Synthesis and bioactivity of 4-methylthio-3-butenylisothiocyanate and raphanusanin, phototropism-regulating substances of radish hypocotyls

Article abstract of DOI:10.1016/j.tetlet.2021.153025

Total syntheses of 4-methylthio-3-butenylisothiocyanate (MTBI, 1) and raphanusanin (2), which are growth inhibitors involved in the phototropism of radish (Raphanus sativus) hypocotyls, were achieved from commercially available thiolane in four and five steps, respectively. MTBI (1) was prepared via the ring opening of thiolane with sodium azide, double bond introduction by oxidation, and isothiocyanation. Cyclization of MTBI to raphanusanin (2) was achieved under mild conditions using sauer Al₂O₃. Synthetic MTBI (1) and raphanusanin (2) showed potent bioactivity in a unilateral administration test and a segment elongation test using radish hypocotyls.

Full text of DOI:10.1016/j.tetlet.2021.153025

Tetrahedron Letters 71 (2021) 153025
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Tetrahedron Letters
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Synthesis and bioactivity of 4-methylthio-3-butenylisothiocyanate and
raphanusanin, phototropism-regulating substances of radish hypocotyls
Yoshiki Noguchi a, Ryoko Watanabe a, Atsushi Arai a, Kosumi Yamada b, Koji Hasegawa b, Taeko Horinouchi c,
Hidenori Watanabe c, Hideyuki Shigemori b,d,
⇑
a Graduate School of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan
b Faculty of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8572, Japan
c Graduate School of Agricultural and Life Sciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
d Microbiology Research Center for Sustainability (MiCS), University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Total syntheses of 4-methylthio-3-butenylisothiocyanate (MTBI, 1) and raphanusanin (2), which are
growth inhibitors involved in the phototropism of radish (Raphanus sativus) hypocotyls, were achieved
from commercially available thiolane in four and five steps, respectively. MTBI (1) was prepared via
the ring opening of thiolane with sodium azide, double bond introduction by oxidation, and isothiocya-
nation. Cyclization of MTBI to raphanusanin (2) was achieved under mild conditions using sauer Al2O3.
Synthetic MTBI (1) and raphanusanin (2) showed potent bioactivity in a unilateral administration test
and a segment elongation test using radish hypocotyls.
Received 23 January 2021
Revised 19 March 2021
Accepted 23 March 2021
Available online 26 March 2021
Keywords:
Phototropism
Bruinsma-Hasegawa theory
Raphanus sativus
4-Methylthio-3-butenylisothiocyanate
(MTBI)
Ó 2021 Elsevier Ltd. All rights reserved.
Raphanusanin
Introduction
Raphanusanin (2) is produced by MTBI (1) cyclization, the
mechanism of which has not been clarified (Fig. 1) [8]. MTBI
Phototropism has long been attributed to growth deviations
caused by auxin (Cholodny–Went theory) [1]. Recently, the
Bruinsma–Hasegawa theory, according to which a phototropism-
regulating substance produced in the illuminated side triggers
the bending of plant seedlings, has been proposed [2,3]. Based on
the latter theory, 4-methylthio-3-butenylisothiocyanate (MTBI)
(1) and raphanusanin (2) were isolated and identified from light-
grown radish hypocotyls as candidate phototropism-regulating
substances in radish hypocotyls. The concentration of these sub-
stances is approximately two times higher in the tissue in the illu-
minated side than that in the shaded side due to phototropic
stimulation [4–6]. It has also been revealed that there is a close
relationship between the production amount of these substances
and the growth rate of hypocotyls [4,7].
(1) is a kind of isothiocyanate specifically contained in Brassi-
caceae, and its action as phytoalexin has been reported.
Raphanusanin (2) is thought to contribute to growth inhibition
by inhibiting auxin-induced changes in the orientation of
microtubes [9].
a
Thus far, MTBI (1) and raphanusanin (2) have been isolated
from radish hypocotyls and evaluated in various bioactivity tests.
However, the isolated yields of compounds 1 and 2 were very poor,
and it was difficult to obtain a sufficient amount for use in various
experiments. In our previously report, raphanusanin (2) has been
obtained by the derivatization of MTBI (1) at extremely low yield
(3.3%) and MTBI (1) itself is not suitable for storage [10]. Therefore,
in this report, we establish total syntheses of MTBI (1) and
raphanusanin (2) in order to stably supply these substances to elu-
cidate the mechanism of phototropism in radish hypocotyls. MTBI
(1) is unstable; however, its precursor (6) is considered a stable
and storable substance. We also examine whether synthetic MTBI
(1) and raphanusanin (2) have the same biological activity as the
natural products.
MTBI (1) is released from 4-methylthio-3-butenyl glucosino-
late (MTBG) (3) via the action of myrosinase (thioglucosidase).
⇑
Corresponding author.
E-mail address: shigemori.hideyuk.fn@u.tsukuba.ac.jp (H. Shigemori).
https://doi.org/10.1016/j.tetlet.2021.153025
0040-4039/Ó 2021 Elsevier Ltd. All rights reserved.

Y. Noguchi, R. Watanabe, A. Arai et al.
Tetrahedron Letters 71 (2021) 153025
Fig. 3. Radish hypocotyl curvature with respect to time after unilateral MTBI (1)
and raphanusanin (2) application.
Fig. 1. Formation of phototropism-regulating substances MTBI and raphanusanin in
radish hypocotyls.
Bioactivity test
Result and discussion
The biological activity of synthetic MTBI (1) and raphanusanin
(2) with regard to the growth of radish hypocotyls was investi-
gated. The ability of these compounds to inhibit hypocotyl bending
and indole-3-acetic acid (IAA)-induced segment elongation was
evaluated in unilateral application and segment elongation tests
[9,11]. Seeds of Sakurajima radish (Raphanus sativus var. hortensis
f. gigantissimus Makino) were immersed in tap water for 30 min
at room temperature and were germinated on moist vermiculite
at 25 °C in the dark over 4 d.
Synthesis of MTBI and raphanusanin
Rahanusanin (2) is thought to be generated by the cyclization
reaction of MTBI (1). MTBI (1) formation was induced by the isoth-
iocyanation of compound 6, which is thought to be obtained via the
oxidation of compound 5, which in turn was obtained by S-methy-
lation of thiolane (4) as a starting material and subsequent ring
opening with azide (Fig. 2).
In the unilateral application test, a small piece of a moist wiping
paper containing MTBI (1) and raphanusanin (2) was attached to
one side of a radish hypocotyl, and the curvature of the hypocotyl
was measured. With MTBI (1), the curvature increased within
10 min, reached 10° in 40–100 min, and then increased slightly
with time (Fig. 3). In contrast, with raphanusanin (2), the curvature
peaked 90 min later and then decreased. Presumably, raphanu-
sanin (2) causes growth inhibition by temporarily inhibiting the
auxin response without altering the mechanical properties of plant
cells. Therefore, it is considered that the growth inhibition may be
released by later metabolism and decomposition.
Thiolane (4) was converted to compound 5 via S-methylation in
the presence of MeOTf at room temperature over 2.5 h, followed by
ring opening by treatment with NaN3 at 60 °C for 15.5 h (86.6%).
Compound 5 was treated with N-chlorosuccinimide at room tem-
perature for 2.5 h, and with triethylamine at 80 °C for 2 h to give
compound 6 (67.0%). MTBI (1) was obtained via isothiocyanation
of compound 6 by treatment with PPh3 and CS2 at room tempera-
ture for 3.5 h (82.0%). E-MTBI (1) was converted to E-raphanusanin
(2) via the cyclization reaction in the presence of sauer Al2O3 at
room temperature for 92 h (41.9%). Sauer Al2O3 is an acidic type
of porous aluminum oxide, and is used as an adsorbent, desiccant,
catalyst, etc.
In the segment elongation test, 5-mm-long radish hypocotyl seg-
ments were incubated on a shaker in a sodium phosphate buffer (pH
6.3) containing IAA and compound 1 or 2, and the final segment
length was measured. The addition of IAA promotes section elonga-
Fig. 4. Effect of MTBI (1) and raphanusanin (2) on IAA-induced segment elongation
Fig. 2. Syntheses of MTBI (1) and raphanusanin (2).
in radish hypocotyls.
2

Y. Noguchi, R. Watanabe, A. Arai et al.
Tetrahedron Letters 71 (2021) 153025
tion compared to the buffer-only condition. However, the addition
of 1 or 2 with IAA suppresses section elongation by IAA. The results
reveal that MTBI (1) (10À4 M) more strongly inhibited segment elon-
gation compared to IAA alone (Fig. 4). Raphanusanin (2) also weakly
inhibited the IAA-induced segment elongation in a dose-dependent
manner. Therefore, these results show that synthetic compounds 1
and 2 have growth inhibiting activity in planta.
In this study, MTBI (1), a phototropism-regulating substance for
radish seedlings, was efficiently synthesized in four steps using thi-
olane (4) as a starting material. MTBI (1) precursor 6 is stable and
suitable for storage. MTBI (1) was successfully converted to
raphanusanin (2) under mild conditions by using alumina. The
synthetic compounds 1 and 2 showed potent phototropism-regu-
lating activity.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.tetlet.2021.153025.
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Declaration of Competing Interest
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Acknowledgments
We thank We would like to thank Chemical Analysis Div. and
the Open Facility Network Office, Research Facility Center for
Science and Technology, University of Tsukuba, for allowing us to
use AVANCE-500 measurement on synthetic compounds. We
would like to thank Editage (www.editage.com) for English lan-
guage editing. This work was partially supported by JSPS KAKENHI
Grant Number JP19K22268.
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