Functional importance of the sugar moiety of jasmonic acid glucoside for bioactivity and target affinity

Article abstract of DOI:10.1039/c4ob02106a

12-O-β-d-Glucopyranosyljasmonic acid (JAG, 1) induces nyctinastic leaf-folding of Samanea saman. The SAR studies of 1 revealed the unique role of its glycone moiety. Biological activity and the target affinity of 1 were affected by the stereochemistry of the glycone moiety. JAG belongs to a unique class of ligands in which the structure of the glycone moiety is involved in the molecular recognition by the target protein. This journal is

Full text of DOI:10.1039/c4ob02106a

Organic &
Biomolecular Chemistry
View Article Online
View Journal | View Issue
COMMUNICATION
Functional importance of the sugar moiety of
jasmonic acid glucoside for bioactivity and
target affinity†
Cite this: Org. Biomol. Chem., 2015,
13, 55
Received 2nd October 2014,
Accepted 15th October 2014
Minoru Ueda,* Gangqiang Yang, Yuuki Nukadzuka, Yasuhiro Ishimaru,
Satoru Tamura and Yoshiyuki Manabe
DOI: 10.1039/c4ob02106a
www.rsc.org/obc
12-O-β-D-Glucopyranosyljasmonic acid (JAG, 1) induces nyctinas-
Three possible roles of the D-glucopyranosyl moiety can be
tic leaf-folding of Samanea saman. The SAR studies of 1 revealed proposed for the target selectivity of 1. First, the conjugation
the unique role of its glycone moiety. Biological activity and the of the D-glucopyranosyl moiety could decrease the lipophilicity
target affinity of 1 were affected by the stereochemistry of the and membrane permeability of the jasmonate and then a
glycone moiety. JAG belongs to a unique class of ligands in which membrane permeable jasmonate targeting COI1-JAZ could be
the structure of the glycone moiety is involved in the molecular converted to a membrane-impermeable 1 targeting membrane
recognition by the target protein.
protein MTJG. Second, the D-glucopyranosyl moiety in 1 could
provide steric hindrance to inhibit binding with the COI1-JAZ
receptor complex. In the crystal structure of the COI1-JAZ-jas-
monoyl L-Ile (JA-Ile) complex,14 JA-Ile is packed tightly between
COI1 and JAZ, and no free space is left for accommodating
additional D-glucopyranosyl moieties. Third, the D-glucopyra-
nosyl moiety in 1 could enhance the affinity with the target
protein as shown in the case of a steroid glycoside, ouabain.
Recently, Cornelius et al. reported an intriguing example in
which the conjugation of a glycone moiety strongly enhanced
the affinity of a ligand with its target protein. The affinity of
ouabain with its target protein, Na,K-ATPase, is enhanced as
much as 26-fold by the glycosidation of the corresponding
aglycone.17 The strong hydrogen bond network between the
glycone moiety of ouabain and the ligand binding pocket of
Na,K-ATPase strongly enhanced their binding. We examined
this third possibility as shown below.
Jasmonic acid (JA) and its derivatives, collectively referred to as
jasmonates, play important roles in the life of higher plants.1–4
We identified 12-O-β-D-glucopyranosyljasmonic acid, also
referred to as jasmonic acid glucoside (JAG, 1), as the bioactive
metabolite that induces nyctinastic leaf-folding of Samanea
saman.5–8 Nyctinastic leaf-folding of plants has been a historic
matter of interest among biologists since Darwin’s era9 and
S. saman has been used as a standard plant material for the
biology of nyctinasty.10,11 Thus, 1 is a key compound for
the molecular understanding of this intriguing biological
phenomenon.
The mode of action of JA has been verified through identifi-
cation of the COI1-JAZ signalling module12–14 in which a
complex of cytosolic COI1 and JAZ proteins functions as the
jasmonate receptor. However, the leaf-folding induction by 1
was revealed to be independent of the COI1-JAZ signalling
pathway.15 Instead, the membrane target protein referred to as
MTJG (membrane target of jasmonic acid glucoside)15,16 is
believed to be involved. Considering that most glycosides are
regarded as biologically inactive derivatives of secondary
metabolites, this is a rare example in which a glucoside of a
plant hormone functions as a ligand totally different from the
parent ligand. Here, we report that 1 belongs to a unique class
of ligands in which the D-glucopyranosyl moiety participates in
the molecular recognition by the target protein.
We first carried out structure activity relationship (SAR)
studies on 1 to estimate the importance of the D-glucopyrano-
syl moiety. We designed ent-1,18 diastereomeric 12-O-β-L-gluco-
pyranosyljasmonic acid (2), diastereomeric
12-O-β-D-
glucopyranosyl-ent-jasmonic acid (ent-2), epimeric 12-O-β-D-
galactopyranosyljasmonic acid (3),18 and open-chain type 12-O-
D-sorbitoyljasmonic acid (4) (Fig. 1). The hydrophilicity of 1
was maintained in ent-1, 2, ent-2, 3, and 4, whereas the struc-
ture recognition of the glycone moiety may be affected by
structural modifications in ent-1, ent-2, 3, and 4. We syn-
thesized 1, ent-1, 2 (Scheme S1†), ent-2 (Scheme S2†), and 3
according to the procedure in ref. 19. The open-chain type 4
was synthesized as shown in Scheme S3.† In the previous
study,15 we demonstrated that 1 induced shrinking of the
extensor motor cell protoplasts isolated from S. saman,
Laboratory of Organic Chemistry, Department of Chemistry, Tohoku University,
6-3 Aramaki-Aza Aoba, Aoba-ku, Sendai 980-8578, Japan.
E-mail: ueda@m.tohoku.ac.jp; Fax: +81-22-795-6553; Tel: +81-22-795-6553
†Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ob02106a whereas ent-1 cannot induce the shrinking. As the shrinking of
This journal is © The Royal Society of Chemistry 2015
Org. Biomol. Chem., 2015, 13, 55–58 | 55

View Article Online
Communication
Organic & Biomolecular Chemistry
Interestingly, clear differences were observed among them.
Only naturally occurring 1 induced cell-shrinking, whereas no
cell-shrinking was observed with the enantiomeric and diastereo-
meric isomers (ent-1, 2 and ent-2) (Fig. 1b). It should be
noted that the stereochemistry of the glycone moiety was
proven to be important for the bioactivity of 1. Additionally,
the substitution of glucopyranosyl into galactopyranosyl (4′-
epimer) in 3 did not affect the cell-shrinking activity or the pre-
viously reported leaf-folding activity (Fig. 1c).18 Subtle struc-
tural modifications, such as epimerization of one hydroxy
group in the sugar moiety, did not affect the biological activi-
ties of 1. In contrast, the glycopyranosyl structure is important
because no cell-shrinking was observed in open-chain type 4.
These results strongly suggested that the structure of the D-gly-
copyranosyl moiety is indispensable for cell-shrinking activity.
Next, we considered that the photoaffinity labeling experi-
ments could provide direct proof that the stereostructure of
the glycone moiety in 1 affected its affinity with its target
protein, MTJG. We previously prepared four stereochemical
hybrids of compact molecular probes19 (CMPs: 5, ent-5, 6, and
ent-6, Fig. 2a) based on 1.20 The CMPs contained D-galactopyra-
nosyl due to its stability against hydrolysis by glucosidases in
living cells. Among them, only the naturally occurring form (5)
could induce the shrinking of Samanea motor cells.20 The
target protein in living cells was labeled by a sterically
minimal azide handle and subsequent introduction of a tag
was achieved by Hüsgen [3 + 2] cycloaddition employing Cu-
catalyzed azide alkyne cycloaddition (CuAAC)21,22 or copper-
free click chemistry (CFCC).23 Thus, the living motor cells were
photo-crosslinked using CMP 5 (1 × 10−4 M) followed by either
CuAAC with 4-ethynyl-2,6-bis(trifluoromethyl)-cinnamamide
(7; 1 × 10−4 M)24,25 or CFCC with the strained difluorinated
cyclooctyne DIFO23 derivative (8; 1 × 10−4 M). In both the
alkyne units, the FLAG peptide was conjugated as a molecular
tag.19,26 As shown in Fig. S1,† CuAAC using 7 gave the better
result. CFCC using
8 gave many non-specific bindings
(Fig. S1†). These nonspecific bindings were also observed
when DIFO 8 was used alone without the addition of CMP 5
(Fig. S1†), and could be due to the nonspecific thiol adducts
because the strained DIFO unit can react with thiol groups in
proteins to give nonspecific adducts.27,28 In contrast, no non-
specific binding was observed in CuAAC, and the result was
very clear because only one target band was observed (Fig. S1†).
The photo-crosslinking against a living Samanea motor cell
was examined using these four CMPs (5, ent-5, 6, and ent-6)
and subsequent cycloaddition was carried out with alkyne 7.
Results of SDS-PAGE analysis and subsequent chemilumine-
scence detection are shown in Fig. 2b and S2.† The order of
target affinity was 5 > ent-6 > 6 = ent-5. The difference between
the results by probe 5 or ent-5 clearly demonstrated the stereo-
specific recognition of the probe by MTJG. Binding of probe 5
with this protein was competitively inhibited by the co-
addition of excess 1 (50-fold), whereas it could not be inhibited
Fig. 1 (a) Structure of JAG (1) and its derivatives; (b) and (c) cell shrink-
ing assays using living Samanea extensor motor cells. (b) Extensor motor
cell protoplasts were treated with 100 μM 2 (white diamonds) or 100 μM
ent-2 (white triangles), and the response was compared to the treat-
ments with 100 μM JAG (1) (black circles), 100 μM ent-1 (white circles),
or mock (black triangles). (c) Extensor protoplasts were treated with
100 μM 3 (cross marks) or 100 μM 4 (black diamonds), and the responses
were compared to the treatments with 100 μM 1 (black circles) or mock
(black triangles). Error bars represent the SE. The a and b labeled
values are statistically different according to analysis of variance fol-
lowed by SNK post-hoc test (a, P < 0.05, n = 8–11, i.e., t = 40 min,
F4, 41 = 20.19, P < 0.001; b, P < 0.05, n = 9, 10, i.e., t = 40 min, F3, 33 = 33.51,
P < 0.001).
motor cells directly causes leaf-folding movement,10 the bio- by the excess co-addition of methyl-D-galactopyranoside (9) or
logical evaluations of 1–4, ent-1, and ent-2 were carried out 12-hydroxy jasmonic acid (10) (Fig. 2c and S3†). Interestingly,
based on this cell-shrinking assay (Fig. 1).
CMP ent-6 consisting of the enantiomeric aglycone and the
56 | Org. Biomol. Chem., 2015, 13, 55–58
This journal is © The Royal Society of Chemistry 2015

View Article Online
Organic & Biomolecular Chemistry
Communication
Fig. 3 (a) Thermodynamic equilibrium of 7S-ent-6 and 7R-ent-6; (b)
UPLC-MS analysis of an equilibrium mixture of 7S-ent-6 (Rt = 14.1 min)
and 7R-ent-6 (Rt = 13.2 min) [detection: ESI (−) m/z = 609.300; column:
Agilent Eclipse Plus C18 (1.8 m, ∅ 2.1 × 50 mm, Agilent Technologies);
flow rate: 0.2 µL min−1; mobile phase: 27% CH3CN (0–12 min), 27–90%
CH3CN (12–14 min), 90% CH3CN (14–18 min) each containing 0.1%
TFA]; (c) NOE correlations (H7–H2, H7–H4, H3–H2a, H7–H2b, and H4–
H2b) on the predominant isomer of 7S-ent-6 in the NMR spectrum.
Fig. 2 (a) Structure of CMPs (5, ent-5, 6, and ent-6) and alkyne tag
units (7 and 8, FLAG = DYKDDDDK); (b) photoaffinity labeling of MTJG
using live Samanea motor cells with CMP (5, ent-5, 6, and ent-6;
4 × 10−4 M) and competitive inhibition using 5 (4 × 10−4 M) and
1 (2 × 10−2 M). Int. Std: 52 kDa in colloidal gold total protein stain detec-
tion; (c) competitive inhibition of binding between 5 (4 × 10−4 M) and
MTJG using 1, 9, or 10 (2 × 10−2 M). Int. Std: 52 kDa in colloidal gold
total protein stain detection.
The 7R-ent-6 is an epimer of 5 at the 3-position, and is
expected to be biologically active and have some affinity with
MTJG. Thus, the presence of 7R-ent-6 (5%) can explain the
weak band of MTJG in Fig. 2b observed when using ent-6.
The results of the photoaffinity labeling experiments
demonstrated that the affinity between a CMP and MTJG can
be affected by a structural modification in the D-galactopyranosyl
moiety. Both the aglycone moiety and the D-galactopyranosyl
moiety participate in the molecular recognition of 5 by MTJG.
D-galactosaminopyranosyl moiety showed a weak affinity with
MTJG. The band was weakly observed using ent-6, whereas it
could not be seen using 6, suggesting the importance of the
D-glycopyranosyl moiety for binding with MTJG. Considering
that no cell-shrinking activity was observed in ent-6,20 the
affinity of ent-6 with MTJG may not be strong enough to
induce cell-shrinking. However, these results provided direct
proof that MTJG recognizes the glycone as well as the aglycone.
The weak affinity of ent-6 with MTJG can be attributed to
thermodynamic equilibrium. JAG 1 is known to exist as an
equilibrium mixture of two isomers at the α-position of the
Conclusions
ketone (7R : 7S = 95 : 5),18 and the bioactive form was revealed Our previous SAR studies suggested that leaf-folding activity of
to be 7R-1.18 UPLC-MS analysis revealed that CMP ent-6 also 1 is strongly affected by the stereochemistry of the aglycone
exists as an equilibrium mixture of 7S- and 7R-forms (Fig. 3a,b moiety.18 In this study it was also revealed that the structure
and S4,† 7S : 7R = 95 : 5). The isomer of ent-6 predominantly and stereochemistry of the D-glucopyranosyl moiety of 1 also
observed in solution was confirmed to be the 7S-form from severely affected the cell-shrinking activity as well as its target
NOE correlations29 in the NMR spectrum (Fig. 3c and S5†). affinity. Our results of the SAR studies of 1 revealed the unique
This journal is © The Royal Society of Chemistry 2015
Org. Biomol. Chem., 2015, 13, 55–58 | 57

View Article Online
Communication
Organic & Biomolecular Chemistry
role of its glycone moiety. Cell-shrinking activity and target 13 B. Thines, L. Katsir, M. Melotto, Y. Niu, A. Mandaokar,
affinity of 1 were severely affected by the stereochemistry of
G. Liu, K. Nomura, S. Y. He, G. A. Howe and J. Browse,
both the aglycone and glycone moieties. The stereochemical
Nature, 2007, 448, 661–665.
hybrid 6 with naturally occurring aglycone and the L-galacto- 14 L. B. Sheard, X. Tan, H. Mao, J. Withers, G. Ben-Nissan,
pyranosyl moiety showed no target affinity in photoaffinity
experiments. In contrast, hybrid ent-6 with enantiomeric agly-
cone and the D-galactopyranosyl moiety containing 5% of 7R-
T. R. Hinds, Y. Kobayashi, F. F. Hsu, M. Sharon, J. Browse,
S. Y. He, J. Rizo, G. A. Howe and N. Zheng, Nature, 2010,
468, 400–405.
ent-6 isomer, a 3-epimer of 5, showed a weaker target affinity 15 Y. Nakamura, A. Mithöfer, E. Kombrink, W. Boland,
than 5 with the naturally occurring stereochemistry. These
results suggested that stereochemistries of both the aglycone
and glycone in 1 contribute to the target affinity.
S. Hamamoto, N. Uozumi, K. Tohma and M. Ueda, Plant
Physiol., 2011, 155, 1226–1236.
16 Y. Nakamura, R. Miyatake and M. Ueda, Angew. Chem., Int.
Ed., 2008, 47, 7289–7292.
JAG belongs to a unique class of ligands in which the
glycone moiety plays an important role in the biological 17 F. Cornelius, R. Kanai and C. Toyoshima, J. Biol. Chem.,
activity as well as the affinity with the target protein.30
2013, 288, 6602–6616.
This work was supported by a Grant-in-Aid for Scientific 18 Y. Nakamura, R. Miyatake, S. Inomata and M. Ueda, Biosci.,
Research on Innovative Areas “Chemical Biology of Natural Biotechnol., Biochem., 2008, 72, 2867–2876.
Products” from MEXT, Japan, and a Grant-in-Aid for Scientific 19 M. Ueda, Y. Manabe, Y. Otsuka and N. Kanzawa, Chem. –
Research (no. 26282207) from JSPS, Japan.
Asian J., 2011, 6, 3286–3297.
20 M. Ueda, G. Yang, Y. Ishimaru, T. Itabashi, S. Tamura,
H. Kiyota, S. Kuwahara, S. Inomata, M. Shoji and T. Sugai,
Bioorg. Med. Chem., 2012, 20, 5832–5843.
21 H. C. Kolb, M. G. Finn and K. B. Sharpless, Angew. Chem.,
Int. Ed., 2001, 40, 2004–2021.
22 C. W. Tornøe, C. Christensen and M. Meldal, J. Org. Chem.,
2002, 67, 3057–3064.
23 J. A. Codelli, J. M. Baskin, N. J. Agard and C. R. Bertozzi,
J. Am. Chem. Soc., 2008, 130, 11486–11493.
Notes and references
1 A. Devoto and J. G. Turner, Ann. Bot., 2003, 92, 329–337.
2 C. Wasternack, Ann. Bot., 2007, 100, 681–697.
3 J. Browse, Phytochemistry, 2009, 70, 1539–1546.
4 C. Wasternack and E. Kombrink, ACS Chem. Biol., 2009, 5,
63–77.
5 M. Ueda and S. Yamamura, Angew. Chem., Int. Ed., 2000,
39, 1400–1414.
24 Y. Manabe, M. Mukai, S. Ito, N. Kato and M. Ueda, Chem.
Commun., 2010, 46, 469–471.
6 M. Ueda and Y. Nakamura, Nat. Prod. Rep., 2006, 23, 548–557.
7 M. Ueda and Y. Nakamura, Plant Cell Physiol., 2007, 48,
900–907.
8 M. Ueda, M. Okazaki, K. Ueda and S. Yamamura, Tetra-
hedron, 2000, 56, 8101–8105.
9 C. Darwin and F. Darwin, The power of movement in plants,
third thousand, John Murray, London, 1882.
10 R. L. Satter and A. W. Galston, Annu. Rev. Plant Physiol.,
1981, 32, 83–110.
11 N. Moran, FEBS Lett., 2007, 581, 2337–2347.
12 A. Chini, S. Fonseca, G. Fernandez, B. Adie, J. M. Chico,
O. Lorenzo, G. Garcia-Casado, I. Lopez-Vidriero,
F. M. Lozano, M. R. Ponce, J. L. Micol and R. Solano,
Nature, 2007, 448, 666–671.
25 Y. Nakamura, S. Inomata, M. Ebine, Y. Manabe, I. Iwakura
and M. Ueda, Org. Biomol. Chem., 2011, 9, 83–85.
26 M. Ueda, Y. Manabe and M. Mukai, Bioorg. Med. Chem.
Lett., 2011, 21, 1359–1362.
27 K. E. Beatty, J. D. Fisk, B. P. Smart, Y. Y. Lu, J. Szychowski,
M. J. Hangauer, J. M. Baskin, C. R. Bertozzi and
D. A. Tirrell, ChemBioChem, 2010, 11, 2092–2095.
28 P. V. Chang, J. A. Prescher, E. M. Sletten, J. M. Baskin,
I. A. Miller, N. J. Agard, A. Lo and C. R. Bertozzi, Proc. Natl.
Acad. Sci. U. S. A., 2010, 107, 1821–1826.
29 H. Seto, E. Nomura, S. Fujioka, H. Koshino, T. Suenaga and
S. Yoshida, Biosci., Biotechnol., Biochem., 1999, 63, 361–
367.
30 M. Ueda, Chem. Lett., 2012, 41, 658–666.
58 | Org. Biomol. Chem., 2015, 13, 55–58
This journal is © The Royal Society of Chemistry 2015