibition 3.5 u 10–10
Frondoside A7-3 (11) 1512 + No inhibition 6.6 u 10-10
Asterosaponin P1 (12) 700 – 1.8 u 10–8 No activation
Tornasteroside A (13) 1266 – 2.9 u 10–8 No activation
Asteriidoside A (14) 1442 – No inhibition No activation
Echinosteroside C 15-O-sulfate (15) 714 – 2.2 u 10–8 No activation
Leviusculoside D (16) 726 – 2.1 u 10–8 No activation

The digestive organs of marine invertebrates are known to contain active arylsulfatases [18]. We analyzed the content
of sulfatase activity in digestive organs of several species of marine mollusks (11 species of bivalves and two species of
gastropods) inhabiting Troits Bay, Poseta Gulf, Sea of Japan (Table 1). A high activity level was observed in the liver of
gastropod mollusks. In Tectonatica janthostoma, it exceeded the activity of bivalve sulfatases by 2–4 times. The sulfatase
activity was determined in five species of gastropod mollusks distributed in the South China Sea on the shore of the Republic
of Vietnam. A low enzyme activity level was observed only in the predatory mollusk Conus rexilum. Species poisonous to
plants contained highly active sulfatases:
Total activity per g of biomass,
Mollusk, species (class)
Pmol/min
Sea of Japan
Tectonatica janthostoma (Gastropoda) 4.13
Littorina sp. (Gastropoda) 2.75
Swiftopecten swifti (Bivalvia) 2.69
Mezuhopecten yessoensis (Bivalvia) 2.33
Chlamys nipponensis (Bivalvia) 1.96
Spisula sachalinensis (Bivalvia) 1.96
Mactra hinensis (Bivalvia) 1.71
Glycymeris yessoensis (Bivalvia) 1.22
Modiolus modiolus (Bivalvia) 1.18
Crenomytilus-grayanus (Bivalvia) 0.68
Peronidia venulosa (Bivalvia) 0.53
Callista brevisiphonata (Bivalvia) 0.44
Mercenaria stimpsoni (Bivalvia) 1.34
S. China Sea
Tectus paramis (Gastropoda) 8.8
Turbo chrysostomus (Gastropoda) 6.5
Lambis scopius (Gastropoda) 5.1
Oliva mniacea (Gastropoda) 3.43
Conus rexilum (Gastropoda) 0.25.
We isolated a sulfatase from the liver of the gastropod mollusk Turbo chrysostomus that catalyzed effectively the
cleavage of potassium p-nitrophenylsulfate. The liver of this mollusk contained in addition to the sulfatase a wide range of
O-glycosidehydrolases (in U/mg of protein): cellulase (0.077), amylase (0.069), laminarinase (0.056), pustulanase (0.006),
N-acetylglucoseaminidase (83.4), E-galactosidase (61.9), D-mannosidase (58.1), and E-glucosidase (27.8). The scheme for
isolating the sulfatase included a combination of sulfate precipitation, ultrafiltration, and hydrophobic and ion-exchange
chromatography. This produced a highly pure sulfatase with a purity that was 300 times greater without accompanying
O-glycosidehydrolases. The solution of highly pure enzyme of specific activity 7.2 u 102 U/mg of protein was stored frozen
at –20°C.
854
 O O
HO O
OH

O
OH
NaO3SO O
NaO3SO
CH2OH CH2OH
NaO3SO CH3 O
O O
OSO3 Na O O
OCH3 O
1 OH
HO HO 2
OH OH
OH
O
O O
O
OH
O
NaO3SO
CH2 OH CH3
O O
O O O
O O O R
OCH3 R
HO
HO H
OH 3, 4 O
OH OH
OH OAc
3: R = OH; 4: R = OSO 3Na O
NaO3SO
CH2 OH
O CH3 O
O O O 5: R =
OCH3 O
OH
HO
HO
OH
OH O 6: R =
O OH
OH
7: R =
HO 5 -7
OH
O O

O
OH OAc
O
R1
CH2R CH2 OSO3 Na
HOCH2 O
O
O O O
OCH3 O
OH
HO 8: R1 = OSO 3Na, R = OH
HO
OH
OH OH 8, 9 9: R1 = OH, R = OSO 3Na

The catalytic and molecular properties of the sulfatases differed considerably and depended on the source and
the functions carried out [19, 20]. The sulfatase from T. chrysostomus exhibited the greatest activity for potassium
p-nitrophenylsulfate at pH 7. According to gel filtration over a column of Superose12 HR 10/30 (FPLC), the molecular weight
of the sulfatase was 35 kDa. The Michaelis constant (Km) for hydrolysis of p-nitrophenylsulfate that was calculated by the
Lineweaver–Burke method was 10 mM. The high Km value could suggest that potassium p-nitrophenylsulfate was not the
actual substrate of sulfatase from T. chrysostomus. The inactivation half-life of the enzyme at 60°C was 30 min.
The specificity of the enzyme was studied using a collection of substrates that included various natural compounds
containing sulfates. An analysis of the reaction products by electrophoresis [21] showed that sulfatase from T. chrysostomus
did not catalyze desulfation of polymeric substrates such as dextran sulfate (Sigma) and fucoidan from the brown alga Fucus
evanescens. Natural fucoidan is 1,3;1,4-D-L-fucan sulfated at the fucose C-2 units and more rarely at C-2–C-4 [22]. According
to mass spectrometry, sulfatase also did not affect fucose sulfated in the C-4 position.

855
 O O
OH
O R
NaO3SO
CH2OSO3Na CH2OSO3Na O H
O CH3 10: R =
O O O OH
OCH3 O HO
OH
HO
HO
OH
OH O
O H
OH
11: R =
HO
OH H OH
10, 11

NaO3SO
H
NaO3SO
OMe O H
CH2OH
HO R2 CH3 O 13: R1 = R 2 = H
HO O
O O
O O
OH O CH2OH
HO
OH O
HO 14: R1 =
CH3 O OH
OH
HO CH3 O
O
OH O OH
OH OR1
OH
HO 14: R 2 = CH 3
OH HO
OH 12 OH 13, 14
OH

OH
OH OH

OSO3Na OSO3Na
OH OH
O O
O OH O OH
OH OH
HO
HO
OMe 15 OMe 16

The action of sulfatase on sulfated natural glycosides of various structures (Table 1) from starfish and holothuria was
evaluated by TLC using the corresponding desulfated glycosides as standards.
The experimental results showed that the enzyme catalyzed cleavage of sulfate located on C-4 of the first
monosaccharide unit (xylose) in the carbohydrate chain of the holothurian triterpene glycosides of the holostane (2–8), lanostane
(11), and norlanostane (10) series. Desulfation of holothurin (2) by sulfatase from T. chrysostomus was also confirmed by
HPLC. Replacement of the quinovose unit in the second position of the holothurian glycosides carbohydrate chain (2–7, 10,
11) by glucose (8, 9) did not affect the enzyme activity. The enzyme did not cleave sulfates located on the glucose C-6 and
3-O-Me-glucose (tertiary and quaternary monosaccharide units, respectively) in carbohydrate chains of the glycosides (8–11)
in addition to sulfates in the C-3 position of quinovose in pseudostichoposide B (4). Thus, it was established that sulfatase was
highly specific for sulfate in the xylose C-4 position.
Starfish glycosides containing sulfates only in the aglycon part of the molecule (13–16) and the sulfated
polyhydroxysteroids halistanol sulfate (1) and asterosaponin (12), which contained a sulfate in the monosaccharide side chain,
were not affected by sulfatase.
It was shown earlier that halistanol sulfate and its derivatives inhibited endo-E-1,3-glucanase from marine mollusks
[23]. It was also shown that several natural glycosides inhibited sulfatase from the marine mollusk Littorina kurila [24].

856
Potassium p-nitrophenylsulfate was used as a substrate to study the action of natural glycosides from marine sources on the
activity of sulfatase from T. chrysostomus.
It was found that glycosides (13, 15, 16) sulfated only on the aglycon that did not undergo enzymatic desulfation
inhibited the activity of sulfatase. Asterosaponin 12, which contained a sulfate in the monosaccharide and yet was not affected
by the enzyme, was also a sulfatase inhibitor. The lack of inhibition by glycoside 14 compared with other glycosides from
starfish could be related to the presence in it of a longer hexasaccharide carbohydrate chain. Studies of the biological activity
of this series of glycosides that were carried out previously also showed that asteriidoside A (14) exhibited the lowest hemolytic
and cytotoxic activity compared with asterosaponins containing a pentasaccharide carbohydrate chain [25, 26].
It was noted that halistanol sulfate, being an effective inhibitor of endo-E-1,3-glucanase of marine mollusks, at
low concentrations activated glucanase with an exo-type of action [23]. We found by studying the effect of the compounds
at various concentrations (0.05–1 mg/mL) on the sulfatase activity that holothurian glycosides at low concentrations
(3.5–8u10–10 M) increased the sulfatase activity by 50%. The exception was holothurin A (2), which did not affect the enzyme
activity against potassium p-nitrophenylsulfate. A peculiarity of the structure of this compound was the additional hydroxyl of
the polycyclic system and the presence of an epoxide.
Thus, a new arylsulfatase (EC 3.1.6.1) was isolated from the marine mollusk T. chrysostomus. Several of its properties
were determined. Its specific action was established. The enzyme catalyzed cleavage of sulfate in the C-4 position of xylose
incorporated into carbohydrate chains of triterpene glycosides of the holostane, lanostane, and norlanostane series.

EXPERIMENTAL

Reagents. Potassium p-nitrophenylsulfate was purchased (Sigma, USA). Holothurian triterpene glycosides and
sulfated polyhydroxysteroids were supplied by staff members of the Laboratory of the Chemistry of Marine Natural Compounds,
G. B. Elyakov Pacific Institute of Bioorganic Chemistry, FEB, RAS. Structures and properties of the compounds were studied
earlier [27–32].
Inorganic salts and acids were commercially available (Reakhim, Russia). Supports for chromatography, BSA (Sigma,
USA), and proteinase inhibitors (Roche Diagnostics, Germany) were commercially available. Marine mollusks were collected
in Troits Bay, Poseta Gulf, Sea of Japan in September 2011 and in the littoral zone of the Socialist Republic of Vietnam (South
China Sea) in May 2011.
Purification of Sulfatase. Mollusk liver (100 g) was homogenized and extracted with Tris-HCl buffer (0.2 M, pH 7.0,
1:3 ratio) with added proteinase inhibitors. The resulting extract was centrifuged for 20 min at 12,000 rpm and 4°C. The
supernatant was treated with NH4SO4 to give 60% saturation. The resulting precipitate was separated by centrifugation and
dissolved in the minimum amount of Tris-HCl buffer (0.1 M, pH 7.0). Enzyme preparation was placed on a column of Phenyl-
Sepharose (12 u 1.5 cm) and eluted stepwise by an ammonium sulfate gradient (2 M, 1 M, 0.5 M, 0.05 M, working buffer,
50 mL each). Fractions containing active enzyme were combined, concentrated three times, and dialyzed against Tris-HCl
buffer (0.025 M, pH 7.0, working buffer). The dialyzed solution was placed on an Econo Pac Q ion-exchange column (2 u 3.5 cm,
Bio-Rad, USA) and eluted by a linear gradient of NaCl from 0 to 1 M (500 mL). Fractions containing sulfatase were combined
and concentrated by ultrafiltration through a PM-10 membrane (Amicon, Holland). Aliquots of sulfatase solution were preserved
at 20°C.
Protein quality was determined by the Bradford method using BSA as a standard [33].
Sulfatase activity was detected by the appearance of p-nitrophenol through the action on potassium
p-nitrophenylsulfate. A reaction mixture containing enzyme solution (50 PL) in working buffer and substrate solution (50 PL,
1 mg/mL) in the same buffer was incubated for 10 min at 37°C and treated with Na2CO3 solution (100 PL, 1 M). The
absorption of the solution was measured at 400 nm. An activity unit was taken as the amount of enzyme that catalyzed the
conversion of 1 Pmol of substrate per min.
Determination of Thermal Stability. Aliquots of enzyme solution in working buffer (50 PL, 10 act. U) were held in
an aqueous thermostat (U-2, Germany) at 20, 37, 45, 55, and 60°C. The stability of the enzyme held at the various temperatures
was checked every 10 min for 1 h. Aliquots (50 PL, 1 mg/mL) were taken every 20 min. The mixture was cooled. A solution
of potassium p-nitrophenylsulfate (50 PL, 1 mg/mL) was added. The residual activity was determined by the method described
above.

857
 Determination of Enzyme pH-optimum. A reaction mixture containing enzyme (10 act. U) in working buffer
(1 PL), potassium p-nitrophenylsulfate solution (50 PL, 1 mg/mL in H2O), and citrate (50 PL, 0.2 M, pH 3.0–6.0) or Tris-HCl
(0.2 M, pH 7.0–9.0) buffer was incubated for 10 min at 37°C. The activity was determined.
Molecular weight was estimated by gel filtration using an AKTA FPLC instrument (Amersham Pharmacia) and
a Superose 12 HR 10/30 column (300 u 1.0 mm, USA). The eluent was working buffer containing NaCl (150 mM). Detection
was made at 280 nm. The proteins human IgG, rabbit IgG, BSA, cytochrome C, and lysozyme were used for calibration. The
yield of sulfatase was determined from the enzyme activity.
Determination of Michaelis Constant. A reaction mixture containing enzyme (10 act. U) in working buffer (50 PL)
and potassium p-nitrophenylsulfate solution (50 PL, 0.02–5 mg/mL) was incubated for 10 min at 37°C. The activity was
determined. The Michaelis constant (Km) was calculated using the Lineweaver–Burke double inverse method.
Enzymatic Hydrolysis of Sulfated Polysaccharides and Fucose. A reaction mixture containing sulfatase solution
in working buffer (50 PL, 10 act. U) and buffer solution (100 PL) of mono- or polysaccharide (1 mg/mL) was held for 16 h.
Then, the reaction was stopped by boiling. The reactions products from fucoidan from the brown alga F. evanescens and
dextran sulfate (Sigma) were detected by electrophoresis [21]. The MALDI-TOF mass spectrum of sulfated glucose was
recorded on a Bruker Biflex III MALDI-TOF mass spectrometer (Germany).
Determination of Inhibiting/activating Action. A reaction mixture containing enzyme solution in working buffer
(50 PL, 10 act. U) and aqueous solutions of inhibitors of various concentration (50 PL; 1, 0.8, 0.6, 0.4, 0.2, 0.1, 0.05 mg/mL)
was held for 10 min. Then, potassium p-nitrophenylsulfate solution (50 PL, 1 mg/mL) was added. The mixture was incubated
for 10 min at 37°C. The reaction was stopped by adding Na2CO3 solution (100 PL, 1 M). Residual enzyme activity was
determined. The concentrations of compounds for which the enzyme activity was inhibited or activated by 50% were taken as
I50 and A50.
Enzymatic Desulfation of Natural Glycosides. A solution of sulfatase in working buffer (20 mL, 10 act. U) was
treated with glycoside calculated so that their final concentration would be less than I50. The mixture was incubated for 16 h.
The reaction was stopped by boiling. The mixture was analyzed using TLC on silica gel and CHCl3:EtOH:H2O (100:100:17)
and using HPLC. Analytical HPLC was carried out on an Agilent Technologies chromatograph (series 1100) using a C-18
column (250 u 4.6 mm, Supelco, Germany) and a refractive-index detector with elution by MeOH (60%).

ACKNOWLEDGMENT

This work was supported by the program of the Russian Academy of Science “Molecular and Cellular Biology”.

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