Discovery of synthetic small molecules that enhance the number of stomata: C-H functionalization chemistry for plant biology

Article abstract of DOI:10.1039/c7cc04526c

The increasing climate changes and global warming are leading to colossal agricultural problems such as abatement of food production and quality. As stomatal development is considered to play a key role in crop plant productivity and water-use efficiency,

Full text of DOI:10.1039/c7cc04526c

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Discovery of synthetic small molecules that
enhance the number of stomata: C–H
Cite this:DOI: 10.1039/c7cc04526c
functionalization chemistry for plant biology†
Received 11th June 2017,
Accepted 7th August 2017
Asraa Ziadi,a Naoyuki Uchida,ab Hiroe Kato,a Rina Hisamatsu,ab Ayato Sato,a
abd
abe
Shinya Hagihara,abc Kenichiro Itami
*
and Keiko U. Torii
*
DOI: 10.1039/c7cc04526c
rsc.li/chemcomm
The increasing climate changes and global warming are leading to stomatal density has been considered as a target trait suitable
colossal agricultural problems such as abatement of food produc- for plant engineering, one idea would be to use such peptides to
tion and quality. As stomatal development is considered to play a control the stomatal number. Indeed, genetic modification to
key role in crop plant productivity and water-use efficiency, studying increase peptide amounts that are known to alter the numbers
stomatal development is useful for understanding the productivity of of stomata could improve the plant water-use efficiency (when
plant systems for both natural and agricultural systems. Herein, we the stomatal numbers are reduced) or photosynthetic capacity
report the first-in-class synthetic small molecules enhancing the (when the numbers of stomata are increased).5 However, growing
number of stomata in Arabidopsis thaliana that have been discovered genetically-modified crop plants in the field may be subject to
by screening of the chemical library and further optimized by the restrictions. An alternative approach would be to use synthetic
Pd-catalyzed C–H arylation reaction. The present study shows not small molecules to manipulate the stomatal number. For example,
only huge potential of small molecules to control the cellular stomata can be increased or decreased by the application of the
and developmental processes of stomata without using genetically synthetic peptide hormones,5e–h though it is not realistic to use
modified plants, but also the power of C–H functionalization peptides for agricultural purpose due to the cost of synthesis. Very
chemistry to rapidly identify the optimized compounds.
recently, a small molecule, bubblin, which increases stomata has
been reported.7 However, this compound exerts severe inhibition
Stomata are epidermal pores essential for the growth and of seedling growth, thereby excluding its potential use for improv-
survival of land plants. They are surrounded by two guard cells, ing the plant productivity. Therefore, the development of small
which act as gates to balance the gas exchange for photosynthesis molecules that increase the stomatal number without side effects
against the loss of water vapor.1 Considering that 40% of the is an urgent issue.
atmospheric CO2 passes through stomata every year,2 mani-
Herein, we report the discovery of small molecules that
pulation of stomatal development and function plays a key role increase the numbers of stomata. Through derivatization of the
in crop plant productivity and water-use efficiency. Previous original hit compound via Pd-catalyzed C–H functionalization, we
reports have shown that the number of stomata is influenced by successfully separated the potency for elevating stomatal differ-
environmental conditions, such as light3 and atmospheric CO2 entiation from the side effects of growth inhibition.
levels.4 To this end, plants use endogenous peptide hormones
We began our study by applying our specially curated ITbM
to regulate the number of stomata on the leaf surface.5 In chemical library including 80 compounds from the LOPACs
addition, the plant steroid hormone brassinosteroids modulate Pfizer chemical library8 toward plant-based phenotypic screening.
stomatal development via acting on peptide signaling.6 As the We counted the number of stomata on the leaves of Arabidopsis
thaliana treated with compounds for 9 days and successfully
identified compounds CL1 and CL2 as stomatal development
enhancing molecules (Fig. 1). Interestingly, these molecules
a Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University,
Chikusa, Nagoya, 464-8601, Japan. E-mail: itami@chem.nagoya-u.ac.jp,
have a similar structure to Celecoxibr, which is a non-steroidal
anti-inflammatory drug. The target of Celecoxib in humans is
cyclooxygenase-2 (COX-2) that produces prostaglandin.9 However,
higher plants including Arabidopsis thaliana possess neither ortho-
logs of the COX-2 gene nor prostaglandin. The stomata increasing
effect of CL1 and CL2 might arise from their interactions with
ktorii@u.washington.edu
b Graduate School of Science, Nagoya University, Chikusa, Nagoya, 464-8602, Japan
c JST, PRESTO, Chikusa, Nagoya, 464-8602, Japan
d JST, ERATO, Itami Molecular Nanocarbon Project, Chikusa, Nagoya, 464-8602,
Japan
e Howard Hughes Medical Institute, University of Washington, Seattle,
WA 98195-1800, USA
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c7cc04526c plant-specific proteins. Although CL1 and CL2 increased the
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Fig. 1 (a) Library-based discovery of compounds CL1 and CL2 that increase
the number of stomata, yet toxic. (b) Summary of the initial structure–activity
relationship study.
number of stomata on the plant leaves, they conferred adverse
effects at high concentrations (Fig. 1a). Seedlings remain smaller
with chlorosis when CL1 was applied at optimal concentrations
for the stomata increasing effect (Fig. 1a). We therefore strate-
gized our efforts to develop new compounds that can increase
the numbers of stomata while minimizing the toxicity at high
concentrations.
We first synthesized the analogues lacking a trifluoromethyl
(CF3) group in the C3-position (Fig. 1b, ZA155) or the aryl group
in the C5-position of the pyrazole core (Fig. 1b, ZA099). In the
case of ZA155, growth inhibition was observed whereas ZA099 did
not inflict any visual growth inhibition on the plant (see ESI† for
details). These results led us to postulate that further derivations
on C5-position of the pyrazole while keeping its CF3 group in the
C3-position may achieve increased number of stomata on plant
leaves without growth inhibition. With our established back-
ground in C–H functionalization chemistry,10–12 we chose to
approach the synthesis of new small molecules with different aryl
groups on the C5-position by C–H arylation of ZA099 rather than
relying on time-consuming conventional multi-step synthesis.13
We initiated our methodology development with systematic
screening of the different experimental variables to achieve
efficient C–H arylation of ZA099 with aryl bromides under
palladium catalysis.14 After extensive screening of the ligand,
base, solvent and temperature, the optimized conditions using
a Pd(OAc)2/BINAP system were identified (Fig. 2a, Tables S1–S3,
Fig. 2 (a) Optimized conditions for the C–H arylation of ZA099 with
bromobenzene and the effect of reaction parameters. (b) Arylpyrazole
derivatives synthesized under standard conditions.
Having established the optimized reaction conditions, we
ESI†). Thus, when ZA099 (1.0 equiv.) was treated with bromo- synthesized a range of arylpyrazole derivatives from ZA099 and the
benzene (2.0 equiv.), KOAc (3.0 equiv.), Pd(OAc)2 (5.0 mol%), corresponding aryl bromides. The conditions are not optimized
and BINAP (7.5 mol%) in DMA at 100 1C, the target phenylated for each derivative and the representative analogues that are
pyrazole ZA138 was obtained in 92% GC yield (Fig. 2a). Listed in useful in the following structure–activity relationship study are
Fig. 2a are the effects of reaction parameters. The use of ligands shown in Fig. 2b. Both electron-withdrawing (compound ZA140)
other than BINAP (PPh3, PCy3, SPhos) resulted in lower yields and electron-donating (compounds ZA141, ZA142, ZA144 and
(entries 1–3). The base exerted a profound influence on the ZA146) substituents were well tolerated under the reaction condi-
reaction, since the absence of it inhibited the reaction (entry 4). tions and were obtained in up to 88% isolated yield. Interestingly,
KOAc was preferred over K3PO4 (entries 5 and 6) possibly even 4-chlorobromobenzene was tolerated and gave access to
indicating a concerted metalation deprotonation (CMD) type ZA139 without losing the C–Cl moiety. Sterically hindered ZA144
mechanism.15,16 The use of polar solvents (DMA or DMF) is also could also be formed, albeit in low yield.
important; less polar solvents such as toluene and cyclohexane
resulted in much lower yields (entries 7–9).
With these analogues in hand, we examined their bioactivity
on stomatal numbers using Arabidopsis seedlings expressing
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substituted compounds (ZA142 and ZA144, Fig. 2b). p-Anisyl
substituted ZA142 did not show promising results (ESI†), but
o-anisyl substituted ZA144, on the other hand, elicited the highest
stomata-increasing activity and did not show any toxicity.
Furthermore, when comparing the dry mass (biomass) of
ZA144 to ZA139 and CL1, we were able to confirm that, in a similar
fashion to DMSO, ZA144 does not confer growth inhibition on
the plant (Fig. 3c). Altogether, we identified ZA144 as the most
effective molecule in increasing the number of stomata without
severe toxicity and could be used to manipulate plant growth and
productivity. While the mode of action of these novel small mole-
cules is yet to be uncovered, this campaign clearly demonstrates
the power of synthetic small molecules in exploring plant biology.
In summary, a rapid C–H functionalization methodology made
it possible to identify a new bioactive small molecule that can
enhance the number of stomata on plant leaves without inhibiting
the plant growth in up to 25 mM concentration. In the new
Pd(OAc)2/BINAP catalytic system, we were able to easily access a
series of derivatized novel small molecules and separate the
desirable bioactivity of promoting stomatal development from
the side effects of growth inhibition. Future identification of
target(s) of ZA144 may provide the molecular basis of pyrazole-
mediated stomatal differentiation and increase in biomass. Our
work emphasizes that, with the recent advent of a number of
game-changing C–H functionalization reactions, there are signifi-
cant opportunities to use these technologies to accelerate plant
biology and agricultural science.
This work was supported by the ERATO program (JPMJER1302)
from JST (K. I.), the PRESTO program (JPMJPR15Q9) from JST
(S. H.), Howard Hughes Medical Institute (HHMI) and Gordon
and Betty Moore Foundation (GBMF3035) (K. U. T.) and JSPS
KAKENHI grant (JP26291057, JP16H01237 and JP17H06476 to
K. U. T.; JP16H01462, JP17H03695 and JP17KT0017 to N. U.;
JP17H06350 to S. H.). ITbM is supported by the World Premier
International Research Center Initiative (WPI), Japan.
Fig. 3 The effect of the different compounds on Arabidopsis thaliana in
terms of (a) toxicity and (b) stomatal numbers. (c) The sum for the dry
weight of 16 seedlings treated with CL1, ZA144, or ZA139 are represented.
Dashes lines in (b) indicate the average values of DMSO-treatment and
ZA099-treatment. Asterisks indicate significant differences by Student’s t-test
(two-tailed) compared to ZA099-treatment in (b) and DMSO-treatment in (c):
ns for P value 40.05, * for P value 40.05, and ** for P value o0.01.
Conflicts of interest
guard-cell specific green fluorescent protein GFP (Fig. 3).17
After allowing the seeds to grow for 9 days in the presence of
these small molecules, the youngest leaves of the newly grown
plants were observed under a fluorescence microscope (Fig. 3b).
Compound ZA139 generated the highest stomatal density (number
of stomata per mmÀ2) on cotyledons compared to the other
compounds tested (Fig. 3b). It was, however, extremely toxic to
the plant with abnormal stomata shape (Fig. 3b and ESI†). The
lower dry weight of seedlings cultured in the presence of ZA139
shows the severe effect of ZA139 on plant growth (Fig. 3a and c).
Another compound worth mentioning is ZA143 (Fig. 1), which
gave a small increase in stomata numbers and was not severely
toxic to the plant (ESI†). In order to increase the stomata
numbers, we thought that perhaps the sulfonamide analogue
(ZA160, Fig. 3a) would perform better. The compound, however,
did not increase the number of stomata on plant leaves and
led to growth inhibition (Fig. 3b). We, therefore, turned our
attention towards synthesizing and testing different anisole
There are no conflicts to declare.
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