Practical synthesis of natural plant-growth regulator 2-azahypoxanthine, its derivatives, and biotin-labeled probes

Article abstract of DOI:10.1039/c4ob00705k

We describe a practical, large-scale synthesis of the "fairy- ring" plant-growth regulator 2-azahypoxanthine (AHX), and its biologically active hydroxyl metabolite (AOH) and riboside derivative (AHXr). AHXr, a biosynthetic intermediate, was synthesized from inosine via a biomimetic route. Biotinylated derivatives of AHX and AHXr were also synthesized as probes for mechanistic studies. This journal is the Partner Organisations 2014.

Full text of DOI:10.1039/c4ob00705k

Organic &
Biomolecular Chemistry
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COMMUNICATION
Practical synthesis of natural plant-growth
regulator 2-azahypoxanthine, its derivatives, and
biotin-labeled probes†
Kazutada Ikeuchi,a Ryosuke Fujii,a Shimpei Sugiyama,a Tomohiro Asakawa,a
Makoto Inai,a Yoshitaka Hamashima,a Jae-Hoon Choi,b Tomohiro Suzuki,b
Hirokazu Kawagishi*b,c,d and Toshiyuki Kan*a
Cite this: Org. Biomol. Chem., 2014,
12, 3813
Received 3rd April 2014,
Accepted 14th April 2014
DOI: 10.1039/c4ob00705k
www.rsc.org/obc
We describe a practical, large-scale synthesis of the “fairy-ring”
plant-growth regulator 2-azahypoxanthine (AHX), and its biologi-
cally active hydroxyl metabolite (AOH) and riboside derivative
(AHXr). AHXr, a biosynthetic intermediate, was synthesized from
inosine via a biomimetic route. Biotinylated derivatives of AHX and
AHXr were also synthesized as probes for mechanistic studies.
Fig. 1 Structures of AHX derivatives.
“Fairy rings” arising from fungus-stimulated plant growth
occur worldwide, and were first reported in 1675, as reviewed
in Nature in 1884.1 In 2010, we reported that, in the case of the
fungus Lepista sordida, the “fairy” is a plant growth stimulator,
which we identified as 2-azahypoxanthine (AHX: 1).2 AHX
exhibited growth-regulating activity towards not only turf
grass, but also other plants tested, even from different
families. Furthermore, this compound increased the seed
yields of rice and wheat in pot-growth and field experiments,2
suggesting that it might find practical application in agricul-
ture. We also showed that 2-aza-8-oxohypoxanthine (AOH: 3) is
a common, biologically active metabolite of AHX (1) in plants
(Fig. 1).3
AHX (1) was chemically synthesized from 5-aminoimida-
zole-4-carboxamide (AICA 2; an intermediate in the purine
metabolic pathway in animals, plants, and microorganisms),
and converted into AOH (3) by xanthine oxidase-mediated reac-
tion. We hypothesized that plants themselves produce AHX (1)
and AOH (3) through a similar pathway, and indeed, we
demonstrated that endogenous AHX (1) and AOH (3) are
formed via the purine pathway in plants.3 We considered that
riboside and/or ribotide derivatives of AICA (5, 7) and AHX (4,
6) might also be involved in the biosynthetic pathway. Further-
more, biotinylated derivatives of AHX and AHX riboside (AHXr:
4) are of interest as potential tools for mechanistic studies to
identify receptor(s) and related proteins. Herein we report
efficient synthetic routes to AHX (2), AOH (3), AHXr (4) and
also biotinylated derivatives of AHX and AHXr.
First, we wished to develop a practical synthesis of AHX (1)
and AOH (3). Although synthesis of 1 has already been
reported,4 optimization for large scale-preparation is necessary
to obtain sufficient material for field experiments. As shown in
Scheme 1, the synthesis was commenced with inexpensive
2·HCl. Upon treatment of 2 with sodium nitrite under acidic
conditions, the desired diazonium formation proceeded
smoothly to provide diazoimidazole carboxamide (DICA: 8).
Although a hundred-gram scale AHX (1) was prepared accord-
ing to the reported procedure (method A),4 we found that the
aSchool of Pharmaceutical Sciences, University of Shizuoka, 52-1 Yada, Suruga-ku,
Shizuoka 422-8526, Japan. E-mail: kant@u-shizuoka-ken.ac.jp;
Fax: +81-54-264-5745; Tel: +81-54-264-5746
bResearch Institute of Green Science and Technology, Shizuoka University, 836 Ohya,
Suruga-ku, Shizuoka 422-8529, Japan. E-mail: achkawa@ipc.shizuoka.ac.jp;
Fax: +81-54-238-4885; Tel: +81-54-238-4885
cGraduate School of Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku,
Shizuoka 422-8529, Japan
dGraduate School of Agriculture, Shizuoka University, 836 Ohya, Suruga-ku,
Shizuoka 422-8529, Japan
†Electronic supplementary information (ESI) available: Experimental procedures
and NMR spectral data and bioassay of 4, 6, 13 and 24. See DOI: 10.1039/
c4ob00705k
Scheme 1 Practical synthesis of AHX (1) and AOH (3).
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triazine ring of 1 can be constructed by heating 8 in methanol
at 60 °C (method B). Furthermore method B was more superior
to method A in terms of yield as well as handling. By means of
this transformation, together with activated carbon treatment,
1 was obtained on an almost decagram scale. Conversion of 1
to 3 was carried out by enzymatic oxidation with xanthine
oxidase.5 Purification of 3 was also accomplished by recrystalli-
zation, and chromatographic purification was unnecessary
(see the ESI† for details).
With the desired biologically active natural products 1 and
3 in hand, we turned our attention to the synthesis of deriva-
tives that would be suitable as probes for mechanistic studies,
following on from our previous work.6 During studies of poly-
phenols such as catechins7 and flavones,8 we had found that a
terminal amine or azide group is favorable for facile incorpor-
ation of a probe moiety without the need for protection of the
phenol group.7b,8a Thus, we decided to incorporate a terminal
azide group onto AHX (1). However, direct alkylation of 1 with
alkyl halide did not proceed smoothly. In order to enhance the
reactivity of the linker, we designed the probe precursor 10, in
which the linker group is connected through the acetal group.
Alkylation reaction was performed after in situ protection of
the nitrogen of imidazole and amide with a TMS group by
treatment with N,O-bis(trimethylsilyl)acetamide (BSA), as
shown in Scheme 2. Upon treatment of 1 with 1-azido-5-(chloro-
methyloxy)pentane 9 in the presence of DBU and BSA, the
desired reaction proceeded smoothly to provide 10 and 11 as a
1.8 : 1 mixture. After separation of each regioisomer, the azide
10 was used as the AHX probe precursor. The Hüisgen conden-
sation reaction9 of azide 10 and acetylene 12 containing a
biotin group in the presence of CuSO4 and sodium ascorbate
proceeded smoothly to afford 13.10 Considering the conven-
ience of the Hüisgen reaction, this synthetic strategy should be
applicable to incorporation of a wide range of functional units,
such as a fluorescent moiety for imaging or a carrier protein
for synthesis of an immunogen.7c We found that the coupling
reaction did not require a tedious purification step, although
purification of biotin-containing probes is sometimes
troublesome.
Scheme 3 Preparation of AHXr (4) and AHXR (6).
14 into the nitrogen atom of 4 seemed to be a challenging
task, a similar aminolysis of the pyrimidine ring of inosine
(14) has been reported.12 After protection of the hydroxyl
groups of 14 with a TBS group, incorporation of a 2,4-dinitro-
phenyl group on the amide nitrogen was carried out via
nucleophilic aromatic substitution reaction of 1-chloro-2,4-
dinitrobenzene to give 15. Upon treatment of 15 with ethylene-
diamine in THF, the desired aminolysis reaction on the imida-
mide ring proceeded smoothly to give 17 with the release of
dihydro-imidazole 16.12 After selective deprotection of the TBS
ether on the primary alcohol of 17 by treatment with NH4F,
construction of the triazine ring of 19 was performed by treat-
ment of 18 with sodium nitrite and hydrochloric acid, as
employed in the preparation of 1. Removal of the 2,4-dinitro-
phenyl protecting group of 19 was carried out by treatment
with ethylenediamine as
a nucleophile. In the original
report,12 treatment of 15 with ethylenediamine in DMF caused
simultaneous ring opening reaction and dinitrobenzene clea-
vage reaction to afford the –CONH2 derivative. However, the
2,4-dinitrophenyl group played a key role in the efficient prepa-
ration of 4, 6 and the probe precursor 23, and a selective ami-
nolysis reaction of 15 was needed. Chemoselective reaction of
15 was accomplished by changing the solvent to THF from
DMF. Finally, TBAF-mediated deprotection of the TBS groups
of 20 provided AHXr (4).10 On the other hand, AHXR (6) was
synthesized by incorporation of phosphate ester into 20 by the
phosphoramidite method,13 followed by the removal of the
TBS groups and the benzyl group to give 6.10
Based on the proposed biosynthetic pathway of AHXr (4)
and AHX-ribotide (AHXR: 6), we next planned to synthesize 4
and 6 from inexpensive inosine (14) as shown in Scheme 3.11
Although conversion of the carbon atom at the C-2 position of
With an efficient synthetic method for AHXr (4) in hand, we
turned our attention to the synthesis of a biotin-labeled AHXr
probe compound, starting from the synthetic intermediate 19.
Since AHXr (4) is a biosynthetic precursor of AHX (2) in plants,
the biotinylated AHXr probe should enable the identification
of proteins involved in the biosynthesis of AHX. Since the
Scheme 2 Hüisgen reaction of azide 10 and alkyne 12.
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Communication
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5 Xanthine oxidase from buttermilk (XOD; 0.28 U mg−1
powder) was purchased from Oriental Yeast Co. (Tokyo,
Japan).
Scheme 4 Synthesis of biotin-labelled AHXr probe 24.
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ribose unit of the AHXr probe (24) can serve as a hydrophilic
linker unit of the AHX probe, an efficient and flexible synthesis
of 24 would be useful. As shown in Scheme 4, the alkylation
reaction of 19 with 9 proceeded smoothly to provide 21. Utiliz-
ing a similar method to that used for preparation of 4, removal
of the 2,4-dinitrophenyl group and the TBS group of 21 pro-
vided a reactive probe precursor 23. Next, coupling with biotin
was examined. Upon treatment of azide 23 and acetylene 12
under the conditions employed for preparation of 13, the
desired Hüisgen reaction proceeded smoothly to provide 2410
without any need for purification. This coupling reaction is
compatible with the reactive hydroxyl group and the amide
group. Synthesis of other kinds of probe molecules from 10
and 23 is in progress in our laboratories, and the details will
be reported in due course.
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Y. Hamashima and T. Kan, Heterocycles, 2014, 88, 1371;
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40, 7; (c) H. C. Kolb, M. G. Finn and K. B. Sharpless, Angew.
Chem., Int. Ed., 2001, 40, 2004.
Conclusion
We have developed a practical, large-scale synthesis of the
plant-growth regulator AHX (1), as well as an efficient synthetic
route for AOH (3), AHXr (4) and AHXR (6), respectively. Biotin-
labeled derivatives of AHX (13) and AHXr (24) were also syn-
thesized. The probe precursors 10 and 23 should also be suit-
able for preparation of other probe molecules.
This work was partially supported by the Programme for
Promotion of Basic and Applied Researches for Innovations in
Bio-oriented Industry, and by a Grant-in-Aid for Scientific
Research on Innovative Areas of “Chemical Biology of Natural
Products” from MEXT.
10 Interestingly, biotin probes 13 and 24, as well as AHXr (4)
and AHXR (6), possessed comparable biological activity to
natural AHX (1). For details, see the ESI.†
11 Direct condensation reaction of AHX (1) and tetraacetyl
ribose was unsatisfactory; for details, see the ESI.†
12 L. D. Napoli, A. Messere, D. Montesarchio and G. Piccialli,
J. Chem. Soc., Perkin Trans. 1, 1997, 2079.
Notes and references
1 H. Evershed, Nature, 1884, 29, 384.
13 For
a review of the phosphoramidite method, see:
2 J.-H. Choi, K. Fushimi, N. Abe, H. Tanaka, S. Maeda,
A. Morita, M. Hara, R. Motohashi, J. Matsunaga, Y. Eguchi,
R. P. Iyer, Tetrahedron, 1992, 48, 2223.
This journal is © The Royal Society of Chemistry 2014
Org. Biomol. Chem., 2014, 12, 3813–3815 | 3815