Synthesis of [15N4] purine labeled cytokinin glycosides derived from zeatins and topolins with 9-β-d, 7-β-d-glucopyranosyl, or 9-β-d-ribofuranosyl group

Article abstract of DOI:10.1002/jlcr.3702

Synthesis of [¹⁵N₄] purine labeled cytokinine glycosides derived from zeatins and topolins containing a 9-β-d, 7-β-d-glucopyranosyl, or 9-β-d-ribofuranosyl group is described. These N⁶-substituted adenine derivatives are i

Full text of DOI:10.1002/jlcr.3702

1
5
Synthesis of [ N ]purine labelled cytokinin glycosides derived from zeatins and topolins
4
with 9-β-D, 7-β-D-glucopyranosyl, or 9-β-D-ribofuranosyl group
1
2
2
1
1*
Lenka Tranová , Jan Buček , Marek Zatloukal , Petr Cankař , and Jakub Stýskala
1
Department of Organic Chemistry, Faculty of Science, Palacký University, 17. listopadu 12,
71 46 Olomouc, Czech Republic
7
2
Department of Chemical Biology and Genetics, Centre of the Region Haná for
Biotechnological and Agricultural Research, Faculty of Science, Palacký University, 783 71
Olomouc-Holice, Czech Republic
*
Corresponding author: Department of Organic Chemistry, Faculty of Science, Palacký
University, 17. listopadu 12, 771 46 Olomouc, Czech Republic. Tel.: +420 585 634 466; Fax:
420 585 634 465, E-mail: jakub.styskala@upol.cz
+
Abstract
15
Synthesis of [ N ]purine labelled cytokinine glycosides derived from zeatins and topolins
4
containing a 9-β-D, 7-β-D-glucopyranosyl, or 9-β-D-ribofuranosyl group is described. These
6
N -substituted adenine derivatives are intended as internal analytic standards for
15
phytohormone analysis. All labelled compounds were prepared from 6-chloro[ N ]purine
4
(1). The equilibrium reaction of 1 with acetobromo--D-glucose gave isomeric 7-β-D (3) and
9
-β-D (4) chloro glucosyl precursors, which were treated with the corresponding amines to
get desired labelled cytokinin 7-β-D (6) and 9-β-D (5) glucopyranosides. Cytokinins
containing 9-β-D-ribofuranosyl group (8) were obtained by direct enzymatic
15
transglycosylation reaction of cytokinins (7) prepared from 6-chloro[ N ]purine (1).
4
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been through the copyediting, typesetting, pagination and proofreading process which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1002/jlcr.3702
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Key words: Labelled purine, labelled adenine, zeatin, topolin, cytokinins, internal standard
1
5
Short title: Synthesis of [ N ]purine labelled cytokinin glycosides
4
Introduction
Cytokinins are plant hormones that play a crucial role in the regulation of plant growth and
6
development. Naturally occurring cytokinins are structurally N substituted adenine
derivatives classified according to the side chain into the isoprenoid family represented by
isopentenyladenine, trans- and cis-zeatin, and aromatic family represented by
1
,2
benzylaminopurine, ortho-topolin, meta-topolin, and para-topolin. Their abundance in
plant cells is not limited only to the free bases, but also to their sugar conjugates. Described
adenine glycosides include β-D-ribofuranoside or β-D-glucopyranoside units attached
9
7
predominantly at the N or, less frequently, N position.
The qualitative and quantitative analysis is a challenging task due to very low concentrations
of cytokinins. The common concentration ranges from the pmol to fmol level per gram of a
fresh plant material. Further, the presence of the structurally similar compounds and
isomers requires efficient purification and separation techniques with a sensitive and high
3
resolution detection. For this reason, the HPLC-MS method is the most frequently used. An
addition of a known quantity of the isotopically labelled cytokinin standard into the plant
tissue extract enables to monitor possible loses during the purification and determine the
amount of the natural cytokinin by the ratio of the area of the MS peaks of the natural and
4
labelled cytokinin.
15
For this reason the synthesis of the [ N ]purine isotopically labelled cytokinin glycosides
4
derived from zeatins and topolins (Figure 1) with the 9-β-D, 7-β-D-glucopyranosyl, or 9-β-D-
ribofuranosyl unit is very important, since these compounds can serve as internal standards
for quantification in the plant materials. The synthesis of these standards usually offers
modification of strategy. The order of glycosylation and aromatic nucleophilic substitution
with amines is crucial, so this paper is devoted to the study of suitable synthetic routes to
prepare labelled cytokinins on a microscale.
Please insert Figure 1.
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Experimental
General
All commercially available reagents were used without further purification and purchased
1
5
15
from standard chemical suppliers. Isotopically labelled 6-chloro[ N ]purine (1) (98 % N)
4
5
was provided by authors of publication, where the improved preparation is described.
Purine nucleotide phosphorylase 120 Unit/mL was purchased from Sigma-Aldrich (Prague,
Czech Republic). Reactions were monitored by LC/MS analyses on a UHPLC-MS system
(Thermo Scientific, Waltham, MA, USA) consisting of a UHPLC chromatograph equipped with
a photodiode array detector and a triple quadrupole mass spectrometer using a C18 column
at 30 °C and flow rate of 800 μL/min. Mobile phases: 10 mM ammonium acetate in HPLC
grade water (A) and HPLC grade acetonitrile (B). A gradient was formed from 5% to 80% of B
1
13
1
in 10 minutes. H and C NMR spectra were measured on an ECA 400II ( H: 399.78 MHz,
1
3
15
C: 100.53 MHz, N: 50.7 MHz ) NMR spectrometer (JEOL Resonance, Tokyo, Japan).
Chemical shifts (δ) are reported in ppm and referenced to the middle peak of the solvent
1
5
signal (DMSO-d : 2.50 ppm, 39.51 ppm; CDCl : 7.27 ppm, 77.00 ppm. N NMR spectra were
6
3
electronically referred to ammonia. All the NMR spectra were acquired at ambient
1
13
temperature. All recorded H and C NMR spectra are available as Supplementary material
online. High resolution mass spectra (HRMS) measurements were performed on an Orbitrap
Elite mass analyzer Thermo Exactive Plus equipped with Heated Electrospray Ionization
(HESI) and Dionex Ultimate 3000 system (Thermo Scientific, MA, USA). Thin layer
chromatography (TLC) was performed on pre-coated silica gel 60 F254 plates and visualized
by exposure to UV light (254 or 366 nm). Column chromatography was carried out by using
silica gel grade 60, meshes size 230 to 400 Å and activated at 140 °C for 48 h prior to use.
Purification of target compounds was carried out by Agilent Technologies 1200 HPLC system
on C18 reverse phase column 19 x 100 mm, 5 μm particles, gradient was formed from 10
mM aqueous ammonium acetate and acetonitrile, flow rate 15 mL/min. For the separation
of 9-β-D-ribofuranosides 8 a mobile phase consisting of 0.1% HCOOH in water and 0.1%
HCOOH in acetonitrile was used.
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Chemistry
1
5
6
-Chloro[ N ]purine-7-β-D-glucopyranoside-2´,3´,4´,6´-tetraacetate
(3)
and
6-
4
1
5
chloro[ N ]purine-9-β-D-glucopyranoside (4). To a glass vial with closure was added 6-
4
15
chloro[ N ]purine (1) (200 mg, 1.26 mmol; dried at 120 °C for 45 min), 2,3,4,6-tetra-O-
4
acetyl--D-glucopyranosyl bromide (1245 mg, 3.03 mmol), finely pulverized anhydrous
K CO (384 mg, 2.78 mmol), and dry acetonitrile (5 mL). The reaction mixture was stirred at
2
3
room temperature for 30 h. The formed suspension was diluted with DCM (2 mL), filtered
off, and washed with DCM (2 x 3 mL). After evaporation of all solvents under reduced
pressure the residue was fractioned by column chromatography on silica gel (55 g, 4 cm ID)
with the following gradient DCM – MeOH (250 mL DCM; 300 mL 150:1; 500 mL 120:1; 300
mL 100:1; 100 mL 70:1). This gradient enabled separation of both isomers: 9-isomer 2 as the
first and 7-isomer 3 as the second one. After evaporation of the solvent, the fractions
containing the pure products were obtained as solid foams to give 80.6 mg (13 %) of 7-
acetate 3 and 332 mg (54 %) of 9-acetate 2, which was deacetylated by dissolving in
ethanolic ammonia (15 mL, saturated at 0 °C) and stirred at 0°C for 3.5 h. All volatiles were
carefully removed under reduced pressure. The residue was dissolved in warm isopropanol
(7 mL) and cooled slowly to 0 °C to give on standing a white crystalline precipitate, which
was filtered off, washed with isopropanol, and dried. The yield of compound 4 was 158 mg
(39 %). Mother liquids, containing the remaining product, were not processed.
1
Data for compound 3: H NMR (400 MHz, CDCl ) δ 8.94 (dd, J = 16.0, 14.4 Hz, 1H), 8.49 (dd, J
3
=
5
11.4, 8.2 Hz, 1H), 6.18 (d, J = 8.4 Hz, 1H), 5.69 (t, J = 9.2 Hz, 1H), 5.47 (t, J = 9.4 Hz, 1H),
.26 - 5.31 (m, 1H), 4.28 (dd, J = 12.6, 5.4 Hz, 1H), 4.19 (dd, J = 12.6, 1.8 Hz, 1H), 4.06-4.10
1
3
(
m, 1H), 2.07 - 2.08 (m, 6H), 2.05 (s, 3H), 1.88 (s, 3H). C NMR (100 MHz, CDCl ) δ 170.4,
3
1
69.9, 169.3, 169.0, 162.2 (br. s), 153.1 (br. s), 146.2 (br. s), 142.8 (br. s), 122.1 (d, J = 14.3
1
5
Hz), 75.0, 72.9, 69.7, 67.5, 61.4, 20.6, 20.5, 20.5, 20.2. N NMR (50.7 MHz, CDCl ) δ 274.3,
3
+
15
2
4
66.4, 246.4, 151.0. HRMS (HESI m/z): [M+H] calcd for C H Cl N O 489.0951; found
1
9
22
4 9
89.0950.
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1
Data for compound 4: H NMR (400 MHz, DMSO-d ) δ 8.92 (dd, J = 12.2, 8.2 Hz, 1H), 8.82 (t,
6
J = 15.4 Hz, 1H), 5.56 (d, J = 9.2 Hz, 1H), 5.39 (d, J = 5.6 Hz, 1H), 5.33 (d, J = 4.8 Hz, 1H), 5.19
(d, J = 4.8 Hz, 1H), 4.59 (t, J = 5.6 Hz, 1H), 4.09 - 4.01 (m, 1H), 3.74 - 3.68 (m, 1H), 3.48 - 3.37
1
3
(
m, 3H), 3.30 - 3.24 (m, 1H). C NMR (100 MHz, DMSO-d ) δ 152.1 (br. s), 151.9 (br. s), 149.2
6
(br. s), 146.5 (d, J = 9.5 Hz), 131.0 (d, J = 9.5 Hz), 83.7 (d, J = 12.4 Hz), 80.2, 76.8, 71.3, 69.7,
1
5
-
6
0.9. N NMR (50.7 MHz, DMSO-d ) δ 277.4, 254.0, 244.5, 174.0. HRMS (HESI m/z): [M-H]
6
1
5
calcd for C H Cl N O 319.0372; found 319.0375.
1
1
12
4 5
General procedure for the preparation of 9-β-D-glucopyranosides 5a-e. A mixture containing
compound 4 (20 mg, 0.062 mmol), corresponding amine (0.08 mmol), TEA (20 μL, 0.14
mmol), and propanol (1.3 mL) was heated with stirring in a sealed vial under nitrogen at 92
°
C for 4 h. After that all volatiles were removed under reduced pressure and the residue was
dissolved in a mixture of methanol (1.5 mL) and water (0.5 mL) to purify the product by the
preparative HPLC method. The combined fractions containing pure glucoside 5 were
partially concentrated under reduced pressure to remove most of acetonitrile and
lyophilized to get a white solid product.
1
5
[
N ]-cis-zeatin-9-β-D-glucopyranoside
4
(5a),
[(Z)-6-((4-hydroxy-3-methylbut-2-en-1-
1
5
yl)amino)-[ N ]purine-9-β-D-glucopyranoside. This compound was prepared according to
4
the general procedure with several exceptions. (Z)-4-amino-2-methylbut-2-en-1-ol
6
hemioxalate (12 mg, 0.04 mmol), TEA (45 μL; 0.35 mmol), and anhydrous ethanol (1 mL) as
a reaction solvent were used. The reaction mixture was heated at 92 °C for 24 h. Yield 17.3
1
mg (72 %). H NMR (400 MHz, DMSO-d ) δ 8.30 (dd, J = 11.9, 8.2 Hz, 1H), 8.19 (t, J = 15.8 Hz,
6
1
H), 7.78 (br. s, 1H), 5.38 (d, J = 9.2 Hz, 1H), 5.36 - 5.21 (m, 3H), 5.14 (d, J = 5.0 Hz, 1H), 4.72
(
t, J = 5.0 Hz, 1H), 4.58 (t, J = 5.5 Hz, 1H), 4.22 - 4.07 (br. s, 1H), 4.02 (d, J = 5.5 Hz, 2H), 3.98
dd, J = 14.0, 8.9 Hz, 1H), 3.70 - 3.66 (m, 1H), 3.26-3.19 (m, 1H), 1.68 (s, 3H). Four signals are
(
+
15
hidden in a signal of water. HRMS (HESI m/z): [M+H] calcd for C H N N O 386.1602;
1
6
24
4 6
found 386.1604.
1
5
[
N ]-trans-zeatin-9-β-D-glucopyranoside (5b), [(E)-6-((4-hydroxy-3-methylbut-2-en-1-
4
1
5
yl)amino)-[ N ]purine-9-β-D-glucopyranoside. This compound was prepared in a similar
4
7
manner as 5a using (E)-4-amino-2-methylbut-2-en-1-ol hemioxalate (12 mg, 0.04 mmol).
1
Yield 20.1 mg (84 %). H NMR (400 MHz, DMSO-d ) δ 8.30 (dd, J = 11.9, 8.2 Hz, 1H), 8.21 (t, J
6
=
16.0 Hz, 1H), 7.85 (br. s, 1H), 5.50 (t, J = 6.0 Hz, 1H), 5.38 (d, J = 9.2 Hz, 1H), 5.34 - 5.22 (m,
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2
3
1
H), 5.14 (d, J = 4.1 Hz, 1H), 4.73 (t, J = 5.6 Hz, 1H), 4.58 (t, J = 5.6 Hz, 1H), 4.10 (br .s, 1H),
.98 (dd, J = 13.3, 9.2 Hz, 1H), 3.76 (d, J = 5.0 Hz, 2H), 3.70 - 3.65 (m, 1 H), 3.27 - 3.20 (m,
+
H), 1.65 (s, 3H). Four signals are hidden in signal of water. HRMS (HESI m/z): [M+H] calcd
1
5
for C H N N O 386.1602; found 386.1603.
1
6
24
4 6
1
5
15
[
N ]-o-topolin-9-β-D-glucopyranoside (5c), [6-((2-hydroxybenzyl)amino)-[ N ]purine-9-β-
4
4
D-glucopyranoside. This compound was prepared according to the general procedure using
8
1
2
-hydroxybenzylamine (9.8 mg). Yield 22.3 mg (88 %). H NMR (400 MHz, DMSO-d ) δ 9.95
6
(br. s, 1H), 8.35 (dd, J = 11.4, 8.2 Hz, 1H), 8.27 - 8.11 (m, 2H), 7.09 -7.01 (m, 2H), 6.79 (d, J =
6
5
3
.9 Hz, 1H), 6.70 (t, J = 7.1 Hz, 1H), 5.39 (d, J = 8.9 Hz, 1H), 5.32 (d, J = 5.5 Hz, 1H), 5.27 (d, J =
.0 Hz, 1H), 5.13 (d, J = 5.6 Hz, 1H), 4.60 - 4.56 (m, 3H), 3.99 (dd, J = 14.4, 8.5 Hz, 1H), 3.70 -
.66 (m, 1H), 3.26 - 3.20 (m, 1H). Three signals are hidden in a signal of water. HRMS (HESI
+
15
m/z): [M+H] calcd for C H N N O 408.1446; found 408.1447.
18
22
4 6
1
5
15
[
N ]-m-topolin-9-β-D-glucopyranoside (5d), [6-((3-hydroxybenzyl)amino)-[ N ]purine-9-
4
4
β-D-glucopyranoside. This compound was prepared according to the general procedure
1
using 3-hydroxybenzylamine (9.8 mg). Yield 23.3 mg (92 %). H NMR (400 MHz, DMSO-d ) δ
6
9
.25 (s, 1H), 8.44 - 8.25 (m, 2H), 8.19 (t, J = 15.6 Hz, 1H), 7.06 (t, J = 7.8 Hz, 1H), 6.73 6.67
m, 2H), 6.56 (dd, J = 8.0, 1.6 Hz, 1H), 5.39 (d, J = 9.2 Hz, 1H), 5.36 - 5.21 (m, 2H), 5.13 (d, J =
.2 Hz, 1H), 4.76 - 4.45 (m, 3H), 4.09 - 3.91 (m, 1H), 3.70 - 3.67 (m, 1H). Four signals are
(
3
+
15
hidden in a signal of water. HRMS (HESI m/z): [M+H] calcd for C H N N O 408.1446;
1
8
22
4 6
found 408.1447.
1
5
15
[
N ]-p-topolin-9-β-D-glucopyranoside (5e), [6-((4-hydroxybenzyl)amino)-[ N ]purine-9-β-
4
4
D-glucopyranoside. This compound was prepared according to the general procedure using
1
4
-hydroxybenzylamine (9.8 mg). Yield 22.0 mg (87 %). H NMR (400 MHz, DMSO-d ) δ 9.22
6
(
br. s, 1H), 8.31 (dd, J = 11.7, 8.0 Hz, 1H), 8.28 - 8.16 (m, 2H), 7.13 (d, J = 8.7 Hz, 2H), 6.65 (d,
J = 8.7 Hz, 2H), 5.38 (d, J = 9.6 Hz, 1H), 5.31 (d, J = 6.0 Hz, 1H), 5.26 (d, J = 3.7 Hz, 1H), 5.13
(d, J = 5.5 Hz, 1H), 4.68 - 4.47 (m, 3H), 3.98 (dd, J = 14.0, 8.9 Hz, 1H), 3.70 -3.66 (m, 1H). Four
+
signals are hidden in a signal of water. HRMS (HESI m/z): [M+H] calcd for
1
5
C H N N O 408.1446; found 408.1445.
1
8
22
4 6
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General procedure for the preparation of 7-β-D-glucopyranosides 6a-e. A mixture containing
peracetylated 3 (17.1 mg, 0.035 mmol), corresponding amine (0.05 mmol), TEA (34 μL, 0.24
mmol), and acetonitrile (1.4 mL) was heated with stirring in a sealed vial under nitrogen at
9
2 °C for 7 h. After that most of acetonitrile was removed with stream of nitrogen. The
residue was diluted with ethanolic ammonia (1 mL, saturated at 0 °C) under stirring at 0°C
for 7 h. Volatiles were removed under reduced pressure to give an oily product, which was
dissolved in a mixture of methanol (2 mL) and water (1 mL) to purify the product by the
preparative HPLC method. The combined fractions containing pure glucoside 6 were
partially concentrated under reduced pressure to remove most of acetonitrile and
lyophilized to get a white solid product.
1
5
[
N ]-cis-zeatin-7-β-D-glucopyranoside
4
(6a),
[(Z)-6-((4-hydroxy-3-methylbut-2-en-1-
1
5
yl)amino)-[ N ]purine-7-β-D-glucopyranoside. This compound was prepared according to
4
the general procedure with several exceptions. (Z)-4-amino-2-methylbut-2-en-1-ol
hemioxalate (7.7 mg, 0.026 mmol), TEA (45 μL; 0.35 mmol), and anhydrous ethanol (1 mL)
as a reaction solvent were used. The reaction mixture was heated at 92 °C for 24 h. Yield 9.0
1
mg (67 %). H NMR (400 MHz, DMSO-d ) δ 8.32 - 8.24 (m, 2H), 7.26 (dd, J = 9.8, 5.0 Hz, 1H),
6
5
5
3
.50 (d, J = 8.7 Hz, 1H), 5.42 (d, J = 5.5 Hz, 1H), 5.39 - 5.30 (m, 2H), 5.26 (d, J = 5.5 Hz, 1H),
.09 (t, J = 4.8 Hz, 1H), 4.73 (t, J = 5.5 Hz, 1H), 4.25 - 4.13 (m, 1H), 4.15 - 3.93 (m, 3H), 3.79 -
.63 (m, 2H), 3.60 - 3.53 (m, 1H), 3.21 - 3.27 (m, 1H), 1.71 (s, 3H). Two signals are hidden in a
+
15
signal of water. HRMS (HESI m/z): [M+H] calcd for C H N N O 386.1602; found
1
6
24
4 6
3
86.1603.
1
5
15
[
N ]-o-topolin-7-β-D-glucopyranoside (6c), [6-((2-hydroxybenzyl)amino)-[ N ]purine-7-β-
4
4
D-glucopyranoside. This compound was prepared according to the general procedure using
2
-hydroxybenzylamine (6.2 mg). The reaction time was prolonged to 22 h. Yield 7.5 mg (56
1
%
). H NMR (400 MHz, DMSO-d ) δ 10.07 (br. s, 1H), 8.36 - 8.24 (m, 2H), 7.76 (dd, J = 10 Hz,
6
5
7
.6 Hz, 1H), 7.15 (d, J = 7.6 Hz, 1H), 7.06 (t, J = 7.6 Hz, 1H), 6.80 (d, J = 8.0 Hz, 1H), 6.71 (t, J =
.2 Hz, 1H), 5.55 (d, J = 8.8 Hz, 1H), 5.48 (br. s, 1H), 5.33 (br. s, 1H), 5.23 (br. s, 2H), 4.68 (dd,
J = 15.4 Hz, 5.6 Hz, 1H), 4.43 (dd, J = 15.4 Hz, 5.6 Hz, 1H), 3.66 (br. s, 2H), 3.60 - 3.56 (m, 1H),
+
3
.51 - 3.42 (m, 1H). Two signals are hidden in a signal of water. HRMS (HESI m/z): [M+H]
1
5
calcd for C H N N O 408.1446; found 408.1445.
1
8
22
4 6
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1
5
15
[
N ]-m-topolin-7-β-D-glucopyranoside (6d), [6-((3-hydroxybenzyl)amino)-[ N ]purine-7-
4
4
β-D-glucopyranoside. This compound was prepared according to the general procedure
1
using 3-hydroxybenzylamine (6.2 mg). Yield 7.2 mg (51 %). H NMR (400 MHz, DMSO-d ) δ
6
9
6
.28 (br. s, 1H), 8.33 - 8.21 (m, 2H), 7.81 (dd, J = 9.8 Hz, 5.8 Hz, 1H), 7.05 (t, J = 8 Hz, 1H),
.80 - 6.73 (m, 2H), 6.58 (dd, J = 7.8 Hz, 1.8 Hz, 1H), 5.54 (d, J = 9.2 Hz, 1H), 5.49 (d, J = 5.6
Hz, 1H), 5.34 (d, J = 3.6 Hz, 1H), 5.24 (d, J = 5.6 Hz, 1H), 5.13 (t, J = 4.4 Hz, 1H), 4.71 (dd, J =
1
3
5.2 Hz, 5.6, 1H), 4.40 (dd, J = 15.2 Hz, 5.4, 1H), 3.67 (br. s, 2H), 3.59 - 3.57 (m, 1H), 3.51 -
+
.42 (m, 1H). Two signals are hidden in a signal of water. HRMS (HESI m/z): [M+H] calcd for
1
5
C H N N O 408.1446; found 408.1447.
1
8
22
4 6
1
5
15
[
N ]-p-topolin-7-β-D-glucopyranoside (6e), [6-((4-hydroxybenzyl)amino)-[ N ]purine-7-β-
4
4
D-glucopyranoside. This compound was prepared according to the general procedure using
1
4
-hydroxybenzylamine (6.2 mg). Yield 7.1 mg (50 %). H NMR (400 MHz, DMSO-d ) δ 9.24
6
(br. s, 1H), 8.31 - 8.23 (m, 2H), 7.70 (dd, J = 9.8, 5.4 Hz, 1H), 7.18 (d, J = 8.4 Hz, 2H), 6.66 (d, J
=
5
8.4 Hz, 2H), 5.52 (d, J = 8.8 Hz, 1H), 5.45 (d, J = 5.6 Hz, 1H), 5.33 (br. s, 1H), 5.24 (br. s, 1H),
.12 (t, J = 4.6 Hz, 1H), 4.70 (dd, J = 14.6, 5.8 Hz, 1H), 4.34 (dd, J = 14.8, 6.0 Hz, 1H), 3.67-3.50
+
(
m, 4H). Two signals are hidden in a signal of water. HRMS (HESI m/z): [M+H] calcd for
1
5
C H N N O 408.1446; found 408.1445.
1
8
22
4 6
Preparation of zeatin-9-β-D-ribofuranoside derivatives 8a-b. 7-Methylguanosine (30 mg, 0.1
1
5
5
mmol) was added to a solution of corresponding labelled [ N ]zeatin derivative 7a or 7b
4
(
10.0 mg, 0.045 mmol) in a mixture of aqueous 0.02 M K HPO (1 mL) and pure 96% ethanol
2
4
(1 mL). This solution reached pH = 7.8 and was not further modified. Further, purine
nucleotide phosphorylase (0.2 mL, 24 Units, 120 U/mL) was added and was kept at 35 °C in a
sealed vial with gentle stirring (150 rpm) for 48 h. Based on HPLC purity a 97% conversion
was reached. To remove most of 7-methylguanine, the reaction mixture was filtered
through a syringe micro filter (0.22 m; 2.5 cm ID), washed with a water – methanol mixture
(1 mL, 1:1) and then water (1 mL). The filtrate was concentrated under reduced pressure to
get a solid, which was dissolved in a mixture of methanol (1 mL) and water (0.5 mL). This
clear solution was used for the preparative HPLC purification. The combined fractions
containing pure riboside 8a or 8b were partially concentrated under reduced pressure to
remove most of acetonitrile and lyophilized to get a white solid product.
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1
5
[
N ]-cis-zeatin-9-β-D-ribofuranoside
4
(8a),
[(Z)-6-((4-hydroxy-3-methylbut-2-en-1-
1
5
1
yl)amino)-[ N ]purine-9-β-D-ribofuranoside. Yield 9.6 mg (60 %). H NMR (400 MHz, DMSO-
4
d6) δ 8.34 (dd, J = 11.9, 8.2 Hz, 1H), 8.19 (t, J = 15.6 Hz, 1H), 7.89 (br. s, 1H), 5.87 (d, J = 6.0
Hz, 1H), 5.43 - 5.37 (m, 2H), 5.33 (t, J = 6.4 Hz, 1H), 5.17 (d, J = 4.6 Hz, 1H), 4.69 (t, J = 5.7 Hz,
1
6
H), 4.59 (dd, J = 12.4, 6.2 Hz, 1H), 4.20 - 4.06 (m, 3H), 4.02 (d, J = 6.0 Hz, 2H), 3.95 (dd, J =
.4, 3.6 Hz 1H), 3.68 - 3.63 (m, 1H), 3.56 - 3.50 (m, 1H), 1.69 (s, 3H). HRMS (HESI m/z):
+
15
[
M+H] calcd for C H N N O 356.1496; found 356.1497.
15
22
4 5
1
5
[
N ]-trans-zeatin-9-β-D-ribofuranoside
4
(8b),
[(E)-6-((4-hydroxy-3-methylbut-2-en-1-
1
5
1
yl)amino)-[ N ]purine-9-β-D-ribofuranoside. Yield 10.3 mg (68 %). H NMR (400 MHz,
4
DMSO-d ) δ 8.33 (dd, J = 11.9, 8.2 Hz, 1H), 8.20 (t, J = 15.1 Hz, 1H), 7.97 (br. s, 1H), 5.86 (d, J
6
=
5
3
6.0 Hz, 1H), 5.50 (t, J = 6.0 Hz, 1H), 5.44 - 5.38 (m, 2H), 5.18 (d, J = 4.4 Hz, 1H), 4.72 (t, J =
.5 Hz, 1H), 4.59 (dd, J = 12.4, 6.2 Hz, 1H), 4.15 - 3.96 (m, 3H), 3.95 (dd, J = 6.4, 3.6 Hz 1H),
.77 (d, J = 5.5 Hz, 2H), 3.68 - 3.63 (m, 1H), 3.56 - 3.50 (m, 1H), 1.65 (s, 3H). HRMS (HESI
+
15
m/z): [M+H] calcd for C H N N O 356.1496; found 356.1497.
15
22
4 5
Preparation of topolin-9-β-D-ribofuranoside derivatives 8c-e. These compounds were
prepared in a similar manner as zeatin-ribosides but with several exceptions. Corresponding
1
5
5
labelled [ N ]topolins 7c-e (10.0 mg, 0.041 mmol) were dissolved in a mixture of aqueous
4
0
.02 M K HPO (1 mL for 7d, 2 mL for 7c and 7e) and pure 96% ethanol (1 mL for 7d, 2 mL
2
4
for 7c and 7e) with slight warming (70 °C) and sonication. After cooling to RT 7-
methylguanosine (30 mg, 0.1 mmol) was added. Further, purine nucleotide phosphorylase
(
0.2 mL, 24 Units, 120 U/mL) was added and kept at 35 °C in a sealed vial with gentle stirring
150 rpm) for 7 days. Insufficient conversion of starting topolins led to extra addition of 7-
(
methylguanosine (15 mg, 0.05 mmol) and purine nucleotide phosphorylase (0.05 mL, 6
Units) to the reaction mixture and stirring at 35 °C for additional 7 days. Based on HPLC
purity, a 98% conversion was reached for 7d, 94% for 7e, and 70% for 7c. To remove most of
7
2
-methylguanine the reaction mixture was filtered through a syringe micro filter (0.22 m;
.5 cm ID), washed with water – methanol mixture (1.5 mL, 1:1) and then water (1 mL). The
filtrate was concentrated under reduced pressure to get a solid, which was dissolved in a
mixture of methanol (1.3 mL) and water (0.6 mL). This clear solution was used for the
preparative HPLC purification. The combined fractions containing a corresponding pure
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riboside 8c-e were partially concentrated under reduced pressure to remove most of
acetonitrile and lyophilized to get a white solid product.
1
5
15
[
N ]-o-topolin-9-β-D-ribofuranoside (8c), [6-((2-hydroxybenzyl)amino)-[ N ]purine-9-β-D-
4
4
1
ribofuranoside. Yield 8.2 mg (53 %). H NMR (400 MHz, DMSO-d ) δ 9.87 (br. s, 1H), 8.38 (dd,
6
J = 11.7, 8.0 Hz, 1H), 8.28 (br. s, 1H), 8.20 (t, J = 15.6 Hz, 1H), 7.09 - 7.02 (m, 2H), 6.79 (d, J =
7
3
.8 Hz, 1H), 6.70 (t, J = 7.6 Hz, 1H), 5.88 (d, J = 5.5 Hz, 1H), 5.54 - 5.00 (m, 3H), 4.60 (br. s,
H), 4.13 (t, J = 3.7 Hz, 1H), 3.95 (dd, J = 7.4, 3.4 Hz, 1H), 3.66 (dd, J = 12.4, 3.7 Hz, 1H), 3.54
+
15
(
dd, J = 11.9, 3.2 Hz, 1H). HRMS (HESI m/z): [M+H] calcd for C H N N O 378.1340; found
1
7
20
4 5
3
78.1342.
1
5
15
[
N ]-m-topolin-9-β-D-ribofuranoside (8d), [6-((3-hydroxybenzyl)amino)-[ N ]purine-9-β-
4
4
1
D-ribofuranoside. Yield 13.1 mg (85 %). H NMR (400 MHz, DMSO- d ) δ 9.24 (s, 1H), 8.40
6
(
br. s, 1H), 8.37 (dd, J = 11.9, 8.2 Hz, 1H), 8.19 (t, J = 15.6 Hz, 1H), 7.06 (t, J = 7.8 Hz, 1H), 6.75
6.68 (m, 2H), 6.57 (dd, J = 7.6, 2.1 Hz, 1H), 5.88 (d, J = 7.3 Hz, 1H), 5.44 (d, J = 6.4 Hz, 1H),
-
5
1
.39 (dd, J = 6.9, 4.6 Hz, 1H), 5.19 (d, J = 4.6 Hz, 1H), 4.62 (br. s, 3H), 4.13 (dd, J = 9.6, 4.1 Hz,
H), 3.95 (dd, J = 7.4, 3.4 Hz, 1H), 3.68 - 3.63 (m, 1H), 3.56 - 3.51 (m, 1H). HRMS (HESI m/z):
+
15
[
M+H] calcd for C H N N O 378.1340; found 378.1341.
17
20
4 5
1
5
15
[
N ]-p-topolin-9-β-D-ribofuranoside (8e), [6-((4-hydroxybenzyl)amino)-[ N ]purine-9-β-D-
4
4
1
ribofuranoside. Yield 12.4 mg (81 %). H NMR (400 MHz, DMSO- d ) δ 9.22 (s, 1H), 8.37 -
6
8
.32 (m, 2H), 8.19 (t, J = 15.6 Hz, 1H), 7.13 (d, J = 8.7 Hz, 2H), 6.66 (d, J = 8.7 Hz, 2H), 5.87 (d,
J = 6.4 Hz, 1H), 5.44 (d, J = 6.0 Hz, 1H), 5.40 (dd, J = 7.2, 4.6 Hz, 1H), 5.18 (d, J = 4.8 Hz, 1H),
4
1
3
.62 - 4.50 (m, 3H), 4.13 (dd, J = 9.6, 4.1 Hz, 1H), 3.95 (dd, J = 7.4, 3.4 Hz, 1H), 3.68 - 3.63 (m,
+
15
H), 3.56 - 3.50 (m, 1H). HRMS (HESI m/z): [M+H] calcd for C H N N O 378.1340; found
1
7
20
4 5
78.1342.
Results and discussion
Syntheses of all desired target [ N ]purine labelled cytokinin glycosides 5, 6, and 8 started
15
4
1
5
15
with completely N labelled 6-chloro[ N ]purine (1), which was prepared by improved
4
5
procedure. However, all pilot studies were performed with unlabelled chloropurine 1.
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Since the Vorbrüggen method was used successfully under thermodynamic controlled
9
reaction conditions in the synthesis of similar compounds, we decided to check this
glycosylation method leading to purine glucosyl 2 before the diversity step involving
nuclephilic aromatic substitution with amines. Subsequently, this derivative was directly
deacetylated into purine glycosyl 4, which is more practical in terms of handling for the final
15
step. This sequence was also approved very useful with 6-chloro[ N ]purine (1) on a micro
4
scale to give 4 in 75 % total yield from 1 over two steps.
Synthesis of 7-regioisomer 3 was challenging. To date, three methods have been described.
The most promising method, where peracetylated 6-chloropurine-7-β-D-glucopyranoside
1
0
was isolated in 60 % yield, was reported in a literature. We repeated this procedure with
unlabelled 1 and found that authors prepared 9-isomer 2 instead of 7-isomer 3. Moreover,
their NMR data correspond to the 9-isomer and are in accordance with the spectra of the 9-
1
1,12
isomer, which was prepared in a different way.
These two glucosylation methods of 6-
chloropurine describe also an equilibrium reaction, where the 7-isomer was obtained as a
minor product together with the more thermodynamically stable 9-isomer, which was
predominant. Alkylation of 6-chloropurine with acetobromo--D-glucose in the presence of
1
1
potassium carbonate seemed to be feasible way to prepare both precursors in one pot.
Unfortunately, the yields of both peracetylated isomers are relatively low (14 % for 7- and
2 % for 9-isomer). This method was modified. Propylene carbonate was replaced with
more practical acetonitrile and a twofold amount of potassium carbonate and acetobromo-
-D-glucose was used to increase the conversion of 1. Application of these modified
4

15
reaction conditions to 6-chloro[ N ]purine (1) allowed to obtain 13 % of 7-isomer 3, which
4
was directly used in the next step to give purine glucosides 6. 9-Isomer 2 was isolated from
the same reaction mixture in 54 % yield. This isomer is possible to use directly in the final
step as well, but for practical reasons was deacetylated to compound 4 (Scheme 1).
Please insert Scheme 1.
The substitution of 4 with amines in the presence of triethylamine gave the target purine
glucosides 5. Similarly, regioisomers 6 were provided after additional one-pot deacetylation
in ethanolic ammonia. Both regioisomers, 5 and 6, were obtained in the good yields after
chromatographic separation.
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Unlabelled cytokinin 9-β-D-glucopyranosides 5 are known, but their 7-β-D-glucopyranosides
represent new compounds, except for trans-zeatin-7-β-D-glucopyranoside 6b, which was
11,13
prepared by a different complicated method.
6
There are several methods to prepare N -substituted adenine 9-β-D-ribofuranoside
derivatives 8 from 6-chloropurine via 6-chloporurine-9-β-D-ribofuranoside intermediate as
1
4,15
their useful direct precursor. This intermediate can be obtained by Vorbrüggen method
in the yields between 36-80 %, or by enzymatic transglycosylation reaction using purine
16–18
nucleotide phosphorylase (PNP) and 7-methylguanosine.
We decided to attempt the
enzymatic method as the most simple and efficient glycosylation at first glance. Preliminary
experiments revealed that these enzymatic methods are suitable for the preparation of 6-
chloropurine-9-β-D-ribofuranoside on a gram scale, but, on a microscale, we observed a
partial hydrolysis resulting in inosine, which contaminated the product. Therefore, we
decided to prove direct enzymatic transglycosylation reaction of 7a-e to give desired
5
ribosides 8a-e. Non-ribosylated labelled derivatives 7a-e were already reported, and only
1
5
4
labelled [ N ]-trans-zeatin-9-β-D-ribofuranoside 8b was provided according to the method
4
1
9
described by Sivadjian, where ribose-1-phosphate was used. To transform cytokinins 7 to
their β-D-ribofuranosides 8, we adapted and modified enzymatic ribosylating method
1
6–18
employing 7-methylguanosine as a ribose-1-phosphate donor.
Since the solubility of
topolins 7c-e in aqueous medium/buffer is low we used a mixture water and ethanol to
increase solubility and improve enzymatic ribosylation equilibrium. This modification
allowed to reach approximately 95 % conversion of all cytokinins 7 (based on HPLC purity)
except for ortho-topolin 7c, where lower 70 % conversion was observed. The conversion of
cis/trans-zeatins 7a and 7b was easy, in contrast to topolins 7c-e, where the excess of
reagents and prolonged reaction time was necessary. This enzymatic method was
developed for unlabelled cytokinins 7* first. Prepared unlabelled cytokinin 9-β-D-ribosides
8
* had identical spectral and physical properties by our method as ribosides obtained from
20
commercial 6-chloropurine-9-β-D-ribofuranoside 9* and the corresponding amine
(Scheme 2).
Please insert Scheme 2.
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Conclusions
1
5
6
Several N labelled cytokinin glycosides derived from N -substituted purine were prepared
to serve as analytical standards. These glycosides with a 9-β-D, 7-β-D-glucopyranosyl, or 9-
1
5
β-D-ribofuranosyl group were prepared from labelled 6-chloro[ N ]purine. Improved
4
1
5
alkylation method of chloro[ N ]purine with acetobromo--D-glucose provided in one-pot
4
two regioisomeric chloro glucosyl precursors 3 and 4. Subsequent nucleophilic substitution
with the corresponding amines afforded 9-β-D 5 and 7-β-D-glucopyranosyl cytokinins 6.
Cytokinins 8, containing 9-β-D-ribofuranosyl group, were prepared by modified direct
enzymatic transglycosylation reaction from cytokinins 7, which were synthesized from 6-
15
chloro[ N ]purine (1). This method also enabled to ribosylate low soluble purine
4
derivatives.
Acknowledgments:
This research was funded by TAČR grant number TE02000058, Palacký University grant
numbers IGA_PrF_2018_029, and IGA_LF_2018_032, and OP PIK grant number
CZ.01.1.02/0.0/0.0/15_019/0004431.
Orcid:
Lenka Tranová: 0000-0002-6266-7340
Jan Buček: 0000-0002-7701-0720
Marek Zatloukal: 0000-0001-9962-2798
Petr Cankař: 0000-0002-7434-6069
Jakub Stýskala: 0000-0003-1726-8604
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Figure 1. Structures of zeatin and topolin cytokinins
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15
Scheme 1. Preparation of [ N ]purine labelled cytokinin glycosides 5, 6 and 8. Reagents and
4
conditions: (i) BSA/DCE, reflux, 30 min then per-O-acetylated -D-glucose, TMSOTf/DCE,
reflux, 2 h; (ii) acetobromo--D-glucose, K CO , ACN, RT, 25 h; (iii) NH -EtOH, 0 °C, 3.5 h; (iv)
2
3
3
R-NH , TEA, PrOH (EtOH), 92 °C, 4-24 h; (v) R-NH , TEA, ACN, (EtOH), 92 °C, 7-24 h then NH -
2
2
3
EtOH, 0 °C, 7 h; (vi) purine nucleotide phosphorylase, 7-methylguanosine, K HPO , water-
2
4
ethanol, 35 °C, 2-14 days.
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Scheme 2. Preparation of unlabelled ribosides 8* by two different methods to confirm their
structural identity. Reagents and conditions: (i) purine nucleotide phosphorylase, 7-
methylguanosine, K HPO , water-ethanol, 35 °C, 2-14 days; (ii) R-NH , TEA, PrOH (EtOH), 82-
2
4
2
9
2 °C, 4-24 h.
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