Preparation of (2?-5?)-oligonucleotides based on 6-N- benzylaminopurineriboside using the Spicaria violacea fungal enzyme complex

Article abstract of DOI:10.1007/s10600-009-9249-6

A method for chemico-enzymatic synthesis of (2?-5?)-oligonucleotides with 6-N-benzylaminopurineriboside as the nucleoside units was proposed. The method consisted of enzymatic hydrolysis of the oligonucleotides with mixed (2?-5?)-(3?-5?)-phosphodiesterbonds that were prepared by polymerization of 6-N-benzyladenosine-2?(3?)-monophosphate by using (3?-5?)-specific nuclease and phosphatase contained in the filtrate of culture medium of the mycelial fungus Spicaria violacea.

Full text of DOI:10.1007/s10600-009-9249-6

Chemistry of Natural Compounds, Vol. 45, No. 1, 2009
PREPARATION OF (2 -5 )-OLIGONUCLEOTIDES BASED ON 6- -
′ ′
N
BENZYLAMINOPURINERIBOSIDE USING THE
FUNGAL ENZYME COMPLEX
Spicaria violacea
T. I. Kulak,1* O. V. Tkachenko,1 O. Yu. Grishan,1 T. A. Kukharskaya,2
L. A. Eroshevskaya,2 E. N. Kalinichenko,1 and A. I. Zinchenko2
UDC 577.113.6:
577.123.2
′ ′
A method for chemico-enzymatic synthesis of (2 -5 )-oligonucleotides with 6-N-benzylaminopurineriboside
as the nucleoside units was proposed. The method consisted of enzymatic hydrolysis of the oligonucleotides
′ ′ ′ ′
withmixed(2 -5 )-(3 -5 )-phosphodiesterbondsthat were prepared by polymerization of 6-N-benzyladenosine-
′ ′ ′ ′
2 (3 )-monophosphate by using (3 -5 )-specific nuclease and phosphatase contained in the filtrate of culture
Spicaria violacea.
medium of the mycelial fungus
Key words: (2′-5′)-oligoadenylates, 6- -benzylaminopurineriboside,
, nuclease, enzymatichydrolysis.
N
Spicariaviolacea
(2′-5′)-Oligoadenylates are oligonucleotides incorporating several adenosine molecules bonded through a
(2′-5′)-phosphodiester bond [ppp5′A2′p(5′A2′p) 5′A, 1] and are one of the principal mediators of interferon antiviral action in
n
mammal cells [1-4]. (2′-5′)-Oligoadenylates are active toward animal and plant viruses [5, 6] and affect on cell growth and
development [7, 8]. The principal types of biological activity are typical of compounds containing an oligonucleotide chain of
at least three nucleoside units. Dephosphorylated oligomers 2 and their synthetic analogs, in particular, those containing
6- -benzyladenosine (3) in various positions of the oligonucleotide chain exhibit interesting biological properties [6, 9]. This
N
nucleoside is a riboside of the natural phytohormone 6- -benzylaminopurine (BAP) and possesses cytokine activity [10].
N
Analogs of the A(2′p5′A) trimer that contain BAP-riboside moieties in various positions of the chain exhibit plant growth-
2
stimulating properties, possess antiviral activity toward plant viruses [6], activate ribonuclease L, and inhibit growth of
syncytium induced by the HIV-1 virus [9].
R O
1
R
Ade
O
O
O
1:
R = H, R = H O P
9 3
1
4
R
2: R = R = H
HO
Ade
O
1
P
O
O
4: R = Bz, R = H
1
OH
O
NHR
R
Ade
HO
P
O
O
N
N
OH
R
Ade =
n
Bz = CH C H
6 5
2
N
N
HO
OH
1, 2, 4
1)InstituteofBioorganicChemistry, NationalAcademyofSciences, Belarus, 220141, Minsk, ul. Akad. V. Kuprevicha,
5/2, e-mail: kulak@iboch.bas-net.by; 2) Institute of Microbiology, National Academy of Sciences of Belarus, 220141, Minsk,
ul. Akad. V. Kuprevicha, 2. Translated from Khimiya Prirodnykh Soedinenii, No. 1, pp. 64-68, January-February, 2009.
Original article submitted July 1, 2008.
0009-3130/09/4501-0074 ©2009 Springer Science+Business Media, Inc.
74

Chemical methods for synthesizing (2′-5′)-oligoadenylates are labor-intensive and expensive. Thus, the literature
4
method for preparing 6-N-benzyladenylyl(2′-5′)-6-N-benzyladenylyl(2′-5′)-6-N-benzyladenosine ( , n = 1) from 6-N-benzyl-
3
adeonsine ( ) is based on the triester method of synthesizing oligonucleotides that includes 11 synthetic steps and requires
performing 9 column chromatographies [6].
2
We have previously shown that short dephosphorylated (2′-5′)-oligoadenylates ( ) can be prepared by enzymatic
hydrolysisoflong-chain oligonucleotidescontainingmixed(2′-5′)-(3′-5′)-internucleotidebondsbyusing(3′-5′)-specificnuclease
and phosphatase in the filtrate of culture medium (FCM) of the mycelial fungus Spicaria violacea [11]. The goal of the present
3
work was todevelop a chemico-enzymatic method for synthesizing (2′-5′)-oligonucleotides containing 6-N-benzyladenosine( )
moieties as the nucleoside units.
Mixed (2′-5′)-(3′-5′)-oligonucleotides are substrates for subsequent enzymatic hydrolysis bythe enzyme complex from
7
FCM of Spicaria violacea and were synthesized bypolymerization of 6-N-benzyladenosine-2′(3′)-monophosphate ( ) obtained
3
5
3
from 6-N-benzyladenosine ( ). 6-N-Benzyl-5′-O-monomethoxytrityladenosine ( ) [6] was prepared by reaction of with
monomethoxytritylchloride (MMTrCl) in pyridine in 85% yield. Then it was treated successively with solutions of
5
tri(imidazolyl)phosphite (PIm ) in THF:pyridine, iodine in THF:pyridine:water, and aqueous HCl. The reaction of with PIm
3
3
6
5
should lead to formation of only cyclic phosphite because the primary 5′-hydroxyl of is blocked by the MMTr group [12,
6
13]. Treatment of the reaction mixture obtained from oxidation of by iodine in the presence of water and pyridine [14] with
HCl (0.2 M) gave first rapid removal of the acid-labile MMTr group and then hydrolysis of the cyclophosphate group that is
7a
7b
also unstable in acidic media [15, 16]. The resulting mixture of 6-N-benzyladenosine 2′(3′)-monophosphates ( and ) was
isolated in 66.7% yield by chromatography over DEAE-Sephadex A-25 in the HCO − -form.
3
Bz
Ade
Bz
Ade
O
RO
Bz
O
MMT rO
Ade
O
HO
O
PIm
3
(PhO) POCl
2
1. I /H O
2
2
+
Bu N
3
2. H
HO
OH
RO
OR
1
O
P
Im
3, 5
7a, b
3: R = H
6
5: R = MMTr
7a: R = H, R = PO H
3 2
1
7b: R = PO H , R = H
Bz
3
2
1
Ade
O
HO
O
O
O
Bz
O
Ade
H,
P
O
O
−
O
O
O
4 + 3
Bz
Ade
H,
P
O
m
O
P
−
O
O
O
−
O
8
O
7a
7b
3
The formation from and of via enzymatic hydrolysis using alkaline phosphatase confirmed that theycontained
7a
7b
a phosphate group. The location of the phosphate on C(2′) or C(3′) of and , respectively, was confirmed by the spin—spin
coupling constants of the corresponding carbohydrate protons with the P atom in the PMR spectrum (3J
= 7.4 Hz for
2′,P
3
7a
7a
7b
2′-monophosphate
3′-monophosphates
(H -form).
and
J
= 7.0 Hz for 3′-isomer ). Two resonances at −1.10 and −0.57 ppm for 2′- and
3′,P
31
7b
and , respectively, were observed in the P NMR spectrum of the resulting mixture of nucleotides
+
7
Phosphates were polymerized bytreatment with diphenylchlorophosphate and tri-n-butylamine in dioxane [16, 17].
8
The resulting 6-N-benzyl derivatives of the oligoadenylates with mixed (2′-5′)-(3′-5′)-phosphodiester bond ( ) by nuclease and
phosphatase in FCM of S. violacea were hydrolyzed at 55°C in sodium acetate buffer (pH 6.0) containing MgCl (10 mM).
2
Aliquots of the reaction mixture were analyzed by TLC during the course of the reaction.
75

Incubation of the (2′-5′)-(3′-5′)-oligoadenylates 8 with FCM of S. violacea led to their hydrolysis and the formation
of 3 and an array of short-chain oligonucleotides with high chromatographic mobility in polar media compared with the values
for the components of the starting mixture of 8.
Enzymatic hydrolysis of 6-N-benzylated oligoadenylates 8 by the enzyme complex from FCM of S. violacea occurred
much slower than for the analogous unmodified adenosine derivatives (the time for completion of the hydrolysis increased by
more than four times even if the amount of FCM used increased by 2.5 times). This was apparently explained by differences
in the three-dimensional structure of the oligoadenylates with free NH groups and their 6-N-benzyl derivatives 8 [6].
2
Preparative separation of the products from enzymatic hydrolysis was carried out by column chromatography over
DEAE-Sephadex A-25. Precipitation by NaI in acetone produced the sodium salts of compounds that were assigned the
following structures based on results from enzymatic hydrolysis and NMR spectroscopy: 6-N-benzyladenylyl(2′-5′)-6-N-
benzyladenosine (4, n = 0), 6-N-benzyladenylyl-(2′-5′)-6-N-benzyladenylyl(2′-5′)-6-N-benzyladenosine (4, n = 1), and
6-N-benzyladenylyl(2′-5′)-6-N-benzyladenylyl-(2′-5′)-6-N-benzyladenylyl-(2′-5′)-6-N-benzyladenosine (4, n = 2) in yields
calculated for 7 of 21.4, 10.1, and 2.4%, respectively.
Treatment ofcompounds4with snake venom phosphodiesterase, which cleavesboth (3′-5′)-and(2′-5′)-internucleotide
bonds, formed 6-N-benzyladenosine (3) and its 5′-monophosphate (9) in the following ratios: 1:1 for 4 (n = 0), 1:2 for 4
(n = 1), and 1:3 for 4 (n = 2). Oligonucleotides 4 were stable to the action of nuclease S of Aspergillus oryzae, which can
1
hydrolyze only (3′-5′)-internucleotide bonds. This indicates that these compounds have (2′-5′) phosphodiester bonds. The
stability to the action of bacterial alkaline phosphatase of the resulting oligomers 4 confirmed that they lacked terminal
phosphates. The results indicate that derivatives 4 are a dinucleosidemonophosphate (n = 0), trinucleosidediphosphate (n = 1),
and tetranucleosidetriphosphate (n = 2), the 6-N-benzyladenosine in which are bonded by (2′-5′)-phosphodiester bonds.
The structures of compounds 4 were confirmed by NMR spectroscopy. Thus, the PMR spectrum of 4 (n = 0) contained
resonances for protons of two nucleosides; of 4 (n = 1), three nucleosides. The PMR spectrum of 4 (n = 0) had spin—spin
coupling constant for proton H-2′ of one of the nucleosides with the P atom (J = 9.1 Hz). The PMR spectrum of 4 (n = 1)
2′,P
agreedwith that for 6-N-benzyladenylyl(2′-5′)-6-N-benzyladenylyl-(2′-5′)-6-N-benzyladenosinethat waspreparedbythetriester
31
method [6]. The P NMR spectrum of 4 (n = 0) had one singlet corresponding to the internucleotide phosphodiester group
whereas that of trimer 4 (n = 1) had two such resonances (−0.97 and −1.39 ppm).
Thus, nuclease from FCM of S. violacea performed selective hydrolysis of (3′-5′)-phosphodiester bonds in mixed
(2′-5′)-(3′-5′)-oligomers 8 that contain nucleoside units of 6-N-benzyladenosine analogous to that which was observed for
compounds based on adenosine [11]. The lack ofreaction products with terminal 2′,3′-cyclophosphates indicates that treatment
of oligomers 8 with the enzyme complex from FCM of S. violacea also caused hydrolysis of terminal 2′,3′-cyclophosphates in
the oligomers. The action of phosphatase in FCM caused dephosphorylation ofthe resulting mono- and oligonucleotides, which
produced nucleoside 3 and short-chain (2′-5′)-oligomers 4.
The described method for synthesizing (2′-5′)-oligonuclotides based on BAP-riboside that uses the enzyme complex
of S. violacea is rather simple and convenient to implement. It does not require isolation of highly pure enzymes and can
produce the target compounds in satisfactory yields.
EXPERIMENTAL
6-N-Benzyladenosine (3) was prepared bymicrobiological transribosylation of 6-N-benzyladenine using uridine as the
carbohydrate donor [18].
6-N-Benzyladenosine-5′-O-monophosphate (9) that was used as a reference for hydrolysis of oligomers 4 by snake
venom phosphodiesterase was prepared by reaction of 3 with POCl in trimethylphosphate [19].
3
Enzymatic hydrolysis ofoligonucleotides was carried out in culture medium offungus strain S. violacea BM-105D that
was preserved in the Belarusian Collection of Nonpathogenic Microorganisms of the Microbiology Institute of the National
Academy of Sciences of Belarus as number BIM F-329. The cultivation conditions for mycelial fungus S. violacea and
preparation of the filtrate of culture medium containing phosphatase and nuclease have been previously described [20].
We used bacterial alkaline phosphatase (Fluka), snake venom phosphodiesterase (Boehringer), and nuclease S of
1
Aspergillus oryzae (NPO Vector).
76

Enzymatic dephosphorylation of nucleotides 7a and 7b by alkaline phosphatase was carried out in Tris-HCl buffer
(0.1 M, pH 9.0). Enzymatic hydrolysis of compounds 4 by snake venom phosphodiesterase was performed in Tris-HCl buffer
(0.1 M, pH 8.7) containing MgCl (10 mM). The action of nuclease S on the resulting oligonucleotides was studied using
2
1
solutions of them in sodium-acetate buffer (0.04 M, pH 4.7) containing ZnCl (0.1 mM) [17]. The composition of the reaction
2
mixtures from enzymatic hydrolysis was analyzed by TLC. Spots of reaction products were eluted by ethanol:water (1:1).
Optical density of the resulting solutions was measured at 270 nm. The amounts of compounds were calculated taking into
account their molar absorption coefficients.
TLC was performed on Kieselgel 60 F254 plates (Merck) using solvent systems CHCl :CH OH (4:1, A) and
3
3
2-propanol:aqueous ammonia (25%):water (7:1:2, B). Column chromatographyused DEAE-Sephadex A-25 anion-exchanger
(Pharmacia).
UV spectra were recorded on a Specord M-400 spectrophotometer. PMRspectra in DMSO-d (TMS internal standard)
6
and D O (t-BuOH internal standard), chemical shifts of protons are given relative to TMS taking the chemical shift of t-BuOH
2
vs. TMS as 1.27 ppm) were recorded on an Avance-500 spectrometer (Bruker). Resonances of protons were assigned using 2D
31
correlation spectroscopy (COSY). P NMR spectra were recorded on the same instrument using H PO (85%) as a standard
3
4
with full decoupling of protons.
Elemental analyses of the products agreed with those calculated.
6-N-Benzyladenosine-2′(3′)-O-monophosphate (7a, 7b). Asolution of6-N-benzyl-5′-O-monomethoxytrityladenosine
(5, 1.26 g, 2 mmol) [6] in THF:pyridine (4:1, 54 mL) was cooled to 0°C; stirred; treated under an Ar atmosphere with a solution
of tri(imidazol-1-yl)phosphine, prepared by addition of PCl (0.53 mL, 6 mmol) to a solution of imidazole (2.45 g, 36 mmol)
3
in THF (48 mL) at 0°C with subsequent filtration of the precipitated imidazole hydrochloride; stirred for 15 min at 0°C; treated
with a solution of iodine (1.52 g, 6 mmol) in THF:H O:pyridine (4:4:1, 45 mL), stored for 30 min, treated dropwise with
2
Na S O solution until colorless, and evaporated to dryness. The solid was evaporated with toluene (2 × 15 mL) in order to
2 2
3
remove traces of pyridine.
The solid after evaporation was dissolved in THF:dioxane:H O (1:1:1, 78 mL), treated with conc. HCl (2.4 mL), stored
2
for 16 h, neutralized with aqueous ammonia (25%) until the pH was 7, and evaporated to dryness. The solid after evaporation
was dissolved in water (400 mL) and extracted with CHCl (2 × 200 mL). The aqueous layer was diluted to 800 mL, placed
3
3
on a column with DEAE-Saphadex A-25 in the HCO − -form (350 cm ), and eluted with water (500 mL) and a gradient of
3
TEAB from 0 to 0.5 M (total volume 3.0 L). Fractions containing a mixture of di(triethylammonium) salts of 2′- and
3′-monophosphates of 6-N-benzyladenosine (7a and 7b) were combined and evaporated. The solid was evaporated with ethanol
to remove traces of TEAB. The solid after evaporation was dissolved in the minimal volume of water:ethanol (1:1), placed on
+
3
a column with cation-exchanger Dowex 50W×4 in the H -form (80 cm ), and eluted with water (200 mL) and water:ethanol
+
(1:1). Fractions containing phosphates 7a and 7b in the H -form were combined and evaporated to afford a mixture of
nucleotides 7a and 7b (0.58 g, 66.7%) as a white powder, R 0.37 (B), UV spectrum (MeOH, λ , nm): 270 (log ε 4.28).
f
max
6-N-Benzyladenosine-2′-O-monophosphate (7a). PMR spectrum (DMSO-d , δ, ppm, J/Hz): 8.52 (1H, m, NH), 8.33
6
( 1H, s, H-2), 8.18 (1H, s, H-8), 7.31-7.16 (5H, m, C H ), 6.06 (1H, d, H-1′, J1′,2′ = 6.53), 5.17 (1H, m, H-2′, J2′,1′ = 6.53,
6
5
J2′,3′ = 4.77, J
= 7.4), 4.68 (2H, m, CH Ph), 4.36 (1H, dd, H-3′, J3′,2′ = 4.77, J3′,4′ = 2.5), 3.98 ( 1H, m, H-4′), 3.64 (1H,
dd, H-5′, J5′,4′ = 3.1, J5′,5′′ = 12.0), 3.52 (1H, dd, H-5′′, J5′′,4′ = 3.0, J5′′,5′ = 12.0). P NMR spectrum (DMSO-d , δ, ppm):
2′,P
2
31
6
−1.10 s.
6-N-Benzyladenosine-3′-O-monophosphate (7b). PMR spectrum (DMSO-d , δ, ppm, J/Hz): 8.52 (1H, m, NH), 8.37
6
(1H, s, H-2), 8.18 (1H, s, H-8), 7.31-7.16 (5H, m, C H ), 5.89 (1H, d, H-1′, J1′,2′ = 7.11), 4.77 (1H, m, H-2′, J2′,1′ = 7.11,
6
5
4
J
J
2′,3′ = 5.3, J < 1.0), 4.68 (2H, m, CH Ph), 4.63 (1H, m, H-3′, J3′,2′ = 5.3, J = 7.0), 4.22 (1H, m, H-4′), 3.67 (1H, dd, H-5′,
2′,P 2 3′,P
31
5′,4′ = 3.0, J5′,5′′ = 12.3), 3.55 (1H, dd, H-5′′, J5′′,4′ = 3.1, J5′′,5′ = 12.3). P NMR spectrum (DMSO-d , δ, ppm): −0.57 s.
6
Polymerization of 6-N-Benzyladenosine-2′(3′)-O-monophosphate (7a, 7b). Phosphates (7a, 7b, 0.57 g, 1.3 mmol)
were treated with methanol:ethanol (1:1, 26 mL) and tri-n-octylamine (0.46 g, 1.3 mmol), refluxed for 15 min until the starting
powder dissolved, cooled, and evaporated to dryness. The solid was evaporated with benzene (3 × 10 mL), dissolved in dioxane
(3.2 mL), stirred, treated with diphenylchlorophosphate (0.39 mL, 1.9 mmol) and tri-n-butylamine (0.91 mL, 3.8 mmol), stirred
for 2 h, treated with diphenylchlorophosphate (0.39 mL, 1.9 mmol) and tri-n-butylamine (0.91 mL, 3.8 mmol), stirred for
20 h, evaporated to half the volume, and poured into ether (150 mL). The powdery white precipitate was filtered off, washed
with ether (2 × 20 mL), and dried in air to afford alkylammonium salts of (2′-5′)-(3′-5′)-oligonucleotides 8 (0.68 g).
77

Enzymatic Hydrolysis of Mixed (2′-5′)-(3′-5′)-Oligonucleotides 8 by Enzyme Complex Nuclease/Phosphatase of
Fungus S. violacea. A mixture of oligomers 8 (0.2 g) was treated with water (17.7 mL), sodium-acetate buffer (6.7 mL, 1 M,
pH 6.0), MgCl solution (0.67 mL, 0.5 M), and FCM of S. violacea (10.4 mL) [20]; stored in a thermostat at 55°C for 6 h;
2
treated with FCM (10.4 mL); stored for another 12 h; heated on a boiling water bath for 3 min; and centrifuged for 10 min at
2,600 rpm. The supernatant was diluted with water to 400 mL, placed on a column with DEAE-Sephadex A-25 in the
3
HCO − -form (120 cm ), and eluted with water until absorption in UV light disappeared and then with a gradient of TEAB from
3
0 to 0.5 M (total volume 2 L). Fractions containing pure oligoadenylates 4 (n = 0-2) were combined and evaporated. The solids
were evaporated with ethanol. Precipitation from methanol solutions by NaI solution (1 M) in acetone produced sodium salts
of the following compounds (in order of elution from the column):
6-N-Benzyladenylyl(2′-5′)-6-N-benzyladenosine (4, n = 0, 32.8 mg, 21.4% calculated for 7a and 7b), R 0.80 (B),
f
UV spectrum (MeOH, λ , nm): 270 (4.52). PMR spectrum (D O, δ, ppm, J/Hz): 8.19 (1H, s), 8.15 (s, 1H), 7.87 (1H, s), 7.82
max
2
(1H, s, 2H-2, 2H-8), 7.41-7.22 (10H, m, 2C H ), 6.17 (1H, d, H-1′, J1′,2′ = 5.22), 5.83 (1H, d, H-1′, J1′,2′ = 3.63), 5.23 (1H, m,
6
5
H-2′, J
= 9.1);
2′,P
6-N-Benzyladenylyl(2′-5′)-6-N-benzyladenylyl(2′-5′)-6-N-benzyladenosine (4, n = 1, 16.0 mg, 10.1%), R 0.78 (B),
f
UV spectrum (MeOH, λ , nm): 270 (4.71). PMR spectrum (D O, δ, ppm, J/Hz): 8.13 (1H, s), 8.08 (1H, s), 8.04 (1H, s), 7.98
max
2
(1H, s), 7.81 (1H, s), 7.75 (1H, s, 3H-2, 3H-8), 7.37-7.18 (15H, m, 3C H ), 6.08 (1H, d, H-1′, J1′,2′ = 5.19), 6.01 (1H, d, H-1′,
6
5
31
J1′,2′ = 4.44), 5.86 (1H, d, H-1′, J1′,2′ = 3.00), 5.07 (1H, m, H-2′, J
= 8.4).
P NMR spectrum (D O, δ, ppm): −1.39 s,
2′,P
2
−0.97 s;
6-N-Benzyladenylyl(2′-5′)-6-N-benzyladenylyl(2′-5′)-6-N-benzyladenylyl-(2′-5′)-6-N-benzyladenosine (4, n = 2,
3.8 mg, 2.4%), R 0.75 (B), UV spectrum (MeOH, λ , nm): 270 (4.74).
f
max
REFERENCES
1.
2.
3.
4.
5.
6.
7.
M. Kerr and R. E. Brown, Proc. Natl. Acad. Sci. USA, 75, 1, 256 (1978).
C. Baglioni, M. A. Minks, and P. A. Maroney, Nature (London), 273, 5664, 684 (1978).
M. R. Player and P. F. Torrence, Pharmacol. Ther., 78, No. 2, 55 (1998).
J.-Q. Han and D. J. Barton, RNA, 8, 4, 512 (2002).
D. K. Liu and G. F. Owens, Biochem. Biophys. Res. Commun. 145, 1, 291 (1987).
E. I. Kvasyuk, T. I. Kulak, A. I. Zinchenko, V. N. Barai, and I. A. Mikhailopulo, Bioorg. Khim., 22, 3, 208 (1996).
J. A. C. Castelli, B. A. Hassel, K. A. Wood, X.-L. Li, K. Amemiya, M. C. Dalakas, P. F. Torrence, and R. J. Youle,
J. Exp. Med., 186, 6, 967 (1997).
8.
9.
Y. Xiang, Z. Wang, J. Murakami, S. Plummer, E. A. Klein, J. D. Carpten, J. M. Trent, W. B. Isaacs, G. Casey, and
R. H. Silverman, Cancer Res., 63, 20, 6795 (2003).
E. I. Kvasyuk, T. I. Kulak, O. V. Tkachenko, S. L. Sentyureva, I. A. Mikhailopulo, R. J. Suhadolnik,
E. E. Henderson, S. E. Horvath, M.-X. Guan, and W. Pfleiderer, Helv. Chim. Acta, 80, 4, 1053 (1997).
D. W. S. Mok and M. C. Mok, Annu. Rev. Plant Physiol. Plant Mol. Biol., 52, 89 (2001).
T. I. Kulak, O. V. Tkachenko, E. B. Rubinova, O. Yu. Yatsyno, T. A. Kukharskaya, L. A. Eroshevskaya,
E. N. Kalinichenko, and A. I. Zinchenko, Khim. Prir. Soedin., 200 (2007).
T. Shimidzu, K. Yamana, N. Kanda, and S. Kitagawa, Bull. Chem. Soc. Jpn., 56, 11, 3483 (1983).
T. Shimidzu, K. Yamana, N, Kanda, and S. Maikuma, Nucl. Acids Res., 12, 7, 3257 (1984).
R. L. Letsinger and W. B. Lunsdorf, J. Am. Chem. Soc., 98, 12, 3655 (1976).
T. Ukita, N. A. Bates, and H. E. Carter, J. Biol. Chem., 216, 2, 867 (1955).
A. M. Michelson, J. Chem. Soc., 4, 1371 (1959).
10.
11.
12.
13.
14.
15.
16.
17.
M. Ya. Karpeiskii, O. K. Mamaeva, S. N. Mikhailov, N. Sh. Padyukova, and G. I. Yakovlev, Bioorg. Khim., 9, 4,
496 (1983).
18.
A. I. Zinchenko, V. N. Barai, V. I. Lyakhovets, T. I. Kulak, E. I. Kvasyuk, and I. A. Mikhailopulo, Bioorg. Khim.,
14, 10, 1401 (1988).
19.
20.
M. Yoshikawa, I. Kato, and T. Takenishi, Tetrahedron Lett., 50, 5065 (1967).
I. A. Severina, V. N. Barai, and A. I. Zinchenko, Izv. Nats. Akad. Nauk Belarusi, Ser. Biol. Nauk, 1, 51 (2002).
78