Facile synthesis of 8-azido-6-benzylaminopurine

Article abstract of DOI:10.1080/15257770.2011.602655

Bromination of 6-benzylaminopurine (1) with Br2 in AcOH in the presence of AcONa afforded 6-benzylamino-8-bromopurine (2) in 59% yield. The position of bromination was confirmed by direct transformation of bromide 2 by reaction with NaN3 in dimethyl sulfoxide to 8-azido-6-benzylaminopurine (3) in a yield of 70% and comparison of its properties with the known compound 2-azido-6- benzylaminopurine (11). Compounds 3 and 11 were checked for their biological activity in specific biotests based on the primary cytokinin effects in living plants. Both synthesized compounds displayed effects similar to the typical cytokinin 6-benzylaminopurine (1). Copyright Taylor and Francis Group, LLC.

Full text of DOI:10.1080/15257770.2011.602655

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Facile Synthesis of 8-Azido-6-
Benzylaminopurine
Mr Mikhail Yu. Steklov a b , Dr Vitali I. Tararov a , Professor Georgy A.
Romanov b & Sergey N. Mikhailov a
a Engelhardt Institute of Molecular Biology, Russian Academy of
Sciences, Moscow, Russia
b Institute of Plant Physiology, Russian Academy of Sciences,
Moscow, Russia
Published online: 02 Sep 2011.
To cite this article: Mr Mikhail Yu. Steklov , Dr Vitali I. Tararov , Professor Georgy A. Romanov &
Sergey N. Mikhailov (2011): Facile Synthesis of 8-Azido-6-Benzylaminopurine, Nucleosides, Nucleotides
and Nucleic Acids, 30:7-8, 503-511
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Nucleosides, Nucleotides and Nucleic Acids, 30:503–511, 2011
Copyright ꢀC Taylor and Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770.2011.602655
FACILE SYNTHESIS OF 8-AZIDO-6-BENZYLAMINOPURINE
Mikhail Yu. Steklov,1,2 Vitali I. Tararov,1 Georgy A. Romanov,2
and Sergey N. Mikhailov1
1Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia
2Institute of Plant Physiology, Russian Academy of Sciences, Moscow, Russia
Bromination of 6-benzylaminopurine (1) with Br2 in AcOH in the presence of AcONa af-
2
forded 6-benzylamino-8-bromopurine (2) in 59% yield. The position of bromination was confirmed
by direct transformation of bromide 2 by reaction with NaN3 in dimethyl sulfoxide to 8-azido-6-
benzylaminopurine (3) in a yield of 70% and comparison of its properties with the known com-
pound 2-azido-6-benzylaminopurine (11). Compounds 3 and 11 were checked for their biological
activity in specific biotests based on the primary cytokinin effects in living plants. Both synthesized
compounds displayed effects similar to the typical cytokinin 6-benzylaminopurine (1).
Keywords Cytokinins; photoaffinity labeling; 8-azido-6-benzylaminopurine
INTRODUCTION
Cytokinins constitute a group of plant hormones and related synthetic
bioregulators that exert multiform effects on plant growth and develop-
ment. Endogenous cytokinins stimulate cell division, photomorphogenesis,
chloroplast development, and pigment biosynthesis, regulate shoot and root
growth and overall plant architecture, and counteract leaf aging and apical
dominance.[1,2]
Cytokinins derivatives with the azido group have been used in many
photoaffinity-labeling experiments.[3–5] Photolysis of the azido group gener-
ates a highly reactive nitrene species that can covalently bind to proteins by
insertion in the nearest O-H, N-H, and S-H bonds. This property is used to
scrutinize the structure of the cytokinin-binding protein active sites.[6] The
modern trend is the application of azido-modified natural compounds for
“click-chemistry” widely used in the modification of nucleic acids, material
Received 29 April 2011; accepted 30 June 2011.
This study was supported by the Program of Presidium RAS “Molecular and Cell Biology” and
grants of the Russian Foundation for Basic Research and the Russian Federal Program (Contract No.
16.512.11.2239).
Address correspondence to Sergey N. Mikhailov, Engelhardt Institute of Molecular Biology, Vavilov
st 32, Moscow, 119991 Russia. E-mail: smikh@eimb.ru
503

504
M. Y. Steklov et al.
science, and drug discovery. Several sensitive methods for detection and
location of biomolecules in living cells have been developed utilizing this
approach.[7–11] Azido-modified cytokinins will be of interest for detection of
cytokinin distribution in plants. Currently, a facile two-step preparation of
8-azido-6-benzyaminopurine (3) from available 6-benzylaminopurine (1) is
disclosed.
RESULTS AND DISCUSSION
The simple methods for preparation of azido-modified cytokinins are
of great value due to the importance of these substances for biological as-
says. We were rather interested in obtaining of 8-azido-6-benzylaminopurine
(3). To our surprise, we could not find in the literature adequate meth-
ods of its synthesis. The first reported strategy starts from hardly available
6,8-dichloropurine.[6] The alternative route from adenosine consists of five
successive steps: bromination of adenosine in position 8, substitution of
bromine with NaN3, 1-N -benzylation with BnBr, subsequent Dimroth re-
arrangement to 8-azido-6-benzyladenosine, and final cleavage of glycoside
bond.[12] The isolated total yield of 3 was 6%. At the same time, the authors
claimed that bromination of 1 with Br2 gave likely 2-bromoderivative, and it
was not possible to substitute bromine atom on azido group.[12]
Bromination of 1 in position 2 seemed to be disputable. Indeed, adeno-
sine was successfully brominated with Br2 in position 8,[13] and the re-
action of adenine with Br2 also gave 8-bromoderivative.[14] It is rather
doubtful to expect that N 6-benzyl substituent in adenine moiety should
dramatically change the site of Br2 attack. As soon as the synthesis of 8-
azido-6-benzylaminopurine (3) via 8-bromo compound 2 is straightforward
(Scheme 1), we focused on bromination of 6-benzylaminopurine (1). A
virtue of this approach is that the photo-sensitive azido group is introduced
in the molecule in the final step.
Bn
N
Bn
N
Bn
N
NH
N
NH
NH
N
N
N
b
a
N3
Br
N
H
N
H
N
H
N
N
3
2
1
SCHEME 1 Two-step synthesis of 8-azido-6-benzylaminopurine (3). Reagents and conditions: a) Br2,
AcOH, AcONa, room temperature, 24 hours, 59%; b) NaN3, dimethyl sulfoxide (DMSO), 75◦C, 36
hours, 70%.
Attempts to utilize the reported methods of bromination of adenine and
adenosine in water solution at ambient temperature failed when applied to
6-benzylaminopurine (1). The possible reason for it was poor solubility of the
starting compound and the product in the reaction media. We have found

Facile Synthesis of 8-Azido-6-Benzylaminopurine
505
out that 6-benzylaminopurine is sufficiently soluble in AcOH. Nevertheless,
the reaction of 6-benzylaminopurine with 1 equivalent of Br2 in AcOH gave
only low yield (20%) of the product 3. Addition of 1 equivalent of AcONa
did not improve the situation. When fourfold excess of Br2 and AcONa was
used, the yield of the product increased significantly to 59%. The coarse of
the bromination can be monitored by thin layer chromatography (TLC) on
silica gel (AcOH-CH2Cl2 1:25). Even by applying the excess of reagents, the
starting compound remained in the reaction mixture.
The reaction of bromide 2 with threefold excess of NaN3 in DMSO at
75◦C was rather slow. A consecutive 36 hours of heating were required to
complete the reaction. The reaction was monitored by 1H NMR spectroscopy
as soon as we were unable to find an appropriate developing system for their
separation on TLC. Aliquots were taken from the reaction mixture after each
8 hours of heating. The samples were diluted with DMSO-d6 before running
the NMR spectrum. The 2-H resonance peak of the bromide 2 at 8.17 ppm
slowly decreased with time, and simultaneously the 2-H peak of the azide 3
at 8.13 ppm increased. Pure azide 3 was isolated in a yield of 70%. Its purity
and identity were confirmed by liquid chromatography/mass spectroscopy
(LC-MS). Chromatograms showed only one peak with z/e 267.06 [M+H+]
(or z/e 311.02 [M-H+ + HCOOH]), which corresponds to azide 3.
To prove the structure of the azide 3, we synthesized 2-azido-6-
benzylaminopurine (11) according to published methods[15–17] starting from
guanine (Scheme 2). This sequence was selected due to availability of gua-
nine, and intermediate compounds could be easily isolated and did not
demand extra purification. Moreover, 2,6-dichloropurine (8) is a key com-
pound in the synthesis of biologically active nucleosides, reversine, and re-
lated compounds.[18–20] We have observed that published procedures are
highly reproducible and may be easily scaled up.
Cl
Cl
Cl
a
N
guanine
N
O
N
N
N
b
N
c
d
4
N
H
N
H
H
N
H
N
N
H
N
N
N
H2N
N
6
5
7
Bn
Bn
N
Bn
N
NH
N
Cl
NH
NH
N
N
N
N
N
N
N
g
e
f
NH2
N
H
N
H
N
H
N
H
N
H
Cl
N
Cl
N
N3
11
8
10
9
SCHEME 2 Synthesis of 2-azido-6-benzylaminopurine (11). Reagents and conditions: a) POCl3,
dimethylformamide (DMF), 80◦C, 8 hours, 72%; b) 12% aqueous AcOH, 70◦C, 4.5 hours, 75%; c)
10% aqueous NaOH, room temperature, 3 hours, 89%; d) NaNO2, conc. HCl, ZnCl2, 5◦C, 1 hour, 54%;
e) benzylamine, isopropanol, reflux, 3.5 hours, 77%; f) 85% hydrazine hydrate in water, reflux, 1.5 hours,
84%; g) NaNO2, HCl, AcOH, room temperature, 96 hours, 82%.

506
M. Y. Steklov et al.
FIGURE 1 The 400 MHz 1H NMR spectra in DMSO-d6 at 32◦C: (top) 8-azido-6-benzyaminopurine (3)
and (bottom) 2-azido-6-benzylaminopurine (11).
The detailed preparation of azide 11 is given in the Experimental part.
1H NMR spectra of azides 3 and 11 are presented in Figure 1. The striking
difference between both compounds is evident.
In DMSO-d6 in the case of the azide 11, formation of two additional cyclic
isomers 11a and 11b is observed (Scheme 3). The ratio 11:11a:11b is equal

Facile Synthesis of 8-Azido-6-Benzylaminopurine
507
Bn
N
Bn
N
Bn
N
NH
NH
NH
N
N
N
N
N
N
N
H
N
H
N
H
N
N
11b
N3
N
N
N
N
11a
11
SCHEME 3 Azido-tetrazolo tautomerism of 2-azido-6-benzylaminopurine (11).
to 0.56:0.41:0.03 (Figure 1). The rate of interconversion of these species is
slow enough to be detected by 1H NMR. Similar tautomeric equilibrium for
2-azidoadenine is well documented.[21] In contrast, the azide 3 at the same
conditions displays simple spectrum in which no other isomer could be
detected, which are in accordance with the literature data.[12] Furthermore,
LC-MS analysis of the azide 11 gives three chromatographic peaks with the
same m/e 267.06 [M+H+], while only one peak with this m/e value was
detected in the case of compound 3.
In conclusion, we have developed a simple and facile two-step method
for the preparation of 8-azido-6-benzylaminopurine (3) starting from avail-
able 6-benzylaminopurine (1) with the crucial step being bromination of 1
with Br2. Compounds 3 and 11 were checked for their biological activity in
specific biotests based on the primary cytokinin effects in living plants. Both
synthesized compounds displayed effects similar to the typical cytokinin 6-
benzylaminopurine (1). These findings are of importance for further use
of azido derivatives of benzylaminopurine for affinity labeling and “click”
reactions.
EXPERIMENTAL
The solvents and materials of reagent grade were used without additional
purification. Melting points were determined by an electrothermal appara-
tus and were uncorrected. TLC was performed on Alugram SIL G/UV254
(Macherey-Nagel) with ultraviolet (UV) visualization. 1H NMR spectra were
recorded on a Bruker AMX 400 NMR instrument at 32◦C. Chemical shifts
in ppm were measured relative to the residual solvent signal as an internal
standard (DMSO-d6, δ = 2.50 ppm). The position of NH resonances was
additionally confirmed by their exchange with the addition of D2O. The UV
spectra were recorded by a Cary300UV/VIS spectrophotometer (Varian).
LC-MS analysis was performed on Surveyor MSQ instrument (Thermo Finni-
gan, USA), operating in atmospheric pressure chemical ionization (APCI)
mode with the detection of positive and negative ions, and equipped with
Onyx Monollithic C18 25 × 4.6 mm Part No CHO-7645 column. The eluent
was a gradient of 0.1% HCOOH aqueous solution in MeCN. Chromato-
graphic peaks were detected simultaneously with an evaporative light scat-
tering detector (ELSD), photodiode array detector (PAD), and total ion

508
M. Y. Steklov et al.
current (TIC) detector. In all cases (except the azide 11), only one peak
was revealed and the chromatographic purity of compounds was more than
99%.
Biological Materials and Methods
Cytokinin activity assays were accomplished by using model plant systems
with Amaranthus (Amaranthus caudatus L.)[22] and transgenic PARR5: GUS
Arabidopsis (Arabidopsis thaliana L.) seedlings.[23] Both assay systems are
specifically sensitive to cytokinins and respond to hormone application in
a few hours, making possible quantitative determinations. For a positive
control, 6-benzyladenine (BA) was applied.
6-Benzylamino-8-bromopurine (2)
To a stirred solution of the compound 1 (200 mg, 0.888 mmol) and
AcONa × 3H2O (534 mg, 3.93 mmol) in AcOH, (5 mL) bromine (0.184 mL,
3.59 mmol) was added in one portion at room temperature. The precip-
itate began to form after 2 hours. The reaction was controlled by TLC
(AcOH/CH2Cl2 1:25). After stirring for 24 hours, the precipitate was fil-
tered and washed thoroughly with acetone until it became colorless, yield-
ing after drying over P2O5 160 mg (59%) of 2 as a colorless solid. Rf = 0.53
(AcOH-CH2Cl2, 1:25). Mp 210◦C–212◦C. 1H NMR (DMSO-d6): 13.65 (br s,
1H, N-H), 8.47 (br s, 1H, N-H), 8.17 (s, 1H, 2-H), 7.18–7.36 (m, 5H, H-Ph),
4.69 (br s, 2H, CH2N). UV (H2O), λmax in nm (ε): pH 2, 211 (14500), 279
(12700); pH 7, 215 (16900), 275 (11600); pH 12, 277 (14200). Anal. Calcd.
for C12H10BrN5: C, 47.39; H, 3.31; N, 23.03. Found: C, 47.25; H, 2.14; N,
22.83.
8-Azido-6-benzyaminopurine (3)
A mixture of NaN3 (543 mg, 8.34 mmol) and bromide 2 (790 mg, 2.61
mmol) in DMSO (11 mL) was heated with stirring at 75◦C for 36 hours.
The reaction was monitored by 1H NMR (a volume of the reaction mixture
containing 1–2 mg of substance was diluted with DMSO-d6). After cooling
at room temperature, the mixture was poured into 200 mL of cold water.
The precipitate was filtered and dried over P2O5 to give 486 mg (70%) of
3 as a pale yellow solid. Rf = 0.53 (AcOH-CH2Cl2, 1:25). Mp 170◦C–172◦C.
Lit.: Mp 175◦C.[12] 1H NMR (DMSO-d6): 13.16 (br s, 1H, N-H), 8.13 (br
s, 2H, 2-H and N-H), 7.07–7.48 (m, 5H, H-Ph), 4.71(br s, 2H, CH2N). MS
(APCI): m/z [M+H+] calcd. for C12H11N8: 267.26, found 267.06; m/z [M-H−
+ HCOOH] calcd. for C13H11N8O2: 311.28, found 311.02. UV (H2O), λmax
in nm (ε): pH 2, 296 (2400); pH 7, 218 (19700), 290 (1800); pH 12, 292
(20400).

Facile Synthesis of 8-Azido-6-Benzylaminopurine
6-Chloro-2-formylaminopurine acetate (6)
509
To a solution of 1,2-dichloroethane (50 mL), DMF (14 mL, 0.18 mol)
and POCl3 (8.4 mL, 0.09 mol) guanine (4.53 g, 30 mmol) was added and the
mixture was stirred at 80◦C for 8 hours. After cooling at room temperature,
the reaction mixture was poured into 120 mL of water. The mixture was
neutralized to pH 5 by adding sodium carbonate (17.5 g, 0.165 mol). The
mixture was stirred for 30 minutes and then was allowed to stand until the
partition of layers ceased. The aqueous layer was separated and solid NaOH
(2.52 g, 0.063 mol) was slowly added. The precipitated product was filtered,
washed with water, and dried over P2O5 to give 4.88 g (72%) of 5 as a
yellow solid. The compound was used in the next step without additional
purification. Rf = 0.70 (EtOH-CH2Cl2, 1:9). 1H NMR (DMSO-d6): 13.15 (br
s, 1H, N-H), 8.57 (s, 1H, 8-H), 8.29 (s, 1H, N=CH-N), 3.14 (s, 3H, Me), 3.03
(s, 3H, Me).
The compound 5 (4.88 g, 21.7 mmol) was mixed with 12% (v/v) AcOH
aqueous solution (52 mL) and stirred at 70◦C for 4.5 hours. After cooling at
20◦C, the precipitate was filtered, washed with water, and dried over P2O5 to
give 3.2 g (75%) of 6 as a yellow solid. Rf = 0.75 (EtOH-CH2Cl2, 1:9). Mp >
260◦C. 1H NMR (DMSO-d6): 12.80 (br s, 2H, N-H + AcOH), 11.07 (d, 3J =
9.6 Hz, 1H, HC=O), 9.32 (d, 3J = 9.6 Hz, 1H, N-H), 8.48 (s, 1H, 8-H), 1.91
(s, 3H, Ac). UV (H2O), λmax in nm (ε): pH 2, 225 (20200), 295 (5000); pH
7, 226 (21300), 294 (5400); pH 12, 239 (15500), 273 (3400), 305 (6000).
2-Amino-6-chloropurine (7)
The compound 6 (3.0 g, 15.2 mmol) was dissolved in 10% (w/w) NaOH
aqueous solution (20 mL) and stirred at room temperature for 3 hours.
Then, the reaction solution was neutralized with conc. hydrochloric acid.
The resulted precipitate was filtered, washed with water, and dried over P2O5
to give 2.3 g (89%) of 7 as a pale yellow solid. Rf = 0.60 (EtOH-CH2Cl2, 1:9).
Mp > 260◦C.[16] 1H NMR (DMSO-d6): 12.81 (br s, 1H, N-H), 8.10 (s, 1H,
8-H), 6.75 (br s, 2H, NH2). UV (H2O), λmax in nm (ε): pH 2, 214 (28600),
238 (6600), 314 (7000); pH 7, 216 (29400), 240 (6200), 307 (7000); pH 12,
271 (4100), 308 (6500).
2,6-Dichloropurine (8)
A solution of ZnCl2 (7.0 g, 110 mmol) in conc. hydrochloric acid (9.2
mL) was cooled down at 10◦C, and finely powdered compound 7 (2.3 g, 13.6
mmol) was added with stirring. The resulting stirred mixture was cooled
further at −5◦C and NaNO2 (1.27 g, 18.4 mmol) was added over a period
of 30 minutes keeping the temperature below 5◦C. The mixture was stirred
for additional 30 minutes and was diluted with 12 mL of water. The product
was extracted with ethyl acetate (4 × 12 mL). The organic phase was washed
with water (2 × 7 mL.), dried over Na2SO4, and evaporated to dryness. The
residue was recrystallized from MeOH to yield after drying over P2O5 1.4 g

510
M. Y. Steklov et al.
(54%) of (8) as slightly yellow crystals. Rf = 0.53 (EtOH-CH2Cl2, 1:25). Mp
178◦C–180◦C. Lit.: Mp 184◦C–186◦C.[17] 1H NMR (DMSO-d6): 14.10 (br s,
1H, N-H), 8.72 (s, 1H, 8-H). UV (H2O), λmax in nm (ε): pH 2, 210 (23900),
273 (9500); pH 7, 211 (23500), 275 (9300); pH 12, 276 (7800).
6-Benzylamino-2-chloropurine (9)
A mixture of compound 8 (0.6 g, 3.17 mmol), benzylamine (1.22 mL,
11.2 mmol), and 2-propanol (9 mL) was heated at 82◦C for 3.5 hours. After
cooling, the volatiles were removed under reduced pressure. Water (27 mL)
was added to the residue, and the mixture was kept at 0◦C for several hours.
The precipitated product was collected by filtration, washed well with water,
and dried over P2O5, yielding 632 mg (77%) of 9 as a colorless solid. Rf =
0.65 (EtOH-CH2Cl2, 1:25). Mp 239◦C–241◦C. Lit.: Mp 245◦C–246◦C.[15] 1
H
NMR (DMSO-d6): 12.93 (br s, 1H, N-H), 8.59 (br s, 1H, N-H), 8.13 (s, 1H,
8-H), 7.42–7.18 (m, 5H, Ph), 4.65 (br s, 2H, CH2N). UV (H2O), λmax in nm
(ε): pH 2, 210 (23500), 274 (16400); pH 7, 210 (24200), 272 (17300); pH
12, 277 (15400).
6-Benzylamino-2-hydrazinopurine (10)
A mixture of compound 9 (600 mg, 2.31 mmol) in 85% solution of hy-
drazine hydrate in water (9 mL) was stirred and refluxed for 1.5 hours. After
cooling to room temperature, a white crystalline precipitate was collected by
filtration, washed well with water, and recrystallized from absolute ethanol
to give after drying over P2O5 492 mg (84%) of 10 as colorless crystals. Rf
= 0.33 (EtOH-CH2Cl2, 1:25). Mp 248◦C–250◦C. Lit.: Mp 252◦C.[15] 1H NMR
(DMSO-d6): 12.26 (br s, 1H, N-H), 7.75 (br s, 1H, N-H), 7.69 (s, 1H, 8-H),
7.09–7.45 (m, 5H, Ph), 4.66 (br s, 2H, CH2N), 3.98 (br s, 1H, N-H). UV
(H2O), λmax in nm (ε): pH 2, 207, (17400), 245 (9800), 277 (10800); pH 7,
206 (17500), 246 (11200), 275 (11400); pH 12, 284 (11300).
2-Azido-6-benzylaminopurine (11)
To a mixture of compound 10 (480 mg, 1.88 mmol) in 10% (v/v) aque-
ous AcOH solution (12 mL) containing sodium nitrite (133 mg, 1.9 mmol)
was added 2 mL of 1 N HCl and the resulting mixture was stirred and
protected from light, for 96 hours at room temperature. The product was
collected by filtration, washed well with water, and dried over P2O5 yielding
410 mg (82%) of 11 as a cream-colored solid. Rf = 0.78 (EtOH-CH2Cl2,
1:10). Mp 218◦C–220◦C. Lit.: Mp 226◦C.[15] 1H NMR (DMSO-d6): 12.94 (br
s, 1H, N-H), 8.52 (br s, 0.97H, N-H), 8.40 (s, 0.56H, 8-H), 8.31 (s, 0,03H,
N-H), 8.12 (br s, 0.03H, 8-H), 8.04 (br s, 0.41H, 8-H), 7.12–7.61 (m, 5H, Ph),
5.41 (d, 3J = 6.4Hz, 0.06H, CH2N), 4.82 (d, 3J = 5.4 Hz, 1.12H, CH2N), 4.63
(br s, 0.82H, CH2N). UV (H2O), λmax in nm (ε): pH 2, 237 (19200), 277
(15300); pH 7, 236 (21800), 273 (15600); pH 12, 236 (26100), 278 (13400).

Facile Synthesis of 8-Azido-6-Benzylaminopurine
REFERENCES
511
1. Sakakibara, H. Plant Hormones. Biosynthesis, Signal Transduction, Action, ed. P.J. Davies, Springer, Dor-
drecht, Netherlands, 2010, pp. 95–114.
2. Romanov, G.A. How do cytokinins affect the cell? Russ. J. Plant Physiol. 2009, 56, 268–290.
3. Brinegar, A.C.; Cooper, G.; Stevens, A.; Hauer, C.R.; Shabanowitz, J.; Hunt, D.F.; Fox, J.E. Charac-
terization of a benzyladenine binding-site peptide isolated from a wheat cytokinin-binding protein:
sequence analysis and identification of a single affinity-labeled histidine residue by mass spectrome-
try. Proc. Natl. Acad. Sci. USA 1988, 85, 5927–5931.
4. Knorre, D.G.; Godovikova, T.S. Photoaffinity labeling as an approach to study supramolecular nu-
cleoprotein complexes. FEBS Lett. 1998, 433, 9–14.
5. Robinette, D.; Neamati, N.; Tomer, K.B.; Borchers, C.H. Photoaffinity labeling combined with mass
spectrometric approaches as a tool for structural proteomics. Expert Rev. Proteomics 2006, 3, 399–408.
6. Mornet, R.; Theiler, J.B.; Leonard, N.J.; Schmitz, R.Y.; Moore, III, F.H.; Skoog, F. Active cytokinins.
Photoaffinity labeling agents to detect binding. Plant Physiol. 1979, 64, 600–610.
7. Moses, J.E.; Moorhouse, A.D. The growing applications of click chemistry. Chem. Soc. Rev. 2007, 36,
1249–1262.
8. Sletten, E.M.; Bertozzi, C.R. Bioorthogonal chemistry: fishing for selectivity in a sea of functionality.
Angew. Chem. Int. Ed. 2009, 48, 6974–6998.
9. Gramlich, P.M.E.; Wirges, C.T.; Manetto, A.; Carell, T. Postsynthetic DNA modification through the
copper-catalyzed azide–alkyne cycloaddition reaction. Angew. Chem. Int. Ed. 2008, 47, 8350–8358.
10. Jewett, J.C.; Carolyn, R.; Bertozzi, C.R. Cu-free click cycloaddition reactions in chemical biology.
Chem. Soc. Rev. 2010, 39, 1272–1279.
11. Cosyn, L.; Palaniappan, K.K.; Kim, S.-K.; Duong, H.T.; Gao, Z.G.; Jacobson, K.A.; Van Calenbergh,
S. 2-triazole-substituted adenosines: a new class of selective A3 adenosine receptor agonists, partial
agonists, and antagonists, J. Med. Chem. 2006, 49, 7373–7383.
12. Sussman, M.R.; Kende, H. The synthesis and biological properties of 8-azido-N 6-benzyladenine, a
potential photoaffinity reagent for cytokinin. Planta 1977, 137, 91–96.
13. Ikehara, M.; Uesugi, S.; Kaneko, M. Nucleic Acid Chemistry, eds. L.B. Townsend, R.S. Tipson, Wiley,
New York, 1970, part 2, pp. 837–841.
14. Laxer, A.; Major, D.T.; Gottlieb, H.E.; Fischer, B. (15N5)-Labeled adenine derivatives: synthesis and
studies of tautomerism by 15N NMR spectroscopy and theoretical calculations. J. Org. Chem. 2001, 66,
5463–5481.
15. Theiller, R.B.; Leonard, N.J. Photoaffinity-labeled cytokinins. Plant Physiol. 1976, 58, 803–805.
16. Yan, Y. 2-amino-6-chloropurine and method for preparing the same. US Patent 6,187,921 B1, 2001.
17. Sakai, M.; Fujiwara, M.; Nakamatsu, T. Process for preparing 2,6-dichloropurine. US Patent Applica-
tion 2006/0235224 A1, 2006.
18. Zeng, Q.; Huang, B.; Danielsen, K.; Shukla, R.; Nagy, T. Facile and practical synthesis of 2,6-
dichloropurine. Org. Proc. Res. Develop. 2004, 8, 962–963.
19. Ciszewski, L.; Waykole, L.; Prashad, M.; Repic, O.
A practical synthesis of 2-arylamino-6-
alkylaminopurines from 2,6-Dichloropurine. Org. Proc. Res. Develop. 2006, 10, 799–802.
20. Koley, M.; Ko¨nig, X.; Hilber, K.; Schnu¨rch, M.; Stanetty, P.; Mihovilovic, M.D. Synthesis and screen-
ing of 2,6-diamino-substituted purine derivatives as potential cardiomyogenesis inducing agents,
ARKIVOC 2011, 45–61.
21. Temple, K.; Kussner, C.L.; Montgomery, J.A. Studies on the azidoazomethine-tetrazole equilibrium.
V. 2- and 6-azidopurines. J. Org. Chem. 1966, 31, 2210–2215.
22. Romanov, G.A.; Getman, I.A.; Schmu¨lling, T. Investigation of early cytokinin effects in a rapid
Amaranthus seedling test. Plant Growth Regul. 2000, 34, 337–344.
23. Romanov, G.A.; Kieber, J.J.; Schmu¨lling, T. A rapid cytokinin response assay in Arabidopsis indicates
a role for phospholipase D in cytokinin signaling. FEBS Lett. 2002, 515, 39–43.