Chemoenzymatic synthesis of cytokinins from nucleosides: Ribose as a blocking group

Article abstract of DOI:10.1039/c8ob00223a

Nucleoside phosphorylases are involved in the salvage pathways of nucleoside biosynthesis and catalyze the reversible reaction of a nucleobase with α-d-ribose-1-phosphate to yield a corresponding nucleoside and an inorganic phosphate. The equilibrium of these reactions is shifted towards nucleosides, especially in the case of purines. Purine nucleoside phosphorylase (PNP, EC 2.4.2.1) is widely used in labs and industry for the synthesis of nucleosides of practical importance. Bacterial PNPs have relatively broad substrate specificity utilizing a wide range of purines with different substituents to form the corresponding nucleosides. To shift the reaction in the opposite direction we have used arsenolysis instead of phosphorolysis. This reaction is irreversible due to the hydrolysis of the resulting α-d-ribose-1-arsenate. As a result, heterocyclic bases are formed in quantitative yields and can be easily isolated. We have developed a novel method for the preparation of cytokinins based on the enzymatic cleavage of the N-glycosidic bond of N⁶-substituted adenosines in the presence of PNP and Na₂HAsO₄. According to the HPLC analysis the conversion proceeds in quantitative yields. In the proposed strategy the ribose residue acts as a protective group. No contamination of the final products with AsO₄^3- has been detected via HPLC-HRMS; simple analytical arsenate detection via ESI-MS has been proposed.

Full text of DOI:10.1039/c8ob00223a

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Chemoenzymatic Synthesis of Cytokinins from Nucleosides:
Ribose as a Blocking Group
Received 00th January 20xx,
Accepted 00th January 20xx
Vladimir E. Oslovsky†, Pavel N. Solyev†, Konstanꢀn M. Polyakov, Cyril S. Alexeev and Sergey N.
Mikhailov*
DOI: 10.1039/x0xx00000x
Nucleoside phosphorylases are involved in the salvage pathways of nucleoside biosynthesis and catalyze the reversible
reaction of the nucleobase with α-D-ribose-1-phosphate to yield the nucleoside and inorganic phosphate. The equilibrium
of these reactions is shifted towards nucleosides, especially in the case of purines. Purine nucleoside phosphorylase (PNP,
EC 2.4.2.1) is widely used in labs and industry for the synthesis of nucleosides of practical importance. Bacterial PNPs have
relatively broad substrate specificity utilizing a wide range of purines with different substituents to form corresponding
nucleosides. To shift the reaction in the opposite direction we have used arsenolysis instead of phosphorolysis. This
reaction is irreversible due to the hydrolysis of the resulting α-D-ribose-1-arsenate. As a result, heterocyclic bases are
formed in quantitative yields and can be easily isolated. We have developed a novel method for preparation of cytokinins
www.rsc.org/
6
based on the enzymatic cleavage of the N-glycosidic bond of N -substituted adenosines in the presence of PNP and
2 4
Na HAsO . According to the HPLC analysis the conversion proceeds in quantitative yields. In the proposed strategy the
3
-
ribose residue acts as a protective group. No contamination of the final products with AsO
HRMS; simple analytical arsenate detection via ESI-MS has been proposed.
4
has been detected via HPLC-
Our aim is to develop ribose as a new blocking group which
can be cleaved enzymatically under very mild conditions in
quantitative (or near to quantitative) yields.
Introduction
Literature analysis reveals that the ribose residue can be
removed by acid or enzymatic hydrolysis. Acid hydrolysis of
cytokinin nucleosides with aromatic residues leads to CKs in
high yields. At the same time, the isoprenyl side chain is rather
labile under these conditions [8]. The enzymatic hydrolysis of
the N-glycosidic bond can be a method of choice for the
Purine is the most widely distributed heterocycle in nature –
the core component of purine alkaloids, cytokinins, purine
nucleoside antibiotics that exhibit profound and diverse
biological activities [1]. Being one of the most promising
heterocyclic scaffolds, purine often serves as a source for drug
design and synthesis of biologically active compounds [2-3].
Cytokinins (CKs) is an important group of phytohormones that
play a crucial role in numerous biochemical processes in
plants: stimulation of cell division, increase of cell volume,
shoot differentiation, activation of metabolite attraction,
influence on the biosynthesis of pigments, etc. Naturally
occurring CKs are adenine derivatives that carry either an
isoprene-derived side chain or an aromatic side chain at the
6
preparation of such CK derivatives starting from N -substituted
adenosines. There are some benefits of using nucleosides as
starting compounds for the preparation of CKs and their
derivatives. In the proposed strategy, the ribose residue acts
9
as a protective group at the N position of purine, increases
6
N -terminus (Figure 1) [4]. The first CK synthesis was
accomplished more than 60 years ago and since that time
efficient chemical procedures of CK preparation have been
developed [5, 6]. One of the most reliable method is the
reaction of 6-chloropurine with benzylamine in n-butanol in
the presence of triethylamine at elevated temperatures [6-7].
Figure 1. Structure of natural cytokinins (iP – isoprenyladenine; cZ – cis-zeatin; tZ –
trans-zeatin; DZ – dihydrozeatin; mT – meta-topolin; oT – ortho-topolin; MeoT – O-
6
methyl-ortho-topolin; MemT – O-methyl-meta-topolin; BAP – N -benzylaminopurine;
KIN – kinetin).
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the regioselectivity of the electrophilic reactions and facilitates they were cooled and neutralized to pH 7 with 25% aqueous
nucleophilic substitutions compared to the unprotected purine ammonia (Scheme 2). It should be noted that the hydrolysis of
6
derivatives. Isolation and purification of the protected N -phenyladenosine (1a) proceeds within 20 min. However,
nucleoside derivatives by adsorption chromatography on silica this method is suitable only for the preparation of aromatic
gel is less laborious than that of the corresponding bases. In and non-labile saturated CK derivatives [8] and cannot be used
addition, the cost of natural nucleosides is comparable or in in case of unstable 6-substituents. The acid hydrolysis of the
6
some cases even lower than the cost of the corresponding fully acetylated N -substituted adenosines (1f
-
h
) proceeds with
6
the formation of N -substituted adenines (2f
heterocyclic bases.
-
h
) during 8 h. The
Recently, we have developed a useful and versatile approach prolongation of the reaction time is needed for the additional
6
for the preparation of N -modified adenosine derivatives by cleavage of the acetyl group at the N -position. Similarly, as in
6
6
the regioselective alkylation of N -acetyl-2′,3′,5′-tri-O- case of the unprotected adenosines, the final products are
acetyladenosine with alcohols under the Mitsunobu reaction precipitated from the reaction mixture after neutralization in
conditions or by the base-promoted reaction with alkyl halides high yields (70-94%).
6
Scheme 1) [9-12]. The main advantage of N -acetyl-2′,3′,5′-tri-
(
O-acetyladenosine as a substrate is the possibility to use both
6
alkyl halides and alcohols for the N -modification, that is Catalyst characteristics
especially important in the case when an amine is not stable or
6
is hardly available. Using this method, several hundred N -
Nucleoside phosphorylases are important biocatalysts for the
reversible phosphorolysis of nucleosides to the corresponding
nucleobases with the formation of α-D-ribose-1-phosphate
substituted adenosines have been synthesized in our
laboratory. The described approach compliments the
(Scheme 3).
6
6
traditional one for the preparation of N -alkylated or N -
arylated adenosines starting from commercially available 6-
chloropurine riboside with alkyl- or arylamines [7] (Scheme 1).
Scheme 3. Reversible phosphorolysis of adenosine. Pi – inorganic phosphate.
6
The equilibrium of these reactions is shifted towards the
nucleosides, especially in the case of purine derivatives [13].
The active site of PNP from Bacillus cereus complex with
adenosine and sulphate is shown on Figure 2. The active
center of the enzyme is formed by two adjacent subunits and
the purine base is bound to the hydrophobic pocket.
Examination of the crystallographic structure of the complex
Scheme 1. Methods of choice for the preparation of N -substituted adenosine
derivatives
Results and discussion
Acid hydrolysis
6
First, we have examined the acid hydrolysis of a number of N - with nucleosides reveals that there is a large space around the
substituted adenines (2a-h) in aqueous 0.5M НСl at 100°С. 2- and 6-positions of purine that can accommodate rather
According to TLC, most of the reactions were complete in 3 h,
bulky substituents (Figure 2) [14].
Figure 2. The active site structure of PNP from Bacillus cereus complex with adenosine
(magenta color) and sulfate (yellow color) (3UAW - PDB ID) [14]. The protein is shown
as solvent access surface coloured by atom type (the carbon atoms for His4 and Arg43
from the second subunit of the dimer are coloured by grey)
6
Scheme 2. Synthesis of N -substituted adenines (2a-h) by acid hydrolysis of
6
unprotected
(1a-e)
and
acetylated
(1f-h)
N -substituted
adenosines.
Reagents and conditions: i – 0.5 M HCl, 100°С, 20 min – 3 h in the case of the
unprotected adenosines, 8 h in the case of the peracetylated adenosines
2
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Scheme 4. Irreversible enzymatic arsenolysis of purine nucleosides; X = NH, O;
2 2 1
R = NH , H; R = aromatic or unsaturated aliphatic substituents
Several complexes of PNP with nucleosides were solved with a other
heterocycles
including
imidazole,
triazole,
resolution of 1.9-2.5Å. As a more accurate model for the benzimidazole, allopurinol and some others [16, 17]. The
interaction of an enzyme with a nucleoside, a PNP structure unprecedentedly broad substrate specificity of PNP was
from Bacillus cereus was selected because this structure is recently extended to the pyrimidine nucleosides [17, 18].
determined with a resolution at 1.2Å and exhibits significant Broad specificity and bulk tolerance of PNP makes it a valuable
similarities with the PNP from E. Coli in overall structure as tool for the preparation of cytokinin nucleosides.
well as in active site composition and substrate-binding Earlier we have used PNP for the preparation of CKs starting
geometry [14].
from the corresponding nucleosides [9]. This reaction is
reversible and the equilibrium is shifted towards the formation
of nucleosides (Scheme 3). To ensure the completeness of
phosphorolysis, alkaline phosphatase is added to the reaction
mixture to hydrolyze α-D-ribose-1-phosphate [9]. Using this
procedure CKs were prepared in moderate yields (50-68%).
Here, we present another approach based on enzymatic
arsenolysis of purine nucleosides with PNP. This reaction is
irreversible due to hydrolysis of the resulting α-D-ribose-1-
arsenate (Scheme 4) [18].
Enzymatic hydrolysis
PNPs are widely used for the preparation of differently
substituted purine nucleosides. Purines with bulky groups in
the 2- and 6-position may be converted by PNP to the
corresponding nucleosides. Bacterial PNPs have broader
specificity than mammalian ones and may utilize a wide range
of purines including adenine, guanine, hypoxanthine and some
We have shown that the PNP-catalyzed arsenolysis reactions
6
of the N -substituted purine nucleoside derivatives in the
2 4 2
presence of Na HAsO ×7H O in subequimolar quantities
proceed with the formation of the corresponding purine
derivatives with quantitative yields according to the HPLC
analysis (Figure 3).
Reactions were conducted in 50 mM Tris-HCl buffer solution,
pH 7.5 at 50°C for 16 h. The increased temperature is
necessary due to the poor solubility of the starting nucleoside.
The enzyme, nevertheless, remains stable and soluble under
these conditions [19]. After cooling to the ambient
temperature the reaction products crystallize directly from the
reaction mixture with high yields (Scheme 5).
The advantage of the proposed method in comparison with
the acid hydrolysis is the possibility of obtaining purines
containing substituents that are unstable under acid
hydrolysis, such as isoprenoid or other labile unsaturated
substituents [8]. Moreover, the proposed method is useful for
6
the preparation of labile O -substituted hypoxanthine and
guanine derivatives, which can also be applied as synthetic
cytokinin analogues. Thus, we have developed mild and
efficient enzymatic method for the cleavage of the D-
6
6
Scheme 5. Synthesis of N -substituted adenines (2b, 2i-k, 2n) and O -substituted ribofuranosyl group which might be of general value for the
hypoxanthines (2l,m) by enzymatic arsenolysis. Reagents and conditions: i – conversion of nucleosides into corresponding heterocycles.
Na
2 4 2
HAsO ×7H O (0.5 eq.), PNP, Tris-HCl buffer, pH 7.5, 50°C, 16 h.
The advantage of the enzymatic arsenolysis as compared to
the enzymatic phosphorolysis is the possibility of carrying out
*
The reaction proceeds quantitatively according to HPLC; yields are given for
compounds after isolation and purification.
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Figure 3. HPLC analysis of the enzymatic arsenolysis of the purine nucleosides 1b, 1i-m. The reaction was triggered by the addition of 2.8 μl of 1 mg/ml solution of PNP E. coli (0.1
activity units) to a nucleoside (0.25-0.29 μmol) in the presence of Na
Experimental section)
2 4 2
HAsO ×7H O (0.5 eq. to nucleoside) in Tris-HCl buffer, pH 7.5 at 50°C (HPLC parameters are described in the
reactions in a short period of time (16 h) using only one We proposed a convenient procedure for the preparation of
enzyme PNP and a small amount of arsenate, while the samples with anionic groups where ionization is very poor: a
phosphorolysis reaction requires an excess of phosphate and dopant of triethylamine to the sample vial (instead of the ion-
an additional use of alkaline phosphatase [9].
pairing additive to the buffer). In our case triethylamine
Products precipitated during the reaction were conveniently additive to a sample dramatically enhanced arsenate and
separated by the filtration and washed with water several phosphate ionization by several orders.
times. To be sure in the absence of toxic sodium arsenate in The optimization of the MS method has been studied in
the products we decided to quantify the possible previous works [23]. We observed the same trends in the
contamination of the CK samples by HPLC-HRMS.
negative ion mode: with higher capillary voltage the intensity
of the arsenate molecular ion was greater.
We empirically optimized some other parameters for our
equipment. There was no marked dependence of RF amplitude
of the ion funnels on the low m/z cutoff, so they were set at
Determination of the product purity via HPLC-HRMS.
All the products obtained by the enzymatic arsenolysis
(Scheme 5) were characterized for purity and homogeneity.
4
00 Vpp. Ion transmission in the collision cell was most
The absence of arsenate was additionally confirmed, since
inorganic arsenic compounds are the most toxic forms of this
element and can be readily absorbed and methylated to
methanearsonate or cacodylate in vivo [20].
effective at 750 Vpp (lower RF voltages produced less intensity
gain, and >1000 Vpp notably reduced the arsenate molecular
ion peak) with collision cell energy of 45 eV. The pre-pulse
storage of 10 μsec ditched most of the unnecessary ions of
higher masses and gained more 100-150 Da peaks. Good
implementation was achieved using quadrupole ion energy of
Currently, a combination of several detectors and methods is
considered as the most powerful strategy for blind detection
of As species [21], and ICP-MS is 3 orders of magnitude more
sensitive than ESI-MS. However, the latter type of
spectrometer is more common in organic chemistry
laboratories. In our case, the nature of the As-containing ion
1
0 eV, no additional MRM or isCID experiments were carried
out.
The sensitivity of the method was evaluated by establishing a
limit of detection (LOD – the lowest concentration of an
analyte that could be detected with a signal-to-noise ratio of at
least 3) and a limit of quantification (LOQ – the same as LOD
but with a signal-to-noise ratio of 10) for arsenate ions. The
linear dynamic range at five positive control concentrations
was known (as arsenate) and there was no need for additional
+
tandem MS/MS experiments for precursor ions ([AsO ] and
3
+
AsO ] ). Instead, we conducted further optimization of
[
2
parameters in the HPLC-MS technique of single ionization for
fast and effective detection.
-5
-6
-7
-8
(
containing 1.0 × 10 M, 1.0 × 10 M, 1.0 × 10 M, 1.0 × 10 M
The addition of quaternary ammonium salts is a known
technique in MALDI mass-spectrometry for proteins and
phosphate group ionization. These dopants create
noncovalent complexes with anionic groups of the molecular
ion and help to reduce alkali metal “messy” ions. Usually
ammonium acetate or formate are added to the buffer
solution, and its pH is generally affected; as a result, a suitable
acid additive is needed to reach the pH range; changes in
retention times should be considered [22].
-9
and 1.0 × 10 M solutions of Na HAsO ) and three repeated
2
4
3-
experiments for each allowed us to assess LOD (AsO4 ) as 12
±
nM concentration, approximately equivalent to 15 ng/ml,
2
3-
and LOQ (AsO4 ) as 50 ng/ml. The samples were treated and
analyzed immediately in 1 h after their dilution.
As a result of the HPLC-HRMS analysis, none of the tested
3-
samples contained AsO4 in it in the quantities exceeding LOQ.
4
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concentrations of A (acetonitrile) in B (water) with the flow
rate of 400 µl/min in the following gradient parameters: 0-15%
A for 6.0 min, 15%-85% A for 1.5 min, 85%-0% A for 0.1 min,
Conclusions
We have shown that nucleosides may be used as the starting
compounds for the synthesis of CKs and their derivatives. In
the suggested strategy, the ribose residue acts as a protective
group. We have compared chemical and enzymatic cleavage of
the N-glycosidic bond in nucleosides.
0
% A for 2.4 min. Retention times were as follows: arsenate
analytical detection) – 0.7 min, 2a – 4.0 min; 2b – 3.9 min; 2c
4.6 min; 2d – 4.9 min; 2e – 4.7 min; 2f – 3.1 min; 2g – 3.1
min; 2h – 4.2 min; 2i – 4.0 min; 2j – 5.5 min; 2k – 4.5 min; 2l
.2 min; 2m – 4.4 min; 2n – 4.0 min.
(
–
–
The chemical hydrolysis results in CKs in high yields. At the
same time it has the evident limit in the case of preparation of
labile CK derivatives. This can be solved by the enzymatic
hydrolysis which proceeds quantitatively under mild conditions
and may be applied to a wide range of nucleosides. The
presence of subequimolar quantities of arsenate in the
reaction mixture makes the enzymatic hydrolysis irreversible.
The final products are wholly separated from arsenate and
other co-reactants – the purity of products has been confirmed
by HPLC-HRMS. Using the arsenolysis reaction the broad
specificity of PNP makes it a valuable tool not only for
preparation of modified nucleosides but also for heterocyclic
bases with bulky aromatic or isoprenoid substituents.
4
HPLC analysis of the test reaction was performed on a Dr.
Maisch Reprosil-Pur C18-AQ column (4×150mm, 5µm, 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%
for 10 min at the flow rate of 1 ml/min with UV detection at
wavelength 260 nm. Retention times of the starting
nucleosides were as follows: 1b – 8.6 min; 1i – 8.6 min; 1j
–
1
3.5 min; 1k – 10.3 min; 1l – 9.7 min; 1m – 10.3 min; 1n – 8.6
min. Retention times of the products were as follows: 2b – 8.9
min; 2i – 8.8 min; 2j – 14.9 min; 2k – 10.9 min; 2l – 10.1 min;
2
m – 10.9 min; 2n – 10.1 min.
In this work, we used recombinant E. coli purine nucleoside
phosphorylase (PNP, EC 2.4.2.1, Sigma) with the concentration
of 1mg/ml and activity 35 units/mg (1 unit corresponds the
phosphorolysis of 1 μmol of inosine to hypoxanthine and
ribose 1-phosphate per minute at pH 7.4 at 25°C).
Experimental
General
The solvents and materials were reagent grade and were used The following nucleosides were obtained according to the
6
6
without additional purification. TLC was performed on previously published protocols: N -benzyladenosine [9], N -
6
6
Alugram SIL G/UV254 (Macherey-Nagel) with UV visualization. isopentenyladenosine, N -geranyladenosine [10], N -phenyl-
6
6
Melting points were determined with Electrothermal Melting adenosine, N -(3-phenylpropan-1-yl)adenosine, N -(4-phenyl-
butane-1-yl)-adenosine, N -(3-phenyl-2-propin-1-yl)adenosine,
6
Point Apparatus IA6301 and are uncorrected.
1
13
6
6
6
H (400 MHz) and C (with complete proton decoupling, 100.6 O -(2-phenylethyl)inosine [11], O -benzylinosine [24], N -(β-
MHz) NMR spectra were recorded on a Bruker AMX 400 NMR naphthylmethyl)adenosine [25], 6-chloro-2-amino-2′,3′,5′-tri-
instrument at 300K. Chemical shifts in ppm were measured O-acetyladenosine [26].
relative to the residual solvent signals as internal standards
1
13
1
(
CDCl
3
3
, H: 7.26 ppm, C: 77.1 ppm; DMSO-d
6
, H: 2.50 ppm,
1
C: 39.5 ppm), spin-spin coupling constants (J) are given in Hz.
Acid hydrolysis
Double resonance technique was applied for assigning the
resonances.
6
N -Phenyladenine (2a)
6
High resolution mass spectra (HRMS) were registered on a The solution of N -phenyladenosine (71 mg, 0.206 mmol) in
Bruker Daltonics micrOTOF-Q II instrument using electrospray 0.5M НСl (2.9 ml, 1.44 mmol) was stirred at 100°С. The
ionization (ESI). The measurements were done in a negative reaction was monitored by TLC (acetonitrile – 25% ammonia,
ion mode. Interface capillary voltage: 4700 V; mass range from 9:1). After 20 min when the traces of initial nucleoside had
m/z 50 to 3000; external calibration (Electrospray Calibrant disappeared, the reaction was cooled and neutralized to pH 7
Solution, Fluka); nebulizer pressure: 0.4 Bar; flow rate: 3 with concentrated NH
o
. The reaction mixture was kept at 0 C
3
µL/min; dry gas: nitrogen (4L/min); interface temperature: for 16 h. The resulting precipitate was filtered, washed with
00°C. Samples were injected into the mass spectrometer cold water (3×5 ml) and dried in vacuum over P . The yield
chamber from the Agilent 1260 HPLC system equipped with of N -phenyladenine (2a) was 37 mg (85%) as white powder.
2
2 5
O
6
1
Agilent Poroshell 120 EC-C18 column (3.0 × 50 mm; 2,7 µm) MP = 243-245°C. H NMR (400 MHz, DMSO-d
and an identically packed security guard, using an
p-m = 7.3 Hz, Hp-Ph), 7.32 (dd, 2H, Jm-o = 7.9 Hz, Jm-p = 7.3 Hz,
autosampler. The samples were injected from the 50% Hm-Ph), 7.95 (d, 2H, Jo-m = 7.9 Hz, Ho-Ph), 8.27 (s, 1H, H2), 8.37
6
): δ = 7.02 (t, 1H,
J
6
13
acetonitrile (LC-MS grade) in water (MilliQ ultrapure water, (s, 1H, H8), 9.69 (s, 1H, N H), 13.03 (br s, 1H, 9NH). C NMR
Merck Millipore KGaA, Germany) solution in the concentration (100 MHz, DMSO-d ): δ = 119.50 (C5), 120.55 (Ph), 122.33 (Ph),
of 0.1 mg/ml (350 μl) with 50 μl dopant solution of 5% 128.35 (Ph), 139.84 (Ph, C8), 150.44 (C4), 151.81 (C2, C6).
6
H]− calculated C11
H
N
−
210.0774, found
triethylamine. The autosampler syringe was washed before HRMS: m/z [M
and after injection two times each with 1% formic acid in 210.0782; m/z [M+Cl] calculated C11
−
8
5
−
−
H
9
N
5
Cl 246.0541, found
acetonitrile (wash 1) followed by 20% methanol in water (wash 246.0550.
2). The column temperature was 30°C and 15 μl of the sample
solution was injected. The column was eluted in a gradient of
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6
−
N -Benzyladenine (2b)
(C8), 149.59 (C4), 150.44 (C2), 153.04 (C6). HRMS: m/z [M−H]
6
−
−
Following the typical procedure for preparation of N - calculated C16
phenyladenine (2a), heating N -benzyladenosine (100 mg, calculated C16
H
12
N
5
274.1087, found 274.1089; m/z [M+Cl]
6
−
H
13
N
5
Cl 310.0854, found 310.0848.
0
.279 mmol) in 3.9 ml of 0.5M НСl (1.95 mmol) during 3 h at
6
6
00°C gave N -benzyladenine (2b) (58 mg, 92%) as white
N -Propargyladenine (2f)
1
6
f
powder. R = 0.50 (acetonitrile – 25% ammonia, 9:1). MP =231– Following the typical procedure for preparation of N -
1
33°C (Lit. [27] 229-231°C) H NMR and C NMR spectra were phenyladenine (2a), heating N -acetyl-N -propargyl-2′,3′,5′-tri-
13
6
6
2
identical to those reported earlier [9].
O-acetyladenosine (1f) (100 mg, 0.211 mmol) in 2.5 ml of 0.5M
6
НСl (1.26 mmol) during 8 h at 100°C gave N -propargyladenine
6
N -(3-Phenylpropan-1-yl)-adenine (2c)
f
(2f) (35 mg, 94%) as white powder. R = 0.67 (acetonitrile –
6
1
Following the typical procedure for preparation of N - 25% ammonia, 9:1). MP = 206-207°C. H NMR (DMSO-d
6
): 3.01
6
6
N -(3-phenylpropan-1- (s, 1H, ≡CH), 4.30 (s, 2H, N CH ), 7.91 (br s, 1H, N H), 8.13 (s,
6
phenyladenine
(
2a),
heating
yl)adenosine (1c) (208 mg, 0.539 mmol) in 7.6 ml of 0.5M НСl 1H, H-2), 8.24 (s, 1H, H-8), 12.94 (br s, 1H, 9NH). C NMR
2
1
3
6
6
(
6 2
3.77 mmol) during 2 h at 100°C gave N -(3-phenylpropan-1- (DMSO-d ): δ = 29.26 (N CH ), 72.38 (HC≡), 82.01 (-C≡), 117.95
yl)adenine (2c) (115 mg, 84%) as white powder. R
f
1
= 0.40 (C-5), 139.51 (C-8), 152.16 (C-2), 153.23 (C-6). HRMS: m/z
−
−
(
acetonitrile – 25% ammonia, 9:1). MP = 193–194°C. H NMR [M
400 MHz, DMSO-d
CH ), 2.65 (t, 2H, JCH2-CH2= 7.4Hz, CH
), 7.13-7.31 (m, 5H, Ph), 7.64 (br s, 1H, N H), 8.06 (H2),
−
H] calculated C
−
8
H
6
N
5
172.0618, found 172.0611; m/z
−
(
6
): δ = 1.91 (p, 2H, JCH2-CH2= 7.4Hz, [M+Cl] calculated C
8
H
7
N
5
Cl 208.0384, found 208.0384.
CH
2
C
H
2
2
2
Ph), 3.52 (br s, 1H,
6
6
6
N -Allyladenine (2g)
N CH
2
1
3
6
8
.16 (H8), 12.84 (br s, 1H, 9NH). C NMR (100 MHz, DMSO-d ): Following the typical procedure for preparation of N -
6
6
6
6
2 2 2
δ = 30.89, 32.63 (CH CH Ph), 38.8-40.3 (N CH -overlapping phenyladenine (2a), heating N -acetyl-N -allyl-2′,3′,5′-tri-O-
with DMSO-d
6
), 118.67 (C5), 125.62, 128.22, 138.49 (C8), acetyladenosine (1g) (100 mg, 0.210 mmol) in 2.5 ml of 0.5M
6
41.84 (Ph), 149.46 (C4), 152.34 (C2), 154.51 (C6). HRMS: m/z НСl (1.26 mmol) during 8 h at 100°C gave N -allyladenine (2g
1
)
−
−
[
M
−
M+Cl] calculated C14
H] calculated C14
−
H
14
N
5
252.1244, found 252.1245; m/z (26 mg, 70%) as white powder. R
f
= 0.45 (acetonitrile – 25%
−
1
Cl 288.1010, found 288.1015; m/z ammonia, 9:1). MP = 224-226°C. H NMR (DMSO-d
[
H
14
N
5
6
): δ = 4.16 (
]− calculated C15
H
N
O
3
−.
br s, 2Н, N СH
7 С=СН-trans), 5.93-6.0 (m, 2H, СH
(s, 1H, H-2), 8.17 (s, 1H, H-8), 12.82 (br s, 1H, 9NH). C NMR
6
), 5.06 (d, 1H, J = 9.5 С=СН-cis), 5.17 (d, 1H, J =
[M+HCO
3
15
5
2
6
1
2
), 7.69 (br s, 1H, N H), 8.09
6
13
N -(4-phenylbutan-1-yl)adenine (2d)
6
6
Following the typical procedure for preparation of N - (DMSO-d
6
): δ = 42.00 (N CH
2 2
), 114.96 (=CH ), 135.91 (CH),
6
N -(4-phenylbutane-1- 138.73 (C-8), 149.83 (C-4), 152.30 (C-2), 154.26 (C-6). HRMS:
phenyladenine
(
2a),
yl)adenosine (1d) (222 mg, 0.55 mmol) in 7.9 ml of 0.5M НСl m/z [M
heating
−
−
−
H] calculated C
8
H
8
−
N
5
174.0774, found 174.0773; m/z
6
−
(
3.89 mmol) during 2 h at 100°C gave N -(4-phenylbutane-1- [M+Cl] calculated C
yl)adenine (2d) (115 mg, 78%) as white powder. R = 0.51
8 9 5
H N Cl 210.0541, found 210.0542.
f
1
6
N -(2-Phenylethyl)adenine (2h)
(
acetonitrile - 25% ammonia, 9:1). MP = 162–164°C. H NMR
400 MHz, DMSO-d ): δ = 1.58-1.67 (m, 4H, CH , CH
CH CH Ph), 3.53 (br s, 2H, N CH
.12-7.29 (m, 5H, Ph), 7.68 (br s, 1H, N H), 8.09 (s, 1H, H2), 2′,3′,5′-tri-O-acetyladenosine (1h) (330 mg, 0.694 mmol) in 8.3
6
(
6
2
2
), 2.61 (t, Following the typical procedure for preparation of N -
6
6
6
2
7
8
H, JCH2-CH2=6.8Hz, CH
2
2
C
H
2
2
2
), phenyladenine (2a), heating N -acetyl-N -(2-phenylethyl)-
6
1
3
6
.19 (s, 1H, H8), 12.88 (br s, 1H, 9NH). C NMR (100 MHz, ml of 0.5M НСl (4.16 mmol) during 8 h at 100°C gave N -(2-
): δ = 28.41, 28.85, 34.85 ( Ph), 38.8-40.3 phenylethyl)adenine (2h) (102 mg, 84%) as white powder. R
-overlapping with DMSO-d ), 117.49 (C5), 125.55, 0.57 (acetonitrile – 25% ammonia, 9:1). MP = 248-250°C. H
DMSO-d
6
C
H
2
C
H
2
C
H
2
f
=
1
6
(N CH
2
6
6
6 2
128.14, 128.24 (Ph), 139.06 (C8), 142.16 (Ph), 150.16 (C4), NMR (DMSO-d ): δ = 2.93 (t, J = 7.5, 2Н, N СH ), 3.73 (br s, 2Н,
H]− calculated СH
6
) 7.15-7.35 (m, 5H, H-Ph), 7.59 (br s, 1H, N H), 8.07 (s, 1H,
13
1
51.93 (C2), 153.88 (C6). HRMS: m/z [M
−
266.1400, found 266.1402; m/z [M+Cl] calculated H-2), 8.19 (s, 1H, H-8), 12.85 (br s, 1H, 9NH). C NMR (DMSO-
2
−
−
C
C
15
H
H
16
N
N
5
5
−
6
15
17
Cl 302.1167, found 302.1164.
d
1
6
2 2
): δ = 35.15 (CH Ph), 41.29 (N CH ), 118.68 (C5), 125.97,
28.26, 128.62 (Ph), 138.78 (C8), 139.57 (Ph), 149.53 (C4),
H]− calculated
6
N -(β-Naphthylmethyl)adenine (2e)
152.34 (C2), 154.27 (C6). HRMS: m/z [M−
238.1087, found 238.1093; m/z [M+Cl] calculated
6
Following the typical procedure for preparation of N -
6
phenyladenine (2a), heating N -(β-naphthylmethyl)adenosine
−
−
C
C
13
13
H
H
12
N
N
5
−
13
5
Cl 274.0854, found 274.0854.
(
1e) (101 mg, 0.248 mmol) in 3.5 ml of 0.5M НСl (1.77 mmol)
6
during 2 h at 100°C gave N -(β-naphtylmethyl)adenine (2e) (65
mg, 94%) as white powder. R
= 0.40 (acetonitrile – 25% Enzymatic arsenolysis
ammonia, 9:1). MP = 245–247°C. H NMR (400 MHz, DMSO-
f
1
Analytical arsenolysis reaction of adenosine
6
d
7
1
=
1
6
): δ = 4.96 (br s, 2H, N CH
2
), 7.43-7.58 (m, 3H, naphthalene),
6
.82-7.92 (m, 5H, N H, naphthalene), 8.31 (s, 1H, H2), 8.38 (s,
The reaction solution contained 0.05 ml of 4 mM adenosine
1
H, H8), 9.00 (br s, 1H, 9NH). C NMR (100 MHz, DMSO-d
3
2 4 2
stock solution (0.2 μmol), 0.025 ml of 4 mM Na HAsO ×7H O
6
): δ
6
43.67 (N CH ), 115.97 (C5), 125.37, 125.62, 125.88, 126.12,
stock solution (0.1 μmol) (1:0.5 ratio) in 0.925ml of 50mM Tris-
HCl buffer, pH 7.5 (total volume 1 ml). The reaction was
2
27.49, 127.89, 132.12, 132.84, 136.83 (naphthalene), 140.65
6
| Org. Biomol. Chem., 2018, 00, 1-3
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Or gP al en ai sc e&d Bo i on mo to al ed cj uu ls at r mC ha re gmi ni ss try
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DOI: 10.1039/C8OB00223A
Organic & Biomolecular Chemistry
triggered by the addition of 0.002 ml of 1 mg/ml solution of
ARTICLE
6
N
-(3-Phenyl-2-propin-1-yl)adenine (2k)
6
PNP E. coli (0.07 activity units). The reaction was incubated at Following the procedure for preparation of N -benzyladenine
o
0 C, and was followed by HPLC (Figure 3).
6
2b), exposure of N -(3-phenyl-2-propin-1-yl)adenosine (1k
2
(
(
)
91 mg, 0.238 mmol) with Na HAsO ×7H O (37 mg, 0.119
2
4
2
mmol) and PNP (0.143 ml, 5 activity units) in 50 mM Tris-HCl
6
buffer pH 7.5 (10 ml) during 16 h at 50°C gave N -(3-phenyl-2-
6
Preparative arsenolysis reaction of N -substituted adenosines
6
N
-Benzyladenine (2b)
propin-1-yl)adenine (2k) (54 mg, 91%) as white powder. R =
f
6
1
To the mixture of N -benzyladenosine (1b) (94 mg, 0.263 0.37 (CH
mmol) and Na HAsO ×7H
O (41 mg, 0.131 mmol) in 50 mM DMSO-d
Tris-HCl buffer pH 7.5 (10 ml) PNP, (0.143 ml, 5 activity units) 8.06 (br s, 1H, N H), 8.13 (s, 1H, H8), 8.26 (s, 1H, H2), 12.97 (s,
2
Cl
2
– EtOH, 95:5). MP = 227-229°C. H NMR (400 MHz,
6
): δ = 4.57 (br s, 2H, N CH
2
), 7.31-7.41 (m, 5H, Ph),
2
4
2
6
6
1
3
6
was added and the mixture was kept at 50°C for 16 h. During 1H, 9NH). C NMR (100 MHz, DMSO-d
the reaction, the initial nucleoside dissolved and the product 81.04, 88.01 (C≡C), 118.93 (C5), 122.43, 128.33, 128.56, 131.28
6 2
): δ = 30.01 (N CH ),
crystallized. The precipitate was filtered, washed with H
2
O
(Ph), 139.16 (C8), 150.01 (C4), 152.20 (C2), 153.68 (C6). HRMS:
−
6
−
(
5×5 ml) and dried in vacuum dessicator over P
benzyladenine (2b) (48 mg, 81%) as white powder. R
CH Cl – EtOH, 95:5). MP = 232–234°C (Lit. [27] 229-231°C). H
2
O
5
to give N - m/z [M
−
H] calculated C14
−
H
10
N
5
248.0931, found 248.0933;
−
= 0.53 m/z [M+Cl] calculated C14
H
11
N
5
Cl 284.0697, found 284.0704.
f
1
(
2
2
1
3
O-6-Benzylhypoxanthine (2l)
6
224.0931, Following the procedure for preparation of N -benzyladenine
NMR and C NMR spectra were identical to those reported
−
−
earlier [9]. HRMS: m/z [M
−
H] calculated C12
H
10
N
5
−
−
found 224.0941; m/z [M+Cl] calculated C12
H
11
N
5
Cl 260.0697,
(
2b), exposure of O-6-benzylinosine (1l) (124 mg, 0.346 mmol)
with Na HAsO ×7H O (54 mg, 0.170 mmol) and PNP (0.143 ml,
activity units) in 50 mM Tris-HCl buffer pH 7.5 (10 ml) during
16 h at 50°C gave O-6-benzylhypoxanthine (2l) (67 mg, 85%) as
= 0.23 (CH Cl – EtOH, 95:5). MP = 174-176°C.
H NMR (400 MHz, DMSO-d ): δ = 5.62 (s, 2H, O CH
mmol) with Na HAsO ×7H O (46 mg, 0.149 mmol) and PNP 7.56 (m, 5H, Ph), 8.38 (s, 1H, H-2), 8.52 (s, 1H, H-8), 13.43 (s,
found 260.0708.
2
4
2
5
6
N
-Isopentenyladenine (2i)
6
Following the procedure for preparation of N -benzyladenine white powder. R
f
2
2
6
1
6
(
2b), exposure of N -isopentenyladenosine (1i) (100 mg, 0.298
6
2
), 7.32-
2
4
2
1
3
(
0.143 ml, 5 activity units) in 50 mM Tris-HCl buffer pH 7.5 (10 1H, 9NH). C NMR (100 MHz, DMSO-d
ml) during 16 h at 50°C gave N -isopentenyladenine (2i) (48 128.08, 128.28, 128.41, 136.36 (Ph), 142.77 (C8), 151.17 (C2)
6
): δ = 67.56 (OCH ),
2
6
mg, 79%) as white powder. R = 0.43 (CH Cl – EtOH, 95:5). MP HRMS: m/z [M
2
−
H]− calculated C12
H
9
N
4
O− 225.0771, found
OCl− 261.0538,
f
2
1
13
−
=
210-212°C (Lit. [28] 216-218°C). H NMR and C NMR 225.0775; m/z [M+Cl] calculated C12
spectra were identical to those reported earlier [9]. HRMS: m/z found 261.0543.
H
10
N
4
−
−
[M−H] calculated C H N 202.1087, found 202.1085; m/z
10 12 5
−
−
O-6-(2-Phenylethyl)hypoxanthine (2m)
Following the procedure for preparation of N -benzyladenine
2b), exposure of O-6-(2-phenylethyl)inosine (1m) (100 mg,
HAsO ×7H O (42 mg, 0.134 mmol) and
2b), exposure of N -geranyladenosine (1j) (100 mg, 0.248 PNP (0.143 ml, 5 activity units) in 50 mM Tris-HCl buffer pH 7.5
mmol) with Na HAsO ×7H
O (39 mg, 0.124 mmol) and PNP (10 ml) during 16 at 50°C gave O-6-(2-
0.143 ml, 5 activity units) in 50 mM Tris-HCl buffer pH 7.5 (10 phenylethyl)hypoxanthine (2m) (58 mg, 90%) as white powder.
[
M+Cl] calculated C H N Cl 238.0854, found 238.0851.
1
0 13 5
6
6
N
-Geranyladenine (2j)
(
6
Following the procedure for preparation of N -benzyladenine 0.268 mmol) with Na
2
4
2
6
(
h
2
4
2
(
6
1
ml) during 16 h at 50°C gave N -geranyladenine (2j) (61 mg,
R
f
= 0.30 (CH
– EtOH, 95:5). MP = MHz, DMSO-d
): δ = 1.54 (s, 3H, Me), (t, 2H, JCH2-CH2 = 6.9 Hz, O CH
.60 (s, 3H, Me), 1.70 (s, 3H, Me), 1.96 (ddd, 2H, JCH2-CH = 6.9 1.4 Hz, Hp-Ph), 7.30 (t, 2H, Jm-o = 7.3 Hz, Hm-Ph), 7.34 (dd, 2H,
CH=CMe ), 2.04
o-m = 7.3 Hz, Ho-Ph), 8.34 (s, 1H, H-2), 8.48 (s, 1H, H-8), 13.37
CH CH=CMe
), (s, 1H, 9NH). C NMR (100 MHz, DMSO-d
), 5.06 (tt, 1H, JCH-CH2 = 6.9 Hz, JCH-CH2 = 1.2 66.77 (O ), 120.02 (C5), 126.32, 128.32, 128.89, 138.02 (Ph),
2
Cl
2
– EtOH, 95:5). MP = 204-206°C. H NMR (400
Ph), 4.75
= 7.0 Hz, J
9
1
1
0%) as white powder. R
f
= 0.56 (CH
2
Cl
2
6
): δ = 3.14 (t, 2H, JCH2-CH2 = 6.9 Hz, CH
2
1
6
62–163°C. H NMR (400 MHz, DMSO-d
6
2
), 7.22 (tt, 1H, J
p
-m
=
p-o
Hz, JCH2CH2 = 7.8 Hz, JCH2-CH2 = 7.2 Hz, CH
2
C
H
2
2
J
1
3
(
dd, 2H, JCH2CH2 = 7.8 Hz, JCH2-CH2 = 7.2 Hz, C
H
2
2
2
): δ = 34.55 (CH Ph),
6 2
6
4
.09 (br s, 1H, N CH
2
CH
2
Hz, C
H
=CMe
=), 7.58 (br s, 1H, N H), 8.05 (s, 1H, H-8), 8.16 (s, 1H, [M
2
), 5.32 (dd, 1H, JCH-CH2 = 5.8 Hz, JCH-CH2 = 6.4 Hz, 142.05 (C8), 151.25 (C2), 153.15 (C4), 159.75 (C6). HRMS: m/z
6
N CH
6
−
−
2
C
H
−
H] calculated C13
−
H
11
N
4
O 239.0927, found 239.0931; m/z
−
1
3
H-2), 12.84 (s, 1H, 9NH). C NMR (100 MHz, DMSO-d
6.10, 17.47 (=CMe -geranyl), 25.37 (Me-geranyl), 25.93 (CH
2
geranyl), 37.65, 37.79 (N CH -geranyl), 118.69 (C5),
6
): δ = [M+Cl] calculated C13
H
12
N
4
OCl 275.0694, found 275.0698.
1
2
2
-
6
6
2
-Amino-
-geranyl), Following the procedure for preparation of N -benzyladenine
2
N -benzyladenine (2n)
2
, CH
22.06 (CH=), 123.92 (CH=), 130.73 (=C(Me)CH
6
1
1
1
6
36.34 (=
C
Me
2
-geranyl), 138.49 (C8), 149.43 (C4), 152.31 (C2),
−
(
2b), exposure of 2-amino-N -benzyladenosine (1n) (100 mg,
−
54.18 (C6). HRMS: m/z [M
−
H] calculated C15
−
H
20
N
5
270.1713, 0.268 mmol) with Na HAsO ×7H O (42 mg, 0.134 mmol) and
2
4
2
−
found 270.1719; m/z [M+Cl] calculated C15
H
21
N
5
Cl 306.1480, PNP (0.143 ml, 5 activity units) in 50 mM Tris-HCl buffer pH 7.5
6
(10 ml) during 16 h at 50°C gave 2-amino-N -benzyladenine
found 306.1481.
(
2n) (48 mg, 75%) as yellow powder. R
f
= 0.40 (CH
2
Cl
2
– EtOH,
1
5:5). MP = 232-233°C. H NMR (400 MHz, DMSO-d
9
6
): δ = 4.67
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Org. Biomol. Chem., 2018, 00, 1-3 | 7
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Organic & Biomolecular Chemistry
Page 8 of 9
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DOI: 10.1039/C8OB00223A
ARTICLE
Organic & Biomolecular Chemistry
6
br s, 2H, N CH
(
2 2 p-m
), 5.65 (2H, s, 2-NH ), 7.19 (t, 1H, J
= 7.0 Hz, 19 A. Bzowska, E. Kulikowska and D. Shugar, Pharmacology &
therapeutics, 2000, 88, 349; T. Araki, I. Ikeda, K. Matoishi, R.
Abe, T. Oikawa, Y. Matsuba, H. Ishibashi, K. Nagahara and Y.
Fukuiri, US Pat., 7629457B2, 2009.
0 C. Yuan, X. Lu, N. Oro, Z. Wang, Y. Xia, T.J. Wade, J. Mumford
and X.C. Le, Clin. Chem., 2008, 54, 163.
Hp-Ph), 7.28 (t, 2H, Jm-o = 7.4 Hz, Hm-Ph), 7.34 (dd, 2H, Jo-m
=
6
.4 Hz, Ho-Ph), 7.58 (br s, 1H, N H), 7.64 (s, 1H, H-8), 12.05 (s,
7
1
1
1
1
3
6
6 2
H, 9NH). C NMR (100 MHz, DMSO-d ): δ = 42.70 (N CH
),
2
13.04 (C5), 126.42 (Ph), 127.24 (Ph), 128.06 (Ph), 135.26 (Ph),
40.68 (C8), 152.03 (C4), 154.65 (C6), 160.15 (C2). HRMS: m/z 21 H. Peng, B. Hu, Q. Liu, Z. Yang, X. Lu, R. Huang, X.F. Li, M.J.
−
−
Zuidhof and X. Le, J. Chromatogr. A, 2014, 1370, 40.
22 S. Banerjee and S. Mazumdar, Int. J. Anal. Chem., 2012,
Article ID 282574; J. Myerson, Increasing ionization efficiency
in mass spectrometry. Patent US7276378. Publication date:
[
M
−
M+Cl] calculated C12
H] calculated C12
−
H
11
N
6
239.1040, found 239.1046; m/z
−
[
H
12
N
6
Cl 275.0806, found 275.0816.
0
4/06/2004.
23 T.D. Ninh, Y. Nagashima and K. Shiomi, Food. Add. Contam.,
007, 23, 1299.
Acknowledgements
2
This work was supported by the Russian Science Foundation
RSF, grant № 16-14-00178).
2
4 S.V. Kolyachkina, V.I. Tararov, C.S. Alexeev, D.M. Krivosheev,
G.A. Romanov, E.V. Stepanova, E.S. Solomko, A.N. Inshakov
and S.N. Mikhailov, Collect. Czech. Chem. Commun., 2011,
(
76, 1361.
2
5 A.A. Orlov, M.S. Drenichev, V.E. Oslovsky, N.N. Kurochkin,
P.N. Solyev, L.I. Kozlovskaya, V.A. Palyulin, G.G. Karganova,
S.N. Mikhailov and D.I. Osolodkin Bioorg. Med. Chem. Lett.,
Notes and references
1
2
H. Rosemeyer, Chemistry & biodiversity, 2004,
M. Gordaliza, Mar. Drugs, 2009, , 833; S.N. Mikhailov, L.
Scotti, R.K. Singla and M.T. Scotti, Curr. Top. Med. Chem.,
016, 16, 2725; C.R. Pye, M.J. Bertin, R.S. Lokey, W.H.
1, 361.
7
2
017, 27, 1267.
6 M.J. Robins and B. Uznański. Canadian Journal of Chemistry,
981, 59, 2601.
7 N.J. Leonard and T.R. Henderson, J. Am. Chem. Soc., 1975,
, 4990.
2
2
2
2
1
Gerwick and R.G. Linington, Proceedings of the National
Academy of Sciences, 2017, 114, 5601.
9
7
3
4
P. Ertl, S. Jelfs, J. Mühlbacher, A. Schuffenhauer and P. Selzer,
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Organic & Biomolecular Chemistry
View Article Online
DOI: 10.1039/C8OB00223A
Cytokinins synthesis based on the irreversible enzymatic cleavage by Purine Nucleoside Phosphorylase
in the presence of Na HAsO has been developed.
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