Structure optimization and bioactivity evaluation of ThDP analogs targeting cyanobacterial pyruvate dehydrogenase E1

Article abstract of DOI:10.1016/j.bmc.2019.115159

Harmful cyanobacteria bloom (HCB) has occurred frequently in recent years and it is urgent to develop novel algicides to deal with this problem. In this paper, a series of novel thiamin diphosphate (ThDP) analogs 5a?5g were designed and synthesized target

Full text of DOI:10.1016/j.bmc.2019.115159

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Structure optimization and bioactivity evaluation of ThDP analogs targeting
cyanobacterial pyruvate dehydrogenase E1
Jiangtao Feng, Haifeng He, Yuan Zhou, Meng Cai, Hao Peng, Honglin Liu,
Lei Liu, Lingling Feng, Hongwu He
PII:
S0968-0896(19)31248-9
https://doi.org/10.1016/j.bmc.2019.115159
BMC 115159
DOI:
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Bioorganic & Medicinal Chemistry
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23 July 2019
2 October 2019
8 October 2019
Please cite this article as: J. Feng, H. He, Y. Zhou, M. Cai, H. Peng, H. Liu, L. Liu, L. Feng, H. He, Structure
optimization and bioactivity evaluation of ThDP analogs targeting cyanobacterial pyruvate dehydrogenase E1,
Bioorganic & Medicinal Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.115159
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Structure optimization and bioactivity evaluation of ThDP analogs
targeting cyanobacterial pyruvate dehydrogenase E1
Jiangtao Feng a, 1, Haifeng He a, b, 1, Yuan Zhou , Meng Cai , Hao Peng , Honglin
a
a
a
a
a
a,
a,
Liu , Lei Liu , Lingling Feng *, Hongwu He *
a
Key Laboratory of Pesticide & Chemical Biology (CCNU), Ministry of Education; College
of Chemistry, Central China Normal University, Wuhan 430079, China.
b
School of Chemistry and Chemical Engineering, Jiangxi Science and Technology
Normal University, Nanchang 330013, PR China
1
These authors equally contributed to this work.
*
Correspondence author:
Lingling Feng Tel: 86-18602731572
Hongwu He Tel & Fax: 86-27-67867960 E-mail: he1208@mail.ccnu.edu.cn
E-mail: fll708@mail.ccnu.edu.cn,
Key words: Thiamin diphosphate; pyruvate dehydrogenase multienzyme component E1;
harmful cyanobacterial blooms; algicides; competitive inhibitors

Abstract
Harmful cyanobacteria bloom (HCB) has occurred frequently in recent years and it is
urgent to develop novel algicides to deal with this problem. In this paper, a series of novel
thiamin diphosphate (ThDP) analogs 5a - 5g were designed and synthesized targeting
cyanobacterial pyruvate dehydrogenase complex E1 (Cy-PDHc E1). Our results showed
that compounds 5a - 5g have higher inhibitory activities against Cy-PDHc E1 (IC50 9.56
µM – 3.48 µM) and higher inhibitory activities against two model cyanobacteria strains
Synechocystis sp PCC6803 (EC50 2.03 µM – 1.58 µM) and Microcystis aeruginosa
FACHB905 (EC50 1.86 µM – 0.95 µM). Especially, compound 5b displayed highest
inhibitory activities (IC = 3.48 µM) against Cy-PDHc E1 and powerful inhibitory activities
50
against cyanobacteria Synechocystis sp PCC6803 (EC50 = 1.58 µM) and Microcystis
aeruginosa FACHB905 (EC50 = 1.04µM). Moreover, the inhibitory activities of compound
5b were even higher than those of copper sulfate (EC50 = 2.02 and 1.71µM separately)
which has been widely used as algicide against cyanobacteria PCC6803 and FACHB905.
The more important was that compound 5b display much higher inhibitory selectivity
between Cy-PDHc E1 (Inhibitory rate 97.4%) and porcine PDHc E1(Inhibitory rate 11.8%)
under the same concentration (100 μM). The inhibition kinetic experiment and molecular
docking research showed that compound 5b can inhibit Cy-PDHc E1 by occupying the
ThDP-binding pocket and then blocking Cy-PDHc E1 bound to ThDP as competitive
inhibitor. The imagines of SEM and TEM showed that cellular microstructures were
heavily destroyed under compound 5b stress. Our results demonstrated compound 5b
could be taken as a potential lead compound targeting Cy-PDHc E1 to obtain
environment-friendly algicide for harmful cyanobacterial blooms control.

1. Introduction
In recent years, harmful cyanobacterial blooms (HCBs) have attracted much attentions for
their increasing frequency, intensity and duration in many aquatic ecosystems around the
world, which were facilitated by multi-factors including water eutrophication and global
warming 1, 2. HCBs were a potential threat to the biodiversity and aquatic food webs of
aquatic ecosystems 3, 4. HCBs also could produce toxic secondary metabolites which
were hazardous to animals and humans 3, 4. Chemical algicides were taken as the most
effective methods, such as herbicides, chemical oxidants and copper-based compounds
5-7.
However, these chemicals were not specially designed to target cyanobacteria, they
displayed strong toxicity to most aquatic organisms or accumulated in the environment
and possessed potential environment risk 5-7. Consequently, it was urgent and essential to
explore novel environment-friendly algicides targeting cyanobacteria.
The pyruvate dehydrogenase complex (PDHc) plays an important role in the
glycolysis pathway which catalyzes the oxidative decarboxylation of pyruvate to
8
acetyl-CoA, CO and NADH . PDHc is comprised of three different component enzymes.
2
Pyruvate dehydrogenase E1 (PDHc E1) is the key enzyme component of PDHc, which is
located at the first one of three PDHc components and catalyzes the rate-limiting step of
the oxidative decarboxylation of the pyruvate in the presence of cofactors ThDP and Mg2+
8.
Considering that cofactor ThDP plays an important role in the oxidative decarboxylation
catalyzed by PDHc E1, many studies have been carried out to design ThDP analogs as E.
coli PDHc E1 inhibitors 9-13. Some ThDP analogs have been designed and synthesized as
E. coli PDHc E1 inhibitors in our laboratory 11-16. For example, HW02 targeting plant PDHc
E1, has herbicide activity 11 and compound I has antifungal activity targeting Escherichia
coli PDHc E1 12 (Fig. 1). However, there were a few reports about developing algicides
targeting cyanobacterial PDHc E1 (Cy-PDHc E1).
In our previous research, IIa - IIi of thiamin diphosphate (ThDP) analogs were
synthesized by introducing substituted benzene ring to replace the pyrophosphate moiety,
and introducing sulfonamide (HN-SO ) groups as a linker to connect triazole ring and
2
benzene ring, respectively 16. We found that IIa - IIi have 77% - 99% inhibitory potency

against cyanobacteria strain (Synechocystis sp PCC6803) when the concentration of
these compounds was 20 μg/mL 16. We hypothesized that the inhibitory effect of these
compounds on cyanobacteria strain might be related to cyanobacterial PDHc E1 (Cy-
PDHc E1). These results led us to search algicides targeting Cy- PDHc E1.
NH2
CH3
S
NH2
N
N
N
N
N
O O
P
O
H3C
N
O
O
O
O
P
O
N
H3C
N
O
P
O
P O
O
O
ThTDP
Triazole-ThTP
NH2
N
I
Cl
Cl
O
P
OCH3
OCH3
N
N
N
O
NO2
O
O
N
H3C
O
HW02
I
Fig. 1 Structures of known ThDP analogs.
In this paper, Cy-PDHc E1 was obtained by cloning, expression and purification
processes. Then we confirmed that IIa - IIi have higher enzymatic inhibitory activities
against Cy-PDHc E1. These results suggested that compounds IIa - IIi were the potential
novel inhibitors against Cy-PDHc E1, and it was feasible to design inhibitors targeting
Cy-PDHc E1 for developing novel algicides to control HCBs. In order to obtain the
compounds with higher inhibitory activities targeting Cy-PDHc E1, a series of 5a – 5g
were obtained by introducing iodine group to the skeleton of IIa - IIi (Fig. 2) because it has
been demonstrated that the iodine group in the 5-position of triazole was beneficial for
increasing the inhibitory activity in our previous works 12. The inhibitory activities (IC ) of
50
IIa – IIi and 5a – 5g were confirmed against Cy-PDHc E1 in vitro and their inhibitory
activities (EC50 values) were examined against two model cyanobacteria Synechocystis
PCC6803 and Microcystis aeruginosa FACHB905 in vivo. Especially, compound 5b
displayed the highest inhibitory activities against Cy-PDHc E1 in vitro and against
cyanobacteria in vivo. Consequently, we analyzed the interaction between compound 5b
and Cy-PDHc E1 by using the inhibition kinetic experiment and molecular docking
research. Meanwhile, we also explored the effect of compound 5b on cellular

microstructures by the imagines of SEM and TEM. Our results demonstrated compound
b could be taken as potential lead compound targeting Cy-PDHc E1 to obtain
environment-friendly algicide against harmful cyanobacterial blooms.
5
NH2
NH2
NH2
I
N
N
N
N
N
N
O
N
N
N
O
O
O
HN
S
O
O
N
N
S
H3C
N
O
O
O
O
H3C
N
N
HN
P
R
H3C
N
R
P
O
Triazole-ThDP
II
5
Fig. 2 Design of novel ThDP analogs targeting cyanobacterial PDHc E1
2. Results and discussion
2.1 Design and synthesis of the novel ThDP analogs targeted Cy-PDHc E1
NH2
CH3
NH2
a
N
N3
HN
O
b
N
N
I
N
O
OH
S
H3C
N
H3C
N
1
N-iodomorpholine
Vitamin B1
NH2
I
I
R
R
R
Cl
O
HN
HN
S
N
N
N
O
c
d
e
S
S
O
O
O
HN
S
N
H3C
N
O
O
O
1
R
5
2
3
4
0
Scheme 1 Reagents and conditions (a) NaN , Na SO , H O, 60-65 C; (b) morpholine, I ,
3
2
3
2
2
MeOH, rt, 1h; (c) CHCCH NH , Et N, DCM, rt; (d) N-iodomorpholine, CuI, THF, rt, 6-8h;
2
2
3
(
e)1, CuI, Et N, THF, rt, 12h
3
In our previous study, we found that IIa - IIi have 77%-99% inhibitory potency against
cyanobacteria strain (Synechocystis sp PCC6803) when the concentration of these
compounds was 20 μg/mL 16. In order to study whether these compounds targeted on
cy-PDHc E1, we obtained Cy-PDHc E1 by cloning, expression and purification processes
and confirmed that IIa - IIi have higher enzymatic inhibitory activities against Cy-PDHc E1.
Furthermore, in order to obtain the compounds with higher inhibitory activities targeting
Cy-PDHc E1 and the interaction between structure and activity, a series of thiamin
diphosphate (ThDP) analogs N-((1-((4-amino-2-methylpyrimidin-5-yl) methyl)-5-iodo-1H-1,
2, 3-triazol-4-yl) methyl) substituted benzenesulfonamide ThDP analogs (5a – 5g) were
obtained by introducing iodine group to the 5-position of triazole ring of IIa - IIi (Scheme 1)

because it has been demonstrated that the iodine group in the 5-position of triazole was
beneficial for increasing the inhibitory activity in our previous works 12.
Table 1 Structures and inhibitory activities of compounds IIa – IIi and 5a – 5g.
NH2
N
N
N
O
NH2
N
I
O
N
HN
S
O
H3C
N
N
N
N
R
O
N
HN
S
H3C
R
5
II
b
Cy-PDHc E1
EC50 (μM)
Compd.
R
a
PCC6803c
8.63 ± 1.07
5.07 ± 0.23
4.38 ± 0.18
9.53 ± 0.73
6.27 ± 0.74
8.96 ± 0.58
4.28 ± 0.14
4.48 ± 0.38
6.29 ± 0.65
1.82 ± 0.16
1.58 ± 0.46
1.97 ± 0.26
1.74 ± 0.11
1.96 ± 0.14
2.03 ± 0.24
1.96 ± 0.19
2.02 ± 0.19
FACHB905c
IC50 (μM)
IIa
IIb
IIc
IId
IIe
IIf
4-H
15.2 ± 1.24
13.0 ± 1.38
4.45 ± 0.24
14.3 ± 0.72
10.4 ± 1.07
8.80 ± 0.48
9.43 ± 0.25
12.0 ± 0.36
4.64 ± 0.10
7.06 ± 0.25
3.48 ± 0.30
9.56 ± 0.67
8.60 ± 0.84
7.75 ± 0.32
6.76 ± 0.83
3.69 ± 0.30
-
5.58 ± 0.97
5.70 ± 0.67
2.68 ± 0.03
5.03 ± 0.33
3.61 ± 0.55
14.1 ± 0.63
4.81 ± 0.50
4.06 ± 0.25
7.27 ± 0.86
1.19 ± 0.09
1.04 ± 0.09
1.66 ± 0.14
1.86 ± 0.13
1.73 ± 0.11
1.53 ± 0.15
0.95 ± 0.04
1.71 ± 0.11
4-Cl
4-NO
2
4-CH
4-F
3
4-Br
IIg
IIh
IIi
2,4,6-(CH
4-OCH
3 3
)
3
2-NO
4-Cl
2
5a
5b
5c
5d
5e
4-NO
2
4- CH
4-F
3
4-Br
5f
3
2,4,6- (CH )3
5g
4-OCH
-
3
CuSO
4
a
IC50 value is defined as the concentration required for 50% inhibition against the activity
of Cy-PDHc E1 in vitro. Values are means ± SD (n = 3).
b
EC50 value is defined as the concentration required for 50% inhibition against
cyanobacteria in vivo. Values are means ± SD (n = 3).
c
PCC6803, Synechocystis sp. PCC6803; FACHB905, Microcystis aeruginosa FACHB905

2.2 Inhibition activities of the novel ThDP analogs against Cy-PDHc E1 and
cyanobacteria
As shown in Table 1, these novel ThDP analogs IIa – IIi and 5a – 5g displayed inhibitory
activities against Cy-PDHc E1 (IC50 15.2 µM – 3.48 µM) in vitro. At the same time, The
EC50 values of compounds IIa – IIi and 5a – 5g against two model cyanobacteria strains
PCC6803 and FACHB905 were further examined in vivo. The results showed that these
compounds had higher inhibitory activities against two model cyanobacteria strains
Synechocystis sp PCC6803 (EC50 9.53 µM – 1.58 µM) and Microcystis aeruginosa
FACHB905 (EC50 14.1 µM – 0.95 µM). Compounds IIa – IIi and 5a – 5g displayed strong
inhibitory activities against Cy-PDHc E1 in vitro, which were consist with their inhibitory
activities against cyanobacteria in vivo. The inhibitory activities of 5a – 5g by introducing
iodine group to the 5-position of triazole ring were higher than those of the corresponding
compounds among IIa – IIi. These results indicated that Cy-PDHc E1 could be as a
potential enzymatic target for developing novel algicides.
Especially, compound 5b displayed highest inhibitory activities (IC50 = 3.48 µM)
against Cy-PDHc E1 and powerful inhibitory activities against cyanobacteria PCC6803
(EC50 = 1.58 µM) and FACHB905 (EC50 = 1.04 µM) (Table 1). Moreover, the inhibitory
activities of compound 5b were even higher than those of copper sulfate which was widely
used as algicide to control HCBs in decades (EC50 = 2.02 and 1.71 µM separately)
against cyanobacteria PCC6803 and FACHB905.
2.3 The selectivity of compound 5b between Cy-PDHc E1 and porcine PDHc E1
It has been reported that pyruvate dehydrogenase complex widely consists not only in
microorganisms but also in animals17, 18. In order to investigate the enzymatic selectivity of
these compounds between Cy-PDHc E1 and animal PDHc E1, the porcine PDHc E1 were
purchased from Sigma and the inhibitory activities of compounds 5a - 5d against porcine
PDHc E1 were examined as shown in Table 2. The more interesting was that the
inhibitory activities of compounds 5a - 5d against Cy-PDHc E1 (94.2% - 97.6%) were
prominently higher than those of compounds 5a - 5d against porcine PDHc E1 (11.8% -

19.0%) at the same concentration (100 µM). These results manifested that compounds 5
had conspicuous enzyme-selectivity between Cy-PDHc E1 and mammal PDHc E1.
Therefore, compounds 5 could have the potential to been used as safe and selective
algicides.
Table 2 Inhibitory potency of 5a – 5d between Cy-PDHc E1 and porcine PDHc E1.
Cy-PDHc E1
Porcine PDHc E1
Compd.
a
a
Inhibitory potency (%)
Inhibitory potency (%)
5a
5b
5c
5d
96.1 ± 3.91
18.5 ± 2.66
11.8 ± 2.35
12.3 ± 4.07
19.0 ± 3.87
97.4 ± 1.13
97.6 ± 0.38
94.2 ± 3.44
a
Inhibition potency (%) of compounds against cyanobacterial or porcine PDHc E1 enzyme
in vitro at 100 μM. Values are means ± SD (n = 3).
2.4 The inhibitory mechanism of compound 5b against Cy-PDHc E1
It has been reported cofactor ThDP owned a crucial role in the reaction catalyzed by
PDHc E18-9, 19. To study the inhibitory mechanism of compound 5b against Cy-PDHc E1,
we firstly clarify the relationship of inhibitor 5b and cofactor ThDP by the inhibition kinetics
analysis. As shown in Fig.3 and Table 3, the maximum velocity (Vmax) and Michaelis
constant (K ) were examined at different concentrations of 5b. All the Vmax values
m
remained nearly constant in the presence or absence of 5b while the K values became
m
higher with increasing concentrations of 5b. These results indicated that compound 5b
were competitive inhibitors of cofactor ThDP 20.
Table 3 Vmax and K values of Cy-PDHc E1 with different concentration of compound 5b
m
5b（μM）
V
max（μM / min）
m
K （μM）
0
2
12.3 ± 0.38
11.6 ± 0.35
12.4 ± 1.90
56.0 ± 7.16

5
12.3 ± 0.55
214.5 ± 34.4
Fig. 3. Enzymatic kinetic of compound 5b against Cy-PDHc E1.
Fig. 4. Binding mode of compound 5b in the ThDP-binding pocket of Cy-PDHc E1, the
protein is shown in cartoon, ligand is shown in stick, some key residues are shown in
green lines and hydrogen bonds are shown in purple lines.
Furthermore, in order to explore the interaction model of compound 5b with ThDP-binding
domain of Cy-PDHc E1, the modeling 3D structure of Cy-PDHc E1 was built on the
Swiss-Model sever 21, which proved to be reliable in our previous works 22. Based on the
3D model structure of Cy-PDHc E1, molecular docking was carried out to analyze the
potential interaction model of compound 5b with Cy-PDHc E1 on the SURFLEX module of
SYBYL software. The ThDP-binding site of Cy-PDHc E1 was shown in Fig. 4, and some
important amino acid residues were selected in green lines. Compound 5b could occupy
the ThDP-binding pocket of Cy-PDHc E1 and display a “V” conformation, which was
similar to the cofactor ThDP binding conformation. Amino acid residues Ala129 and Ile131

could form strong hydrogen bonds with the aminopyrimidine moiety of 5b. Meanwhile, π-π
stacking interaction could be found between the aminopyrimidine moiety and amino acid
residue Phe 85 of Cy-PDHc E1. Moreover, Tyr80 and Arg81 formed powerful hydrogen
bonds interaction with sulfonamide group (HN-SO ) of compound 5b, and Asp167 formed
2
hydrogen bonds interaction with the nitro group (NO ) of compound 5b. It could be
2
concluded that these powerful hydrogen bonds and π-π stackings accounted for the
strong inhibitory activities of 5b against Cy-PDHc E1. These results indicated that 5b
might occupy the ThDP-binding pocket and then suppress the activity of Cy-PDHc E1.
2.5 The effect of compound 5b on growth of cyanobacteria
Fig. 5. The effect of 5b on the growth of Synechocystis sp. PCC6803 (a, c) and
Microcystis aeruginosa FACHB905 (b, d) at different concentrations of 5b. The optical
density at 680 nm (OD680) was detected to represent the cells number, both
Synechocystis sp. PCC6803 and Microcystis aeruginosa FACHB905 were respectively
treated with 0, 2, 4, 8 and 10 µM compound 5b and cultured for 10 day. Values are
represented as the mean ± SD (n = 3).
As shown in Fig. 5, compound 5b could dramatically suppress the growth of
Synechocystis sp. PCC6803 and Microcystis aeruginosa FACHB905 at all tested
concentrations, and the inhibitory potency was concentration-dependent. As shown in Fig.
5, the growth of Synechocystis sp. PCC6803 and Microcystis aeruginosa FACHB905
were completely inhibited by compound 5b at 10 and 8 µM during the testing period. The

inhibitory activities of compound 5b against PCC6803 at 7 day were 96.2%, 90.7%, 74.6%
and 56.3% at concentrations of 10, 8, 4 and 2 µM, respectively (Fig. 5a). Meanwhile, after
treated for 7day, 5b displayed inhibitory rations of 97.8%, 87.4%, 68.0% and 59.8%
against cyanobacteria FACHB905 at corresponding concentration 10, 8, 4 and 2 µM,
respectively (Fig. 5b). These results indicated that compound 5b were highly effective to
control the growth of two model cyanobacterial strains Synechocystis sp. PCC6803 and
Microcystis aeruginosa FACHB905.
2.6 The effect of compound 5b on the cellular microstructure of cyanobacteria
Fig. 6. The effect of 5b on the cellular microstructure of cyanobacteria. (a) Cellular
microstructure of the control and 5b treated cyanobacteria cells analyzed by SEM. (b)
Subcellular microstructure of the control and 5b treated cyanobacteria cells analyzed by
TEM. c: cytoderm; n: nucleoid; t: thylakoid.
As shown in Fig. 6, the effect of compound 5b on the microstructure of the
FACHB905 cells was analyzed by SEM and TEM. The SEM imagine showed that cells of
the control were plump, unbroken and the surface of cells was smooth (Fig. 6a). In
contrast, cells being treated by 5b became obviously shrunken, misshapen, and the
surface of cells was more wrinkled compared to the control group. The subcellular
microstructure of cells was observed by TEM in Fig. 6b. The results of TEM showed that
the cytoderm of control cells were smooth and displayed a regular circle shape. On the

contrary, the shape of the treated cells was obviously distorted and the cytoderm was
rough. Compared to the intact nucleoid of control cells, the nucleoid of treated cells was
dramatically broken up and the content of lamellar thylakoids of treated cells were
obviously reduced. These results indicated that 5b can heavily damage the microstructure
of cyanobacteria cells and have strong anti-cyanobacteria activities. PDHc E1 is a key
enzyme that links glycolysis and tricarboxylic acid cycle. It plays an important role in the
energy metabolism of cyanobacteria cells. Compound 5b inhibited the activity of
cyanobacteria PDHc E1 and then resulted in the accumulation of pyruvate as a result, the
energy supply was interrupted in the cyanobacteria cells, which might lead to the cleavage
of the nucleic acid material of cyanobacteria cells and the destruction of the cell
membrane. In addition, the structure of lamellar thylakoids was further destroyed. As
shown in Fig. 5 a and b, the color of cyanobacteria cells gradually was changed from
blue-green to yellow, suggesting that the photosynthesis system of cyanobacteria cells
was destroyed under compound 5b stress. The results mentioned above indicated that
compound 5b could indirectly result in the destruction of cyanobacterial cell morphology
and substructure by inhibiting the activity of cyanobacterial PDHc E1, then ultimately
resulted in the death of cyanobacteria cells.
3. Conclusion
In this paper, we designed and synthesized a series of novel thiamin diphosphate
(
ThDP) analogs 5a - 5g targeting cyanobacterial pyruvate dehydrogenase complex E1
Cy-PDHc E1). Compounds 5a - 5g showed higher inhibitory activities against Cy-PDHc
(
E1 and higher inhibitory activities against Synechocystis sp PCC6803 and Microcystis
aeruginosa FACHB905. Among these compounds, 5b displayed highest inhibitory
activities (IC50 = 3.48 µM) against Cy-PDHc E1 and powerful inhibitory activities against
cyanobacteria PCC6803 (EC50 = 1.58 µM) and FACHB905 (EC50 = 1.04 µM), and showed
much higher inhibitory selectivity between Cy-PDHc E1 and porcine PDHc E1 under the
same concentration. Furthermore, our results showed that 5b served as competitive
inhibitor of ThDP to inhibit Cy-PDHc E1 by occupying the ThDP-binding pocket and then

blocking Cy-PDHc E1 bound to ThDP. The cellular microstructures were heavily
destroyed under compound 5b stress. Our results demonstrated compound 5b could be
taken as a potential lead compound targeting Cy-PDHc E1 to obtain environment-friendly
algicide for harmful cyanobacterial blooms control.
4. Experimental procedures
4.1. Chemical synthesis procedures
Unless otherwise noted, all reagents and chemicals were obtained from commercial
suppliers and used without further purification. TLC was performed to monitor all reactions
by using pre-coated glass plates. The structures of compounds 5a - 5g were
characterized by H NMR and 13C NMR spectra at 400 MHz and 100 MHz on a Varian
Mercury-Plus 400 spectrometer using DMSO-d6 as solvent. Chemical shifts were
recorded in parts per million (ppm) with TMS as the internal standard. High resolution
electron impact mass spectra (HR-EIMS) were recorded under electron impact (70 eV)
condition using a MicroMass GCT CA 055 instrument, and signals were given in m/z.
1
4
.1.1.
-(azidomethyl)-2-methylpyrimidin-4-amine 1 and N-iodomorpholine.
The important intermediate 5-(azidomethyl)-2-methylpyrimidin-4-amine 1 was
General
procedure
for
preparation
of
5
synthesized as reported methods 23. Vitamin B1 (30.6 mmol, 10.3 g) and NaN (75.4
3
mmol, 4.9 g) were dissolved in 100 mL water, and then Na SO (3.0 mmol) was added.
2
3
0
The mixture was stirred at 60 – 65 C for 5 h and cooled to room temperature, citric acid
21 g, 100 mmol) was then added to adjust pH to 4. The mixture was stirred at room
(
temperature for 1 h and then potassium carbonate was added to adjust pH to 10. The
mixture was stirred until white precipitate appeared. The white sediment was collected
through filtration, washed with water (20 mL) and dried to obtain
5-(azidomethyl)-2-methylpyrimidin-4-amine 1.
N-iodomorpholine was synthesized by the reaction of morpholine (100 mmol, 8.7 g)
and iodine (100 mmol, 25.4 g) in 400 mL; methanol solution.24 The mixture was stirred at
room temperature for 1 h and the sediment was collected by filtration and dried to get
intermediate N-iodomorpholine (94%).

4.1.2. General procedures for preparation of intermediates 3 and 4
Intermediates 3 were synthesized by the reaction of substituted benzenesulfonyl chloride
with prop-2-yn-1-amine. The solution of substituted benzenesulfonyl chloride (5 mmol) in
dichloromethane (10 mL) was added dropwise to the dichloromethane (10 mL) solution of
prop-2-yn-1-amine (0.33 g, 6 mmol) and triethylamine (0.66 g, 6 mmol) with ice-salt bath.
Then the reaction mixture was stirred at room temperature and monitored by TLC. The
reaction mixture was then added water (20 mL) and extracted three times with
dichloromethane. The organic phases were washed with 10% NaOH solution, brine, dried
with Na SO and evaporated under reduced pressure to furnish compounds 3.
2
4
Intermediates 4 were synthesized by the reaction of intermediates 3 (2 mmol) with
intermediates N-iodomorpholine (1.02 g, 3 mmol) by using CuI (19 mg, 0.1 mmol) as
catalyst in the tetrahydrofuran solution 25. The reaction mixture was stirred for 6 - 8 h at
room temperature. The mixture was then treated with vacuum filtering, and the filtrate was
evaporated under reduced pressure to obtain intermediates 4.
4.1.3. General procedures for preparation of title compounds 5
Intermediates 4 (1.5 mmol) were added to the mixture of intermediate 1 (0.21 g, 1.5 mmol),
CuI (14 mg, 0.075 mmol) and Et N (0.3 mg, 3 mmol) in tetrahydrofuran (10 mL) solution.
3
The mixture was stirred for 12 h at room temperature and then poured into water until the
reaction was completed based on TLC monitoring, the precipitate was collected by
filtration, washed with DCM (10 mL) and dried to obtain title compounds 5.
4.1.3.1N-((1-((4-amino-2-methylpyrimidin-5-yl)methyl)-5-iodo-1H-1,2,3-triazol-4-yl) methyl)
o
1
-
4-chlorobenzenesulfonamide 5a. Yellow solid; Yield 75%; m.p. 151-153 C; H NMR (400
MHz, DMSO-d ) δ(ppm): 9.26 (s, 1H), 8.66 (s, 1H), 8.30 (s, 1H), 7.90 (s, 1H), 7.78 (d, J =
6
8.3 Hz, 2H), 7.66 (d, J = 8.4 Hz, 2H), 5.42 (s, 2H), 4.00 (d, J = 5.0 Hz, 2H), 2.56 (s, 3H);
13
C NMR (100 MHz, DMSO-d ) δ(ppm): 163.45, 161.80, 147.66, 144.40, 142.94, 139.60,
6
1
37.74, 129.77, 129.07, 109.71, 47.44, 38.70, 21.80; HR-EIMS: calcd. for C H ClIN O S
15
15
7
2
+
[
M+H] 519.98139, found: 519.98212.

4.1.3.2 N-((1-((4-amino-2-methylpyrimidin-5-yl)methyl)-5-iodo-1H-1,2,3-triazol-4-yl)methyl)
o
1
-
4-nitrobenzenesulfonamide 5b. Light yellow solid; Yield 67%; m.p. 155-157 C; H NMR
(
400 MHz, DMSO-d ) δ(ppm): 9.25 (s, 1H), 8.66 (d, J = 32.7 Hz, 2H), 8.38 (d, J = 8.2 Hz,
6
2
H), 8.02 (d, J = 7.9 Hz, 2H), 7.78 (s, 1H), 5.42 (s, 2H), 4.04 (s, 2H), 2.50 (s, 3H); 13C
NMR (100 MHz, DMSO-d ) δ(ppm) 162.41, 160.81, 149.09, 146.62, 145.49, 141.67,
6
+
1
5
4
27.93, 124.19, 109.01, 46.63, 37.95, 21.00; HR-EIMS: calcd. for C H IN O S [M+H]
15 15 8 4
31.00544, found: 531.00501.
.1.3.3 N-((1-((4-amino-2-methylpyrimidin-5-yl)methyl)-5-iodo-1H-1,2,3-triazol-4-yl)methyl)
o
1
-
4-methylbenzenesulfonamide 5c. Light yellow solid; Yield 81%; m.p. 150-153 C; H
NMR (400 MHz, DMSO-d ) δ(ppm): 9.26 (s, 1H), 8.70 (s, 1H), 8.06 (s, 1H), 7.82 (s, 1H),
6
7
2
1
.68 (d, J = 7.8 Hz, 2H), 7.38 (d, J = 7.9 Hz, 2H), 5.44 (s, 2H), 3.93 (s, 2H), 2.52 (s, 3H),
.38 (s, 3H); 13C NMR (100 MHz, DMSO-d ) δ(ppm) 163.30, 161.69, 143.18, 142.63,
6
37.57, 130.14, 127.18, 109.85, 47.49, 38.78, 21.90, 21.76; HR-EIMS: calcd. for
+
C H IN O S [M+H] 500.03601, found: 500.03620.
16
18
7
2
4.1.3.4 N-((1-((4-amino-2-methylpyrimidin-5-yl)methyl)-5-iodo-1H-1,2,3-triazol-4-yl)methyl)
o
1
-
4-fluorobenzenesulfonamide 5d. Yellow solid; Yield 83%; m.p. 164-168 C; H NMR(400
MHz, DMSO-d ) δ(ppm): 9.27 (s, 1H), 8.68 (s, 1H), 8.31 (t, J = 5.5 Hz, 1H), 7.88 (s, 1H),
6
7
2
1
.79 (d, J = 8.5 Hz, 2H), 7.66 (d, J = 8.6 Hz, 2H), 5.43 (s, 2H), 4.01 (d, J = 5.2 Hz, 2H),
.53 (s, 3H); 13C NMR (100 MHz, DMSO-d ) δ(ppm) 163.43, 161.75, 147.49, 142.83,
6
39.61, 137.73, 129.72, 129.05, 109.73, 87.52, 47.36, 38.68, 21.64; HR-EIMS: calcd. for
+
C H FIN O S [M+H] 504.01094, found: 504.01147.
15
15
7
2
4.1.3.5 N-((1-((4-amino-2-methylpyrimidin-5-yl)methyl)-5-iodo-1H-1,2,3-triazol-4-yl)methyl)
o
1
-
4-bromobenzenesulfonamide 5e. White solid; Yield 74%; m.p. 161-164 C; H NMR (400
MHz, DMSO-d ) δ(ppm): 9.27 (s, 1H), 8.69 (s, 1H), 8.32 (t, J = 5.5 Hz, 1H), 7.87 (s, 1H),
6
7
2
1
.81 (d, J = 8.6 Hz, 2H), 7.71 (d, J = 8.6 Hz, 2H), 5.44 (s, 2H), 4.00 (d, J = 5.3 Hz, 2H),
.53 (s, 3H); 13C NMR (100 MHz, DMSO-d ) δ(ppm) 163.39, 161.71, 147.40, 142.68,
6
40.01, 132.62, 129.14, 126.72, 109.79, 87.80, 47.35, 38.67, 21.55; HR-EIMS: calcd. for
+
C H BrIN O S [M+H] 563.93088, found: 563.93064.
15
15
7
2
4.1.3.6 N-((1-((4-amino-2-methylpyrimidin-5-yl)methyl)-5-iodo-1H-1,2,3-triazol-4-yl)methyl)

o
1
-
2,4,6-trimethylbenzenesulfonamide 5f. White solid; Yield 85%; m.p. 195-197 C; H NMR
(
400 MHz, DMSO-d ) δ(ppm): 9.35 (s, 1H), 8.73 (s, 1H), 8.02 (t, J = 5.4 Hz, 1H), 7.82 (s,
6
1
3
1
H), 7.05 (s, 2H), 5.49 (s, 2H), 4.02 (d, J = 5.1 Hz, 2H), 2.60 (d, J = 7.8 Hz, 9H), 2.31 (s,
H); 13C NMR (100 MHz, DMSO-d ) δ(ppm) 163.16, 161.63, 147.79, 142.17, 141.86,
6
38.95, 138.72, 134.45, 132.03, 110.01, 47.20, 37.87, 23.16, 21.60, 20.95; HR-EIMS:
+
calcd. for C H IN O S [M+H] 528.06731, found: 528.06810;
18
22
7
2
4.1.3.7 N-((1-((4-amino-2-methylpyrimidin-5-yl)methyl)-5-iodo-1H-1,2,3-triazol-4-yl)methyl)
o
1
-
4-methoxybenzenesulfonamide 5g. Light green solid; Yield 62%; m.p. 139-142 C; H
NMR (400 MHz, DMSO-d ) δ(ppm): 9.30 (s, 1H), 8.76 (s, 1H), 8.01 (s, 1H), 7.78 (d, J =
6
2
6.3 Hz, 3H), 7.11 (s, 2H), 5.48 (s, 2H), 3.94 (s, 2H), 3.86 (s, 3H), 2.55 (s, 3H); 13C NMR
100 MHz, DMSO-d ) δ(ppm) 163.36, 162.59, 161.74, 144.35, 142.72, 132.24, 132.11,
(
6
1
29.33, 114.80, 109.80, 56.40, 47.42, 38.74, 21.78; HR-EIMS: calcd. for C H IN O S
16 18 7 3
+
[
M+H] 516.03093, found: 516.03114.
4.2. Inhibitory activity evaluation of compounds against Cy-PDHc E1 and porcine
PDHc E1 in vitro
The protein of Cy-PDHc E1 was prepared through cloning, expression and purification
procedures in our laboratory as previous report 22. In order to evaluate the inhibitory
activity of compounds against Cy-PDHc E1, the half maximal inhibitory concentration (IC )
50
values of these compounds were determined in vitro. For IC50 determination, a standard
reaction mixture containing 50 mM K PO (pH 7.2), 0.375 mM 2,
-dichlorophenolindophenol (DCIP), 0.8 mM sodium pyruvate, 0.1 mg/mL purified
3
4
6
Cy-PDHc E1 enzyme, 200 µM ThDP and compounds. The reaction mixture was
incubated for 5 min at 37°C and then the ThDP was added to initiate the reaction.
Inhibitory activity was determined by monitoring the reduction of DCIP at 600 nm through
a microplate reader (Bioteck Synergy2, USA). The concentrations of compounds were
arranging from 0 to 100 μM. The IC50 values were determined by nonlinear least-squares
fitting of the data using the logistic kinetic equations in Growth/sigmoidal model from origin
7.5 software.

To evaluate the inhibitory activities of compounds against porcine PDHc E1, a
standard reaction mixture containing 1.0 mM MgCl , 0.2 mM ThDP, 0.5 mM
2
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, 6.5 mM phenazine
methosulfate, 2 mM sodium pyruvate, 50 mM K PO , pH 7.0, 5 μL porcine PDHc E1
3
4
enzyme and 100 μM compounds. The reaction mixture was incubated for 3 min at 37°C,
and then substrate pyruvate was added to initiate the reaction, the optical density at 566
nm (OD566) was recorded. The inhibitory activities of compounds at 100 μM were
determined.
4.3. Inhibitory activity evaluation of compounds against cyanobacteria in vivo
To evaluate the inhibitory activity of compounds against cyanobacteria. The half maximal
effective concentration (EC ) were determined. Two model cyanobacteria strains
50
Synechocystis sp PCC6803 and Microcystis aeruginosa FACHB905 were chosen and
o
cultured with BG11 medium at 28 C for 7 days at a 12 : 12 h of light/dark cycle until the
logarithmic growth phase. The 1% cultured cells were then transferred to a fresh medium
containing 0 - 100 μM compounds. The optical density at 680 nm showed a linear relation
with cyanobacteria cell number and was used to monitor the growth of cyanobacteria. The
half maximal effective concentration (EC ) were calculated after cultured for 7 days as
50
described previously 26.
4.4. Inhibitory kinetic analysis
In order to investigate the inhibitory type of compound 5b against Cy-PDHc E1, the
maximum velocity Vmax and Michaelis constant Km constant were examined by kinetic
experiments 27. The procedures and reaction mixture were same as the enzymatic
activities testing experiment. The concentration of compound 5b was fixed and ThDP in
different concentrations (0 - 6 mM) were added to trigger the reaction. The reaction
velocity was defined as the reduction of DCIP in one minute. The kinetic parameters
Vmax and Km were fitted using the hill kinetic equation model of origin 7.5 software.

4
.5. Homology modeling and molecular docking
Homology modeling of the model 3D structure of Cy-PDHc E1 was performed according
our previous works 22. The result of amino acid sequence alignment of Cy-PDHc E1 with
geobacillus stearothermophilus PDHc E1(Geo-PDHc E1) displayed 38.4% identity.
SWISSMODEL server (https://swissmodel.expasy.org/) has been performed to build the
modeling 3D structure of Cy-PDHc E1 by taking the crystal structure of Geo-PDHc E1
(
PDB ID: 1W88) as template 28. The cofactors ThDP and Mg2+ were derived from the
template crystal structure. The model 3D structure of Cy-PDHc E1 had been proved to be
reliable in our previous works 16.
The model 3D structure of Cy-PDHc E1 was used for molecular docking. The
structure of compound 5b was prepared with SYBYL7.0 and optimized. The 3D structure
of Cy-PDHc E1 was prepared with SYBYL7.0. The water and metal ion in the model were
deleted. Then the SURFLEX module of SYBYL7.0 package was carried out to investigate
the interaction model of Cy-PDHc E1 with compound 5b. All amine acid residues located
in the cofactor ThDP-binding pocket were selected, the corresponding amino acid
residues, such as Tyr80, Asp81, Phe85, Ala129, Ile131 and Asp167, were thus involved
with the active site. All calculations were performed on the CCNU Grid website
http://www.202.114.32.71:8090/ccnu/chem/platform.xml.
4.6. Observation of cellular microstructure
SEM and TEM were applied to observe the morphological changes of cyanobacteria
FACHB905 cells after compound 5b treatment. The treatment groups were cultured under
stress of 4 μM compound 5b for 7 days, cyanobacteria cells of the control and treatment
groups were harvested, fixed, dehydrated and dried according to reported methods 29.
The samples were analyzed on a Field Emission Scanning Electron Microscope S - 4800
(Hitachi, Japan). Meanwhile, sections used for TEM observation were prepared as
previous reports 30. Subcellular microstructures were detected by a Hitachi Model
HT-7700 transmission electron microscope (Tokyo, Japan).

Acknowledgments
The work was supported by the Natural Science Foundation of China (No. 21877047,
21877046, 21867011, 21472062 and 21472061), National Research and Development
Plan (No. 2017YFD0200506), and the Project of the Science Fund of Jiangxi Education
Office (GJJ170672), the Project of Hubei Natural Science Foundation (2017CFB232),
China postdoctoral science foundation (2017M622486). The work was also financially
supported by self-determined research funds of CCNU from the colleges’ basic research
and operation of MOE (CCNU18ZDPY02) and the Program of Introducing Talents of
Discipline to Universities of China (111 program, B17019).
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Graphical abstract

Declaration of interests
☒
The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be
considered as potential competing interests: