Synthesis and inhibitory activity of deoxy-d-allose amide derivative against plant growth

Article abstract of DOI:10.1080/09168451.2018.1445521

1,2,6-Trideoxy-6-amido-d-allose derivative was synthesized and found to exhibit higher growth-inhibitory activity against plants than the corresponding deoxy-d-allose ester, which indicates that an amide group at C-6 of the deoxy-d-allose amide enhances i

Full text of DOI:10.1080/09168451.2018.1445521

Bioscience, Biotechnology, and Biochemistry
ISSN: 0916-8451 (Print) 1347-6947 (Online) Journal homepage: http://www.tandfonline.com/loi/tbbb20
Synthesis and inhibitory activity of deoxy-d-allose
amide derivative against plant growth
Md. Tazul Islam Chowdhury, Hikaru Ando, Ryo C. Yanagita & Yasuhiro
Kawanami
To cite this article: Md. Tazul Islam Chowdhury, Hikaru Ando, Ryo C. Yanagita & Yasuhiro
Kawanami (2018): Synthesis and inhibitory activity of deoxy-d-allose amide derivative against plant
growth, Bioscience, Biotechnology, and Biochemistry, DOI: 10.1080/09168451.2018.1445521
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Bioscience, Biotechnology, and Biochemistry, 2018
https://doi.org/10.1080/09168451.2018.1445521
Synthesis and inhibitory activity of deoxy-d-allose amide derivative against
plant growth
Md. Tazul Islam Chowdhurya, Hikaru Andob, Ryo C. Yanagitab and Yasuhiro Kawanamib
adivision of applied Bioresource science, the United graduate school of agricultural sciences, ehime University, matsuyama, Japan; bFaculty
of agriculture, Kagawa University, miki-cho, Japan
ABSTRACT
ARTICLE HISTORY
received 17 January 2018
accepted 20 February 2018
1,2,6-Trideoxy-6-amido-d-allose derivative was synthesized and found to exhibit higher growth-
inhibitory activity against plants than the corresponding deoxy-d-allose ester, which indicates
that an amide group at C-6 of the deoxy-d-allose amide enhances inhibitory activity. In addition,
the mode of action of the deoxy-d-allose amide was significantly different from that of d-allose
which inhibits gibberellin signaling. Co-addition of gibberellin GA3 restored the growth of rice
seedlings inhibited by the deoxy-d-allose amide, suggesting that it might inhibit biosynthesis of
gibberellins in plants to induce growth inhibition.
KEYWORDS
rare sugar; d-allose;
gibberellin biosynthesis;
inhibitory activity
Abbreviations: GA: gibberellin; DMF: N,N-dimethylformamide; TsCl: 4-toluenesulfonyl chloride
Rare sugars are monosaccharides present in limited
amounts in nature [1]. Although the biological activ-
ities of almost all rare sugars are largely unknown,
some biological activities of d-allose (1), a C-3 epimer
of d-glucose, have been reported. d-Allose showed an
immunosuppressive effect [2] and a protective effect
against liver damage in animals [3], anti-proliferative
effect through cell cycle arrest and apoptosis [4–6], sup-
pressive effect for development of salt-induced hyper-
tension [7], and anti-oxidative effects [8,9]. d-Allose
has also been demonstrated to scavenge reactive oxygen
species (ROS) generated by the hypoxanthinexanthine
oxidase system to inhibit the production of ROS from
neutrophils [10], and retard plant growth [11]. However,
its concentration required for inhibition of plant growth
is relatively high (more than 3 mM). erefore, it is nec-
essary to improve its activity by chemical modification.
We previously reported that 6-O-acyl-d-allose showed
six times higher growth-inhibitory activity against lettuce
than d-allose [12]. Furthermore, we described the effect
of fatty acid chain length on growth-retarding activity of
6-O-acyl derivatives of d-allose, d-gulose, and d-altrose
on rice seedlings [13,14], and the role of hydroxyl groups
at C-1 and C-2 of d-allose on the biological activity [15].
e inhibitory activity of 6-O-decanoyl-2-deoxy-d-
allose was comparable to that of 6-O-decanoyl-d-allose
(2), whereas 6-O-decanoyl-1,2-dideoxy-d-allose (4)
showed little activity. In general, amide groups are more
stable than ester groups that are susceptible to hydroly-
sis by esterases in vivo and, therefore, we expected that
amide replacement would improve inhibitory activity
on plant growth. However, we previously reported that
the amide replacement of 2 resulted in lower inhibi-
tory activity [16], which prompted us to investigate the
amide replacement effect on the other allose ester. In this
study, we synthesized a new deoxy derivative 6 having
amide group at C-6 to examine the effect of replacement
of the ester group of 4 with an amide group on plant
growth-inhibitory activity (Figure 1).
Results and discussion
6-(Decanoylamino)-1,2,6-trideoxy-d-allose (6) was pre-
pared from the known compound 1,2-dideoxy-d-allose
(3) [17] as shown in Scheme 1. First, regioselective tosyl-
ation of 3 at low temperature gave a monotosylate 7 in
68% yield. en, azidation of 7 with sodium azide in
DMF followed by catalytic hydrogenation afforded an
amine 5. Finally, acylation of 5 with decanoyl chloride
gave the amide 6 in 63% yield in three steps.
e biological activity of 6 was evaluated using let-
tuce, cress, Italian ryegrass, and rice seedlings (Figure
2). e amide 6 inhibited the growth of four plants
in a concentration-dependent manner ranging from
0.03 to 1 mM, and interestingly, slight growth promo-
tion (119%) for lettuce roots was observed at 0.1 mM
(Figure 2(a)). Of the plant species tested, rice seedlings
were most susceptible to 6; the rice growth was com-
pletely inhibited by 6 at a concentration greater than
1 mM (Figure 2(d)). e concentrations required for
CONTACT yasuhiro Kawanami
kawanami@ag.kagawa-u.ac.jp
© 2018 Japan society for Bioscience, Biotechnology, and agrochemistry

2
M. T. I. CHOWDHURY ET AL.
OH
1
1
2
HO
HO
2
3
O
O
O
3
5
5
OR
OR
NHR
HO
HO
4
4
6
6
OH
OH
OH
1: R = H
2: R = CO(CH2)8CH3
3: R = H
4: R = CO(CH2)8CH3
5: R = H
6: R = CO(CH2)8CH3
Figure 1. structures of d-allose and deoxy-d-allose derivatives 1–6.
Figure 2. Biological activity of the amide 6 on (a) lettuce, (b) cress, (c) italian ryegrass, and (d) rice seedlings in 0.01–3.0 mm. the
plant growth was completely inhibited at 3 mm of (a) and (b), and 1 and 3 mm of (d). Values are mean se from three independent
experiments.
50% inhibition (IC50) of lettuce, cress, Italian ryegrass,
and rice hypocotyls growth are listed in Table 1. e
IC50 values for the four plants range from 0.2 to 1 mM.
Among the four species, 6 showed the highest activity
for cress for both shoots and roots with IC50 values of
0.21 and 0.25 mM, respectively. e IC50 values of other
d-allose derivatives for cress are summarized in Table 2.
e activity of the amide 6 was at least 14 times higher
than that of the amine 5, which demonstrated the same
effect of acylation with medium-chain fatty acids at the
C-6 on inhibitory activity as the modification of d-allose
1 [12]. Furthermore, comparing the IC50 values of the
amide 6 with that of the ester 4 having the same decanoyl
group, 6 exhibited significantly higher activity than 4.
ese results indicate that the replacement of an ester
group with an amide group enhances inhibitory activity
in the 1,2-dideoxy-d-allose series.
Further, we investigated the mode of action of 6
on plant growth. In plants, four different types of growth
inhibitors are known. Of these, prohexadione-calcium,
trinexapac-ethyl, daminozide (N-dimethylaminosuccinic
acid), and 16,17-dihydro-GA5 block 3β-hydroxylation of
GA20 to GA1 in gibberellin (GA) biosynthesis [18,19]. It is
alsoreportedthatgrowthsuppressionbythesecompounds
can be reversed by co-addition of the active gibberellin,
GA3 [20,21]. As reported previously, co-addition of GA3

BIOSCIENCE, BIOTECHNOLOGY, AND BIOCHEMISTRY
3
(a)
(b)
Figure 3. (a) effect of the amide 6, daminozide (dam, 0.25 mm), and ga3 (1 μm) on rice seedlings and (b) effect of uniconazole (Uni,
0.001 mm), ga3 (1 μm), and 6 (0.2 mm) on rice seedlings. relative lengths (% of control) of shoot and second leaf sheath of rice at
7 days after treatment. Values are mean sem (n = 14), and bars with different letters indicate significant difference as determined
by tukey’s honestly significant difference comparison (p < 0.05).
also restored the growth of rice seedlings treated with
O
O
TsCl, Py, CH3CN
OH
OTs
d-allose ester [13,14], whereas the monosaccharide form
of d-allose did not inhibit GA biosynthesis but inhibited
GA-signaling pathway [11]. erefore, we examined the
effect of co-addition of GA3 on growth inhibition by 6.
Daminozide was used as a positive control. As shown in
Figure 3(a), rice growth was restored (183%) by co-ad-
dition of GA3 (1 μM) with 6. (0.25 mM). Neither with-
ered shoots nor roots were observed. We found a similar
tendency with daminozide. ese results suggest that the
amide 6 as well as the ester 2 inhibits gibberellin bio-
synthesis to cause growth suppression. Furthermore, we
examined whether 6 affects a GA-signaling pathway in
rice, using another known growth retardant, unicona-
zole, which inhibits the conversion of ent-karuene to
ent-karuonic acid in an earlier stage of gibberellin biosyn-
thesis [21] (Figure 3(b)). For this experiment, rice seeds
were pre-treated with 0.001 mM of uniconazole at 30 °C
in the dark for 24 h [22]. We observed similar growth
recovery for both of uniconazole and GA3-treated groups
(210 and 213%, respectively, for shoot length) regard-
less of presence or absence of 6. ese results suggest
that, unlike d-allose, the amide 6 does not inhibit GA
signaling.
HO
HO
0 oC to rt
68%
OH
OH
3
7
1) NaN3, DMF, 90 oC
2) H2, Pd/C, EtOH, rt
O
H
N
(CH2)8CH3
HO
3) decanoyl chloride, Et3N
CH2Cl2, rt
OH
O
6
63% (3 steps)
Scheme 1. synthesis of deoxy-d-allose amide (6).
Table 1. ic50 values of the amide 6 for four plant species.
IC50 (mM)
Plant
Shoot
Root
lettuce
cress
1.0
0.82
0.25
0.33
nd
0.21
0.51
0.25
italian ryegrass
rice
nd = not determined.
Table 2. ic50 values of 1, 2, 4, 5, and 6 for cress.
In summary, 6 was synthesized from 1,2-dide-
oxy-d-allose (3) and its biological activity on plant
growth was evaluated. We found that the replacement
of the ester group of 4 to an amide group led to enhance-
ment of plant growth-inhibitory activity. Further, co-ad-
dition experiments with GA3 imply that 6 may inhibit
GA biosynthesis in rice seedling like daminozide. It
is noteworthy that 6 exhibit higher inhibitory activity
than d-allose 1 inhibiting GA signaling. us, the allose
amide 6 could be a new class of plant growth regulators
which has higher activity and more safety compared to
IC50 (mM)
Compound
Shoot
Root
1
2
4
5
6
1.5
0.71
0.36
2.7
>3.0
0.25
0.32
>3.0
>3.0
0.21
commercially available daminozide having dimethylhy-
drazine-structure which is suspected of being carcino-
genic in mice [23].

4
M. T. I. CHOWDHURY ET AL.
J = 7.2 Hz), 3.22 (1H, dd, J = 9.6, 2.8 Hz), 3.27 (1H, dd,
J = 14.4, 6.8 Hz), 3.52 (1H, dd, J = 14.4, 2.0 Hz,), 3.56
(1H, ddd, J = 9.6, 6.8, 2.8 Hz), 3.62 (1H, dd, J = 11.0,
5.5 Hz), 3.71 (1H, td, J = 12.4, 2.8 Hz), 4.02 (1H, br
d, J = 2.8 Hz). 13C-NMR (150 MHz, CD3OD) δ: 14.42,
23.70, 27.07, 30.26, 30.39, 30.45, 30.62, 33.03, 33.73,
36.95, 42.36, 62.71, 68.00, 70.81, 75.87, 176.95. [α]22
+ 24.2 (c 0.480, MeOH). Rf 0.45 (ethyl acetate/methaD-
nol = 30:1). HR-ESI-TOF-MS m/z (M + Na)+: calculated
for C16H31NO4Na, 324.2151; found, 324.2150.
Experimental
d-Allose was provided by the Rare Sugar Research Center
at Kagawa University, Japan. H and 13C NMR spectra
1
were measured at 600 MHz and 150 MHz, respectively,
with a Jeol JNM-ECA600 spectrometer in CD3OD at
room temperature using TMS as an internal standard.
Optical rotation data were measured with a Jasco P-1010
optical rotation polarimeter using methanol. High res-
olution mass spectrums (HRMS) were taken using a
Waters Xevo G2-XS-TOF mass spectrometer.
Biological assay
Synthesis of 6-O-(4-toluenesulfonyl)-1,2-dideoxy-
d-allose (7)
e biological activity of amide (6) was tested on four
different plant species: lettuce (Lectuca sativa), cress
(Lepidium sativum), Italian ryegrass (Lolium multi-
florum), and rice (Oryza sativa L. cv. Nihonbare) seed-
lings. Statistical tests were carried out using R (version
3.3.3) statistical sofware.
Bioassay on lettuce, cress, and Italian ryegrass: e
amide 6 was dissolved in a small volume of methanol,
which was added to a sheet of filter paper (Toyo No. 2) in
a 3.5 cm Petri dish and dried. e filter paper in the Petri
dish was then moistened with 0.8 mL of a 0.05% (v/v)
aqueous solution of Tween 20. Ten sets of test plants
were arranged on the filter paper and grown in the dark
at 25 °C. e control groups were treated only with a
solution of Tween 20. e lengths of the hypocotyls or
shoots and roots of the lettuce, cress, and Italian ryegrass
seedlings were measured afer 48 h and the percentage
of shoot length and root length were calculated with
reference to the shoot and root lengths of the seedlings
in the control groups.
Tosyl chloride (77 mg) and pyridine (50 μL) were added to
1,2-dideoxy-d-allose (3, 19.5 mg, 0.13 mmol) in CH3CN
(0.9 mL) at 0 °C, and the reaction mixture was stirred at
room temperature for 18 h. e solvent was evaporated and
the residue was then purified by column chromatography
on silica gel to give a monotosylate 7[24] as a colorless liquid
(26.6 mg, 68%). 1H-NMR (600 MHz, CD3OD) δ: 1.70 (1H,
m), (1H, tdd, J = 12.4, 5.3, 2.4 Hz),2.46 (3H, s), 3.34 (1H, dd,
J = 10.0, 2.9 Hz), 3.56 (1H, ddd, J = 10.0, 5.3, 1.4 Hz), 3.66
(1H, ddd, J = 12.4, 2.2, 2.4 Hz), 3.68 (1H, m), 3.99 (1H, br
dd, J = 6.0, 2.9 Hz), 4.09 (1H, dd, J = 10.4, 6.0 Hz), 4.26 (1H,
dd, J = 10.4, 2.0 Hz), 7.78 (1H, d, J = 8.3 Hz), 7.43 (2H, d,
J = 8.3 Hz). 13C-NMR (150 MHz, CD3OD) δ: 21.75, 34.83,
66.57, 71.83, 72.91, 73.82, 79.48, 129.08, 130.96, 134.39,
146.43. Rf 0.51 (ethyl acetate/hexane = 4:1).
Synthesis of 6-(decanoylamino)-1,2,6-trideoxy-d-
allose (6)
Bioassay on rice seedlings: Following Ref. [22], rice
seeds were sterilized with ethanol for 5 min and then
washed with water. e seeds were then sterilized for
30 min with 1% sodium hypochlorite and washed again
with water. e sterilized seeds were soaked in water for
2 d at 30 °C under fluorescent light. Seven germinated
seeds were transplanted into tubes containing a test solu-
tion of 0.05% Tween 20 (2 mL each). Afer incubating
for 7 d under light, the lengths of shoot and the second
leaf sheath of each rice seedling were measured and the
growth ratios against control were calculated.
NaN3 (34 mg, 0.52 mmol) was added to a solution of 7
(39.5 mg, 0.13 mmol) in DMF (1.0 mL) at 90 °C, and
the reaction mixture was stirred for 48 h. e resulting
mixture was extracted with ethyl acetate (5 mL × 3), and
the residue was purified by column chromatography on
silica gel to give an azide 8 (20.8 mg) as a colorless liquid,
Rf 0.42 (ethyl acetate/methanol = 10:1), which was used
without further purification. Pd/C (17.5 mg) was added
to 8 in ethanol (1.0 mL), and the reaction mixture was
stirred under a H2 atmosphere for 48 h. e resulting
mixture was filtered through Celite and concentrated
in vacuo to give an amine 5 (16.6 mg) as a colorless liq-
uid, Rf 0.05 (ethyl acetate/methanol = 10:1), which was
used without further purification. Triethylamine (54 μL,
0.39 mmol) and decanoyl chloride (81 μL, 0.40 mmol)
were added to 5 in dichloromethane (1.3 mL) at room
temperature, and the reaction mixture was stirred for
5 h. Afer the usual work-up, the residue was purified by
column chromatography on silica gel to give the amide
Author contributions
M.T.I.C. and Y.K. designed the synthetic route and wrote
the manuscript with the aid of R.C.Y. M.T.I.C. and H.A.
conducted the synthetic experiment and bioassay.
Acknowledgements
1
(6) as a colorless liquid (24.6 mg, 63% in 3 steps). H-
e first author is grateful to the Ministry of Education,
Culture, Sport, Science and Technology (MEXT), Japan for
providing a scholarship and to the Sher-e-Bangla Agriculture
University, Dhaka, Bangladesh for granting study in Japan.
NMR (600 MHz, CD3OD) δ: 0.88 (3H, t, J = 6.9 Hz),
1.29 (12H, m), 1.58 (2H, br t, J = 6.9 Hz), 1.72 (1H,
m), 1.80 (1H, tdd, J = 14.4, 5.5, 2.8 Hz), 2.19 (2H, t,

BIOSCIENCE, BIOTECHNOLOGY, AND BIOCHEMISTRY
5
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Disclosure statement
No potential conflict of interest was reported by the authors.
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