Use of Nucleoside Phosphorylases for the Preparation of Purine and Pyrimidine 2′-Deoxynucleosides

Article abstract of DOI:10.1002/adsc.201701005

Enzymatic transglycosylation – the transfer of the carbohydrate moiety from one heterocyclic base to another – is being actively developed and applied for the synthesis of practically important nucleosides. This reaction is catalyzed by nucleoside phosphorylases (NPs), which are responsible for reversible phosphorolysis of nucleosides to yield the corresponding heterocyclic bases and monosaccharide 1-phosphates. We found that 7-methyl-2′-deoxyguanosine (7-Me-dGuo) is an efficient and novel donor of the 2-deoxyribose moiety in the enzymatic transglycosylation for the synthesis of purine and pyrimidine 2′-deoxyribonucleosides in excellent yields. Unlike 7-methylguanosine, its 2′-deoxy derivative is dramatically less stable. Fortunately, we have found that 7-methyl-2′-deoxyguanosine hydroiodide may be stored for 24 h in Tris-HCl buffer (pH 7.5) at room temperature without significant decomposition. In order to optimize the reagent ratio, a series of analytical transglycosylation reactions were conducted at ambient temperature. According to HPLC analysis of the transglycosylation reactions, the product 5-ethyl-2′-deoxyuridine (5-Et-dUrd) was obtained in high yield (84–93%) by using a small excess (1.5 and 2.0 equiv.) of 7-Me-dGuo over 5-ethyluracil (5-Et-Ura) and 0.5 equiv. of inorganic phosphate. Thymidine is a less effective precursor of α-d-2-deoxyribofuranose 1-phosphate (dRib-1p) compared to 7-Me-dGuo. We synthesized 2′-deoxyuridine, 5-Et-dUrd, 2′-deoxyadenosine and 2′-deoxyinosine on a semi-preparative scale using the optimized reagent ratio (1.5:1:0.5) in high yields. Unlike other transglycosylation reactions, the synthesis of 2-chloro-2′-deoxyadenosine was performed in a heterogeneous medium because of the poor solubility of the initial 2-chloro-6-aminopurine. Nevertheless, this nucleoside was prepared in good yield. The developed enzymatic procedure for the preparation of 2′-deoxynucleosides may compete with the known chemical approaches. (Figure presented.).

Full text of DOI:10.1002/adsc.201701005

Title: Use of Nucleoside Phosphorylases for the Preparation of Purine
and Pyrimidine 2′-Deoxynucleosides
Authors: Mikhail S. Drenichev, Cyril S. Alexeev, Nikolay N. Kurochkin,
and Sergey Mikhailov
This manuscript has been accepted after peer review and appears as an
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content of this Accepted Article.
To be cited as: Adv. Synth. Catal. 10.1002/adsc.201701005
Link to VoR: http://dx.doi.org/10.1002/adsc.201701005

Advanced Synthesis & Catalysis
10.1002/adsc.201701005
FULL PAPER
DOI: 10.1002/adsc.201((will be filled in by the editorial staff))
Use of Nucleoside Phosphorylases for the Preparation of Purine
and Pyrimidine 2′-Deoxynucleosides
a†
a†
a
Mikhail S. Drenichev , Cyril S. Alexeev , Nikolay N. Kurochkin ,
a
Sergey N. Mikhailov *
a
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov str. 32, Moscow, 119991 Russia.
Correspondence to smikh@eimb.ru, sergey.mikhailov1949@gmail.com, tel. +7-499-135-9733.
The authors contributed equally.
*
†
Received: ((will be filled in by the editorial staff))
Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/adsc.201######.
Abstract. Enzymatic transglycosylation - a transfer of the deoxyuridine (5-Et-dUrd) was obtained in high yield (84-
carbohydrate moiety from one heterocyclic base to another - 93%) by using a small excess (1.5 and 2.0 equiv.) of 7-Me-
is being actively developed and applied for the synthesis of dGuo over 5-ethyluracil (5-Et-Ura) and 0.5 equiv. of
practically important nucleosides. This reaction is catalyzed
by nucleoside phosphorylases (NPs), which are responsible
for reversible phosphorolysis of nucleosides to yield the
corresponding heterocyclic bases and monosaccharide 1-
phosphates.
We found that 7-methyl-2′-deoxyguanosine (7-Me-dGuo) is
an efficient and novel donor of the 2-deoxyribose moiety in
the enzymatic transglycosylation for the synthesis of purine
and pyrimidine 2'-deoxyribonucleosides in excellent yields.
Unlike 7-methylguanosine, its 2ʹ-deoxy derivative is
dramatically less stable. Fortunately, we have found that 7-
methyl-2′-deoxyguanosine hydroiodide may be stored for 24
h in Тris-HCl buffer (pH 7.5) at room temperature without
significant decomposition.
inorganic phosphate. Thymidine is a less effective
precursor of α-D-2-deoxyribofuranose-1-phosphate (dRib-
1
p) compared to 7-Me-dGuo.
We synthesized 2'-deoxyuridine,
5-Et-dUrd,
2'-
deoxyadenosine and 2'-deoxyinosine on a semi-preparative
scale using the optimized reagents ratio (1.5:1:0.5) in high
yield.
Unlike other transglycosylation reactions, the synthesis of
2
-chloro-2'-deoxyadenosine
heterogeneous medium because of the poor solubility of
initial 2-chloro-6-aminopurine. Nevertheless, this
was
performed
in
nucleoside was prepared in good yield. The developed
enzymatic procedure for the preparation of 2'-
deoxynucleosides may compete with the known chemical
approaches.
In order to optimize the reagent ratio, a series of analytical
transglycosylation reactions were conducted at ambient
Keywords:
enzymes,
nucleoside
phosphorylases
temperature.
According
to
HPLC
analysis
of
nucleosides, phosphorolysis, transglycosylation.
transglycosylation reactions, the product 5-ethyl-2′-
completely replaced the old procedures.[4] This
method consists in the condensation of trimethylsilyl
derivatives of heterocyclic bases with peracylated
monosaccharides in the presence of Lewis acids.
Stereoselectivity of the reaction is determined by the
Introduction
Several drugs based on modified nucleosides and
possessing antitumor (Cladribine, Fludarabine,
Pentostatin, Nelarabin, Vidaza, Decitabin, etc.) or
antiviral (hepatitis C, herpes, HIV, etc.) activity
2-O-acyl group, which is involved in the formation of
the acyloxonium ion. Thus, natural β-nucleosides, in
which the heterocyclic base is in the trans position to
the 2-O-acyl group, are produced in the case of D-
ribose, whereas the absence of the 2-O-acyl group, as
in 2-deoxyribose, results in the formation of a
mixture of α,β-isomers. This greatly complicates the
purification of the target compounds.
(
Ribavirin, Zidovudine, Lamivudine, etc.) are
[1, 2]
currently used in clinical practice.
Two main
methods are employed to prepare nucleoside
analogues. One of them is based on the modification
of natural compounds. In another one, heterocycle or
monosaccharide are modified followed by the
formation of N-glycosidic bond. The glycosylation
method developed by Vorbrüggen et al. almost
[3]
Enzymatic methods for the formation of a glycosidic
bond complement chemical procedures and, in some
1
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Advanced Synthesis & Catalysis
10.1002/adsc.201701005
cases, have obvious advantages.[5-7] The ability of
NPs to cleave 2'-deoxynucleosides and β-D-
arabinofuranosyl nucleosides and to catalyze
reversible phosphorolysis[ is widely used for the
The overall transglycosylation process may be
represented as two consecutive equilibrium reactions
(Scheme 1), where Keq1 and Keq2 are the equilibrium
constants of the corresponding phosphorolysis
reactions. One or two different NPs may be used in
this process; their choice depends on the starting
nucleoside (Nuc-1) and heterocyclic base (B2) and
the resulting nucleosides (Nuc-2) (Scheme 1).
5]
[8-9]
preparation of nucleosides and their analogues.
The equilibrium of these reactions is shifted towards
nucleosides,[ the shift being more pronounced for
purine nucleoside. It may be considered as a driving
force for the overall transglycosylation process.
NPs are involved in the salvage pathway of
nucleoside biosynthesis. This class of enzymes
includes thymidine phosphorylase (TP; EC 2.4.2.4),
uridine phosphorylase (UP; EC 2.4.2.3), and purine
nucleoside phosphorylase (PNP; EC 2.4.2.1). These
enzymes are found in virtually all organisms and
share similar primary structures. The most substrate-
specific enzyme is TP, which is active only towards
thymidine. UP catalyzes the phosphorolysis of both
thymidine and uridine, PNP may cleave the N-
glycosidic bond in purine ribo- and 2'-
deoxyribonucleosides. [
10]
Scheme 1. Enzymatic phosphorolysis of nucleosides and
transglycosylation.
NP NP
1
,
2
are
nucleoside
phosphorylases, B
OH.
1
and B are heterocyclic bases, R - H or
2
11, 12]
The analysis of these reactions shows that the highest
yield of Nuc-2 may be expected when the equilibrium
of step 1 is shifted towards the formation of α-D-2-
deoxyribose-1-phosphate (dRib-1-p), and the
equilibrium of step 2 is shifted towards the target
nucleoside (Nuc-2). Therefore, dRib-1-p is the
optimal substrate for the preparation of the target
nucleoside in the highest yield. There are three
sources of the key dRib-1-p:
The enzymatic transglycosylation, a transfer of the
carbohydrate moiety from one heterocyclic base to
another, has been applied for the synthesis of
nucleosidic drugs (Cladribine, Fludarabine, Nelarabin,
[8, 9]
and Vidarabin) (Fig. 1).
The term
“
transglycosylation” was adopted from the
carbohydrate field to describe the enzymatic transfer
of sugar from an oligosaccharide to another
[13]
carbohydrate acceptor.
[14]
1
2
. Commercially available dRib-1-p;
dRib-1-p synthesized according to known
.
[15, 16]
procedures;
. dRib-1-p synthesized by the enzymatic reaction
through the intermediate formation of D-2-
3
[17, 18]
deoxyribose-5-phosphate;
Methods 2 and 3 are rather laborious; besides,
commercially available dRib-1-p is rather expensive.
We supposed that dRib-1-p may be prepared by the
enzymatic phosphorolysis of 7-Me-dGuo in the
presence of PNP (step 1, Scheme 1). The resulting
dRib-1-p was used without the isolation, in the
condensation with the heterocyclic base B2 in the
presence of NP (step 2, Scheme 1).
We suggest this procedure as the most simple and
cost-effective one. The further advantage of this
method is that it allows the combination of steps 1
and 2 (Scheme 1) without the isolation of labile dRib-
1-p.
Figure 1. Structures of Cladribine, Fludarabine,
Nelarabine, and Vidarabine.
7
-Methylguanosine is often used for the preparation
[18-22]
of purine ribonucleosides.
It was shown that
Here, we developed a new effective method for the
preparation of 2'-deoxynucleosides by NP-catalyzed
transglycosylation starting from 7-methyl-2′-
deoxyguanosine.
phosphorolysis of 7-methylguanosine in the presence
of PNP produces α-D-ribofuranose-1-phosphate and
7
-methylguanine (7-Me-Gua) in quantitative yields.
[
23]
Thus, 7-methylguanosine is
a
low-cost
replacement for α-D-ribose-1-phosphate. We
Results and Discussion
2
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Advanced Synthesis & Catalysis
10.1002/adsc.201701005
supposed that its phosphorolysis will proceed in a
similar way affording phosphate in a high yield.
To the best of our knowledge, the use of 7-
methyl-2′-deoxyguanosine for the enzymatic
synthesis of nucleosides is not described in the
literature probably because of its instability in
aqueous solutions. [
20°C in 50 mM Tris-HCl buffer, pH 7.5; the degree
of decomposition was about 30% after two days at
20°C (Figure 16, Supporting Info).
As follows from the analysis of UV spectra, the
decomposition of compound 1 in Tris-HCl buffer at
pH 7.5 is similar in character to its phosphorolysis in
the presence of PNP (Figures 13-14, Supporting Info)
and significantly differs from the cleavage of the
imidazole ring of 1 under basic conditions (pH >7.5,
Figure 15, Supporting Info). Thus, it can be
concluded that the imidazole ring of 1 is stable in
Tris-HCl buffer at pH 7.5, while the N-glycosidic
bond is slowly hydrolyzed under such conditions.
The stock solution of 1 remains stable during storage
at 7°C in 50 mM Тris-HCl buffer, pH 7.5 (its
hydrolysis is ca. 5% after one week, Figure 17,
Supporting Info). The transglycosylation conditions
were chosen based on the properties of 1. Thus, the
conditions of choice are room temperature and Тris-
24]
The methylation of guanosine using an original
[25]
method
gave, in our hands, 7-methyl-2′-
deoxyguanosine hydroiodide (1) with some visible
impurities. Therefore, we modified this procedure by
performing
the
methylation
in
N,N-
dimethylformamide in the presence of barium
1
carbonate. The purity of 1 was estimated by H NMR
spectroscopy at > 98%. The developed protocol
allowed the preparation of this compound on a
multigram scale (Scheme 2, step i).
HCl buffer (pH 7.5). The degree of decomposition of
o
1
increases at elevated temperature (37 C).
The use of 1 as the initial substrate makes it possible
to simplify the general scheme of enzymatic
transglycosylation (Scheme 1) because step 1
becomes irreversible. Compound 1 was nearly
quantitatively converted into dRib-1-p in the presence
Scheme 2. Synthesis of 2′-deoxyribonucleosides by
transglycosylation. Reagents and conditions. (i)
MeI/BaCO
potassium dihydrophosphate
E. coli PNP (E. coli TP for pyrimidine bases Ura, 5-F-Ura,
3
, DMF, 5.5 h, r.t., 65%. (ii) heterocyclic base B, of PNP (Scheme 1).
Tris-HCl buffer, pH 7.5,
,
To optimize the reagent ratio, a series of analytical
transglycosylation reactions were conducted at room
temperature (Table 1). The formation of purine
nucleosides proceeded in the presence of PNP, and
the formation of pyrimidine nucleosides requires the
presence of two enzymes, namely PNP and TP. When
using 5-Et-Ura at a concentration of about 0.2 mM,
the reaction is usually completed within 1 h. In the
presence of a large excess of inorganic phosphate
over heterocyclic base (≥5 equiv.), the yield of the
target nucleoside decreased. This reflects the
reversible feature of 5-Et-Urd production. On the
other hand, the phosphate concentration should be
sufficient for the transglycosylation rate to overcome
the hydrolysis rate of 1 or dRib-1-p.
5
9
-Et-Ura), r.t., 24 h, isolated yields: dAdo, 93%; dIno,
5%; 2-Cl-dAdo, 70%; dUrd, 80%; 5-F-dUrd, 81%; 5-Et-
dUrd, 81%.
2
'-Deoxynucleoside 1 is stable in the solid state for
several weeks at ambient temperature and for at least
months at -20°C. Compound 1 is highly soluble in
6
water and buffer solutions at ambient temperature
that makes it possible to use 1 as a substrate for
enzymatic reactions, where aqueous media and mild
conditions are preferable. According to the literature
data, the 7-methylated guanosine derivative is stable
in aqueous solutions at high temperature up to 90°C
in the pH range from 2 to 6. The N-glycosidic bond is
cleaved in strong acids (pH<2). The cleavage of the
imidazole ring of guanine proceeds under basic
conditions (pH >8)
to 7.1.
dramatically less stable compared to 7-
methylguanosine that limits its use for the synthesis
of nucleosides. Moreover, dRib-1-p is also unstable
under acidic conditions and at elevated temperatures
According to the HPLC data, 5-Et-dUrd can be
obtained in high yield (84-93%) by using a small
excess (1.5 and 2.0 equiv.) of 1 over 5-Et-Ura in the
presence of 0.5 equiv. of inorganic phosphate (Table
[24]
just above its pK
a
that is close
[26]
7-Methyl-2'-deoxyguanosine (1) is
1).
The use of a large excess of 1 results in a quantitative
yield of the product (Table 1, entry 1). However, a
large excess of 1 complicates the purification of the
product and is not cost-effective. Therefore, a small
excess of 1 (1.5 equiv.) was chosen for the further
development of the translycosylation protocol.
Thymidine (Thd) is a less effective precursor of
dRib-1-p compared to 1 (Table 1, entries 2,4). A low
yield of transglycosylation when using Thd as a
glycosyl donor is apparently associated with
o
[27]
(
30-60 C).
We have investigated the stability of 1 by HPLC and
UV spectroscopy (Figures 10-17, Supporting Info).
Compound 1 appeared to be unstable in aqueous
solution at pH 4.1 and 20°C. According to HPLC
analysis, compound 1 proved to be quite stable at
3
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Advanced Synthesis & Catalysis
10.1002/adsc.201701005
reversibility of its phosphorolysis reaction (Step 1 in
Scheme 1).
The conduction of enzymatic transglycosylation
using a small excess of 1 (Table 1, entries 6 and 7)
makes the chromatographic purification of the target
products easier and reduces their cost.
These conditions were extended to the preparative
synthesis of several purine and pyrimidine
nucleosides on a larger scale (Table 2).
was established (Fig. 2B). We have not observed any
formation of 5-isopropyluridine starting from 5-
isopropyluracil (entry 3, Table 2) probably due to the
steric interactions of the bulky substituent with the
catalytic site of TP.
This protocol was tested on the synthesis of
cladribine (Table 2, entry 5). Because of the low
solubility of initial 2-chloro-6-aminopurine in Tris-
HCl buffer, the reaction was performed in
heterogeneous medium (see the Experimental
Section) as opposed to the other transglycosylation
reactions, which were performed in homogeneous
solutions. According to this procedure, cladribine was
isolated in 70% yield.
Table 1. Analytical reactions resulting in the 5-Et-dUrd
formation from 5-Et-Ura, 1 (or Thd) and potassium
ad)ihydrophosphate at 20°С at different reagent ratios for 1 h
Glycosyl
donor
Yield,
%
Reagent ratio
1 (Thd):5-Et-Ura:Pi
Entry
1
2
3
4
5
6
7
8
1
Thd
1
Thd
1
1
1
1
100
80
95
56
78
93
84
65
5:1:0.5
5:1:0.5
3:1:0.5
1.5:1:0.5
2:1:1
2:1:0.5
1.5:1:0.5
1:1:0.5
a)
The reactions were performed in 1 mL volume at 0.2 μM
concentration of 5-Et-Ura and 20°C for 1 h until the
equilibrium of the transglycosylation reaction was
established; HPLC analysis was performed on the
®
reversed-phase sorbent Luna C18
.
The enzymatic reactions were controlled using the
reversed-phase HPLC sorbent Dr. Maisch 5µm
Reprosil-Pur C18-AQ 120 Å (Fig.2). In the
preparative reactions, the equilibrium was established
within 20 h at room temperature.
The only one peak of 5-ethyluracil was observed in a
Figure 2. Preparative synthesis of 5-Et-dUrd using
chromatogram before the addition of the enzyme (Fig. transglycosylation reaction. HPLC analysis of the reaction
2
A, t
reversed-phase sorbent Luna C18 column with RT ~
.00-5.50 that is difficult to integrate (Figure 18,
0
). Compound 1 gave a very broad peak on a
mixture was performed on the reversed-phase sorbent Dr.
Maisch 5µm, Reprosil-Pur C18-AQ 120Å (А) before the
addition of PNP and TP (t ); (В) after the establishment of
0
the equilibrium (teq 24 h). 1, 5-Et-Ura; 2, 7-Me-Gua; 3, 5-
Et-dUrd in a linear gradient of acetonitrile in deionized
water from 2 to 12% for 10 min at a flow rate of 1 ml/min
with UV detection at a wavelength of 260 nm.
®
4
Supporting Info). We could not detect 1 on the
reversed-phase sorbent Dr. Maisch 5µm, Reprosil-
Pur C18-AQ 120Å (Figure 11, Supporting Info).
Therefore, we performed reaction analysis using a
pair of 5-ethyluracil and 5-ethyl-2′-deoxyuridine.
We have successfully employed the developed
protocol for the preparation of 2'-deoxyuridine, 5-
ethyl-2'-deoxyuridine, 2'-deoxyadenosine, and 2'-
deoxyinosine on a semi-preparative scale using the
optimized reagent ratio (1.5:1:0.5) in high yield.
A variety of enzymatic methods for cladribine
synthesis using 2-chloro-6-aminopurine and 2ʹ-
deoxynucleosides as a starting compounds has been
[28-34]
described in the literature.
According to HPLC
the analytical yields were determined in the range
from 80 to 95%. At the same time the isolated yields
of cladribine were substantially lower (35-88%). The
best results were obtained when using 2-chloro-6-
aminopurine and 2ʹ-deoxyguanosine in the presence
of intact cells of GA E. coli BMT 4D/1A strain (81%)
(
Table 2). 7-Me-Gua is practically insoluble in water
and precipitates from the reaction mixture. The
formation of 7-methylguanine and 5-ethyl-2′-
deoxyuridine was accompanied by a decrease in the
intensity of the 5-ethyluracil signal as the equilibrium
4
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Advanced Synthesis & Catalysis
10.1002/adsc.201701005
[
31]
and using dRib-1-p in the presence of E. coli PNP
In
conclusion,
7-methyl-2′-deoxyguanosine
88%). [
35]
hydroiodide (1) was shown for the first time to be a
source of 2′-deoxyribose in enzymatic synthesis of
nucleosides. It was determined on the basis of
analytical data, chromatographic purification and
prime cost that the optimal ratio of the initial
compounds 7-Me-dGuo : base : Pi is 1.5:1:0.5. This
developed methodology allows the preparation of
both purine and pyrimidine 2′-deoxyribonucleosides
in high yield.
(
We prepared cladribine using a small excess of
glycosyl-donor 1 in 70% yield within the range
reported in the literature. From our point of view, the
main problem of cladribine synthesis by
transglycosylation is a low solubility of 2-chloro-6-
aminopurine in aqueous media. [32] Analysis of patent
literature has revealed that one of a possible
improvements might be the dropwise addition of
alkali solution of 2-chloro-6-aminopurine to the
buffered reaction mixture. [36] Even in this case the
yield does not exceed 80% and is comparable with
the method described above (Table 2).
Experimental Section
Table 2. Preparative synthesis of nucleosides from 7-
methyl-2′-deoxyguanosine by transglycosylation
a)
The solvents and materials were of reagent grade and were
used as received.
HPLC analysis was performed using an Akvilon (Russia)
HPLC gradient system (2×Stayer pumps (2nd series),
Stayer MS16 dynamic mixer, and Stayer 104M UV-Vis
detector).
The stability of 1 was analyzed by HPLC on a 4.6×250
®
mm chromatographic column (sorbent Luna NH
2
, average
particle diameter 5 μm, pore diameter 100 Å, catalogue
number 00G-4378-E0, Phenomenex (USA)) equipped with
a protective column of EC-standart (4×3.0 mm, average
®
particle diameter 5 μm, sorbent Luna NH
2
, catalogue
number AJ0-4302, Phenomenex (USA)), in a linear
gradient of acetonitrile in deionized water from 2 to 12%
for 10 min at a flow rate of 1 ml/min with UV detection at
a wavelength of 280 nm.
HPLC analysis of transglycosylation reactions was
performed on
a
4×150 mm Dr. Maisch HPLC
chromatographic column (5µm, Reprosil-Pur C18-AQ 120
Å, Part No r15.aq.s1504, Dr. Maisch HPLC GmbH
(
Germany)) in a linear gradient of acetonitrile in deionized
water from 2 to 12% (from 2 to 25% for cladribine) for 10
min (flushing with 12-80% acetonitrile/deionized water for
1
0-10.1 min, then 80-2% for 10.1-10.8 min for 5-ethyl-2′-
deoxyuridine, 2′-deoxyadenosine) at a flow rate of 1
ml/min with UV detection at a wavelength of 260 nm.
The pH values were determined with a microprocessor-
based pH (mV- C) bench meter 211 (Hanna Instruments,
Germany) equipped with an HI1131B double junction
combination pH electrode and a HI7662 stainless steel
temperature sensor for pH compensation.
a)
Reactions were performed at 20°C for 20 h using
1
.5:1:0.5 ratio of the initial substrates 1 : heterocyclic
base : Pi until the equilibrium of transglycosylation was
established.
reversed-phase sorbent Dr. Maisch 5µm, Reprosil-Pur C18
AQ 120Å.
b)
HPLC analysis was performed on the
-
Preparative chromatography was performed using
Kieselgel (0.040-0.063 mm, Merck).
TLC was carried out on Alugram SIL G/UV254
The use of a small excess of 1 in preparative
(
Macherey-Nagel) with UV visualization ( = 254 nm).
enzymatic transglycosylation reactions makes it
UV spectra were recorded on a Cary 300 UV/VIS
instrument (Varian, Australia).
possible
to
simplify
the
isolation
and
chromatographic purification of the target products
and provides nearly quantitative yields in most cases.
The structures of all products were confirmed by
NMR and UV spectroscopy.
1
H NMR spectra were recorded on a Bruker AMX 300
13
NMR instrument (Germany) at 300 MHz and 303 K.
C
NMR spectra (with complete proton decoupling) were
recorded on a Bruker AMX 400 NMR instrument
(
Germany) at 100 MHz and 303 K. Chemical shifts were
measured in ppm relative to the residual solvent signals as
Conclusion
1
13
internal standards (DMSO-d
6
, H: 2.50 ppm, C: 39.5
5
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Advanced Synthesis & Catalysis
10.1002/adsc.201701005
1
ppm; D
2
O, H: 4.79 ppm). Spin-spin coupling constants (J)
spectroscopy and HPLC at 20°C. Compound 1 was
hydrolyzed by 73% after two days and by 96% in a
week at 20°C according to HPLC. The retention time
are given in Hz.
-Chloro-6-aminopurine (2-chloroadenine) was prepared
by amination of 2,6-dichloropurine.
2
[37]
5-Ethyluracil and
(
3
RT) of 7-methylguanine is 6.50 min; compound 1 -
.10 min on chromatographic column 4.6×250 mm
5
-isopropyluracil were prepared according to the literature
[38]
procedure.
®
(
Luna NH , 5 μM, 100 Å) in a linear gradient of
2
acetonitrile in deionized water from 2 to 12% for 10
min at a flow rate 1 ml/min with UV detection at
wavelength 280 nm, injection volume was 20 μL.
Enzymes
E. coli purine nucleoside phosphorylase (PNP, 295
U/ml, 32 mg/ml solution) and E. coli thymidine
phosphorylase (1197 U/ml, 17.2 mg/ml solution)
were purchased from Sigma–Aldrich (United States).
Study of stability of 1 in Tris-HCl buffer. pH 7.5
To study the stability of 1 at 20°C and 7°C, a 1mM
stock solution of 1 (40.9 mg) in 100 mL of 50 mM
Tris-HCl buffer at pH 7.5 was used. After 1 h, 1, 2, 5,
and 7 days aliquots were taken from the solution,
diluted 50-fold with Tris-HCl buffer (pH 7.5), and
analyzed by UV spectroscopy and HPLC at 20°C.
The retention time (RT) of 7-methylguanine is 6.50
7
-Methyl-2′-deoxyguanosine hydroiodide (1)
′
To a solution of 2-deoxyguanosine monohydrate (5 g,
7.5 mmol) in dry DMF (100 mL) barium carbonate
6.9 g, 35 mmol) was added. Then iodomethane (10.9
1
(
mL, 175 mmol) was added dropwise to the resulting
suspension with vigorous stirring. The reaction
mixture was stirred for 5.5 h at r.t. and then allowed
to stand for 30 min in an open flask. The reaction
mixture was filtered off from barium carbonate
through celite (25 mL). The celite was washed with
DMF (50 mL). The transparent liquid filtrates were
collected, diluted with trichloromethane (0.5 L), and
allowed to stand at 0°С for 16 h. The precipitate was
filtered, washed with ethanol (100 mL) and
trichloromethane (100 mL), and dried on a vacuum
pump at r.t. for 1 h. Yield 4.6 g (65%) as a white
min; compound 1 - 3.10 min on chromatographic
®
column 4.6×250 mm (Luna NH
2
, 5 μM, 100 Å) in a
linear gradient of acetonitrile in deionized water from
to 12% for 10 min at a flow rate 1 ml/min with UV
2
detection at wavelength 280 nm, injection volume 20
μL.
5-Fluoro-2′-deoxyuridine
5-Fluorouracil (227 mg, 1.75 mmol) was dissolved in
100 mL of 50 mM Tris-HCl buffer (pH 7.5) on
heating and cooled to r.t. Then potassium
powder that is stable on storage at -20°С for 6 months. dihydrophosphate (119 mg, 0.875 mmol) and 1
R
f
0.1 (dichloromethane – ethanol, 1:1, v/v).
1H NMR (300 MHz, DMSO-d
): 11.63 br s (1H, NH
-Me-Gua), 9.27 s (1H, H8 7-Me-Gua), 7.15 br s (2H,
(1.074 g, 2.62 mmol) were added to the solution, and
the resulting mixture was diluted with 50 mM Tris-
HCl buffer (pH 7.5) to a volume of 350 mL. Then
solutions containing enzymes E. coli PNP (10 µl of
6
7
′
NH 7-Me-Gua), 6.19 t (1H, J1′,2′a=J1′,2′b=6.0, H1),
2
32 mg/ml solution of E. coli PNP (2.95 U)) and
5.50-5.25 m (1H, OH), 5.25-4.75 m (1H, OH), 4.37
E. coli TP (10 µl of 17.2 mg/ml solution of E. coli TP
diluted 10-fold (1.2 U)) were added to the mixture
using a dispenser and the resulting mixture was
carefully stirred for 5 min and allowed to stand
overnight at r.t. without stirring. The reaction mixture
was filtered through a Phenomenex membrane (0.2 µ,
′
ddd (1H, J3′,2′a= 5.0, J3′,2′b= 4.8, J3′,4′= 3.9, H3), 4.00 s
), 3.92 td (1H, J4′,3′= 3.9, J4′,5′a= 4.2, J4′,5′b
(
3H, CH
3
=
′
′
4
3
2
.2, H4), 3.62 dd (1H, J5′a,4′ = 4.2, J5′a,5′b = -12.0, H5a),
.57 dd (1H, J5′b,5′a = 4.2, J5′b,5′a = -12.0, H5b), 2.55-
.45 m (1H, H2a), 2.40 ddd (1H, J2′b,2′a = -13.4, J2′b,1′
′
′
′
=
6.0, J2′b,3′ = 4.8, H2b). UV (H
2
O): λmax (ε) = 256 nm
47 mm) and concentrated in vacuo to the volume of
(
11000); λmax (ε) = 281 nm (7600); UV (50 mM Tris-
HCl buffer, pH 7.5): λmax (ε) = 256 nm (7100); λmax (ε)
281 nm (7600).
ca. 50 mL. The suspension was repeatedly filtered off
from the precipitate of 7-methylguanine through a
Phenomenex membrane (0.2 µ, 47 mm) and
concentrated in vacuo to near dryness. The residue
was dissolved in ethanol (50 mL), and then silica gel
=
Study of stability of 1 in aqueous solution. pH 4.1
(
20 mL) was added to the solution. The resulting
To study the stability of 1 at 20°C, 10.2 mg of 1 were
dissolved in 25 mL of deionized water (Milli-Q ).
mixture was evaporated to dryness, co-evaporated
with ethanol (2×50 mL), and the dry residue was
applied on a chromatographic column (4×10 cm)
packed with silica gel (150 mL) for purification. The
®
The pH value of the resulting 1 mM solution of 1 was
estimated to be 4.1. After 1 h, 1, 2, 3, 4, and 7 days,
aliquots were taken from the solution, diluted 50
times for UV spectroscopy and 5 times for HPLC
analysis with deionized water, and analyzed by UV
column was washed with
a
mixture of
dichloromethane and ethanol (95:5 v/v, 200 mL), the
product was eluted with dichloromethane:ethanol
6
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Advanced Synthesis & Catalysis
10.1002/adsc.201701005
(
90:10 v/v) and dichloromethane : ethanol (85:15 v/v). presence of potassium dihydrophosphate (119 mg,
The fractions containing the product were collected
and evaporated in vacuo to dryness. The resulting
residue was repeatedly purified on a chromatographic
column (2×10 cm) packed with silica gel (50 mL).
The column was washed with a dichloromethane :
ethanol mixture (95:5 v/v, 200 mL), the product was
eluted with dichloromethane : ethanol (90:10 v/v).
The fractions containing the product were collected,
evaporated in vacuo, co-evaporated 5 times with
dichloromethane and dried on a vacuum pump at r.t.
for 1 hour. Yield 349 mg (81%) as white crystals.
0.875 mmol), E. coli PNP and E. coli TP at r.t. gave
319 mg (80%) of 2′-deoxyuridine as a white powder.
Yield (according to HPLC) 90%. NMR and UV
spectra are identical to those of the commercially
available 2′-deoxyuridine.
2
-Chloro-2′-deoxyadenosine (cladribine)
2-Chloro-6-aminopurine (21 mg, 0.124 mmol) was
suspended in 150 mL of 50 mM Tris-HCl buffer (pH
7.5) in an ultrasonic bath at r.t. Then potassium
dihydrophosphate (8.5 mg, 0.062 mmol) and 1 (76
mg, 0.186 mmol) were added to the suspension. Then
a solution containing E. coli PNP (10 µl of 32 mg/ml
solution of E. coli PNP (2.95 U)) was added to the
mixture and the resulting suspension was carefully
stirred for 6 days at r.t. The reaction mixture was
filtered through a Phenomenex membrane (0.2 µ, 47
mm) and concentrated in vacuo to the volume of ca.
10 mL. The suspension was repeatedly filtered from
the precipitate of 7-methylguanine through a
Phenomenex membrane (0.2 µ, 47 mm) and
concentrated in vacuo to near dryness. The residue
was dissolved in ethanol (10 mL), and then silica gel
(1.5 mL) was added to the solution. The resulting
mixture was evaporated to dryness, co-evaporated
with ethanol (2×10 mL), and the dry residue was
applied on a chromatographic column (1×10 cm)
packed with silica gel (5 mL) for purification. The
Yield (according to HPLC analysis) 95%. R
f
0.23
1
(
dichloromethane : ethanol, 9:1, v/v). H NMR (300
3
MHz, D O): 8.09 d (1H, JH,F=6.5, H6 5-F-Ura), 6.32
td (1H, J1′,2′a=J1′,2′b=6.6, JH,F=6.6, H1), 4.51 ddd (1H,
J
J
J
4
J
J
2
5
′
′
3′,2′a= 6.5, J3′,2′b= 4.2, J3′,4′= 4.8, H3), 4.10 ddd (1H,
′
4′,3′= 4.8, J4′,5′a= 3.5, J4′,5′b = 4.9, H4), 3.92 dd (1H,
′
5′a,4′ = 3.5, J5′a,5′b = -12.5, H5a), 3.82 dd (1H, J5′b,5′a
=
′
.9, J5′b,5′a = -12.5, H5b), 2.48 ddd (1H, J2′b,2′a = -14.2,
′
2′b,1′ = 6.6, J2′b,3′ = 4.2, H2b), 2.38 ddd (J2′a,2′b = -14.2,
2′a,1′ = 6.6, J2′a,3′ = 6.5, H2a). 13C NMR (100 MHz,
′
2
D
2
O): 162.41 d ( JC,F=26, 4-C=O), 153.02 (5-C=O),
1
2
1
8
4
43.49 d ( JC,F=233, 5-C), 128.44 d ( JC,F=34, C6),
′
′
′
′
9.57 (C1), 88.40 (C4), 73.17 (C3), 63.92 (C5),
′
1.58 (C2). UV (H O): pH 2-7: λmax (ε) = 269 nm
2
(
8200); pH 13: λmax (ε) = 269 nm (6300).
5
-Ethyl-2′-deoxyuridine
Following the procedure for the preparation of 5-
fluoro-2′-deoxyuridine, the reaction of 5-ethyluracil
column was washed with
a
mixture of
dichloromethane and ethanol (95:5 v/v, 100 mL), the
product was eluted with dichloromethane : ethanol
(
245 mg, 1.75 mmol) with 1 (1.074 g, 2.62 mmol) in
the presence of potassium dihydrophosphate (119 mg,
(
90:10 v/v). The fractions containing the product
0
3
.875 mmol), E. coli PNP and E. coli TP at r.t. gave
63 mg (81%) of 5-ethyl-2′-deoxyuridine as a white
were collected, evaporated in vacuo, co-evaporated 5
times with dichloromethane.
powder. Yield (according to HPLC) 90%. R 0.31
f
The fractions containing the product were collected,
evaporated in vacuo, co-evaporated 5 times with
dichloromethane and dried on a vacuum pump at r.t.
for 1 hour to obtain 25 mg (70%) of cladribine as
white crystals.
(
dichloromethane : ethanol, 9:1, v/v).
1H NMR (300 MHz, D
O): 7.68 t (1H, J = 1.1, H6 5-
4
2
′
Et-Ura), 6.36 t (1H, J1′,2′a=J1′2′b=6.7, H1), 4.53 td (1H,
′
J
3′,2′a=J3′,2′b=5.5, J3′,4′=4.4 H3), 4.09 ddd (1H, J4′,5′a
.5, J4′,5′b=4.7, J4′,3′=4.4, H4), 3.91 dd (1H, J5′a,5′b= -
2.5, J5′a,4′= 3.5, H5a), 3.83 dd (1H, J5′b,5′a= - 12.5,
5′b,4′= 4.7, H5b), 2.44 dd (2H, J2′a,1′=J2′b,1′=6.7,
=
′
1
3
1
J
J
CH
Yield (according to HPLC) 81%. H NMR (300 MHz,
′
2
D O): 8.27 s (1H, H8 – 2-Cl-Ade), 6.38 dd (1H,
′
′
J
J
J
J
1′,2′a=7.4, J1′,2′b= 6.6, H1), 4.68 ddd (1H, J3′,2′a=6.2,
′
3
4
′
2′a,3′=J2′b,3′=5.5, H2), 2.37 qd (2H, J = 7.5, J = 1.1,
3′,2′b=3.4, J3′,4′=3.0, H3), 4.22 ddd (1H, J4′,3′=3.0,
4′,5′a=3.3, J4′,5′b=4.3 H4), 3.90 dd (1H, J5′a,5′b= -12.6,
5′a,4′=3.3, H5a), 3.82 dd (1H, J5′b,5′a= -12.6, J5′b,4′=4.3,
H5b), 2.81 ddd (1H, J2′a,2′b= -13.9, J2′a,1′=7.4,
2′a,3′=6.2, H2a), 2.60 ddd (1H, J2′b,2′a= -13.9,
2′b,1′=6.6, J2′b,3′=3.4, H2b). UV (H O): pH 2: λmax (ε)
3
13
′
2
), 1.14 t (3H, J = 7.5, CH
3
). C NMR (100 MHz,
′
D
2
O): 168.90 (C=O), 154.37 (C=O), 139.53 (C6),
′
′
′
′
1
6
19.84 (C5), 89.30 (C1), 87.97 (C4), 73.15 (C3),
′
′
′
3.80 (C5), 41.42 (C2), 22.28 (CH
O): pH 2-7: λmax (ε) = 266 nm (9800); pH 13:
max (ε) = 266 nm (7400).
2
), 14.68 (CH
3
).
J
J
′
UV (H
2
2
λ
= 265 nm (13600); pH 7-13: λmax (ε) = 265 nm
14100).
(
2
′-Deoxyuridine
Following the procedure for the preparation of 5-
fluoro-2′-deoxyuridine, the reaction of uracil (196 mg, Following the procedure for the preparation of 5-
2′-Deoxyadenosine
1.75 mmol) with 1 (1.074 g, 2.62 mmol) in the
fluoro-2′-deoxyuridine, the reaction of adenine (236
7
This article is protected by copyright. All rights reserved.

Advanced Synthesis & Catalysis
10.1002/adsc.201701005
mg, 1.75 mmol) with 1 (1.074 g, 2.62 mmol) in the
presence of potassium dihydrophosphate (119 mg,
A. Stepchenko, Y. A. Sokolov, A. I. Miroshnikov, I.
A. Mikhailopulo. Chemistry–A Eur. J. 2015, 21,
1
3401-13419.
0
2
.875 mmol) and PNP at r.t. gave 407 mg (93%) of
′-deoxyadenosine as a white powder. Yield
[
[
17]
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D. V. Chuvikovsky, R. S. Esipov, Yu. S.
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Bioorg. Med. Chem., 2006, 14, 6327–6332.
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(
according to HPLC) 100%. NMR and UV spectra
are identical to those of the natural compound.
2
′-Deoxyinosine
Following the procedure for the preparation of 5-
fluoro-2′-deoxyuridine, the reaction of hypoxanthine
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W. J. Hennen, C. H. Wong. J. Org. Chem. 1989,
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This work was supported by Russian Science
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10.1002/adsc.201701005
FULL PAPER
Use of nucleoside phosphorylases for the
preparation of purine and pyrimidine 2′-
deoxynucleosides
Adv. Synth. Catal. Year, Volume, Page – Page
Mikhail S. Drenichev, Cyril S. Alexeev, Nikolay
N. Kurochkin, Sergey N. Mikhailov*
TOC Graphic
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