Synthesis and inhibitory activity of mechanism-based 4-coumaroyl-CoA ligase inhibitors

Article abstract of DOI:10.1016/j.bmc.2018.04.006

4-Coumaroyl-CoA ligase (4CL) is ubiquitous in the plant kingdom, and plays a central role in the biosynthesis of phenylpropanoids such as lignins, flavonoids, and coumarins. 4CL catalyzes the formation of the coenzyme A thioester of cinnamates such as 4-coumaric, caffeic, and ferulic acids, and the regulatory position of 4CL in the phenylpropanoid pathway renders the enzyme an attractive target that controls the composition of phenylpropanoids in plants. In this study, we designed and synthesized mechanism-based inhibitors for 4CL in order to develop useful tools for the investigation of physiological functions of 4CL and chemical agents that modulate plant growth with the ultimate goal to produce plant biomass that exhibits features that are beneficial to humans. The acylsulfamide backbone of the inhibitors in this study was adopted as a mimic of the acyladenylate intermediates in the catalytic reaction of 4CL. These acylsulfamide inhibitors and the important synthetic intermediates were fully characterized using two-dimensional NMR spectroscopy. Five 4CL proteins with distinct substrate specificity from four plant species, i.e., Arabidopsis thaliana, Glycine max (soybean), Populus trichocarpa (poplar), and Petunia hybrida (petunia), were used to evaluate the inhibitory activity, and the half-maximum inhibitory concentration (IC₅₀) of each acylsulfamide in the presence of 4-coumaric acid (100 μM) was determined as an index of inhibitory activity. The synthetic acylsulfamides used in this study inhibited the 4CLs with IC₅₀ values ranging from 0.10 to 722 μM, and the IC₅₀ values of the most potent inhibitors for each 4CL were 0.10–2.4 μM. The structure–activity relationship observed in this study revealed that both the presence and the structure of the acyl group of the synthetic inhibitors strongly affect the inhibitory activity, and indicates that 4CL recognizes the acylsulfamide inhibitors as acyladenylate mimics.

Full text of DOI:10.1016/j.bmc.2018.04.006

Accepted Manuscript
Synthesis and inhibitory activity of mechanism-based 4-coumaroyl-CoA ligase
inhibitors
Bunta Watanabe, Hiroaki Kirikae, Takao Koeduka, Yoshinori Takeuchi,
Tomoki Asai, Yoshiyuki Naito, Hideya Tokuoka, Shinri Horoiwa, Yoshiaki
Nakagawa, Bun-ichi Shimizu, Masaharu Mizutani, Jun Hiratake
PII:
S0968-0896(18)30152-4
https://doi.org/10.1016/j.bmc.2018.04.006
BMC 14289
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
25 January 2018
2 April 2018
3 April 2018
Please cite this article as: Watanabe, B., Kirikae, H., Koeduka, T., Takeuchi, Y., Asai, T., Naito, Y., Tokuoka, H.,
Horoiwa, S., Nakagawa, Y., Shimizu, B-i., Mizutani, M., Hiratake, J., Synthesis and inhibitory activity of
mechanism-based 4-coumaroyl-CoA ligase inhibitors, Bioorganic & Medicinal Chemistry (2018), doi: https://
doi.org/10.1016/j.bmc.2018.04.006
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Synthesis and inhibitory activity of mechanism-based 4-coumaroyl-CoA ligase inhibitors
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Bunta Watanabea, *, Hiroaki Kirikaea, Takao Koedukaa, 1, Yoshinori Takeuchia, Tomoki Asaia,
Yoshiyuki Naitoa, Hideya Tokuokab, Shinri Horoiwab, Yoshiaki Nakagawab, Bun-ichi Shimizua, 2,
Masaharu Mizutania, 3, Jun Hiratakea
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a Institute for Chemical Research, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan
b Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto
606-8502, Japan
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* Corresponding author; Tel.: +81-774-38-3233; Fax.: +81-774-38-3229; E-mail address:
bunta@scl.kyoto-u.ac.jp
Present addresses: 1 Graduate School of Sciences and Technology for Innovation, Yamaguchi
University, Yamaguchi 753-8515, Japan; 2 Graduate School of Life Sciences, Toyo University,
1-1-1-Izumino, Itakura-machi, Oga-gun, Gunma 374-0193, Japan; 3 Graduate School of
Agricultural Science, Kobe University, Rokkodai 1-1, Nada-ku, Kobe, Hyogo 657-8501, Japan
Keywords: 4-coumaroyl-CoA ligase; mechanism-based inhibitor; ANL superfamily; acylsulfamide
Abbreviations: ANL, acyl- and aryl-CoA synthetases, nonribosomal peptide synthetase adenylation
domains, luciferase; CDI, 1,1′-carbonyldiimidazole; 4CL, 4-coumaroyl-CoA ligase; CoA,
coenzyme A; DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; MEM, 2-methoxyethoxymethyl; rt, room
temperature; TMS, tetramethylsilane; Z, benzyloxycarbonyl.
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Abstract
4-Coumaroyl-CoA ligase (4CL) is ubiquitous in the plant kingdom, and plays a central role in the
biosynthesis of phenylpropanoids such as lignins, flavonoids, and coumarins. 4CL catalyzes the
formation of the coenzyme A thioester of cinnamates such as 4-coumaric, caffeic, and ferulic acids,
and the regulatory position of 4CL in the phenylpropanoid pathway renders the enzyme an
attractive target that controls the composition of phenylpropanoids in plants. In this study, we
designed and synthesized mechanism-based inhibitors for 4CL in order to develop useful tools for
the investigation of physiological functions of 4CL and chemical agents that modulate plant growth
with the ultimate goal to produce plant biomass that exhibits features that are beneficial to humans.
The acylsulfamide backbone of the inhibitors in this study was adopted as a mimic of the
acyladenylate intermediates in the catalytic reaction of 4CL. These acylsulfamide inhibitors and the
important synthetic intermediates were fully characterized using two-dimensional NMR
spectroscopy. Five 4CL proteins with distinct substrate specificity from four plant species, i.e.,
Arabidopsis thaliana, Glycine max (soybean), Populus trichocarpa (poplar), and Petunia hybrida
(petunia), were used to evaluate the inhibitory activity, and the half-maximum inhibitory
concentration (IC50) of each acylsulfamide in the presence of 4-coumaric acid (100 µM) was
determined as an index of inhibitory activity. The synthetic acylsulfamides used in this study
inhibited the 4CLs with IC50 values ranging from 0.10 to 722 µM, and the IC50 values of the most
potent inhibitors for each 4CL were 0.10 to 2.4 µM. The structure-activity relationship observed in
this study revealed that both the presence and the structure of the acyl group of the synthetic
inhibitors strongly affect the inhibitory activity, and indicates that 4CL recognizes the acylsulfamide
inhibitors as acyladenylate mimics.
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1. Introduction
4-Coumaroyl-CoA ligase (4CL, EC 6.2.1.12) plays a pivotal role in the phenylpropanoid pathway
via the formation of coenzyme A (CoA) thioesters of cinnamates, i.e., key precursors for various
classes of important secondary metabolites such as lignins, flavonoids, and coumarins. The 4CL
proteins, which are widespread in the plant kingdom, have been obtained from angiosperm dicot
families such as Apiaceae,1 Brassicaceae,2-4 Fabaceae,5, 6 Lamiaceae,7 Moraceae,8 Rosaceae,9, 10
Rutaceae,11 Salicaceae,12-14 Solanaceae,15-17 as well as from the monocot families Poaceae,18-20
gymnosperm Pinaceae,21, 22 and bryophyte plants,23, 24 and their enzymatic properties have been
characterized.
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In the aforementioned plants, more than two isoforms of 4CL are identified frequently. These are
often considered to play distinct plant-specific physiological functions, and these functions are
sometimes correlated with the localization of isoforms. In Populus tremuloides (aspen), for example,
the expression of Pt4CL1 is specific in lignifying xylem and associated with the lignin biosynthesis,
whereas Pt4CL2 mainly expresses in the epidermal cells of stem and leaf, and is involved in the
biosynthesis of phenylpropanoids except lignins.12 To date, 4CL proteins from dicot plants are
generally categorized into two evolutionary divergent classes:2 while class I 4CLs are regarded as
key enzymes in the lignin biosynthesis, class II 4CLs concern the biosynthesis of other
phenylpropanoids such as flavonoids, volatile benzenoids,16 and coumarins that confer protection
from UV, wounding, and pathological fungi, as well as the attraction of pollinators. A phylogenetic
classification of 4CLs specific for monocot,18, 20, 25, 26 gymnosperm,22 and bryophyte plants23 has
been proposed.
As suggested by the name, almost all 4CLs recognize 4-coumaric acid as a good to excellent
substrate. Caffeic and ferulic acids generally serve as good substrates, but strong preferences are
sometimes observed. For example, At4CL1 and At4CL2 from Arabidopsis thaliana both belong to
class I and catalyze the ligation of caffeic acid, although only the former also reacts with ferulic
acid.2 Moreover, only a minority of 4CL enzymes recognizes cinnamic acid as a good substrate.9, 11
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The CoA thioester formation of cinnamic acid is catalyzed predominantly by cinnamic acid:CoA
ligase, which is evolutionally distinct from 4CL.16 To our best knowledge, only two 4CLs, i.e.,
Gm4CL1 from Glycine max (soybean)5 and At4CL4 from A. thaliana,3 can effectively catalyze the
ligation of sinapic acid, and the physiological role of these sinapic acid-activating 4CLs still
remains unclear. The crystal structures of 4CL clearly show that the substrate specificity mainly
arises from the size of the binding pocket of the cinnamates,27, 28 and that it is possible to broaden
the reactivity of 4CL based on protein-engineering techniques.29-31
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The regulatory position of 4CL in the biosynthesis of phenylpropanoids renders the enzyme an
attractive target that controls the composition of the products of the phenylpropanoid pathway in
plants. Among the phenylpropanoids lignins have attracted the most attention, and the decrease of
lignin content induced by the suppression of the functions of 4CL has been reported for A.
thaliana,32-35 Nicotiana tabacum (tobacco),36, 37 Oryza sativa (rice),18 Panicum virgatum
(switchgrass),19 Pinus radiate (radiata pine),38 Populus spp. (poplars),39-42 and Saccharum spp.
(sugarcane).26 These results are sometimes correlated to the improvement of the biomass quality,19,
26, 34 since the rigidity of lignins may render the digestion of biomass difficult during the production
of biofuels. The decrease of the content of other phenylpropanoids such as eugenol,7 chlorogenic
acid, and tannins42 via the suppression of the 4CL functions has also been reported. Therefore, the
control of the 4CL functions potentially represents a promising approach to generate plants, which
contain modified contents/compositions of a wide variety of phenylpropanoids, and thus offer
properties that are beneficial to humans.
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To date, almost all attempts to regulate the functions of 4CL have been conducted via genetic
techniques, and chemical approaches using 4CL-specific inhibitors have not yet been developed
(vide infra). However, the chemical regulation of enzyme functions using specific inhibitors usually
offers advantages over genetic methods. One can simply start and stop the regulation of target
enzymes by choosing the time and the duration of the application of the inhibitor, while the
intensity of the application can be controlled via the dose of the inhibitor. Moreover, simultaneous
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regulation of more than two kinds/isoforms of enzymes can be easily achieved by employing an
appropriate set of inhibitors. Therefore, 4CL-specific inhibitors should be useful tools for the
investigation of the physiological roles of 4CL, as well as for the regulation of the phenylpropanoid
pathway in order to generate plants with properties that are beneficial to humans.
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It has been reported that one substrate of a 4CL enzyme inhibits the catalytic reactions of the 4CL
enzyme with other substrates. In case of the poplar 4CLs, for example, caffeic acid strongly inhibits
the ligation of 4-coumaric acid and ferulic acid catalyzed by Pt4CL1 from P. tremuloides 43 and
Ptr4CL3/5 from P. trichocarpa.14 However, such inhibitions cannot suppress the pathways starting
from the substrates used as inhibitors. Yogo and co-workers have reported that the herbicides
propanil and swep inhibit a tobacco 4CL (Nt4CL1) expressed in Escherichia coli, as well as other
4CLs extracted from several plants.44, 45 Products of the phenylpropanoid pathway such as chalcone,
naringenin, and coniferin have also been reported to inhibit 4CLs.17, 21, 46 However, these
non-substrate inhibitors are not specific to 4CL, as propanil and swep are well-known inhibitors of
the photosystem II,45 while chalcone also inhibits phenylalanine ammonia lyase.47
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In order to develop 4CL-specific inhibitors, we focused on the catalytic mechanism of 4CL. 4CL
belongs to the ANL superfamily of enzymes (acyl- and aryl-CoA synthetases, nonribosomal peptide
synthetase adenylation domains, and firefly luciferase).48 The characteristic aspect of this family is
that these members activate carboxylate substrates with ATP to form acyladenylate intermediates,
which are used in a diverse set of second partial reactions.49 The schematic illustration of the
ligation reactions via acyladenylate intermediates catalyzed by ANL enzymes are depicted in Figure
1. The enzymes accept carboxylate substrates and ATP in the adenylate-forming conformation; the
resulting acyladenylate intermediates react with CoA in the thioester-forming conformation. Due to
this common catalytic mechanism, mimics of the acyladenylate intermediates are expected to act as
inhibitors specific to the ANL superfamily of enzymes. In fact, acylsulfamide analogues synthesized
as acyladenylate intermediate mimics, have been reported to inhibit ANL enzymes in vivo50 and in
vitro.51 In these acylsulfamide analogues, the phosphonate moiety of the acyladenylate
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intermediates is replaced with a sulfamide moiety, which is resistant to further enzymatic reactions.
In the present study, we adopted this acylsulfamide strategy, and designed novel acylsulfamide
analogues as mechanism-based 4CL inhibitors (Figure 1). In order to unveil the effect of
substituents on the benzene ring of the cinnamate moiety against the 4CL inhibitory activity, the
acyl groups of 1a–d were varied to include cinnamic (1a), 4-coumaric (1b), ferulic (1c), and sinapic
(1d) acid. Moreover, 1e, which bears an octanoic acid as an acyl group, and AMP mimic 1e, were
designed to determine the presence of the cinnamate-specific recognition mechanism of 4CL. The
inhibitory activity of the synthetic acylsulfamide analogues was evaluated using five distinct 4CL
proteins with different substrate specificity. It is worth mentioning that the substrate specificity of
each 4CL should correlate with the structure of the cinnamate binding pocket in the
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adenylate-forming conformation. As 1a–d are expected to act as mimics of the acyladenylate
intermediate, the inhibitory activity of 1a–d should reflect the affinity of the 4CLs against the
corresponding acyladenylate intermediates in the thioester-forming conformations.
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Figure 1. Schematic illustration of the ligation reactions via acyladenylate intermediates catalyzed
by ANL enzymes, and structures of the acylsulfamide analogues (1a–f) used in this study.
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2. Results and discussion
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2.1 Synthesis
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The synthetic route to 1a–d is outlined in Scheme 1. The synthesis of 1a is straightforward, i.e.,
1-cinnamoylimidazole (2a), prepared from cinnamic acid and 1,1′-carbonyldiimidazole (CDI), was
condensed with the protected AMP analogue 3 in the presence of
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1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) to furnish fully protected intermediate 4a, which was
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hydrolyzed under acidic conditions to afford 1a.
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Scheme 1. Synthesis of 1a–d; reagents and conditions: (a) DBU, MeCN, rt, 57–70%; (b)
HCO2H/H2O (4:1, v/v), rt, 33–98%.
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In the synthesis of 1b–d, the phenolic hydroxy groups of the 4-coumaric, ferulic, and sinapic acid
moieties were protected prior to condensation (Scheme 2). For that purpose, these cinnamates were
treated with an excess of 2-methoxyethoxymethyl chloride (MEMCl) in the presence of
ethyldiisopropylamine. The crude products, in which the carboxy groups were esterified, were
converted into the carboxylic acids 5b–d via basic hydrolysis prior to treatment with CDI. The thus
obtained acylimidazoles (2b–d) were condensed with 3, and subsequently subjected to acidic
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hydrolysis, which furnished inhibitors 1b–d (Scheme 1).
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Scheme 2. Synthesis of acylimidazoles 2b–d; reagents and conditions: (a) MEMCl, (i-Pr)2NEt,
CH2Cl2, rt; (b) NaOH, MeOH/H2O, rt, 72–84% (two steps); (c) CDI, MeCN, rt, 76%–quant.
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The synthesis of 1e and 1f is illustrated in Scheme 3. A procedure similar to that for the formation
of 1a was employed for the synthesis of 1e, i.e., 1-octanoylimidazole52 was condensed with 3 to
generate 4e, which was treated with aqueous formic acid to furnish 1e. The corresponding AMP
analogue (1f) was directly synthesized from 3 via acidic hydrolysis.
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Scheme 3. Synthesis of 1e and 1f; reagents and conditions: (a) 1-octanoylimidazole, DBU, MeCN,
rt, 79%; (b) HCO2H/H2O (4:1, v/v), rt, 36–97%.
As illustrated in Scheme 4, 3, which is the key synthetic intermediate in this study, was synthesized
from 2′,3′-O-isopropylideneadenosine (6). According to previous reports,53 6 was converted into
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amine 7 in two steps. Previously, 7 was directly transformed into 3 by the treatment of sulfamoyl
chloride.54 Even though this preparation requires only a single step, chromatographic purification is
required, and the reported yield (34%) is not satisfactory. In this study, 7 was initially treated with
N-(benzyloxycarbonyl)sulfamoyl chloride, which was prepared in situ by the reaction between
chlorosulfonyl isocyanate and benzyl alcohol, to give the protected sulfamide 8 in 82% yield.
Subsequently, the benzyloxycarbonyl (Z) group on the sulfamide nitrogen atom of 8 was removed
by hydrogenolysis to afford 3 in 81% yield. In this procedure, chromatographic purification was not
necessary in either step, due to the high purity of the products, and the overall yield (66%) was
improved by a factor of ~2 relative to the previously reported method.
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Scheme 4. Synthesis of 3; reagents and conditions: (a) ref. 53; (b) BnOH, OCNSO2Cl, CH2Cl2, 0
ºC–rt, 82%; (c) H2, 10% Pd-C, AcOH, rt, 81%.
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2.2. Evaluation of the inhibitory activity and structure-activity relationship
The inhibitory activity of the synthetic acylsulfamide analogues was evaluated using five 4CLs with
distinct substrate specificity, i.e., At4CL1 and At4CL2 from A. thaliana,2 Gm4CL1 from G. max,5
Ptr4CL3 from P. trichocarpa,14 and Ph4CL1 from Petunia hybrida (petunia).16 These 4CL proteins
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were conventionally expressed in E. coli and purified. The 4CL activity was measured via a
previously described spectrophotometric assay.55 4-Coumaric acid (100 µM), ATP, and CoA (250
µM each) were used as substrates. The 4CL activity was recorded in the presence of inhibitors at
various concentrations, and the half-maximum inhibitory concentration (IC50) determined from
dose-response curves was used as an index of inhibitory activity. The corresponding results are
summarized in Table 1. Recently, Li and Nair have reported the crystal structures of Nt4CL2
complexed with the acyladenylate intermediates in the thioester-forming conformations.28 Based on
their results we designed our synthetic inhibitors to mimic the acyladenylate intermediates, the
inhibitory activity of the synthetic acylsulfamides should reflect the affinity of the 4CLs against the
acyladenylate intermediates in the thioester-forming conformations, whereas the substrate
specificity of each 4CL should correlate with the structure of the binding pocket of the cinnamates
in the adenylate-forming conformation.
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Table 1. Inhibitory activity of 1a–f against 4CLs.
IC50 (µM)a
Inhibitor
Acyl group
At4CL1
At4CL2
Gm4CL1
69.5 ± 0.5
0.55 ± 0.01
0.10 ± 0.01
0.18 ± 0.03
249 ± 13
Ptr4CL3
Ph4CL1
cinnamoyl
4-coumaroyl
feruloyl
sinapoyl
octanoyl
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25.1 ± 2.8
0.26 ± 0.02
5.5 ± 0.3
58.2 ± 4.6
361 ± 39
> 1000
38.0 ± 4.3
2.4 ± 0.3
34.5 ± 3.4
56.8 ± 3.5
722 ± 54
> 1000
24.9 ± 3.3
0.87 ± 0.07
3.9 ± 0.1
55.2 ± 4.1
92.6 ± 4.2
> 1000
4.8 ± 0.9
0.38 ± 0.04
1.5 ± 0.2
62.2 ± 1.5
25.1 ± 0.8
> 1000
1a
1b
1c
1d
1e
1f
> 1000
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a) Mean values ± standard deviation (n = 3).
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Against At4CL1, 1b exhibited a strong inhibitory activity (IC50: 0.26 µM), and 1c also acted as a
potent inhibitor (IC50: 5.5 µM). Against At4CL2, 1b was the most potent inhibitor (IC50: 2.4 µM),
while 1c was less active. Compared to At4CL1, the sensitivity of At4CL2 against 1b decreases by
approximately one order of magnitude. These results should reflect the substrate specificity of
At4CL1/2:2 i) the preference of At4CL1 for 4-coumaric acid is ten times higher than that for ferulic
acid, ii) the interaction of At4CL1 with 4-coumaric acid approximately seven times higher than that
of At4CL2, and iii) At4CL2 does not react with ferulic acid. Both 1a and 1d weakly inhibit
At4CL1/2, which uses cinnamic acid as a poor substrate and shows no activity against sinapic acid.2
Replacement of the substrate-type acyl groups of 1a–d with an octanoyl group renders 1e almost
inactive against At4CL1/2. These results indicate that the structure of the cinnamate binding pocket
of At4CL1/2 in the thioester-forming conformation merely accepts the octanoyl group, and,
therefore, the ligation reactions catalyzed by At4CL1/2 should be specific to cinnamates.
As expected form the wide substrate specificity of Gm4CL1, which is one of the few 4CLs that
exhibits high activity against sinapic acid, 1c (IC50: 0.10 µM), 1d (IC50: 0.18 µM), and 1b (IC50:
0.55 µM) showed strong inhibitory activity. It is known that Gm4CL1 efficiently catalyzes the
ligation reactions of ferulic, sinapic, and 4-coumaric acids.5 The low inhibitory activity of 1a
against Gm4CL1 is consistent with the poor reactivity of the enzyme against cinnamic acid,5 and
these results suggest that the hydroxy groups at the 4-position of the benzene ring of 1b–d play an
important role in the substrate recognition of the enzyme. The ligation reactions of Gm4CL1 can be
cinnamate specific, as in the cases of At4CL1/2, considering that 1e only shows a marginal activity
against Gm4CL1.
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Against Ptr4CL3, 1b (IC50: 0.87 µM) and 1c (IC50: 3.9 µM) exhibit strong and potent inhibitory
activity, respectively, while that of 1d is less active. These results are consistent with the substrate
specificity of Ptr4CL3, for which 4-coumaric acid is the most preferable and ferulic acid is a good
substrate, while sinapic acid shows no reactivity.14 However, no data was reported with respect to
cinnamic acid, even though cinnamic acid is expected to be a poor substrate for Ptr4CL3, given that
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1a does not inhibit the enzyme effectively. The inhibitory activity of 1e against Ptr4CL3 is very
weak (IC50: 92.6 µM), although Ptr4CL3 is approximately 2.5–7 times more sensitive against 1e
compared to At4CL1/2 and Gm4CL1. This result suggests that the structure of the cinnamate
binding pocket of Ptr4CL3 in the thioester-forming conformation is slightly different from that of
At4CL1/2 and Gm4CL1.
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Compared to the other 4CLs, Ph4CL1 is more sensitive against 1a (IC50: 4.8 µM), which is
approximately 13-fold weaker than the most potent 1b (IC50: 0.38 µM). The difference is consistent
with the substrate specificity of Ph4CL1, i.e., the catalytic activity of Ph4CL1 against 4-coumaric
acid is approximately 13 times higher than that against cinnamic acid.16 With respect to 4-coumaric
and ferulic acids, a slight difference between the sensitivity toward the acylsulfamide inhibitors and
the substrate specificity was observed. Ph4CL1 shows the highest reactivity against ferulic acid and
the catalytic efficiency of the enzyme against 4-coumaric acid is about 60% of that against ferulic
acid.16 However, 1b was the most potent inhibitor for Ph4CL1, followed by 1c (IC50: 1.5 µM). This
difference should arise from the structural differences with respect to the cinnamate binding site of
Ph4CL1 between the adenylate- and the thioester-forming conformations, and the degree of the
difference between the two conformations should be higher than for other 4CLs. Among the tested
4CLs, Ph4CL1 is the most sensitive enzyme against 1e (IC50: 25.1 µM), indicating that the structure
of the cinnamate binding pocket of Ph4CL1 in the thioester-forming conformation should
substantially differ from that of the other 4CLs. Even though the ligation reaction catalyzed by
Ph4CL1 should be specific against cinnamates, the inhibitory activity of 1e against the enzyme is
still ca. 65-fold less potent than that of 1b. From a phylogenetic perspective,16 Ph4CL1 is more
closely related to Ptr4CL3 (designated as Pt1s07400 in the literature) than to Gm4CL1, while
At4CL1/2 is even more distinct. Therefore, the differences in the cinnamate binding pocket
structures could potentially arise from the evolutionary diversity of the 4CL proteins.
The presence of the acyl group of the sulfamide moiety is critical for the 4CL inhibitory activity,
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given that the AMP mimic 1f was inactive against all 4CLs tested. This result indicates that 4CL
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recognizes the acylsulfamide inhibitors 1a–e as mimics of the acyladenylate intermediates, and the
affinity of 4CL toward the acyladenylate intermediates that bear cinnamates as the acyl group is
superior to that of AMP. Compared to 1f, 1d exhibited weak but clear inhibitory activity against
At4CL1/2, Ptr4CL3, and Ph4CL1, which exhibit no catalytic activity against sinapic acid. This
result also supports the notion that 1d acts as the acyladenylate intermediate mimic, i.e., that the
structures of the cinnamate binding pocket in the adenylate-forming conformations that are unable
to accommodate sinapic acid could be enlarged in the thioester-forming conformations, where the
binding acyladenylate intermediates react with CoA.
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3. Conclusions
In this study, we designed and synthesized acylsulfamide inhibitors for 4CL as mimics of
acyladenylates, which represent the key intermediates in the enzymatic reaction of 4CL. The target
inhibitors and the important synthetic intermediates were fully characterized using two-dimensional
NMR spectroscopy techniques. The inhibitory activity of synthetic acylsulfamides was evaluated
using five 4CL proteins with distinct substrate specificity, and the highest inhibitory activity against
each 4CL, represented by IC50 values, ranged from 0.10 to 2.4 µM. The tested 4CLs are very
sensitive against the presence/structure of the acyl group of the sulfamide moiety, and inhibitors that
carry better substrates as the acyl group tend to inhibit 4CL with higher potency. The
structure-activity relationship established in this study indicates that 4CL recognizes the
acylsulfamide inhibitors as mimics of the acyladenylate intermediates. Investigations into the
biological activity of the acylsulfamide inhibitors in vivo are currently in progress.
4. Material and methods
4.1. Synthesis of 4CL inhibitors
4.1.1. General
Meltingpoints (mp) were measured with a Mettler-Toledo FP62 meltingpoint apparatus and are
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uncorrected. NMR spectra were obtained on a JEOL JNM-AL300 (300 MHz for 1H) or a Bruker
AVANCEIII 600 (600 MHz for 1H) spectrometers. Chemical shifts are reported in parts per million
relative to tetramethylsilane as the internal standard unless otherwise noted. The 1H and 13C NMR
signals of 4c–e, 8, and 1a–f were fully assigned using two-dimensional NMR measurements based
on 1H-1H correlation (COSY), heteronuclear single quantum coherence (HSQC), and heteronuclear
multiple bond correlation (HMBC) techniques. These results, together with the assignment of 4a
and 4b, are summarized in Tables S1–S3 (Supporting Information). Elemental analyses were
performed on a Yanaco MT-5. High resolution mass spectrometry (HRMS) was performed on a
JOEL JMS-700 spectrometer.
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All reagents and solvents were used as received from commercial sources. Silica gel flash column
chromatography was carried out using a Yamazen W-prep 2XY system with Hi-Flash columns
(silica gel, 40 µm). Reverse-phase medium pressure column chromatography was carried out using
a Büchi Sepacore chromatography system with Nacalai COSMOSIL 5C18-PAQ column (φ20 mm ×
250 mm). 1-Octanoylimidazole52 and 753 were prepared according to the literature methods.
4.1.2. Synthesis of protected cinnamates 5b–d
4.1.2.1. (E)-3-{4-[(2-Methoxyethoxy)methoxy]phenyl}acrylic acid (5b)
The following procedure is typical for 5b–d. To an ice-cooled solution of 4-coumaric acid (5.06 g,
30.8 mmol) and (i-Pr)2NEt (21.5 mL, 123 mmol) in anhydrous CH2Cl2 (40 mL) was added MEMCl
(11.0 mL, 96.3 mmol) dropwise for 5 min. The mixture was stirred for 14 h at room temperature
and refluxed for 9 h. The reaction mixture was diluted with CHCl3 (60 mL) and washed with
saturated aqueous NaHCO3 solution (40 mL). The organic layer was dried over anhydrous MgSO4
and concentrated under reduced pressure to give the (2-methoxyethoxy)methoxy ester of 5b as an
orange viscous oil (13.0 g). 1H NMR (300 MHz, CDCl3) δH: 3.37 (3H, s), 3.40 (3H, s), 3.54–3.60
(4H, m), 3.81–3.86 (4H, m), 5.30 (2H, s), 5.46 (2H, s), 6.33 (1H, d, J = 16.2 Hz), 7.07 (2H, d, J =
8.7 Hz), 7.48 (2H, d, J = 8.7 Hz), 7.70 (1H, d, J = 16.2 Hz). This crude material was dissolved in
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MeOH (60 mL) followed by addition of 10% (w/v) aqueous NaOH solution (20 mL). The reaction
mixture was stirred for 1.5 h at room temperature and concentrated in vacuo. The residue was
dissolved in water (60 mL) and washed with Et2O (20 ml × 3). The water layer was acidified with
2M HCl and a precipitated solid was collected by suction filtration. The precipitate was washed
with water and hexane respectively, and dried under reduced pressure to give 5b (6.56 g, 84%) as a
colorless solid. This was used without further purification. Mp 102–103 ºC (EtOH). 1H NMR (300
MHz, CDCl3) δH: 3.38 (3H, s), 3.55–3.58 (2H, m), 3.81–3.84 (2H, m), 5.31 (2H, s), 6.33 (1H, d, J =
15.9 Hz), 7.07 (2H, d, J = 8.7 Hz), 7.50 (2H, d, J = 8.7 Hz), 7.74 (1H, d, J = 15.9 Hz). Anal. Calcd
for C13H16O5: C, 61.90; H, 6.39. Found: C, 61.89, H, 6.43.
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4.1.2.2. (E)-3-{3-Methoxy-4-[(2-methoxyethoxy)methoxy]phenyl}acrylic acid (5c)
Yield = 84%. A colorless solid. Mp 82–84 ºC (EtOH). 1H NMR (300 MHz, CDCl3) δH: 3.38 (3H, s),
3.55-3.58 (2H, m), 3.86–3.89 (2H, m), 3.92 (3H, s), 5.37 (2H, s), 6.34 (1H, d, J = 15.6 Hz),
7.08–7.13 (2H, m), 7.22 (1H, d, J = 8.1 Hz), 7.73 (1H, d, J = 15.6 Hz). Anal. Calcd for C14H18O6: C,
59.57; H, 6.43. Found: C, 59.34; H, 6.48.
4.1.2.3. (E)-3-{3,5-Dimethoxy-4-[(2-methoxyethoxy)methoxy]phenyl}acrylic acid (5d)
Yield = 72%. A colorless solid. Mp 83–93 ºC (EtOH). 1H NMR (300 MHz, CDCl3) δH: 3.37 (3H, s),
3.55–3.59 (2H, m), 3.87 (6H, s), 3.98–4.01 (2H, m), 5.25 (2H, s), 6.36 (1H, d, J = 15.8 Hz), 6.77
(2H, s), 7.70 (1H, d, J = 15.8 Hz). Anal. Calcd for C15H20O7: C, 57.69; H, 6.45. Found: C, 57.51; H,
6.44.
4.1.3. Synthesis of acylimidazoles 2a–d.
4.1.3.1. 1-Cinnamoylimidazole (2a)
The following procedure is typical for 2a–d. To a solution of cinnamic acid (201 mg, 1.36 mmol) in
anhydrous MeCN (10 mL) was added CDI (267 mg, 1.65 mmol). The reaction mixture was stirred
for 2.5 h at room temperature and concentrated in vacuo. The residue was dissolved in toluene (20
mL) and the resultant solution was washed successively with water (10 mL) and brine (10 mL). The
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organic layer was dried over anhydrous MgSO4 and evaporated to give 2a (233 mg, 86%) as a
colorless solid. This was used without further purification.1H NMR (300 MHz, CDCl3) δH: 7.04 (1H,
d, J = 15.5 Hz), 7.12 (1H, s), 7.38–7.43 (3H, m), 7.59–7.63 (3H, m), 8.03 (1H, d, J = 15.5 Hz), 8.32
(1H, s).
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4.1.3.2. 1-[(E)-3-{4-[(2-methoxyethoxy)methoxy]phenyl}acryloyl]imidazole (2b)
Yield = quantitative. A colorless solid. 1H NMR (300 MHz, CDCl3) δH: 3.38 (3H, s), 3.55–3.58 (2H,
m), 3.82–3.85 (2H, m), 5.33 (2H, s), 6.94 (1H, d, J = 15.3 Hz), 7.12 (2H, d, J = 8.7 Hz), 7.16 (1H,
dd, J = 1.4, 0.8 Hz), 7.61 (2H, d, J = 8.7 Hz), 7.63 (1H, d, J = 1.4 Hz), 8.03 (1H, d, J = 15.3 Hz),
8.32 (1H, br s).
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4.1.3.3. 1-[(E)-3-{3-methoxy-4-[(2-methoxyethoxy)methoxy]phenyl}acryloyl]imidazole (2c)
Yield = 86%. A colorless solid. 1H NMR (300 MHz, CDCl3) δH: 3.38 (3H, s), 3.55–3.58 (2H, m),
3.87–3.90 (2H, m), 3.96 (3H, s), 5.40 (2H, s), 6.97 (1H, d, J = 15.3 Hz), 7.17–7.30 (4H, m), 7.65
(1H, s), 8.03 (1H, d, J = 15.3 Hz), 8.44 (1H, br s).
4.1.3.4. 1-[(E)-3-{3,5-dimethoxy-4-[(2-methoxyethoxy)methoxy]phenyl}acryloyl]imidazole (2d)
Yield = 76%. A pale yellowish solid. 1H NMR (300 MHz, CDCl3) δH: 3.37 (3H, s), 3.55–3.58 (2H,
m), 3.91 (6H, s), 3.99–4.02 (2H, m), 5.27 (2H, s), 6.87 (2H, s), 6.97 (1H, d, J = 15.2 Hz), 7.18 (1H,
br s), 7.65 (1H, br s), 7.99 (1H, d, J = 15.2 Hz), 8.42 (1H, br s).
4.1.4. N-Benzyloxycarbonyl-N′-(2′,3′-O-isopropylidene-5′-deoxyadenosin-5′-yl)sulfamide (8)
To an ice-cooled solution of chlorosulfonyl isocyanate (0.95 mL, 10.9 mmol) in anhydrous CH2Cl2
(12.5 mL) was added benzyl alcohol (1.1 mL, 10.6 mmol) dropwise for 3 min and the resultant
mixture was further stirred for 1 h at the same temperature. This solution was added to an
ice-cooled mixture of 7 (3.01 g, 9.83 mmol) and Et3N (1.5 mL, 10.8 mmol) in anhydrous CH2Cl2
(30 mL) dropwise for 10 min. The reaction mixture was stirred for 1 h at room temperature and the
precipitate was collected by suction filtration to give 8 (4.19 g, 82%) as a colorless solid. This was
used without further purification. Mp 162–164 ºC. 1H NMR (600 MHz, CD3SOCD3) δH: 1.33 (3H,
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s), 1.57 (3H, s), 3.20 (1H, ddd, J = 13.5, 6.9, 5.1 Hz), 3.30 (1H, ddd, J = 13.5, 5.1, 5.1 Hz), 4.36
(1H, ddd, J = 5.1, 5.1, 3.0 Hz), 5.01 (1H, dd, J = 6.3, 3.0 Hz), 5.09 (2H, s), 5.35 (1H, dd, J = 6.3,
3.3 Hz), 6.15 (1H, d, J = 3.3 Hz), 7.29–7.36 (5H, m), 7.45 (2H, br s), 8.22 (1H, s), 8.33 (1H, s),
8.77 (1H, dd, J = 6.9, 5.1 Hz), 11.38 (1H, br s). 13C NMR (151 MHz, CD3SOCD3) δC: 25.1, 27.0,
44.7, 66.6, 81.6, 82.6, 83.4, 90.0, 113.4, 119.4, 127.8 (2C), 128.1, 128.3 (2C), 135.6, 140.1, 148.2,
151.5, 152.6, 156.2. HRMS-FAB (m/z): [M+H]+ calcd for C21H26N7O7S, 520.1614; found,
520.1623.
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5
6
7
8
9
4.1.5. N-(2′,3′-O-Isopropylidene-5′-deoxyadenosin-5′-yl)sulfamide (3)
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Into a mixture of 8 (4.19 g, 8.07 mmol) and 10% (w/w) Pd-C (420 mg) in AcOH (30 mL) was
bubbled hydrogen gas for 4 h with stirring. The catalyst was filtered off and the filtrated was
concentrated under reduced pressure. The residue was recrystallized from MeOH to give the title
compound (2.52 g, 81%) as a colorless solid. Mp 150 ºC dec [lit.54 mp 150–152 ºC (EtOH)]. 1H
NMR (300 MHz, CD3SOCD3) δH: 1.34 (3H, s), 1.57 (3H, s), 3.01–3.28 (2H, m), 4.34–4.38 (1H, m),
5.03 (1H, dd, J = 6.3, 2.7 Hz), 5.37 (1H, dd, J = 6.3, 3.3 Hz), 6.11 (1H, d, J = 3.3 Hz), 6.60 (2H, s),
7.35 (2H, br s), 7.35–7.40 (1H, m), 8.17 (1H, s), 8.30 (1H, s). HRMS-FAB (m/z): [M+H]+ calcd for
C13H20N7O5S, 386.1247; found, 386.1237.
4.1.6. Condensation of acylimidazoles and sulfamide 3.
4.1.6.1. N-Cinnamoyl-N′-(2′,3′-O-isopropylidene-5′-deoxyadenosin-5′-yl)sulfamide (4a)
The following procedure is typical for 4a–d. To an ice-cooled suspension of 2a (100 mg, 0.504
mmol) and 3 (212 mg, 0.550 mmol) in anhydrous MeCN (10 mL) was added DBU (0.25 mL, 1.67
mmol) dropwise and the mixture was stirred for 22 h at room temperature followed by addition of
AcOH (0.15 mL, 2.62 mmol). After stirring for 3 min, silica gel (2.5 g) was added and the solvent
was evaporated. The residue was subjected to silica gel flash column chromatography
(CHCl3/MeOH = 96:4) to give 4a (183 mg, 70%) as a colorless solid. Mp 136–139 ºC. 1H NMR
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(300 MHz, CD3OD) δH: 1.34 (3H, s), 1.60 (3H, s), 3.40 (1H, dd, J = 13.7, 3.8 Hz), 3.47 (1H, dd, J =
13.7, 3.8 Hz), 4.48–4.52 (1H, m), 5.04 (1H, dd, J = 6.0, 2.1 Hz), 5.31 (1H, dd, J = 6.0, 4.2 Hz), 6.04
(1H, d, J = 4.2 Hz), 6.53 (1H, d, J = 15.8 Hz), 7.36–7.40 (3H, m), 7.52–7.55 (2H, m), 7.65 (1H, d, J
= 15.8 Hz), 8.19 (1H, s), 8.40 (1H, s). 13C NMR (75 MHz, CD3OD, solvent: 49.0) δC: 25.5, 27.6,
46.2, 83.1, 84.2, 84.7, 93.2, 115.6, 119.1, 120.8, 129.1 (2C), 129.8 (2C), 131.5, 135.2, 141.8, 145.6,
149.5, 154.2, 157.2, 165.9. HRMS-FAB (m/z): [M+H]+ calcd for C22H26N7O6S, 516.1665; found,
516.1665.
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6
7
8
4.1.6.2.
9
N-(2′,3′-O-Isopropylidene-5′-deoxyadenosin-5′-yl)-N′-[(E)-3-{4-[(2-methoxyethoxy)methoxy]p
henyl}acryloyl]sulfamide (4b)
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Yield = 61%. A colorless solid. Mp 105–125 ºC. 1H NMR (300 MHz, CD3OD) δH: 1.34 (3H, s),
1.60 (3H, s), 3.32 (3H, s), 3.37 (1H, dd, J = 13.5, 4.2 Hz), 3.44 (1H, dd, J = 13.5, 4.2 Hz),
3.53–3.56 (2H, m), 3.78–3.81 (2H, m), 4.46–4.50 (1H, m), 5.04 (1H, dd, J = 6.3, 2.7 Hz), 5.29 (2H,
s), 5.31 (1H, dd, J = 6.3, 4.2 Hz), 6.05 (1H, d, J = 4.2 Hz), 6.39 (1H, d, J = 15.6 Hz), 7.05 (2H, d, J
= 8.7 Hz), 7.50 (2H, d, J = 8.7 Hz), 7.59 (1H, d, J = 15.6 Hz), 8.20 (1H, s), 8.37 (1H, s). 13C NMR
(75 MHz, CD3OD, solvent: 49.0) δC: 25.5, 27.6, 46.3, 59.1, 68.9, 72.7, 83.2, 84.3, 84.8, 93.2, 94.2,
115.6, 117.0, 117.5 (2C), 120.9, 129.1, 130.9 (2C), 141.9, 145.3, 149.6, 154.3, 157.3, 160.6, 166.3.
HRMS-FAB (m/z): [M+H]+ calcd for C26H34N7O9S, 620.2139; found, 620.2151.
4.1.6.3.
N-(2′,3′-O-Isopropylidene-5′-deoxyadenosin-5′-yl)-N′-[(E)-3-{3-methoxy-4-[(2-methoxyethoxy)
methoxy]phenyl}acryloyl]sulfamide (4c)
Yield = 57%. A pale yellow solid. Mp 99–120 ºC. 1H NMR (600 MHz, CD3OD) δH: 1.33 (3H, s),
1.59 (3H, s), 3.32 (3H, s), 3.39 (1H, dd, J = 13.2, 3.6 Hz), 3.45 (1H, dd, J = 13.2, 3.6 Hz),
3.53–3.55 (2H, m), 3.81–3.82 (2H, m), 3.85 (3H, s), 4.48 (1H, ddd, J = 3.6, 3.6, 1.8 Hz), 5.03 (1H,
dd, J = 6.0, 1.8 Hz), 5.28 (2H, s), 5.31 (1H, dd, J = 6.0, 4.2 Hz), 6.04 (1H, d, J = 4.2 Hz), 6.40 (1H,
d, J = 15.9 Hz), 7.09 (1H, dd, J = 8.4, 1.8 Hz), 7.11 (1H, d, J = 8.4 Hz), 7.13 (1H, d, J = 1.8 Hz),
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7.58 (1H, d, J = 15.9 Hz), 8.19 (1H, s), 8.38 (1H, s). 13C NMR (151 MHz, CD3OD) δC: 25.6, 27.6,
46.3, 56.4, 59.1, 69.0, 72.7, 83.2, 84.3, 84.8, 93.3, 95.3, 112.3, 115.7, 117.4, 117.5, 120.9, 123.3,
130.0, 141.9, 145.5, 149.6, 150.0, 151.4, 154.3, 157.4, 166.2. HRMS-FAB (m/z): [M+H]+ calcd for
C27H36N7O10S, 650.2244; found, 650.2245.
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5
4.1.6.4.
6
N-(2′,3′-O-Isopropylidene-5′-deoxyadenosin-5′-yl)-N′-[(E)-3-{3,5-dimethoxy-4-[(2-methoxyeth
oxy)methoxy]phenyl}acryloyl]sulfamide (4d)
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8
Yield = 64%. A colorless solid. Mp 117–127 ºC. 1H NMR (600 MHz, CD3OD) δH: 1.34 (3H, s),
1.60 (3H, s), 3.33 (3H, s), 3.38 (1H, dd, J = 13.8, 3.6 Hz), 3.45 (1H, dd, J = 13.8, 3.6 Hz),
3.53–3.55 (2H, m), 3.84 (6H, s), 3.93–3.95 (2H, m), 4.48 (1H, ddd, J = 3.6, 3.6, 2.4 Hz), 5.03 (1H,
dd, J = 6.0, 2.4 Hz), 5.14 (2H, s), 5.31 (1H, dd, J = 6.0, 4.2 Hz), 6.05 (1H, d, J = 4.2 Hz), 6.43 (1H,
d, J = 15.6 Hz), 6.85 (2H, s), 7.57 (1H, d, J = 15.6 Hz), 8.20 (1H, s), 8.37 (1H, s). 13C NMR (151
MHz, CD3OD) δC: 25.6, 27.6, 46.4, 56.6 (2C), 59.1, 69.5, 72.8, 83.3, 84.4, 84.9, 93.3, 98.0, 106.5
(2C), 115.7, 118.6, 121.0, 131.7, 137.9, 142.0, 145.7, 149.7, 154.4, 154.9 (2C), 157.5, 166.1.
HRMS-FAB (m/z): [M+H]+ calcd for C28H38N7O11S, 680.2350; found, 680.2344.
4.1.6.5. N-(2′,3′-O-Isopropylidene-5′-deoxyadenosin-5′-yl)-N′-octanoylsulfamide (4e)
To a mixture of 3 (2.00 g, 5.19 mmol) and 1-octanoylimidazole (1.11 g, 5.71 mmol) in anhydrous
MeCN (100 mL) was added DBU (1.6 mL, 10.7 mmol) dropwise. The mixture was stirred for 80
min and the solvent was removed in vacuo. The residue was taken up in EtOAc and washed
successively with 3% (w/v) citric acid/brine solution and brine. The organic layer was dried over
anhydrous Na2SO4 and concentrated in vacuo to give a viscous oil. This material was dissolved in
acetone (100 mL), and hexane (250 mL) was slowly added to the solution. After stirring for 1 h, a
precipitate was collected by suction filtration to give the title compound (2.10 g, 79%) as a colorless
solid. Mp 157–158 ºC. 1H NMR (600 MHz, CD3OD) δH: 0.86 (3H, t, J = 7.2 Hz), 1.19–1.30 (8H,
m), 1.36 (3H, s), 1.53–1.58 (2H, m), 1.61 (3H, s), 2.18–2.26 (2H, m), 3.34 (1H, dd, J = 13.5, 3.9
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Hz), 3.38 (1H, dd, J = 13.5, 3.9 Hz), 4.47 (1H, ddd, J = 3.9, 3.9, 2.4 Hz), 5.05 (1H, dd, J = 6.3, 2.4
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Hz), 5.31 (1H, dd, J = 6.3, 4.2 Hz), 6.06 (1H, d, J = 4.2 Hz), 8.21 (1H, s), 8.35 (1H, s). 13C NMR
(151 MHz, CD3OD) δC: 14.4, 23.6, 25.6, 26.0, 27.7, 30.06, 30.08, 32.8, 36.8, 46.3, 83.3, 84.5, 85.1,
93.3, 115.7, 121.0, 142.1, 149.7, 154.4, 157.5, 174.3. Anal. Calcd for C21H33N7O6S: C, 49.30; H,
6.50; N, 19.16. Found: C, 49.48; H, 6.45; N, 18.93.
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6
4.1.7. Synthesis of inhibitors 1a–f
7
4.1.7.1. N-Cinnamoyl-N′-(5′-deoxyadenosin-5′-yl)sulfamide (1a)
8
The following procedure is typical for 1a–f. A solution of 4a (400 mg, 0.776 mmol) in 80% (v/v)
aqueous HCO2H solution (10 mL) was stirred for 5.5 h at room temperature followed by
evaporation. The residue was purified by reverse-phase medium pressure column chromatography
(H2O/MeOH = 100:0–25:75) to give the title compound (284 mg, 77%) as a colorless solid. Mp 166
ºC dec. 1H NMR (600 MHz, CD3SOCD3) δH: 3.20 (1H, ddd, J = 13.5, 3.6, 3.6 Hz), 3.25 (1H, ddd, J
= 13.5, 8.4, 3.6 Hz), 4.11–4.13 (1H, m), 4.15 (1H, ddd, J = 3.6, 3.6, 1.8 Hz), 4.77 (1H, ddd, J = 6.9,
6.9, 5.4 Hz), 5.33 (1H, br d, J = 3.6 Hz), 5.51 (1H, br d, J = 6.9 Hz), 5.83 (1H, d, J = 6.9 Hz), 6.65
(1H, d, J = 15.6 Hz), 7.41–7.46 (5H, m), 7.58–7.60 (2H, m), 7.63 (1H, d, J = 15.6 Hz), 8.28 (1H, s),
8.30 (1H, s), 9.39 (1H, dd, J = 8.4, 3.6 Hz), 11.66 (1H, br s). 13C NMR (151 MHz, CD3SOCD3) δC:
45.1, 71.4, 72.1, 83.7, 88.7, 119.1, 119.6, 127.9 (2C), 128.9 (2C), 130.3, 133.9, 140.7, 142.9, 148.3,
152.5, 156.2, 163.4. HRMS-FAB (m/z): [M+H]+ calcd for C19H22N7O6S, 476.1352; found,
476.1353.
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4.1.7.2. N-(5′-Deoxyadenosin-5′-yl)-N′-[(E)-3-(4-hydroxyphenyl)acryloyl)]sulfamide (1b)
Yield = 33%. Mp 225 ºC dec. 1H NMR (600 MHz, CD3SOCD3) δH: 3.18 (1H, ddd, J = 13.5, 3.6,
3.6 Hz), 3.22 (1H, ddd, J = 13.5, 8.4, 3.6 Hz), 4.10–4.12 (1H, m), 4.14–4.15 (1H, m), 4.75–4.78
(1H, m), 5.31 (1H, br d, J = 3.0 Hz), 5.50 (1H, br d, J = 6.6 Hz), 5.82 (1H, d, J = 7.2 Hz), 6.43 (1H,
d, J = 15.6 Hz), 6.81 (2H, d, J = 8.7 Hz), 7.42 (2H, br s), 7.43 (2H, d, J = 8.7 Hz), 7.53 (1H, d, J =
15.6 Hz), 8.27 (1H, s), 8.29 (1H, s), 9.29–9.31 (1H, m), 10.03 (1H, br s), 11.49 (1H, br s). 13C NMR
(151 MHz, CD3SOCD3) δC: 45.1, 71.4, 72.1, 83.7, 88.7, 115.2, 115.8 (2C), 119.6, 125.0, 129.9 (2C),
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140.7, 143.1, 148.3, 152.5, 156.2. 159.7, 163.8. HRMS-FAB (m/z): [M+H]+ calcd for C19H22N7O7S,
492.1301; found, 492.1302.
3
4.1.7.3. N-(5′-Deoxyadenosin-5′-yl)-N′-[(E)-3-(4-hydroxy-3-methoxyphenyl)acryloyl]sulfamide
(1c)
4
5
Yield = 52%. A pale green solid. Mp 214 ºC dec. 1H NMR (600 MHz, CD3SOCD3) δH: 3.17 (1H,
ddd, J = 13.5, 3.6, 3.6 Hz), 3.23 (1H, ddd, J = 13.5, 9.0, 3.6 Hz), 3.80 (3H, s), 4.11–4.13 (1H, m),
4.15 (1H, ddd, J = 3.6, 3.6, 1.8 Hz), 4.77 (1H, ddd, J = 6.9, 6.9, 4.8 Hz), 5.29 (1H, d, J = 3.6 Hz),
5.48 (1H, d, J = 6.9 Hz), 5.83 (1H, d, J = 6.9 Hz), 6.46 (1H, d, J = 15.6 Hz), 6.81 (1H, d, J = 8.4
Hz), 7.04 (1H, dd, J = 8.4, 1.8 Hz), 7.14 (1H, d, J = 1.8 Hz), 7.40 (2H, br s), 7.52 (1H, d, J = 15.6
Hz), 8.27 (1H, s), 8.28 (1H, s), 9.30 (1H, br s), 9.61 (1H, s), 11.47 (1H, br s). 13C NMR (151 MHz,
CD3SOCD3) δC: 45.1, 55.4, 71.4, 72.1, 83.8, 88.8, 111.3, 115.6 (2C), 119.7, 122.2, 125.5, 140.7,
143.3, 147.7, 148.3, 149.2, 152.5, 156.3, 163.9. HRMS-FAB (m/z): [M+H]+ calcd for C20H24N7O8S,
522.1407; found, 522.1421.
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4.1.7.4.
N-(5′-Deoxyadenosin-5′-yl)-N′-[(E)-3-(4-hydroxy-3,5-dimethoxyphenyl)acryloyl]]sulfamide
(1d)
Yield = 98% (without purification). A pale yellow solid. Mp 144 ºC dec. 1H NMR (600 MHz,
CD3SOCD3) δH: 3.19 (1H, ddd, J = 12.6, 3.0, 3.0 Hz), 3.25 (1H, ddd, J = 12.6, 9.0, 3.0 Hz), 3.80
(6H, s), 4.14 (1H, dd, J = 4.8, 1.8 Hz), 4.18 (1H, ddd, J = 3.0, 3.0, 1.8 Hz), 4.79 (1H, dd, J = 6.9,
4.8 Hz), 5.84 (1H, d, J = 6.9 Hz), 6.50 (1H, d, J = 15.6 Hz), 6.89 (2H, s), 7.40 (2H, br s), 7.54 (1H,
d, J = 15.6 Hz), 8.27 (1H, s), 8.31 (1H, s), 9.01 (1H, br s), 9.38 (1H, dd, J = 9.0, 3.0 Hz), 11.41 (1H,
br s). 13C NMR (151 MHz, CD3SOCD3) δC: 45.1, 55.9 (2C), 71.5, 72.2, 83.8, 88.9, 105.8 (2C),
116.0, 119.7, 124.3, 138.3, 140.7, 143.7, 147.9 (2C), 148.3, 152.6, 156.3, 163.8. HRMS-FAB (m/z):
[M+H]+ calcd for C21H26N7O9S, 552.1513; found, 522.1515.
4.1.7.5. N-(5′-Deoxyadenosin-5′-yl)-N′-octanoylsulfamide (1e)
Yield = 36% (recrystallized from hot EtOAc). A colorless solid. Mp 172 ºC. 1H NMR (600 MHz,
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CD3SOCD3) δH: 0.83 (3H, t, J = 6.9 Hz), 1.17–1.26 (8H, m), 1.46–1.51 (2H, m), 2.17–2.25 (2H, m),
3.15–3.22 (2H, m), 4.12 (1H, br s), 4.15 (1H, ddd, J = 3.6, 3.6, 1.8 Hz), 4.75–4.78 (1H, m), 5.32
(1H, br d, J = 1.8 Hz), 5.51 (1H, br d, J = 6.6 Hz), 5.84 (1H, d, J = 7.2 Hz), 7.43 (2H, br s), 8.25
(1H, s), 8.29 (1H, s), 9.16 (1H, dd, J = 6.0, 6.0 Hz), 11.37 (1H, br s). 13C NMR (151 MHz,
CD3SOCD3) δC: 13.8, 22.0, 24.3, 28.27, 28.32, 31.0, 35.1, 45.0, 71.4, 72.2, 83.7, 88.8, 119.7, 140.7,
148.4, 152.4, 156.2, 171.6. HRMS-FAB (m/z): [M+H]+ calcd for C18H30N7O6S, 472.1978; found,
472.1973.
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4
5
6
7
8
4.1.7.6. N-(5′-Deoxyadenosin-5′-yl)sulfamide (1f)
9
Yield = 97% (without purification). A colorless solid. Mp 136–151 ºC (lit.54 mp 136–139 ºC). 1H
NMR (600 MHz, CD3SOCD3) δH: 3.20 (1H, ddd, J = 12.9, 8.7, 3.9 Hz), 3.27 (1H, ddd, J = 12.9, 3.9,
3.9 Hz), 4.18 (1H, ddd, J = 3.9, 3.9, 2.1 Hz), 4.20 (1H, dd, J = 5.1, 2.1 Hz), 4.77 (1H, dd, J = 6.9,
5.1 Hz), 5.87 (1H, d, J = 6.9 Hz), 6.63 (2H, br s), 7.40 (2H, br s), 7.81 (1H, dd, J = 8.7, 3.9 Hz),
8.20 (1H, s), 8.30 (1H, s). 13C NMR (151 MHz, CD3SOCD3) δC: 44.8, 71.2, 72.3, 83.9, 88.6, 119.6,
140.6, 148.6, 152.3, 156.2. HRMS-FAB (m/z): [M+H]+ calcd for C10H16N7O5S, 346.0934; found,
346.0926.
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4.2. Expression of 4CL proteins in E. coli and its purification.
Full length cDNAs of the 4CL genes from A. thaliana, G. max, P. trichocarpa, and P. hybrida were
amplified with polymerase chain reactions using the designed primers, based on the published
sequences on the website of NCBI (https://www.ncbi.nlm.nih.gov). The amplified DNAs were
cloned into the multi-cloning site in pET28a for At4CL1, At4CL2, Ph4CL1, and Ptr4CL3 or pCold I
for Gm4CL1. Resulting constructs were transformed into E. coli Rosetta2 (DE3) pLysS. The
recombinant 4CL proteins were induced by adding isopropyl--D-thiogalactopyranoside to 0.5 mM.
Harvesting and proteins purification by affinity chromatography with COSMOGEL His-Accept
(Nacalai) were performed using previously described methods.56
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4.3. 4CL activity assay.
The 4CL activity was measured using a spectrophotometric assay as described by Knobloch and
Hahlbrock.55 4CL reaction mixtures contained 0.1 mM 4-coumaric acid, 0.25 mM CoA, 2.5 mM
ATP, 2.5 mM MgCl2, 0.2 M Tris-HCl at pH 8.0, and test compounds (0.5% v/v dimethyl sulfoxide,
final). The blank as background contained the same components but without CoA. The purified
4CL proteins were added to start the enzymatic reaction and its activity was measured as the
increase in absorbance at the absorption maximum (333 nm) of 4-coumaroyl CoA ester. The
extinction coefficient of the ester was used to calculate enzyme activities.32 Half-maximum
inhibitory concentration (IC50) were determined from fitting the data to a sigmoidal dose-response
model against percentage inhibition.
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Acknowledgements
This work was supported by JSPS KAKENHI grant JP 23510278 (to J. H.) and a research grant for
Exploratory Research on Sustainable Humanosphere Science from the Research Institute for
Sustainable Humanosphere (RISH), Kyoto University (to B. W.). Parts of the experimental
measurements were carried out using a Bruker 600 MHz NMR and a JEOL JMS-700 mass
spectrometer in the Joint Usage/Research Center (JURC) at the Institute for Chemical Research,
Kyoto University. The authors are greatly indebted to Ms. Kyoko Ohmine, Ms. Toshiko Hirano, and
Ms. Akiko Fujihashi for performing instrumental analyses.
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