Fungicide chinomethionate as a new family of photoinducible DNA-cleaving agents

Article abstract of DOI:10.1016/S0960-894X(03)00745-5

It is demonstrated for the first time in this report that chinomethionate is capable of causing efficient DNA cleavage under mild irradiation conditions, a fungicide molecule that processes the simple group of 1,3-dithio-2-one as its reactive functionality.

Full text of DOI:10.1016/S0960-894X(03)00745-5

Bioorganic & Medicinal Chemistry Letters 13 (2003) 3561–3563
Fungicide Chinomethionate as a New Family of Photoinducible
DNA-Cleaving Agents
Jianying Qi, Tianhu Li* and Albert S. C. Chan*
Open Laboratory of Chirotechnology of the Institute of Molecular Technology for Drug Discovery and Synthesis
and Department of Applied Biology and Chemical Technology, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong
Received 23 April 2003; accepted 30 June 2003
Abstract—It is demonstrated for the first time in this report that chinomethionate is capable of causing efficient DNA cleavage
under mild irradiation conditions, a fungicide molecule that processes the simple group of 1,3-dithio-2-one as its reactive func-
tionality.
# 2003 Elsevier Ltd. All rights reserved.
Chinomethionate (6-methyl-2,3-quinoxalinedithiol cyc-
lic carbonate), a major ingredient of Morestan, is an
effective fungicide against mites, aphids, white flies and
powdery mildew on many species.1 One of the structural
distinctiveness of this fungicide is the possession of 1.3-
dithio-2-one as its reactive functionality. It was reported
in the past that field workers using chinomethionate had
experienced server skin irradiation when they were
exposed to sunlight.2 In addition, it was found that the
half-life of this fungicide on apple leaves in the sun was
much shorter than those in the shady areas.3 Chemical
studies, on the other hand, revealed that irradiation of a
dilute benzene solution of chinomethionate by UV light
gave rise to products arising from the reaction of inter-
mediate radicals with the surrounding oxygen and 1,3-
dithio-2-one was the reactive functionality.4,5 With the
aim of identifying new types of photoinducible DNA-
cleaving functionality that are readily activated under
mild irradiation conditions, the photochemical proper-
ties of chinomethionate on target DNA were examined
recently in our laboratory. In this communication, we
report that fungicide chinomethionate with the func-
tionality of 1,3-dithio-2-one embedded in its molecular
structure was capable of causing efficient DNA cleavage
under mild irradiation condition, a mode of action that
might underlie the previously observed biological beha-
viors of this fungicide.1À3 In addition, our studies
showed that both carbon-centered and hydroxyl radi-
cals were most likely accountable for the DNA-cleaving
process by chinomethionate, a pathway that was
consistent with the chemical properties of this fungicide.
The DNA-cleaving ability of chinomethionate under
irradiation was examined in our studies by monitoring
the conversion of circular supercoiled (form I) to cir-
cular relaxed (form II) DNA. For this purpose, this
fungicide was irradiated at 365 nm by using a Blak-Ray
Bench Lamp (15 W) at various concentrations of chino-
methionate for 10 min in the presence of fw174RF
NDA (30 mM/bp) in acetonitrile: Na2HPO4 buffer (25
mM, pH 7.0): triton [10:20:2.5 (v/v/v)]. As shown in
Figure 1, single-stranded DNA cleavage was observed
at concentration as low as 10 mM and in a concen-
tration-dependent fashion while no DNA cleavage
occurred in the absence of chinomethionate (lane 1, Fig.
1). In addition, the yield of this DNA-cleaving reaction
increased with the increase of irradiation time (Fig. 2).
Under the conditions designed for our investigation, the
yield of this DNA-cleaving reaction could reach up to
nearly completion in 12 min, illustrating the relatively
high efficiency of this DNA-cleaving process by chino-
methionate. As a positive control, the reaction of DNA
with methyl quinoxalinecarboxylate, a known highly
efficient photoinducible DNA-cleaving agent,6 was
*Corresponding author. Tel.: +852-2766-5607; fax: +852-2364-9932;
e-mail: bcachan@polyu.edu.uk
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00745-5

3562
J. Qi et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3561–3563
carried out under the similar reaction conditions to the
one designed for chinomethionate. As can be seen from
Figure 3, the DNA-cleaving activity of methyl quinox-
alinecarboxylate was nevertheless more efficient than
the sulfur-containing agent, chinomethionate.
and Loeffler in 19804 in which carbon-centered and
peroxide radicals were generated in the reactions.
It has been recognized previously that simple phenyl
radicals are capable of causing efficient single-stranded
scissions to DNA.9 In addition, it was well established
in the past that hydroxyl radical generated from oxygen,
superoxide radical as well as from hydrogen peroxide
could initiate a DNA cleaving process effectively.10 In
order to examine whether the carbon-centered and
hydroxyl radicals were indeed the active species
accountable for the DNA-cleaving process by chino-
methionate, the inhibitory effects of 2,2,6,6-tetra-
methylpiperidinooxy (TEMPO, carbon-centered radical
scavenger) and sodium benzoate (hydroxyl radical sca-
venger)9 on this DNA-cleaving process were examined
accordingly. As shown in Figure 4, the DNA-cleaving
process by chinomethionate was efficiently inhibited by
the addition of TEMPO and this inhibitory effect
increased with the increase of the concentration of
TEMPO (lanes 3–5). These observations could accord-
ingly be considered as the experimental evidence of
involvement of carbon-centered radical in the DNA-
cleaving reactions. Similarly, addition of sodium
benzoate to the corresponding reaction mixture slowed
down the rate of the DNA-cleaving reactions (lanes 6–
10), indicating that hydroxyl radicals were probably the
additional active species involved in the DNA-cleaving
process by chinomethionate.
As a reference point for this study, the photolyse of 30
mg of chinomethionate in tetrahydrofuran as solvent
was carried in our laboratory by using the same irra-
diation source set up for our DNA cleavage studies.7
After 15 h irradiation at room temperature, for exam-
ple, 6-methyl-1,2,3,4-tetrahydroquinoxaline-2,3-dione
(5, Scheme 1) was obtained8 in 65% yield from the cor-
responding reaction. This experimental result of ours
was compatible with the outcome of photodecomposi-
tion studies on chinomethionate conducted by Clark
Figure 1. DNA cleavage by chinomethionate activated by UV irradia-
tion.9 Reactions were incubated in 25 mM Na2HPO4 buffer solution
(pH 7.0) containing DNA (30 mM/bp), quinomethionate, 10% (v/v)
CH3CN and 2.5% (v/v) triton-X and were irradiated at room tem-
perature for 10 min. Lane 1, DNA alone; lane 2, DNA+ 10 mM of 1;
lane 3, DNA+ 50 mM of 1; lane 4, DNA+ 100 mM of 1; lane 5,
DNA+ 250 mM of 1; lane 6, DNA+ 500 mM of 1; lane 7, DNA+ 750
mM of 1.
In summary, our studies demonstrate that the simple
functionality of 1,3-dithio-2-one embedded in fungicide
Figure 2. Irradiation-time dependence of DNA-cleavage by chino-
methionate. Reactions were incubated in 25 mM Na2HPO4 buffer
solution (pH 7.0) containing DNA (30 mM/bp), 250 mM of 1, 10% (v/v)
CH3CN and 2.5% (v/v) triton-X and were irradiated at room tem-
perature. Lane 1, DNA alone without irradiation; lane 2, DNA+ 1
without irradiation; lane 3, DNA alone irradiated for 8 min; lane 4,
DNA+ 1 irradiated for 8 min; lane 5, DNA alone irradiated for 10
min; lane 6, DNA+ 1 irradiated for 10 min; lane 7, DNA alone irra-
diated for 12 min; lane 8, DNA+ 1 irradiated for 12 min; lane 9,
DNA alone irradiated for 14 min; lane 10, DNA+ 1 irradiated for 14
min.
Scheme 1. Proposed decomposition mechanism of chinomethionate
under UV irradiation.4
Figure 4. DNA cleavage by chinomethionate inhibited by radical sca-
vengers. Reactions were incubated in 25 mM Na2HPO4 buffer solution
(pH 7.0) containing DNA (30 mM/bp), 250 mM of 1, 10% (v/v)
CH3CN and 2.5% (v/v) triton-X and were irradiated at room tem-
perature for 8 min. Lane 1, DNA alone; lane 2, DNA+1; lane 3,
DNA+1+1 mM of TEMPO; lane 4, DNA+1+10 mM of TEMPO;
lane 5, DNA+1+100 mM of TEMPO; lane 6, DNA+1+0.1 mM of
sodium benzoate; lane 7, DNA+1+1 mM of sodium benzoate; lane 8,
DNA+1+10 mM of sodium benzoate; lane 9, DNA+1+100 mM of
sodium benzoate; lane 10, DNA+1+200 mM of sodium benzoate.
Figure 3. DNA cleavage by methyl 2-quinoxalinecarboxylate (MQ)
activated by UV irradiation. Reactions were incubated in 25 mM
Na2HPO4 buffer solution (pH 7.0) containing DNA (30 mM/bp), MQ,
10% (v/v) CH3CN and 2.5% (v/v) triton-X and were irradiated at
room temperature for 10 min. Lane 1, DNA alone; lane 2, DNA+ 1
mM of MQ; lane 3, DNA+ 2 mM of MQ; lane 4, DNA+ 4 mM of
MQ; lane 5, DNA+ 8 mM of MQ; lane 6, DNA+ 12 mM of MQ.

J. Qi et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3561–3563
3563
chinomethionate was capable of causing efficient DNA
cleavage, a process that can readily be activated via mild
UV irradiation. In addition, our results indicate that the
DNA-cleaving process by chinomethionate was see-
mingly through the generation and reaction of carbon-
centered and hydroxyl radicals. In consideration of the
structural simplicity of 1,3-dithio-2-one and the mild
irradiation condition as the activating force, this newly
discovered photoinducible DNA-cleaving functionality
could have certain implications in the design of new
diagnostic probes and therapeutic agents.11
Staiff, T. C. Abstracts, 10th Annual Meeting of the Society of
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7. A Blak-Ray Bench Lamp (Model: XX-15L, 365 nm, 15 W,
UVP, Inc., USA) was used as the irradiation source of our
investigations. All DNA-cleaving reactions by chinomethio-
nate were analyzed by electrophoresis on 1% agarose gels in
1 M of Tris–acetate buffer (100 V, 1 h). The resulting gels were
subsequently photographed on a UV transilluminator after
attaining with ethidium bromide. The amount of DNA in each
band of ethidium-stained gels was quantified using Lumi-
Imager (LumiAnalyst 3.1).
Acknowledgements
8. For 5, dH (DMSO-d6): 11.873 (s, 1H, NH); 11.848 (s, 1H,
NH); 6.910 (s, 1H, ArH); 7.008 (d, J=8 Hz, 1H, ArH); 6.901
(d, J=8 Hz, 1H, ArH); 2.265 (s, 3H, CH3).
We thank the University Grants Committee of Hong
Kong (Areas of Excellence Scheme, AOEP/10-01) and
the Hong Kong Polytechnic University ASD Fund for
the financial support of this study.
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