Synthesis of Cantharidin Derivatives as Photo-Initiated DNA Cleavage Agents

Introduction

Cantharidin

Cantharidin, a traditional Chinese medicine, is a poisonous chemical compound secreted by many species of blister beetle, and the most notable one is secreted by the Spanish fly, Mylabris phalerata Pall or M. cichorii L. The false blister beetles and cardinal beetles also have cantharidin. It was first isolated by Pierre Robiquent in 1810. Dadamen confirmed the structure of cantharidin in 1914. Cantharidin is also called exo, exo-2,3-dimethyl-7oxobicyclo [2.2.1] heptane-2,3-dicarboxylic acid anhydride (Figures 1.1 & 1.2) and secreted by the male blister beetle which will be given to the female during the mating. Afterwards the female beetle will cover its eggs with it as a defense against predators. Researchers of Harvard University, such as Woodward R B, et al. they synthesize desoxycantharidine in 1941 [1]. A total synthesis of cantharidin (I) from the butadiene adduct (X) of dimethyl 3,6-epoxy-3,4,5,6-tetrahydrophthaIate (IX) was described by Stork G, et al. [2, 3]. Efficient total synthesis of Cantharidin via a High-Pressure Diels-Alder Reaction was carried out by Dauben W. G. and Krabbenhoft H O [4, 5]. We use the method which is developed by Lin P. Y. in our laboratory because High-Pressure Diels-Alder Reaction is too dangerous. We use dried bodies of those beetles to extract cantharidin (Scheme 1) [6].

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Scheme 1: Preparation of cantharidin.

Cantharidin has been reported to be active which can inhibit cancer cell proliferation in 1264. Recently, some reports indicate cantharidin and cantharidin derivatives have clinical potential, which can be seen as protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) inhibitors [7]. This activity is necessary for the growth inhibition activity of these compounds. Protein phosphatases are involved, among others, in the regulation of multiple cellar processes including signal transduction pathway, cell cycle progression, glucose metabolism, and calcium transport [8]. Cantharidin can also induce leukemia cell apoptosis by p53-dependent mechanism [9]. A previous study suggested that treatment with several analogs of cantharidin increase the activity of xanthine oxidase which would result in an increased production of reactive oxygen species (ROS) [10]. ROS are well known as DNA damaging agents [11]. Via comet assay, DNA single- and double-strands can be broken by treating cantharidin. Thus, although the biochemical target of cantharidin and its derivative is known, but the critical molecular pathways about how cantharidin and its derivative cause growth inhibition of cell death are unclear.

Cantharidin Derivatives: Cantharidin has been reported to be active against various human cancers, especially hepatocarcinoma; however, its severe renal toxicity limits the development as chemotherapeutic agents [12, 13]. Lin P Y, et al. synthesized a series of novel cantharidinimides and cantharidin derivatives by using high pressure and evaluated their cytotoxicities against human carcinoma cells [14, 15] (Scheme 2).

Scheme 2: Synthesis of novel cantharidinimides by using high pressure.14 (a): TEA, Toluene, c.a. 200°C reflux, R = Amines.

Other Chemical Materials

Many mutagens fit into the space between DNA two adjacent base pairs of DNA, this is called intercalating. Most intercalators are aromatic and planar molecules. In order for an intercalator to fit between base pairs, the bases must be separated and distorted the DNA strands by unwinding of the double helix. This inhibits both transcription and DNA replication, causing toxicity and mutations. As a result, DNA intercalators are often carcinogens. Acridines, aflatoxin and ethidium bromide are well-known examples [16, 17, 18] (Figures 1.3-1.7).

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Deoxyribonucleic Acid (DNA)

Deoxyribonucleic acid (DNA) is nucleic acid that contains the genetic instructions used in the development and functioning of all known living organisms and some viruses. The main role of DNA molecules is the long-term storage of information. It store the genetics messages to lead with the features of the life such as replication, metabolism and physiological processes of bio-organisms, Of course, to facilitate the construction of other components of cells, such as proteins and RNA molecules are also guided by the genetic codes of DNA. DNA is a long polymer made from repeating units which called nucleotides [21]. The DNA chain ranges from 22 to 26 Angstroms wide (2.2 to 2.6 nanometers), and one nucleotide unit is 3.3 Å (0.33nm) long.

In living organisms, DNA does not usually exist as a single molecule, but instead as a tightly-associated pair of molecules [22]. These two long strands entwine like vine, in the shape of a double helix. The nucleotide repeats contain both the segment of the backbone of the molecule, which holds the chain together, and a base linked to a sugar is called a sugar and one or more phosphate groups is called a nucleotide. If many nucleotides are linked together, as in DNA, the nomenclature of the corresponding polymer is a polynucleotide [23].

The backbone of DNA strand is made from alternating phosphate and sugar residues [24]. The sugar in DNA is 2-deoxyribose which is a pentose (five-carbon) sugar. The deoxyribose contains seven hydrogen atoms attached to the 5-membered ring, and generally designated these atoms as H-5’, H-5”, H-4’, H-3’, H-2’, H-2”, and H-1’ (Figure 1.8) [25]. The sugar is joined together by phosphate groups derived from phosphodiester bonds between the third and fifth carbon atoms of adjacent sugar rings. The sugar-phosphate backbone is highly vulnerable to be attacked by free radical. Abstraction of a hydrogen atom from deoxyribose produces a carbon-based sugar radical that can rearrange, and culminate in scission of the nucleic acid strand [26]. Those asymmetric bonds mean a strand of DNA as a direction. In a double helix the direction of the nucleotide in one strand is opposite to their direction in the other strand. This arrangement of DNA strands is called anti-parallel. The asymmetric ends of DNA strands are referred to as the 5’ (five prime) and 3’ (three prime) ends. The 5’ end is with a terminal phosphate group and the 3’ end is with a terminal hydroxyl group (Figure 1.9). One of the major differences between DNA and RNA is sugar, 2-deoxyribose is replaced by the alternative pentose sugar ribose in RNA [27].

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The DNA double helix is stabilized by hydrogen bonds between the bases attached to the two strands. These bases are classified into two types; adenine (A) and guanine (G) are fused five-and six-membered heterocyclic compounds called purines, whereas cytosine (C) and thymine (T) are six-membered rings called pyrimidine’s (Figure 8). Another fifth pyrimidine base, called uracil (U), usually takes place of thymine in RNA and differs from thymine by lacking a methyl group on its ring. Uracil is not usually found in DNA, occurring only as a breakdown product of cytosine. Those four bases (A, T, C, and G) are attached to the sugar/phosphate to form the complete nucleotide, and each type of bases on one strand forms hydrogen bonding with just another specific type of base on the other strand. Here, purines form hydrogen bonds to pyrimidines, where A bonding only to T with two hydrogen bonds, and C bonding only to G with three hydrogen bonds.

This arrangement of two nucleotides binding together across the double helix is called base pair. As a result of this complement, all the information in the double-stranded sequence of a DNA helix is duplicated on each strand, which is vital in DNA replication. Indeed, this reversible and specific interaction between complementary base pair is critical for all the functions of DNA in living organisms [28]. Purine and pyrimidine hetrerocycles containing rich electrons are prime targets for reaction with electrophiles: alkylating agents, oxidizing agents and halogens. These reagents rarely lead to direct (or frank) strand scission, but allow DNA or RNA cleavage to arise site specifically in second chemical step, usually including heat, base, or enzyme treatment to effect deglycosylation and β-elimination of the 3’-phosphate (Figure 1.10) [29].

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Normally, the double helix is right-handed spiral. As the DNA strands wind around each other, they leave gaps between each set of phosphate backbones to combine bases inside together. There are two of these grooves twisting around the surface of the double helix: one groove is 22 Å wide and the other, the minor groove, is 12Å wide [30]. The narrowness of the minor groove means that the edges of the bases are more accessible than the major groove. As a result, proteins like transcription factors can bind to specific sequences in double-stranded DNA which usually make contacts to the sides of the bases exposed in the major groove [31]. This situation varies in unusual conformations of DNA within the cell, but the major and minor grooves are always named to reflect the differences in size that would be seen if the DNA is twisted back into the ordinary B form.

DNA Damage

Sources of DNA damage can be sub-divided into endogenous damage and exogenous damage. DNA endogenous damage such as attacked by reactive oxygen species produced from normal metabolic byproducts (spontaneous mutation) can cause oxidation and deamination. Exogenous damage caused by external agents such as ultraviolet radiation from the sun, other radiation frequencies (x-rays and gamma rays), hydrolysis or thermal disruption, certain plant toxins and artificial mutagenic chemicals etc. Artificial mutagenic chemicals are able to cause DNA damage such as deoxyribose decomposed or nucleo base site oxidation. Furthermore, artificial mutagenic chemicals can cooperate with DNA discrimination systems to cleavage specific base site and hydrolyse phosphodiester bond to form direct strand scission via irradiation, redox reaction or pH regulation. According to the conception of antisense genes, a single strand of DNA or RNA is purposely designed to prevent some translation from injurious proteins and artificial mutagenic chemicals which reveal the potential for the molecular engineering of DNA for genetic manipulation and gene therapy in biomedicine [32].

Types of DNA Damage

For DNA damages, there are four major types are corresponded to the base lesion and listed as follow: Oxidation: Reactive oxygen species can cause base oxidation and interrupt DNA strand. Reactive oxygen species, such as hydroxyl radical, can be produced in various ways (Figure 1.11). Hydroxyl radical tends to attack guanine nucleobase. There are three major intermediates that result from hydroxyl radical oxidizing a guanine. These effects cause the formation of C4 adduct (1), C5 adduct (2), and the C8 adduct (3) (Figure 1.12).

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Alkylation: Methylation is the most common type of alkylation. DNA methylation typically occurs at CpG sites (cytosine-phosphate-guanine sites; where a cytosine is directly followed by guanine in DNA sequence); this methylation results in the conversion of the cytosine to 5-methylcytosine (Figure 1.13&1.14).

Hydrolysis: Depurination or depyrimidination are DNA alteration in which the hydrolysis of theβ-N-glycocidic link between purine or pryimidine base and yield the deoxyribose-phosphate backbone [33].

Deamination: Spontaneous deamination is the hydrolysis reaction of cytosine into uracil which can release ammonia during the process. Deamination of 5-methylcytosine results in thymine and ammonia. This is the most common single nucleotide mutation and if it does not affect the function of the gene, the mutation will persist. Deamination of guanine results in the formation xanthine and producing hypoxanthine in adenine (Figure 1.15).

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Another DNA damage type is C-H abstraction on sugar moiety. For example, Rhodium (III) complex’s (Figure 1.16) is a metal complexes that has been established to cleave DNA through abstraction of 3’-hydrogen. Rhodium (III) coordinated by well-known intercalating ligands is able to bind to duplex DNA and generate strand breaks in presence. Previous studies revealed the products of this reaction to be, as shown in scheme 3. 5’-phosphate-terminated DNA strands, a DNA strand terminating in a 3’-phosphoglycaldehyde group and base propenoic acid.

Scheme 3: Proposed H-3¢-Abstraction Pathway for DNA Strand Scission Mediated by [Rh(phen)2(phi)]3+ or [Rh(phi)2bpy]3+ under Aerobic Conditions (adapted from Sitlani et al. [34])

Correlative Analysis Methods

Scientists have used different techniques such as HPLC, PAGE or reaction with thiobarbituric acid to detect DNA damage products (Table 1). Gel electrophoresis is employed to identify DNA strands lesion and Maxam-Gilbert sequencing reaction is used to determine DNA sequence. Recently, MALDI-TOF provides rapid and sensitive method to directly analyze not only site specificity but also DNA cleavage products [35].

Principle of Gel Electrophoresis: DNA is a negatively charged molecule. If put DNA into an electric field, DNA will move from the negative to the positive pole. A gel serves as the matrix for the movement of the DNA molecule. The gel is a semisolid material with certain size pores. Plasmid DNA structure consists of three forms, Form I, supercoiled; Form II, relaxed and Form III, linear (Figure 1.17). The supercoiled form is able to move faster through the pores of the gel than linear and relaxed form. Because the different mobility, we can identify the plasmid DNA structure in gel. Furthermore, we can use the method to test the ability of compound cleavage DNA.

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Principle of Maxam-Gilbert Sequencing: Around 1977, Wally Gilbert (Harvard) and his technician Allan Maxam developed chemical sequencing. This method requires radioactive labeling at one end and purification of DNA fragment sequenced. Chemical treatment generates breaks at a small proportion of one or two of the four nucleotide bases in each of four reactions (G, A+G, C, C+T) (Table 2). Thus a series of labeled fragments is generated, from the radiolabled end to the first ‘cut’ site in each molecule. The fragments in the four reactions are arranged side by side in gel electrophoresis for size separation. To visualize the fragment, the gel is exposed to X-ray film for autoradiography which yields a series of dark bands and each corresponds to a radiolabeled DNA fragment, from which the sequence may be inferred (Figure 1.18).

Motive

Previous report indicates that cantharidin causes oxidative stress to provoke DNA damage in vivo, so we try to use cantharidin and its derivatives to trigger DNA cleavage by irradiation. Some of materials exhibiting planet aromatic compound were also determined to test whether it can cleave DNA strand or not. In the mean-time, we would like to endeavor on the mechanism of the critical molecular pathways of DNA damages. Ultimately, we would like to develop chemical nucleases to apply to anticancer or gene therapy in the future.

Materials and Methods

Extract Cantharidin from Beetles (Table 3)

Instrument:

• Hot plate : IKA LABORTECHNIK
• Heating Bath B-490
• Rotavapor R-200
• Low Temperature Thermostatic Water baths The preparation of Cantharidin (Figure 2.1).

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We added Mylabris (0.5Kg) into 50% EtOH and concentrated it by evaporator. After concentrating, we extract H2O layer from the mixture and filtrated the layer by celite filter. The filtrate will partition into H2O layer and EtOAc layer by added EtOAc. Finally, we collected the EtOAc layer and concentrated the layer by evaporator. The residues were being recrystallized with MeOH and produced cantharidin (0.5g). Finally, we got white crystal. Yield: 0.1% m.p: 216-218°C (MeOH); 1H NMR (CDCl3, 500MHz): δ 1.24 (s, 6H, CH3×2), δ 1.74-1.81 (m, 4H, CH2×2), δ 4.73 (t, 2H, J=3.0, 1.8Hz, OCH×2).

Synthesis (Table 4)

Instrument:

• 2-Decimal Balance: METTLER YOLEDO, AG 245
• 4-Decimal Balance: METTLER YOLEDO, AG 204
• Hot plate : IKA LABORTECHNIK
• Thick-walled glass
• Teflon tank
• Seal steel-made container
• Microprocessor temperature controller: MAXTHERMO MC-2838 The preparation of 2,6-Dimethyl-4-[4-(6-methyl- benzothiazol-2-yl)-phenyl]-10-oxa-4-aza-tricyclo [5.2.1.0] decane-3,5-dione (compound D) (Figure 2.2).

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Figure 2.2: Structure of 2,6-Dimethyl-4-[4-(6-methyl- benzothiazol-2-yl)-phenyl]-10-oxa-4-aza-tricyclo [5.2.1.0] decane-3,5-dione (compound D) We added cantharidin 196 mg (196.2 g/mol) and 2-4-(Aminophenyl)-6-methy-benzothiazole 264 mg (240.33g/mol) in 2ml triethanolamine and sealed those mixture into a thick-walled glass tube. After heating the tube to 200°C for 2.0 h, open the lid and cool the tube to room temperature. Continue heating to 190°C for 1.0h to evaporate H2O. Finally, we will get black and sticky mixture (337mg). The mixture was separated by PTLC.

The Layer chromatography was performed under dichloromethane/ ethyl acetate 18:1(by vol.) as its solvent system. Then, the PTLC plate was sunk into solvent to separate products. After heating the plate, the sample was able to identify as dark-yellow band on PTLC plates. Scrape of the products from PTLC and mix with CH2Cl2. Finally, we filtered off the silica and dried the CH2Cl2 solvent to obtain crystals (yellow solid) Yield: 48%; MS m/z (rel. int.) 418.14[M]+ (100) PTLC R_f_: 0.85 (Figure 2.3).

The Preparation of N-(4-Amino-Phenyl)-Benzo[De] Isoquinoline-1,3-Dione (Compound K)

We add 198 mg 1,8-Naphthalic anhydride (198.17 g/ mol) and 119 mg p-phenylenediamine (108.14 g/mol) into 2 ml triethanolamine and 8 ml toluene. Then, we seal those mixture into a Teflon tank and heated it to 180°C for

3hr. Cooled it to room temperature slowly. After cooling, we discarded the solvent and get precipitate. The precipitate was transferred into a clean tank and added CH2Cl2 to dissolve it. The dissolved mixture was separated by PTLC. The products (148 g) were obtained in 75% yield.

The Layer chromatography was performed under dichloromethane/ hexane 1:1 (by vol.) as its solvent system. Then, the PTLC plate was sunk into solvent to separate produce. After heating the plate, the sample was able to identify as brown band on PTLC plates. Scrape of the products from PTLC and mix with CH2Cl2. Finally, we filtered off the silica and dried the CH2Cl2 solvent to obtain crystals.

White crystal. Yield: 75% m.p: 295-297°C (MeOH);

1H NMR (CDCl3, 500MHz): δ 6.80(2H,d, J=8.8Hz,phenyl

H3’,H5’), 7.06(2H,d, J=8.6Hz,phenyl H-2’,H-6’), 7.77(2H,t,

$$ \begin{array}{l} h 3, h 5 J, 7. 0 6 (2 H, d, J = 8. 0 H z, p h e n y l H - 2, H - 6 J, 7. 7 7 (2 H, t, J = 7. 7 H z, H - 5, H - 8), 8. 2 3 (2 H, d, J = 8. 0 H z, H - 6, H - 7), 8. 6 2 (2 H, t, J = 6. 7 H z, H - 4, H - 9). \\ \end{array} $$ The Growth of Escherichia coli (pUC19 DH5α) (Table 5)

Instrument: • 2-Decimal Balance: METTLER YOLEDO, AG 245

• 4-Decimal Balance: METTLER YOLEDO, AG 204

• Water Purification systems: BIBBY-A400 (RO) & Millipore Milli-Q Plus (DI)

• Oven: DENG YNG, Drying Oven DOS60

• Autoclave: TOMIN, TM-329,121°C, 1.2 Kg/cm2

• Orbital Shaker Incubator: FIRSTEK SCIENTIFIC, S300R

• BSC: 4BH-24

The pUC19 plasmid was transformed to E.coli. We added 5μl pUC19 plasmid to a 50μl solution of DH5α competent cell, put in an ice bath for 5 min, and in a 42°C bath for 2 min with heat stock. Finally 100μl LB was added, and the mixture was incubated at 37°C, 10 min. We collected 50μl broth and spread it on a LB(Amp) plate by glass beads at 37°C incubate it overnight. After incubating the plate, we selected one survival colony to transfer it to 3ml LB and incubate it for 16~18 hr. We added the 3 ml LB mixture to 100 ml LB and incubate it in 37°C, 220 rpm condition till OD= 0.8.

Purification Plasmid DNA (Table 6)

Instrument:

• Water Purification systems: BIBBY-A400 (RO) & Millipore Milli-Q Plus (DI)

• Oven: DENG YNG, Drying Oven DOS60

• Autoclave: TOMIN, TM-329,121°C, 1.2 Kg/cm2

• Vortex: FINEVORTEX

• Low Temperature Freezer: 4°C, FIRSTEK SCIENTIFIC, CC-2; -20°C: White-Westinghouse
• Spin
• Centrifuge: Labnet MODLE:24D; Beckman, Coulter Optima L-90K Ultracentrifuge
• Rotor: SLA 1500
• Ultraviolet-Visible Spectrophotometer: HP 8453 The harvested cells were separated by centrifuge (8000rpm, 10min). Pellet, which contained cells, we used 10ml Midiperp Kit solution I to resuspend and react it for 10 min in room temperature. The 10ml Midiperp Kit solution II was added to mixture and reacted it for 10 min. After reaction, 10ml Midiperp Kit solution III was added to mixture and denatured proteins, cell remains, and chromosome DNA etc. By centrifuging (8000 rpm, 10min), we can obtain supernatant liquor, which contained pUC19 plasmid DNA. In order to precipitate the rough pUC19 plasmid DNA, we added 24 ml isopropanol and centrifuged (12000 rpm, 10 min). We used 100μl D.I. Water to dissolve the rough pUC19 plasmid (white pellet) and added 100μl phenol & chloroform mixture. After centrifuging, we got supernatant liquor and added 100μl chloroform to precipitate impurities. We selected supernatant liquor and used 80μl isopropanol to precipitate pure pUC19 plasmid. Finally, we collected the pellet by centrifuging and used D.I. Water to dissolve it. We also processed dialysis to obtain more pure pUC19 plasmid DNA and used UV-vis spectrometry (Ratio=260/280) to identify the degree of purity (Figure 2.4).

Photoreaction & DNA Cleavage Studies via Supercoiled Plasmid (Table 7 & 8)

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Instrument: • 4-Decimal Balance: METTLER YOLEDO, AG 204

• Water Purification systems: BIBBY-A400 (RO) & Millipore Milli-Q Plus (DI)

• Oven: DENG YNG, Drying Oven DOS60

• Agar Horizontal gel Electrophoresis

• Electrophoresis Power Source: GE 100

• Photo-documentation system: Electronic case; MITUBISHI, P91W; DOC-print CCD F590 180x240
• Photochemical Reactor PR 2000
• pH Meter: SUNTEX pH/ORP Meter
• Microwave oven: SAMPO Explore the DNA Cleavage with Site Specificity (Table 9)

Supercoiled pUC19 plasmid (1mg/mL) was reacted with varieties of concentration of those compounds (1μM~1000 μM) by irradiating UV light (352nm) at room temperature for 1hr. After photoreaction, we analyzed the cleaving abilities of those compounds by gel electrophoresis. To the reaction mixture was added gel-loading buffer (2μL containing 0.25% bromophenol blue, 0.25% xylene cyanol, and 30% glycerol). Then the reaction mixture was loaded on 1% agarose gel with ethidium bromide staining. The electrophoresis tank was attached to a power supply at current (100mA). The gel was visualized by 312-nm UV transilluminator and photographed by photo documentation system. Quantitation of DNA cleavage was performed by integration of the optical density as function of the band area by use of Scion Image program.

Instrument:

• 4-Decimal Balance: METTLER YOLEDO, AG 204
• Water Purification systems: BIBBY-A400 (RO) & Millipore Milli-Q Plus (DI)
• Oven: DENG YNG, Drying Oven DOS60
• Spin
• SDS Vertical Slab Gel Electrophosis
• Electrophoresis Power Sourse: Bio-Rad
• Orbital Shaker: FIRSTEK S-101
• pH Meter: SUNTEX pH/ORP Meter
• PCR machine: Biometra T3 Thermo cycler
• pH Meter: SUNTEX pH/ORP Meter
• Low Temperature Freezer: 4°C, FIRSTEK SCIENTIFIC, CC-2; -20°C: White-Westinghouse
• ECL Semi-dry Blotters: Amersham Biosciences
• CL-1000 Ultraviolet Crosslinker: UVP
• Typhoon: 9410 To explore the DNA cleavage with site specificity, we use the HIV-27 DNA, which labeled the 5’-terminus of oligonucleotide (5’-GCAGATCTGAGCCTGGGAGCTCTCTGC-3’) with biotin [36]. The reaction mixture containing 5’-end labeled biotin-DNA solution (50μM), a phosphate buffer (0.10 M, pH 5.0) and compound D (1mM) in Pyrex vial were irradiated with 352-nm UV at room temperature for 1.0hr, the reaction mixtures were quenched with 95% EtOH. The solutions were then subjected to piperidine treatment and ethanol precipitation. The DNA pellet was re-suspended in aqueous piperidine solution (10% 70μL) and maintained at 95°C for 30 min. Subsequently, the reaction mixtures were sequentially lyophilized, treated with water (30μL), lyophilized, and re-suspended in the formamide loading buffer (80% formamide, 0.25% bromophenol blue, and 0.25% xylene cyanol). The DNA solution and Maxam-Gilbert markers were analyzed by use of 20% polyacrylamide/7M urea gel. The electrophoresis was performed at voltage of 250 V for 1 hr. The 20% polyacrylamide/8M urea gel was transferred to nylon membrane by ECL semi-dry blotters. The DNA solution and Maxam-Gilbert markers produced chemiluminescence by Panomics’ EMSA kit protocol. The cleavage positions were visualized by use of Kodak X-Omat Ar-5 film.

We also use the HIV-27 DNA labeled 5’-terminus of oligonucleotide with 6-FAM (6-Caboxyfluorescein) to proceed experiment. The reaction mixture containing 5’-end

labeled 6-FAM-DNA solution (20μM), a phosphate buffer (0.10 M, pH 5.0) and compound D (100μM) in Pyrex vial were irradiated with 352-nm UV at room temperature for 1.0hr, the reaction mixtures were quenched with 95% EtOH. The solutions were then subjected to piperidine treatment and ethanol precipitation. The DNA pellet was resuspended in aqueous piperidine solution (10% 70μL) and maintained at 95°C for 30 min. Subsequently, the reaction mixtures were sequentially lyophilized, treated with water (30μL), lyophilized, and resuspended in the formamide loading buffer (80% formamide, 0.25% bromophenol blue, and 0.25% xylene cyanol). The DNA solution and Maxam-Gilbert markers were analyzed by use of 20% polyacrylamide/7M urea gel. The electrophoresis was performed at voltage of 250 V for 1 hr. After electrophoresis, we use 488-nm light to excite 6-FAM and detected the emission wave light (518-nm) by typhoon.

Results and Discussion

Synthesis

The cantharidin was isolated from dried bodies and prepared cantharidin derivatives using the pressure technique synthesis. We utilized the hydrothermal method to synthesize 8-Naphthalic anhydride derivatives. The method of extract cantharidin from Mylabris phalerata Pall is safer than High-Pressure Diels-Alder Reaction, Here, we could obtain 0.5g cantharidin and identified by NMR (Appendix 1). In addition, we use a simple procedure for preparation of cantharidin derivatives 1,8-Naphthalic anhydride derivatives and identify those compound by H-NMR (Appendix 2-5 ).

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Appendix 1: 1H-NMR spectrum of cantharidin (CDCl3, 500 MHz).

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Appendix 2: 1H-NMR spectrum of compound D (CDCl3, 300 MHz).

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Appendix 3: LRMS spectrum of Compound D (EI).

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Appendix 4: LRMS spectrum of Compound D (FAB).

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Appendix 5: 1H-NMR spectrum of compound K (CDCl3, 400 MHz).

Prepare Super-Coiled DNA

The E. coli (DH5α) containing pUC19 plasmid was cultured in LB medium with amplicillin for 12~16h. After incubating and centrifuging, we got about 0.29 mg/ml bacteria pellet (wet weight). Then, we used different solvents to extract the plasmid. Notably, the extracted plasmid was affected with irradiating with UV light (352-nm) (Figure 3.1).

We surmised that the plasmid contained some impurities which may cause radiation- sensitivity. To remove the impurities, the extracted plasmid was dialysised. Compare with the commercial plasmid, we could use the easier and cheaper method to get a great quantity of plasmid with high purity.

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DNA Cleavage Studies Mediated by Cantharidin Derivatives Using Supercoiled Plasmid pUC19 According to the previous literature, Cantharidin was found that it could cleavage DNA in cell. But the detail mechanism is still not clear. Here, we use cantharidin and its derivatives (compound A~L) to explore the mechanism and also want to know all of them could be serves as photo-induced DNA cleavage agents. The pUC19 plasmid was used as DNA source for investigating the behavior of these compounds. All of these compounds were dissolved in DMSO as stoke solution. Then, the stoke solutions were added to the supercoiled plasmid (Form I) solution to the final concentration of 1mM and irradiated with or without irradiation (λ=352-nm) at room temperature for 1hr.

After reaction, the results were analyzed by gel electrophoresis. The results from gel electrophoresis showed that cantharidin and compound A, B, C, E, F, G, H, K and L could not cleavage pUC19 plasmid by single-strand scission with irradiation (Appendix 6-17). These indicated that they may adopt other mechanism to cleavage DNA. Compound D, I, and J could cleavage the plasmid through single-strand scission and produce the relaxed circular DNA (from II). Compare the efficiency, compound D exhibited the highest cleaving efficiency (Figure 3.2-3.5) and compound I and compound J (Appendix14 and 15) only exhibited a little cleaving efficiency under acidic condition (pH=5.0 and pH=6.0).

Land 1: Marker. Land 2: Control. Land 3: 10mM phosphate buffer, pH=5.0, dark, 1hr. Land 4: 10mM phosphate buffer, pH=5.0, UV=352 nm, 1hr. Land 5: 10mM phosphate buffer, pH=6.0, dark, 1hr. Land 6L 10mM phosphate buffer, pH=6.0, UV=352nm, 1hr. Land 7: 10mM phosphate buffer, pH=7.0, dark，1hr. Land 8: 10mM phosphate buffer, pH=7.0, UV=352 nm, 1hr. Land 9: 10mM phosphate buffer, pH=8.0, dark, 1hr. Land 10: 10mM phosphate buffer, pH=8.0, UV=352 nm, 1hr. Land 11: 10mM phosphate buffer, pH=9.2, dark, 1hr. Land 12: 10mM phosphate buffer, pH=9.2, UV=352 nm, 1hr.

Appendix 6: Gel retardation assay of plasmid pUC19 in presence of 1mM.

Land 1: Marker. Land 2: Control. Land 3: 10mM phosphate buffer, pH=6.0, dark, 1hr. Land 4: 10mM phosphate buffer, pH=6.0, UV=352 nm, 1hr. Land 5: 10mM phosphate buffer, pH=7.0, dark, 1hr. Land 6: 10mM phosphate buffer, pH=7.0, UV=352 nm, 1hr. Land 7: 10mM phosphate buffer, pH=8.0, dark, 1hr. Land 8: 10mM phosphate buffer, pH=8.0, UV=352 nm, 1hr.

Appendix 7: Gel retardation assay of plasmid pUC19 in presence of 1mM.

Land 1: Marker. Land 2: Control. Land 3: 10mM phosphate buffer, pH=6.0, dark, 1hr. Land 4: 10mM phosphate buffer, pH=6.0, UV=352 nm, 1hr. Land 5: 10mM phosphate buffer, pH=7.0, dark, 1hr. Land 6: 10mM phosphate buffer, pH=7.0, UV=352nm, 1hr. Land 7: 10mM phosphate buffer, pH=8.0, dark, 1hr. Land 8: 10mM phosphate buffer, pH=8.0, UV=352 nm, 1hr.

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Appendix 8: Gel retardation assay of plasmid pUC19 in presence of 1mM.

Land 1: Marker. Land 2: Control. Land 3: 10mM phosphate buffer, pH=6.0, dark, 1hr. Land 4: 10mM phosphate buffer, pH=6.0, UV=352 nm, 1hr. Land 5: 10mM phosphate buffer, pH=7.0, dark, 1hr. Land 6: 10mM phosphate buffer, pH=7.0, UV=352 nm, 1hr. Land 7: 10mM phosphate buffer, pH=8.0, dark, 1hr. Land 8: 10mM phosphate buffer, pH=8.0, UV=352 nm, 1hr.

Appendix 9: Gel retardation assay of plasmid pUC19 in presence of 1mM.

Land 1: Marker. Land 2: Control. Land 3: 10mM phosphate buffer, pH=6.0, dark, 1hr. Land 4: 10mM phosphate buffer, pH=6.0, UV=352 nm, 1hr. Land 5: 10mM phosphate buffer, pH=7.0, dark, 1hr. Land 6: 10mM phosphate buffer, pH=7.0, UV=352 nm, 1hr. Land 7: 10mM phosphate buffer, pH=8.0, dark, 1hr. Land 8: 10mM phosphate buffer, pH=8.0, UV=352 nm, 1hr.

Appendix 10: Gel retardation assay of plasmid pUC19 in presence of 1mM.

Land 1: Marker. Land 2: Control. Land 3: 10mM phosphate buffer, pH=6.0, dark, 1hr. Land 4: 10mM phosphate buffer, pH=6.0, UV=352 nm, 1hr. Land 5: 10mM phosphate buffer, pH=7.0, dark, 1hr. Land 6: 10mM phosphate buffer, pH=7.0, UV=352 nm, 1hr. Land 7: 10mM phosphate buffer, pH=8.0, dark, 1hr. Land 8: 10mM phosphate buffer, pH=8.0, UV=352 nm, 1hr.

Appendix 11: Gel retardation assay of plasmid pUC19 in presence of 1mM.

Land 1: Marker. Land 2: Control. Land 3: 10mM phosphate buffer, pH=6.0, dark, 1hr. Land 4: 10mM phosphate buffer, pH=6.0, UV=352 nm, 1hr. Land 5: 10mM phosphate buffer, pH=7.0, dark, 1hr. Land 6: 10mM phosphate buffer, pH=7.0, UV=352 nm, 1hr. Land 7: 10mM phosphate buffer, pH=8.0, dark, 1hr. Land 8: 10mM phosphate buffer, pH=8.0, UV=352 nm, 1hr.

Click to enlarge

Appendix 12: Gel retardation assay of plasmid pUC19 in presence of 1mM.

Land 1: Marker. Land 2: Control. Land 3: 10mM phosphate buffer pH=6.0, dark, 1hr. Land 4: 10mM phosphate buffer, pH=6.0, UV=352 nm, 1hr. Land 5: 10mM phosphate buffer, pH=7.0, dark, 1hr. Land 6: 10mM phosphate buffer, pH=7.0, UV=352 nm, 1hr. Land 7: 10mM phosphate buffer, pH=8.0, dark, 1hr. Land 8: 10mM phosphate buffer, pH=8.0, UV=352 nm, 1hr.

Appendix 13: Gel retardation assay of plasmid pUC19 in presence of 1mM.

Land 1: Marker. Land 2: Control. Land 3: 10mM phosphate buffer, pH=5.0, dark, 1hr. Land 4: 10mM phosphate buffer, pH=5.0, UV=352 nm, 1hr. Land 5: 10mM phosphate buffer, pH=6.0, dark, 1hr. Land 6: 10mM phosphate buffer, pH=6.0, UV=352nm, 1hr. Land 7: 10mM phosphate buffer, pH=7.0, dark, 1hr. Land 8: 10mM phosphate buffer, pH=7.0, UV=352 nm, 1hr. Land 9: 10mM phosphate buffer, pH=8.0, dark, 1hr. Land 10: 10mM phosphate buffer, pH=8.0, UV=352 nm, 1hr. Land 11: 10mM phosphate buffer, pH=9.2, dark, 1hr. Land 12: 0mM phosphate buffer, pH=9.2, UV=352 nm, 1hr.

Appendix 14: Gel retardation assay of plasmid pUC19 in presence of 1mM.

Land 1: Marker. Land 2: Control. Land 3: 10mM phosphate buffer, pH=5.0, dark, 1hr. Land 4: 10mM phosphate buffer, pH=5.0, UV=352 nm, 1hr. Land 5: 10mM phosphate buffer, pH=6.0, dark, 1hr. Land 6: 10mM phosphate buffer, pH=6.0, UV=352nm, 1hr. Land 7: 10mM phosphate buffer, pH=7.0, dark, 1hr. Land 8: 10mM phosphate buffer, pH=7.0, UV=352 nm, 1hr. Land 9: 10mM phosphate buffer, pH=8.0, dark, 1hr. Land 10: 10mM phosphate buffer, pH=8.0, UV=352 nm,1hr. Land 11: 10mM phosphate buffer, pH=9.2, dark, 1hr. Land 12: 0mM phosphate buffer, pH=9.2, UV=352 nm, 1hr.

Appendix 15: Gel retardation assay of plasmid pUC19 in presence of 1mM.

Land 1: Marker. Land 2: Control. Land 3: 10mM phosphate buffer, pH=6.0, dark, 1hr. Land 4: 10mM phosphate buffer, pH=6.0, UV=352 nm, 1hr. Land 5: 10mM phosphate buffer, pH=7.0, dark, 1hr. Land 6: 10mM phosphate buffer, pH=7.0, UV=352 nm, 1hr. Land 7: 10mM phosphate buffer, pH=8.0, dark, 1hr. Land 8: 10mM phosphate buffer, pH=8.0, UV=352 nm, 1hr.

Click to enlarge

Appendix 16: Gel retardation assay of plasmid pUC19 in presence of 1mM.

Land 1: Marker. Land 2: Control. Land 3: 10mM phosphate buffer, pH=6.0, dark, 1hr. Land 4: 10mM phosphate buffer, pH=6.0, UV=352 nm, 1hr. Land 5: 10mM phosphate buffer, pH=7.0,dark, 1hr. Land 6: 10mM phosphate buffer, pH=7.0, UV=352 nm, 1hr. Land 7:10mM phosphate buffer, pH=8.0, dark, 1hr. Land 8: 10mM phosphate buffer, pH=8.0, UV=352 nm, 1hr.

$$ \mathrm {C} _ {2 4} \mathrm {H} _ {2 2} \mathrm {N} _ {5} \mathrm {O} _ {5} \mathrm {S}, \mathrm {M W} = 4 1 8. 5 $$ Compound D

Appendix 17: Gel retardation assay of plasmid pUC19 in presence of 1mM.

Figure 3.2: DNA cleavage Studies using plasmid pUC19 in presence of 1mM Compound D. Land 1: Marker. Land 2: Control. Land 3: 10mM phosphate buffer, pH=6.0, dark, 1hr. Land 4: 10mM phosphate buffer, pH=6.0, UV=352 nm, 1hr. Land 5: 10mM phosphate buffer, pH=7.0, dark, 1hr. Land 6: 10mM phosphate buffer, pH=7.0, UV=352 nm, 1hr. Land 7: 10mM phosphate buffer, pH=8.0, dark, 1hr. Land 8: 10mM phosphate buffer, pH=8.0, UV=352 nm, 1hr.

Figure 3.3: DNA cleavage studies using supercoiled plasmid pUC19 in the presence of 1mM Compound D (different pH value). Land 1: Marker. Land 2: Control. Land 3: 10mM phosphate buffer, pH=5.0, dark, 1hr. Land 4: 10mM phosphate buffer, pH=5.0, UV=352 nm, 1hr. Land 5: 10mM phosphate buffer, pH=6.0, dark, 1hr. Land 6: 10mM phosphate buffer, pH=6.0, UV=352 nm, 1hr. Land 7: 10mM phosphate buffer, pH=7.0, dark, 1hr. Land 8: 10mM phosphate buffer, pH=7.0, UV=352 nm, 1hr. Land 9L 10mM phosphate buffer, pH=8.0, dark, 1hr. Land 10: 10mM phosphate buffer, pH=8.0, UV=352 nm, 1hr. Land 11: 10mM phosphate buffer, pH=9.2, dark, 1hr. Land 12: 0mM phosphate buffer, pH=9.2, UV=352 nm, 1hr.

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Figure 3.4: DNA cleavage studies using supercoiled plasmid pUC19 in presence of 1µM~1000µM concentrations of Compound D, pH=7.0. Land 1: Marker. Land 2: Control. Land 3: 1000µM compound, dark, 1hr. Land 4: 1000µM compound, UV=352 nm, 1hr. Land 5: 100µM compound, dark, 1hr. Land 6: 100µM compound, UV=352 nm, 1hr. Land 7: 10µM compound, dark, 1hr. Land 8: 10µM compound, UV=352nm, 1hr. Land 9: 1µM compound, dark, 1hr. Land 10: 1µM compound, UV=352 nm, 1hr.

Explore the DNA Cleavage with Site Specificity

Based on above results, we know the compound D has the good cleaving efficiency but the results cannot show if the compound D has the ability of recognize specific nucleobase. Previous literature indicates that the bulged structure is one of the important motifs in DNA recognition. Its unpaired nucleobase are capable of forming complexes with nucleic acid-binding-proteins as well as acting as the bind site for small molecules [37, 38]. They were believed to be the intermediates in the fragment shift mutagenesis then lead to some genetic diseases [39, 40]. Since DNA stores genetic information, a change in sequences or structures has the potential to induce erroneous gene expression resulting in the information of non-functional proteins.

In order to explore the site-specific DNA cleavage of the compound D, HIV-27 DNA with bulged structure were treated as DNA template. Dimethyl sulfate (G reaction) and 88% formic acid (A+G reaction) were also used to treat with the HIV-27 DNA to serve as control experiments to help us to check the cleavage site. Here we used different methods to detect the results of experiment. First method is ethidium bromide staining. We added dimethyl sulfate (G reaction) and 88% formic acid (A+G reaction) to HIV-27 DNA that without any labeling respectively, and then added hot piperidine to generate the cleaving patterns. After incubating for 1h, the mixtures were analyzed by gel electrophoresis and stained with ethidium bromide (Figure 3.6). The result displayed two DNA cleavage fragments and indicated the G reaction indeed proceeded. But the results could not identify the cut site of DNA cleavage directly.

To improve the limitation, we use the chemiluminescence for detection. The HIV-27 DNA was labeled with biotin at 5’ end. After analyzing the reaction by gel electrophoresis, the patterns from gel were transferred to the nylon membrane and hybrid with streptavidin-HRP. The biotin and streptavidin have the high affinity. Then, inducing the HRP to produce chemiluminescence and used film to capture the signal. Although it also displayed the cleavage fragment, the single of cleft DNA fragments was too weak to detected.

According to those results, we decided to use the fluorescence for detection. The fluorescence is more sensitive than the two methods describing previously. HIV- 27 DNA was labeled with 6 FAM-labelled at 5’- terminus (6 FAM, 6-carboxy-fluorescein). After separating the fragment by gel electrophoresis, we use laser (λ= 488 nm) to excite the 6 FAM and detect the emission at λ=518 nm (green light). As the Figure 3-6 shown, it was clearer to see the fragments of G reaction and photoreaction with compound D. The results also indicated that the compound D can recognize guanine residue and cleavage guanine residue directly and proceed the site-specific DNA cleavage. Therefore, compound D might be a candidate for using in anticancer via site-specific DNA cleavage of some important genes. Currently, we are investigating the site at which the DNA was cleaved by compound D.

Conclusion

In this thesis, we apply cantharidin and its derivatives to the pUC19 plasmid or HIV-27 DNA to investigate if they have the function of site-specific DNA cleavage. The previous studies have displayed that cantharidin could cleavage DNA in cell. Hence, scientists tried to design many cantharidin derivatives to cleavage DNA. Base on above, we try to utilize our synthesized cantharidin derivative serving as DNA cleavage agent to cleavage DNA in tumor cells. Our experimental results indicated that compound D possesses the ability to cleavage DNA. In addition, the experiment of site-specific DNA cleavage indicated that compound D can recognize guanine residue and cleavage guanine residue directly. Therefore, cantharidin derivatives could be developed as new photo-induced DNA-cleaving agents via combine with different ligands.