Synthesis and DNA-cleaving activity of a series of substituted arenediazonium ions

Article abstract of DOI:10.1134/S1068162008040158

We investigated the reactions of substituted aryl radicals and aryl cations derived from arenediazonium ions and their ability to cause cleavage of supercoiled DNA and their tendency toward free radical or cation formation in the presence and absent of co

Full text of DOI:10.1134/S1068162008040158

ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2008, Vol. 34, No. 4, pp. 488–498. © Pleiades Publishing, Ltd., 2008.
Original Russian Text © B. Çeken, M. Kízíl, 2008, published in Bioorganicheskaya Khimiya, 2008, Vol. 34, No. 4, pp. 546–557.
Synthesis and DNA-Cleaving Activity of a Series
of Substituted Arenediazonium Ions1
B. Çeken and M. Kízíl2
Department of Chemistry, Science and Art Faculty, Dicle University, 21280 Diyarbakir, Turkey
Received May 3, 2007; in final form September 28, 2008
Abstract—We investigated the reactions of substituted aryl radicals and aryl cations derived from arenediazo-
nium ions and their ability to cause cleavage of supercoiled DNA and their tendency toward free radical or cat-
ion formation in the presence and absent of copper (I) chloride. It was found that the substituted arenediazonium
salts can cleave supercoiled DNA to the open circular form II DNA and linear form III DNA. Results method-
ical studies indicate that both carbon-centered radicals and aryl cations participate in the cleavage pathways.
Keywords: Agaricus bisporus, dediazoniation, DNA cleavage, substituted aryl radicals, substituted aryl ca-
tions
DOI: 10.1134/S1068162008040158
INTRODUCTION
nylazo)-guanine. The ability of arendiazonium ions to
cleave DNA might result from the simultaneous action
of arendiazonium ions and aryl radicals or aryl cation
derived from them [7] (Scheme 1).
Arendiazonium ions (Ar–N+2 ), such as those found
in the common mushroom Agaricus bisporous were
shown to be mutagenic and carcionogenic [1–12].
A direct attack on DNA by aryl radicals would
However, the mechanisms involved in their carcinoge- explain the carcinogenic activity attributed to arenedia-
nicity remain unclear. Arenediazonium ions are chemi- zonium ions in the light of in vitro aryl cation caused by
cally different from alkanediazonium ions: they are aryl radicals to both natural and synthetic nucleobases,
more stable. Their reactions include azo coupling, sub- nucleosides, and polynucleotides [20] and their ability
stitution of nitrogen by nucleophiles, or induction of to produce unspecific DNA cleavage [21–25]. It must
free radical processes [13–14]. The degradation of are- be remembered that a number of reducers will increase
nediazonium ions, known as dediazoniation, may occur the chance of aryl radical formation in the biological
via two mechanisms, either heterolytic to form aryl cat- medium.
ion or homolytic to form aryl radical. The latter
Some other pathways have been also suggested, for
requires the transfer of one electron from a reducing
example the capacity of carbon-centered radicals
agent. Both processes can occur simultaneously; the
heterolytic one can be activated thermally or photo-
derived from the p-methylbenzenediazonium ion to
activate the AP-1 system and thus stimulate the phos-
chemically, whereas the second one is favoured by
phorylation of ERK1, ERK2, and p38 proteins in simi-
strong reducers [15].
lar way to that observed for superoxide anion and
hydroxyl radicals [26].
Stock et al. [16] found that the reaction of benzene-
diazonium ion with adenine, adenosine, or adenylic
acid resulted in the formation of N6 triazenes, while that
with guanine and its derivatives, in the formation of C-
8 azo coupled or C-8 arylated products and unstable N2
triazenes [17]. The formation of a triazene adduct has
also been considered to be an intermediate step in the
arylation of adenine residues in DNA [18]. Stiborova
et al. [19] determined that the reaction of benzenediaz-
onium ion with guanine results in the formation of not
only a C-8 azo-coupled product, 8-(phenylazo)guanine,
but also in the formation of another product, 8-(biphe-
It is evident that the toxicity of arenediazonium ions
derives from the appearance during the dediazoniation
processes of very reactive compounds, the aryl radical
and aryl cation, which are intermediates in the
homolytic or heterolytic dediazoniation mechanisms,
respectively [27]. It was suggested by many authors
[28] that both mechanisms may operate simultaneously
and competitively, depending on the experimental con-
ditions under which dediazoniation occurs [29].
p-Hydroxybenzenediazonium is an arenediazonium
ion present in the inedible mushroom A. xanthodermus
[30]. It cleaves DNA [22] and is responsible for the for-
mation of the C8-guanine adduct [31] and also causes
tumors when repeatedly administered as a sulfate salt
1
The text was submitted by the authors in English.
Corresponding author; phone: +90 412 2488406; fax: +90 412
2488039; e-mail: muratk@dircle.edu.tr
2
488

SYNTHESIS AND DNA-CLEAVING ACTIVITY
N2+BF4–
489
NH2
R5
R4
R1
R2
R5
R1
Isoamyl nitrite, HBF (aq)
4
EtOH, 0°C, 30 min
R4
R2
R3
R3
(1)–(10)
(1) R1 = R2 = CH3
(2) R1 = R5 = CH3
(3) R1 = R5 = CH2CH3 (4) R1 = CH2CH3
R2 = R3 = R4 = H R2 = R3 = R4 = R5 = H
R3 = R4 = R5 = H
R2 = R3 = R4 = H
(5) R1 = R3 = R4 = R5 = H (6) R1 = R2 = R4 = R5 = H (7) R1 = Cl
(8) R1 = NO2
R2 = R4 = R5 = H
R3 = Cl
R2 = CH2CH3
R3 = CH2CH3
R3 = NO2
R2 = R4 = R5 = H
(9) R1 = CH2CH2OH
(10) R1 = R2 = R4 = R5 = H
R2 = R3 = R4 = R5 = H
R3 = CH2OH
Scheme 1. Synthesis of arenediazonium salts (1)–(10).
by subcutaneous injection [32]. Moreover, it is known
However, the chemical behavior of phenyl radicals
that the edible mushroom A. bisporus contains several toward DNA and DNA components was not been
arylhydrazides, arylhydrazines, and arenediazonium extensively investigated [23]. A better understanding of
ions [33]. Arenediazonium ions occur naturally as the the structural features and other factors that control the
secondary metabolites. These ions can also be formed reactivity of phenyl radicals toward DNA and its com-
from other precursors, such as azo dyes [34–35] and ponents could greatly benefit the rational design of syn-
aryl nitrosamines [13]. For example, 4-(hydroxyme- thetic DNA cleavers and, hence, facilitate the develop-
thyl)benzenediazonium (HMBD) induces sucutis and ment of more efficient and less toxic pharmaceuticals.
skin cancer in mice [36–37]. Hiramoto et al. [24] found
that arenediazonium ions are reductively decomposed
and form the carbon-centered aryl radicals.
Obviously, the side reactions of radical intermedi-
ates with proteins, which may cause cytotoxicity and
other unwanted side effects, are of great interest. There-
fore, further studies of the ability of phenyl radical to
damage DNA, amino acids, peptides, and eventually
proteins, are clearly warranted. There are several
aspects regarding arenediazonium ions chemistry and
the mechanism of interaction with biological macro-
molecules, which remain partially unknown and
become the subject of investigation nowadays. Among
the aspects are the interaction between diazonium
group and the rest of the molecule, the identity of the
genotoxic agent, the exact site where the genotoxic
agent induces the DNA damage and whether the dam-
age is produced as a result of a direct attack of the geno-
toxic agent or occurs in a secondary process. It is also
unknown, whether the DNA damages induced by are-
nediazonium ions really cause subsequent carcinogenic
effects. The present study was devoted to explore the
reactions of a series of substituted aryl radicals and aryl
cations which are derived from arenediazonium salts,
toward supercoiled DNA.
Ganett et al. [38] have shown that arenediazonium
ions react with cellular DNA, forming mainly
C8-arylguanine adducts, including p-methyl, p-meth-
oxymethyl, and p-hydoxymethyl derivatives. It was
also suggested that the phenyl radicals and biradicals
are generated in biological systems a intermediates of
some potential drug candidates, whose target is DNA
[39–41]. Radical attack on DNA is one of the main rea-
sons for DNA damage [42–43]. It was found that aro-
matic σ,σ-biradicals play an important role in the
action of some antitumor drugs, such as the enediyne
type antitumor antibiotics. The key step in the DNA
cleavage by such radicals is hydrogen atom abstraction
from the sugar moiety in DNA [42–43]. In addition to
aromatic biradical, many monoradicals formed upon
metabolic conversion of organic compounds can also
attack DNA by hydrogen atom abstraction and also by
addition to a nucleobase. It was shown that OH and
alkoxyl radicals can attack the C5=C6 double bond of
the pyrimidine base moiety via addition or the sugar
moiety via hydrogen abstraction in nucleosides, nucle-
otides, and DNA [42–44]. It was also reported that the
thiyl radical can attack the C6 position of a pyrimidine
nucleosides [45]. Furthermore, benzoyl peroxide can
RESULTS
Synthesis of Arenediazonium Salts
We prepared various substituted arenediazonium
be metabolized to phenyl radicals, which can damage tetrafluoroborates (1)–(10) in 22–89% yields by the
DNA [46].
reaction of the corresponding amine with isoamyl
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 34 No. 4 2008

490
ÇEKEN, KÍZÍL
(a)
Form I
100
80
60
40
20
0
Form II
1
1
2
2
3
3
4
4
5
5
6
6
7
7
8
8
(b)
Form II
Form I
Fig. 1. a The quantified band intensity for the sc-DNA (form I), oc-DNA (form II), and l-DNA (form III) with Quantity One 4.5.2
version software. b Single-strand cleavage of pBluescript M13+ (3.2 kb) supercoiled DNA (form 1) to relaxed circular (form II) by
arenediazonium salts (1)–(3). Lane 1, control DNA; lane 2, DNA + CuCl (3.1 mM); lane 3, DNA + (1) (1.5 mM); lane 4, DNA +
(1) (1.5 mM) + CuCl (3.1 mM); lane 5, DNA + (2) (1.5 mM); lane 6, DNA + (2) (1.5 mM) + CuCl (3.1 mM); lane 7, DNA +
(3) (1.5 mM); lane 8, DNA + 3 (1.5 mM) + CuCl (3.1 mM).
nitrite and aqueous fluoroboric acid in ethanol [46]. neither copper(I) chloride nor arenediazonium salt (1)
(Scheme 1).
themselves can effect DNA cleavage.
The net DNA cleavage values in the presence of
one-electron donor for the arenediazonium salts (1)–(3)
with 2,3- and 2,6-dimethyl groups and 2,6-diethyl
groups are 41.97%, 44.78% and 37.00%, respectively
(table). This net DNA cleavage is due to DNA single-
strand cleavage caused by (1)–(3) and copper(I) chlo-
ride. It is known that a nick or a single stand cleavage
of the form I DNA converts it to the form II DNA. The
absence of a band for the linear form III DNA suggests
that the DNA cleavage is single rather than double-
stranded. Otherwise, the linear DNA band would have
appeared between the form I DNA and the form II DNA
bands.
The cleaving efficiencies of (1)–(3) in TAE buffer in
the presence of coper(I) chloride were found to be sim-
ilar to each other, excect the arenediazonium salts (1)
and (2) were more efficient for the induction of DNA
relaxation than (3). The high potency of (1 and (2) can
be attributed to their methyl groups, which improve
their ability to cleave DNA by facilitating the genera-
tion of aryl radical.
DNA Cleavage
The DNA cleaving activity of arenediazonium salts
(1)–(10) was evaluated by monitoring the conversion of
circular supercoiled DNA (form I) to circular relaxed
DNA (form II) or linear (form III) DNA.
pBluescript M13+ supercoiled DNA (Form I) was
incubated with various arenediazonium salts in the
presence and absence of copper(I) chloride at 37°C for
2 h in dark, and then the reaction mixtures were analy-
sed by gel electrophoresis in 1%Agarose gel (Ethidium
Bromide staining). Figure 1b shows the agarose gel pic-
ture of DNA cleavage induced by arenediazonium salts
(1)−(3). The upper band represent the circular relaxed
DNA (form II). The columns above the gel picture
(Fig. 1‡) correspond to the quantitified band intesity for
the circular supercoiled DNA (form I) and the circular
relaxed DNA (form II). Lane 1 is the control DNA; it
represents the original structure of DNA with 98.42%
of form I DNA and 1.58% of form II. Lane 2 represents
Our gel electrophoresis results indicate that the
a DNA incubation with an aqueous solution of cop- attachment of various electron-donating groups in o-,
per(I) chloride to determine, whether copper(I) ions m-, and p-positions of benzene ring of an arenediazo-
promotes the strand scission. Lane 3 represent the nium salt could enhance the DNA-cleaving activity,
effect of arenediazonium salt (1) on DNA without cop- ethyl groups demonstrating the strogest effect among
per(I) chloride. Cleavage of the DNA would indicate the groups. As shown in Fig. 2 and table, these are
either the photolytic formation of aryl cation or direct ethyl-containing arenediazonium salts (4), (5), and (6).
alkylation of purines. DNA cleavage was not observed Moreover, a direct comparision of net DNA cleavage
in this lane. Lane 4 represents an incubation of DNA allowed us to find that the net DNA cleavage was
with an aqueous solution of copper(I) chloride and 80.03% for (4), 100% for (5), and 100% for (6). Thus,
aqueous solution of arenediazonium salt (1). This in the presence of copper(I) chloride, the corresponding
experiment was carried out to investigate the reduction ethyl-containing aryl radical generated in situ acted at
of arenediazonium ion to aryl radical leading to the the DNA cleavage more potently, than the aryl cation.
cleavage of the DNA strand. Lanes 2 and 3 show that During the investigation of a series of arenediazonium
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 34 No. 4 2008

SYNTHESIS AND DNA-CLEAVING ACTIVITY
491
(a)
100
Form I
80
60
40
20
0
Form II
Form III
1
1
2
2
3
3
4
4
5
5
6
6
7
7
8
8
(b)
Form II
Form III
Form I
Fig. 2. a The quantified band intensity for the sc-DNA (form I), oc-DNA (form II), and l-DNA (form III) with Quantity One 4.5.2
version software. b Single- and double-strand cleavage of pBluescript M13+ (3.2 kb) supercoiled DNA (form 1) to relaxed circular
(form II) and linear (form III) by arenediazonium salts (4)–(6). Lane 1, control DNA; lane 2, DNA + CuCl (3.1 mM); lane 3, DNA +
(4)(1.5 mM); lane 4, DNA + (4)(1.5 mM)+ CuCl (3.1 mM); lane 5, DNA + (5)(1.5 mM); lane 6, DNA + (5)(1.5 mM) + CuCl
(3.1 mM); lane 7, DNA + (6) (1.5 mM); lane 8, DNA + (6) (1.5 mM) + CuCl (3.1 mM).
(a)
100
Form I
80
Form II
60
Form III
40
20
0
1
1
2
2
3
3
4
4
5
5
6
6
(b)
Form II
Form III
Form I
Fig. 3. a The quantified band intensity for the sc-DNA (form I), oc-DNA (form II), and l-DNA (form III) with Quantity One 4.5.2
version software. b. Single- and double-strand cleavage of pBluescript M13+ (3.2 kb) supercoiled DNA (Form 1) to relaxed circular
(form II) and linear (form III) forms by arenediazonium salts (7) and (8). Lane 1, control DNA; lane 2, DNA + CuCl (3.1 mM);
lane 3, DNA + (7)(1.5 mM); lane 4, DNA + (7)(1.5 mM) + CuCl (3.1 mM); lane 5, DNA + (8) (1.5 mM); lane 6, DNA + (8)
(1.5 mM) + CuCl (3.1 mM).
salts with alkyl groups in benzene ring, it was noticed that study, the authors observed DNA cleavage and the
reaction was shown to be light-dependent.
that compounds (4), (5), and (6) are unstable in day-
light; these compounds showed a light dependent activ-
ity in plasmid relaxation assay for DNA cleavage. It is
important to remind that we controlled all our experi-
ments to protect all our samples from the exposure to
light; in this way, we avoided the formation of carboca-
tion from diazonium salts via a photochemical path-
way. To avoid the effects of photoexcitation of samples,
all the reactions were carried out in Eppendrof tubes
protected from light by means of foil covering. Never-
theless, DNA cleavage was still observed for (4), (5),
and (6) in the absence of copper(I) chloride (Fig. 2,
lanes 3, 5, and 7, and table). This confirms that the plas-
mid relaxation does not require an added electron donor
(mediating homolytic dediazoniation of diazonium
Aqueous solutions of arenediazonium salts are
known to be converted very easily on illumination into
aryl cations, which are good alkylating agents. Alkyla-
tion of purine bases in DNA is widely used to trigger
DNA cleavage. In this study, an enhanced DNA cleav-
age by (4), (5), and (6) (relative to the absence of cop-
per(I) chloride) was observed (Fig. 2, cf. lanes 4, 6 and
8 with lanes 3, 5 and 7).
We carried out a comparative study of dediazonia-
tion of o- nitro- and p-nitrochloroarenediazonium ions
on DNA cleavage. The electron-withdrawing and
donating properties of NO2- and Cl groups strongly
determine the reactivity of both compounds, thus exert-
group) as shown for other diazonium reagents [20]. In ing different influences upon dediazoniation reaction
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 34 No. 4 2008

492
ÇEKEN, KÍZÍL
(a)
100
80
60
40
20
0
Form I
Form II
1
1
2
2
3
3
4
4
5
5
6
6
(b)
Form II
Form I
Fig. 4. a The quantified band intensity for the sc-DNA (form I), oc-DNA (form II), and l-DNA (form III) with Quantity One 4.5.2
version software. b Single-strand cleavage of pBluescript M13+ (3.2 kb) supercoiled DNA (form 1) to relaxed circular (form II) by
arenediazonium salts (9) and (10). Lane 1, control DNA; lane 2, DNA + CuCl (3.1 mM); lane 3, DNA + (9) (1.5 mM); lane 4,
DNA + (9) (1.5 mM) + CuCl (3.1 mM); lane 5, DNA + (10) (1.5 mM); lane 6, DNA + (10) (1.5 mM) + CuCl (3.1 mM).
(a)
100
Form I
80
Form II
60
40
20
0
1
1
2
2
3
3
4
4
5
6
6
7
7
8
8
9
9
10
10
(b)
5
Form II
Form I
Fig. 5. a The quantified band intensity for the sc-DNA (form I), oc-DNA (form II), and l-DNA (form III) with Quantity One 4.5.2
version software. b Effects of ethanol, cysteine, and β-mercaptoethanol on the single-strand cleavage of pBluescript M13+ (3.2 kb)
supercoiled DNA induced by arenediazonium salt. Lane 1, control DNA; lane 2, DNA + CuCl (3.1 mM); lane 3, DNA + (1)
(1.5 mM); lane 4, DNA + (1) (1.5 mM) + CuCl (3.1 mM); lane 5, DNA + ethanol (2.5 mg/ml); lane 6, DNA + (1) (1.5 mM) + ethanol
(2.5 mg/ml); lane 7, DNA + cystein (2.5 mg/ml); lane 8, DNA + (1) (1.5 mM) + cysteine (2.5 mg/ml); lane 9, DNA + β-mercapto-
ethanol (2.5 mg/ml); lane 10, DNA + (1)(1.5 mM) + β-mercaptoethanol (2.5 mg/ml).
[46]. Figure 3 shows the DNA cleavage activity of com-
pounds (7) and (8). The net DNA cleavage activity for
(7) was found to be 100% and 79.69% in the presence
and absence of copper(I) chloride, respectively. The net
DNA cleavage activity for (8) was found to be 100%
and 14.00% in the presence and the absence of cop-
per(I) chloride, respectively (table). A chloro substitu-
ent is a strong π donor and a very strong σ electron
acceptor, both π- and σ effects are important for stabili-
zation of triplet aryl cations. Electron donor substitu-
ents always increase the electron density within the
ring, thus stabilising the cation; of these the best are
π electron donors in the order para and ortho > meta
[47].
The most extensively consumed species of mush-
room A. bisporous contains a number of aromatic
hydrazines, among which the most abundant is agar-
itine, β-N-(γ-L(+)-glutamyl-4-(hydroxymethyl)phe-
nylhydrazine. It occurs at concentrations as high as
1.7 mg/g of raw mushroom [48]. The postulated ulti-
mate carcinogenic metabolite of agaritine is believed
to be the highly reactive and mutagenic
4-(hydroxymethyl)benzenediazonium ion (HMBD)
[49]. For this intermediate to be formed, the
glutamyl moiety must be removed to release the free
hydrazine that can presumably be oxidised to form a
diazonium ion (Scheme 2).
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 34 No. 4 2008

SYNTHESIS AND DNA-CLEAVING ACTIVITY
493
O
N
COOH
+N
HN NH2
HN
N
H
NH2
(GGT)
Cytochrome P450
γ-Glutamyl transpeptidase
OH
OH
OH
4-
4-
Agaritine
(Hydroxyme-
(Hydroxymethyl)ben-
zenediazonium ion
thyl)phenylhydrazine
Scheme 2. Putative pathways of the agaritine bioactivation.
It was demonstarted that the 4-(hydroxymethyl)ben- mercapto ethanol. Inhibition of DNA cleavage by etha-
zenediazonium ion found in edible mushroom
A. bisporous [33] and p-hydroxybenzenediazonium ion
in the inedible mushroom A. xanthodermus [31] cleave
DNA and are responsible for the sucutis and skin can-
cer in mice [36]. Thus, a detailed knowledge of the
mechanisms associated with the degradation process of
arenediazonium ions would appear to be important for
understanding of the genotoxic properties of this kind
of compounds, particularly when the hemolytic path-
way may compete with the heterolytic one for the pro-
ducing potentially genotoxic agents. In addition, p-
hydroxybenzenediazonium ion has a good electron-
releasing group in p-position and an electron-attracting
diazonium group, so that an effective electron transfer
from the donor group to the acceptor might be
nol may be due to its scavenging activity toward the
carbon-centered radical [24], but the mechanism of
inhibition of cystein and β-mercapto ethanol may be
different. We think that cystein and β mercapto ethanol
reacted with arenediazonium salt (1) to form an unsta-
ble adduct, which may prevent the generation of the
carbon-centered radical [51]. At 2.5-mg/ml concentra-
tion, cystein, a general radical trap, inhibited 72.13% of
DNA cleavage. Similarly, ethanol and β-mercapto eth-
anol inhibited 49.57% and 57.33% of DNA cleavage,
respectively (Fig. 6). The trapping studies are con-
sisitent with the participation of carbon-centered radi-
cals in the cleavage process by arenediazonium ion (1).
Effects of ethanol, cystein, and β-mercaptoethanol
expected. It must be remembered that arenediazonium on the single-strand cleavage of pBluescript M13+
compounds are genearlly very unstable and that they
become more stable according to the nature of the sub-
stituents, especially if these are electron donors [50].
Hydroxy substitution contributes to the strength of the
Ar−N2 bond by means of a resonance mechanism, par-
ticularly, if the OH group is deprotonated.
(3.2 kb) supercoiled DNA induced by arenediazonium
salt (1). Percent of inhibition of the single-strand cleav-
age of pBluescript M13+ (3.2 kb) supercoiled DNA
was calculated as follows [51]: inhibition (%) =
Inhibition percent
100
The DNA cleavage ability of 2-(hydroxyethyl)ben-
zenediazonium ion (9) and 4-(hydroxymethyl)benzene-
diazonium ion (10) was investigated. DNA cleavage
was observed for (9) only in the presence of copper(I)
chloride (Fig. 4, lane 4); thus, in the absence of cop-
per(I) chloride, no DNA cleavage was observed for (9)
(Fig. 4, lane 3). The net DNA cleavage value for (9) was
found to be 57.34% in the presence of copper(I) chlo-
ride (table). On the other hand, DNA cleavage was
observed for 10 both in the presence and absence of
copper (I) chloride (Fig. 4, lanes 5 and 6). The net DNA
cleavage value for (10) was found to be 100% and
69.88 % in the presence and absence of copper(I) chlo-
ride, respectively (table).
80
60
40
20
0
Ethanol
Cystein
β-mercapto-
ethanol
Fig. 6. Effects of ethanol, cystein, and β-mercaptoethanol
on the single-strand cleavage of pBluescript M13+ (3.2 kb)
supercoiled DNA induced by arenediazonium salt (1). Per-
cent of inhibition of the single-strand cleavage of pBlue-
script M13+ (3.2 kb) supercoiled DNA was calculated as
follows [51]: inhibition (%) = 100–[(Sm+a – Sc/(Sm–Sc)]
where Sm+a is % remaining supercoiled after treatment
with mix without agent, Sc is percent of supercoiled DNA
remaining in control untreated plasmid, and Sm is percent
of remaining supercoiled DNA in mixture without agent.
The potential involvement of carbon-centered radi-
cals into DNA cleavage by arenediazonium salt (1) was
investigated by trapping experiments using ethanol,
cystein, and β-mercapto ethanol (Fig. 5, lanes 6, 8, and
10). It was found that DNA cleavage by arenediazo-
nium salt (1) 1 was inhibited by ethanol, cystein, and β-
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 34 No. 4 2008

494
ÇEKEN, KÍZÍL
Net DNA cleavage by arenediazonium salts (1)–(10) in the presence and absence of one-electron donor (CuCl)
Donor
(1)
(2)
(3)
(4)
(5)
(6)
(7)
(8)
(9)
(10)
–
2.61
4.00
5.17
18.66
80.03
18.05
86.57
79.69
14.00
0
69.88
+
41.97
44.78
37.00
100
100
100
100
57.34
100
Note: The percentage of net DNA cleavage was calculated by the following equation: {[(Form II) + (Form III) ] – [(Form II) +
s
s
c
(Form III) ]/[(Form II) + (Form III) ]} × 100%. The subscripts 's' and 'c' refer to the samples and controls, respectively.
c
s
s
1−[(Sm+a – Sc/(Sm–Sc)] where Sm+a is % remaining DNA cleavage, depending upon the substitutents
supercoiled after treatment with mix without agent, Sc attached to the aryl unit. The electrical effects [52]
is percent of supercoiled DNA remaining in control resulting from substituents attached to the aromatic
untreated plasmid, and Sm is percent of remaining rings of arenediazonium salts could play important
supercoiled DNA in mixture without agent.
roles in the DNA cleaving process.
The net DNA cleavage by arenediazonium salt (6)
was found to be 86.57 % in the absence of one electron
donor, copper(I) chloride. This value was the highest
among the all tested arenediazonium salts in the
absence of copper(I) chloride (table). Therefore, to
access the potential role of carbon-centered radicals in
the reaction that lead to DNA cleavage by (6), radical
trapping experiments using ethanol, cystein, and β-
mercaptoethanol were carried out. The DNA cleavage
was inhibeted by none of these radical scavengers. This
suggests that carbon-centered cation may be involved
in the pathway(s) leading to the strand scission (data
not shown). These results suggest that DNA damage
induced by arenediazonium ions may arise not only due
to aryl radicals, but also aryl cation production.
Among the arenediazonium salts (1)–(10), only
salts (6)–(8) were the molecules that performed the
double-strand scission in the presence of copper(I)
chloride (Fig. 2, lane 8, Fig. 3, lanes 4, 6). In compari-
sion with single-strand cleavers (1)–(5) and (9), (10),
stronger potency for compounds (6), (7), and (8) could
be due to the great reactivity associated with the result-
ing radicals.
Aryl substitution does affect the reactivity and rate
of deprotonation by aryl radicals [53]. A carboxylic
acid or carboxylate substitution was calculated to result
in a bond dissociation energy of the p-hydrogen of
113.8 kcal/mol and 112.3 kcal/mol, respectively [54],
which is comparable to the experimental bond dissoci-
ation energy value of 110.6 3.4 kcal/mol for the p-
hydrogen of phenyl radical [55] in gas phase. The influ-
ence of the environment on the reaction of the thermal
dediazotization in acidic methanol solutions was inves-
tigated by Bunnett et al. [56]. In nitrogen atmosphere,
the dediazotization is homolytical, whereas, under oxy-
gen, the reaction proceeds heterolytically via the for-
mation of an arene cation. The presence of strong elec-
tron acceptors in the phenyl ring favors the homolytical
type of the reaction via the intermediate formation of
phenyl radical. Due to the presence of diazo group, are-
nediazonium salts are highly reactive and widely used
in the chemical synthesis of azodyes [57], in prepara-
tive synthesis [58], and in analytical methods [59]. Are-
nediazonium ions can undergo either thermal or photo-
chemical heterolytic dediazoniation, yielding the aryl
cation [60]. These ions can also be dediazoniated in a
homolytic process via one-electron reduction, thus gen-
erating aryl radicals.
Arenediazonium salts
Net DNA cleavage in the absence of one electron
donor (%)
Net DNA cleavage in the presence of one electron
donor (%)
Although most arenediazonium ions are attributed
to oxidants, such structural characteristics as the elec-
tron-donating and withdrawing properties of substitu-
ents in the aromatic ring, together with interference
from solvent interactions, other reducing and oxidising
compounds, light, or the conditions of the reaction
medium may substantially modify their reactivity, giv-
DISCUSSION
We prepared a series of substituted arenediazonium ing rise to different patterns of decomposition and
salts in order to explore the role of substituted aryl rad- resulting in different biological effects. In this context,
ical and aryl cation on the DNA-cleavage efficiency. heterolytic dediazoniation has been reported to occur
Consequently, we used in investigation arenediazonium with p-methylbenzenediazonium and p-nitrobenzene-
salts containing an electron-donating (+M) group, such diazonium ions in an aqueous medium [61–62], while
as methyl and ethyl, and also both electron-donating other authors interpreted their results as indicating het-
(+M) group and electron-withdrawing (–M) group, erolytic and homolytic processes during the thermal
such as NO2 and Cl, in the same molecule. We have also and photochemical dediazoniation of several arenedia-
investigated the DNA cleavage ability of 4-(hydroxym- zonium ions in trifluoroethanol and ethanol [63, 64].
ethyl)benzenediazonium ion [36] (9) and o-(hydroxy- Furthermore, in certain cases, the reducing capacity of
ethyl)benzenediazonium ion (10). Our results in table water was sufficient to reduce arenediazonium to the
and table show that the efficiency, indicated by net aryl radical [65]. It is therefore obvious that the reaction
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 34 No. 4 2008

SYNTHESIS AND DNA-CLEAVING ACTIVITY
495
conditions need to be chosen carefully when trying to in the same molecule exhibit an appealing potency.
establish the mechanisms involved in the decomposi- Moreover, it is possible that the damage of DNA may
tion of such reactive compounds as arenediazonium be genotoxic, in addition to acute toxicity. There are
ions.
very little data available for the toxicity of the substi-
tuted arenediazonium salts. Our results indicate that the
carbon-centered aryl radical and aryl cations, generated
by arenediazonium salts, are active species for DNA
scission. Application of the process to specific DNA
sequences promises to lead to new possibilities in the
design of anticancer and chemical nucleases.
N+2BF4–
R1
•
R5
R4
R1
R2
R5
R4
–N2
+ Cu+
R2
path ‡
R3
R3
EXPERIMENTAL
path b –N2
DNA
Caution: Arenediazonium ions are tumorigenous
and should be handled with care. The completly dry
arenediazonium ions are shock sensitive any may
explode.
+
Damage of purine
or pirimidine base
or deoxyribose
R5
R4
R1
R2
Melting points were measured on a Gallenkamp
Model apparatus in open capillaries. IR spectra were
recorded in KBr pellets using a FTIR Unicam spectro-
photometer. 1H- and 13C NMR spectra were measured
in CD3COCD3 at 400/100 MHz on a Bruker NMR
spectrometer; chemical shifts are reported in δ scale
(ppm). A DNA Agarose Gel Electrophoresis was per-
formed on a Bio Rad Gel Doc Electrophoresis appara-
tus (Bio-Rad Laboratories, Hercules, CA United
States). Quantitation of bands in the gels were accom-
plished with Quantity one 4.5.2 version software.
General procedure for synthesis of arenediazo-
nium salts (1–10). The diazonium tetrafluoroborates
were synthesised according to the procedure described
in [46] and stored at –18°ë in darkness before use. The
corresponding to (1)–(10) amine (2.0 mmol) was dis-
solved in ethanol (1 ml) and aqueous fluoroboric acid
(48% solution, 6.0 mmol) was added to the solution; the
mixture was then cooled to –5°ë. Isoamyl nitrite (2.50
mmol) was added dropwise to the reaction mixture, and
stirring continued for 0.5 h. Dilution of the mixture
with diethyl ether (30 ml) led to precipitation. The solid
was collected by filtration under nitrogen and washed
with cold diethyl ether. The crude products were recrys-
tallised from acetone–diethyl ether to give a pure sam-
ple. The physical and spectra data of compounds
(1)−(10) are as follows:
R3
DNA
Single of Double
Strand Cleavage
Scheme 3. The possible reaction of arenediazonium salts
with DNA through generation of aryl radicals (path a) or
aryl cation (path b).
The possible mechanism for DNA strand–breaking
activity of arenediazonium salts are shown in
Scheme 3. The degradation of arenediazonium ions,
known as dediazoniation, may ocur via two mecha-
nisms, either heterolytic (Scheme 3, path b) or
homolytic (Scheme 3, path a). The latter requires the
transfer of electron from a reducing agent. In the
absence of catalyst, with weakly basic nucleophiles, in
the presence of O2, in aqueous acid, and in the dark, the
dediazoniation reactions are believed to proceed via
rate-determining loss of N2, generating a highly reac-
tive phenyl cation, which reacts rapidly and with a very
low selectivity with available nucleophiles [66]. The
DNA cleavage by aryl radical involves a deprotonation
from deoxyribose sugar, wheraes DNA cleavage by
aryl cations, which are capable of behaving as alkylat-
ing agents, includes alkylation of purine bases. The
alkylation of purine bases in DNA triggers the DNA
cleavage.
2,3-Dimethylbenzenediazonium tetrafluoborate
(1); colorless crystals (0.34 g, 74%); mp 69–71°C; 69–
71°C; νmax (cm–1): 3043, 2932, 2264, 1609, 1562, 1094,
and 812; 1H NMR (400 MHz, CD3COCD3): 7.12 (1 H,
dd, J 8.2, 1.1 Hz, ArH), 7.05 (1 H, ddd, J 8.0, 1.1 Hz,
ArH), 6.9 (1 H, dd, J 8.2, 1.1 Hz, ArH), 2.28 (3 H, s,
CH3), 2.16 (3 H, s, CH3); 13C NMR (100 MHz,
CD3COCD3): 9.7 (q), 18.6 (q), 112.2 (d), 122.9 (d),
125.2 (d), 139.1 (s), 159.9 (s), 162.3 (s).
CONCLUSIONS
The ability of substituted arenediazonium ion to
cleave DNA was studied. It is suggested that the toxic-
ity of the arenediazonium salts may be due to genera-
tion of reactive aryl radicals damaging biomolecules.
All the arenediazonium salts tested can cause a certain
amount of DNA cleavage, although their cleaving
potency is different from each other. Among various
substituents in the benzene ring of an arenediazonium
2,6-Dimethylbenzenediazonium tetrafluoborate
salt, our results on gel electrophoresis indicate that (2); colorless crystals (0.10 g, 22%). mp 86–87°C; νmax
1
attachment of an electron-donating (+M) group, such as (cm–1): 3063, 2932, 2257, 1610, 1584, 1080, 822; H
ethyl and also both electron-donating (+M) group and NMR (400 MHz, CD3COCD3): 7.06 (1 H, ddd, J 8.0,
electron-withdrawing (–M) group such as NO2 and Cl 8.0, 1.2 Hz, ArH), 6.6 (2 H, dd, J 8.3, 1.5, ArH), 2.9
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 34 No. 4 2008

496
ÇEKEN, KÍZÍL
(6 H, s, CH3); 13C NMR (100 MHz, CD3COCD3): 14.6 1.5 Hz, ArH), 7.7 (1 H, ddd, J 8.1, 8.1, 1.6 Hz, ArH),
(q), 119.4 (d), 123.6 (d), 128.2 (s), 129.5 (s).
7.6 (1 H, dd, J 8.2, 1.1 Hz, ArH), 7.5 (1 H, ddd, J 8.0,
8.0, 1.2 Hz, ArH), 3.9 (2 H, t, J 4.8 Hz, CH2CH2OH),
3.48 (2 H, m, CH2CH2OH), 1.13 (1 H, t, CH2OH); 13C
NMR (100 MHz, CD3COCD3): 54.86 (t), 55.9 (t), 57.1
(s), 60.9 (d), 123.25 (d), 130.5 (d), 134.95 (d), 141.02
(s).
4-(Hydroxymethyl)benzenediazonium tetraflu-
oborate (10) [46]; mustard colour crystals (0.40 g,
89%). mp119–121°C; νmax (cm−1): 2932, 2594, 2270,
1589, 1516, 1067, 830; 1H NMR (400 MHz,
CD3COCD3): 8.7 (1 H, dd, J 8.3, 1.5 Hz, ArH), 7.9
(1 H, dd, J 8.1, 8.1, 1.6 Hz, ArH), 7.6 (1 H, dd, J 8.2,
1.1 Hz, ArH), 7.5 (1 H, dd, J 8.0, 8.0, 1.2 Hz, ArH), 4.9
(2 H, d, J 7.5, CH2OH), 1.2 (1 H, t, CH2OH); 13C NMR
(100 MHz, CD3COCD3): 62.7 (t), 112.3 (s), 128.5 (d),
132.9 (d), 158.67 (s).
Purification of plasmid DNA. The plasmid pBlue-
script M13+ DNA was prepared and isolated according
to standard protocols using Qiagene plasmid mini prep-
aration kit (Qiagene) [67]. The purity of pBluescript
M13+ was confirmed via both Agarose Gel Electro-
phoresis and UV spectroscopy by determining the ratio
of absorbances at 260 and 280 nm [68]. The concentra-
tion of DNA was determined from the absorbance at
260 nm (A260 = 1.0 for 50 µg/ml).
Densitometric analysis of treated and control
pBluescript M13+ plasmid DNA Gel was scanned by
a Gel documentation system (Gel-Doc-XR, BioRad,
Hercules, CA, United States). Bands in the gel were
quantified using discovery series Quantity One pro-
gramme (version 4.5.2, BioRad Co.).
2,6-Diethylbenzenediazonium
tetrafluoborate
(3); colorless crystals (0.26 g, 51%); mp 99–101°C;
ν
max (cm–1): 3063, 2932, 2251, 1630, 1576, 1086, 816;
1H NMR (400 MHz, CD3COCD3): 7.06 (1 H, ddd, J
8.0, 8.0, 1.2 Hz, ArH), 6.9 (2 H, dd, J 8.3, 1.5, ArH), 2.9
(4 H, m, CH2), 2.6 (4 H, m, CH3); 13C NMR (100 MHz,
CD3COCD3): 13.5 (q), 15.6 (q), 119.4 (d), 123.6 (d),
128.2 (s) 128.9 (s).
2-Ethylbenzenediazonium tetrafluoborate (4);
light blue crystals (0.30 g, 65%). mp 73–74°C; νmax
(cm–1): 2978, 2937, 2264, 1641, 1562, 1094, 776; H
1
NMR (400 MHz, CD3COCD3) 9.5 (1 H, dd, J 8.3, 1.5
Hz, ArH), 9.3 (1 H, ddd, J 8.1, 8.1, 1.6 Hz, ArH), 9.2 (1
H, dd, J 8.2, 1.1 Hz, ArH), 9.0 (1 H, ddd, J 8.0, 8.0, 1.2
Hz, ArH), 3.4 (2 H, m, CH2CH3) 1.6 (3H, t, J 4.8
CH2CH3); 13C NMR (100 MHz, CD3COCD3): 13.7 (q),
22.9 (t), 114.7 (d), 119.5 (d), 126.6 (d), 129.1 (d), 130.2
(s), 209.0 (s).
3-Ethylbenzenediazonium tetrafluoborate (5);
pink crystals (0.25 g, 55 %). mp 68–70°C; νm1ax (cm–1):
2970, 2932, 2270, 1642, 1470, 1080, 803; H NMR
(400 MHz, CD3COCD3): 8.8 (1 H, dd, J 8.3, 1.5 Hz,
ArH), 8.7 (1 H, ddd, J 8.1, 8.1, 1.6 Hz, ArH), 7.2 (1 H,
dd, J 8.2, 1.1 Hz, ArH), 6.9 (1 H, ddd, J 8.0, 8.0, 1.2 Hz,
ArH), 2.9 (1 H, s, J 7.5, ArH) 2.6 (2 H, m, CH2CH3),
1.2 (3 H, t, J 4.8 CH2CH3); 13C NMR (100 MHz,
CD3COCD3): 14.0 (q), 112.4 (d), 114.6 (d), 186.9 (d),
129.2 (d), 145.7 (s), 157.3 (s).
4-Ethylbenzenediazonium tetrafluoborate (6);
yellow crystals (0.20 g, 44%); mp 87–88°C; νmax
(cm−1): 2978, 2932, 2264, 1641, 1584, 1086, 849; H
1
DNA cleaving activity. The ability of compounds
(1)–(10) to cleave DNA was investigated using a plas-
mid relaxation assay to monitor the conversion of cir-
cular supercoiled pBluescript M13+ DNA (form I) into
the relaxed circular (form II) and linear DNA (form III).
Plasmid relaxation require an added electron donor
(mediating homolytic dediazoniation of diazonium
group) as shown for other diazonium reagents. Like in
similar to previous studies [21, 40], we examined inor-
ganic and organic one-electron donors such as cop-
per(I) chloride, sodium iodide, and ascorbic acid in
order to reduce the arenediazonium salts to aryl radicals
and to optimize conditions for efficient DNA cleavage.
It was found that copper(I) chloride works the best [21]
(data not shown).
To investigate their DNA-cleaving potency, super-
coiled pBluescript M13+ DNA (200 ng) was placed in
0.5-ml Eppendorf tubes protected from light by means
of a foil cover and used in dark room. The DNA
(200 ng) was treated with a solution of diazonium salt
(1.5 mM) in dark in the presence of copper(I) chloride
(1.5 mM), which acted as a one-electron donor. The
NMR (400 MHz, CD3COCD3): 7.8 (2 H, dd, J 8.3,
1.5 Hz, ArH), 7.5 (2 H, dd, J 8.1, 1.5 Hz, ArH), 3.0
(2 H, m, CH2CH3) 1.3 (3 H, t, J 4.8, CH2CH3); 13C NMR
(100 MHz, CD3COCD3): 13.9 (q),105.0 (t), 111.7 (s)
131. (d), 133.0 (d), 160.7 (s).
2-Chloro-4-nitrobenzenediazonium tetrafluobo-
rate (7); colorless crystals (0.15 g, 60%); mp 162–
163°C; νmax (cm−1): 3063, 2991, 2276, 1641, 1562,
1
1080, 843; H NMR (400 MHz, CD3COCD3): 9.27
(1 H, dd, J 8.3, 1.5 Hz, ArH), 9.01 (1 H, dd, J 8.3,
1.5 Hz, ArH), 8.82 (1 H, s, ArH); 13C NMR (100 MHz,
CD3COCD3): 121.78 (s), 124.88 (s), 127.33 (d), 136.88
(d), 138.52 (d), 154.48 (s).
4-Chloro-2-nitrobenzenediazonium tetrafluobo-
rate (8); colorless crystals (0.17 g, 68%). mp 125–
127°C; νmax (cm−1): 3063, 2967, 2284, 1649, 1562,
1
1344, 1173, 849; H NMR (400 MHz, CD3COCD3):
9.2 (1 H, dd, J 8.3, 1.5 Hz, ArH), 8.7 (1 H, dd, J 8.3,
1.5 Hz, ArH), 7.4 (1 H, s, ArH); 13C NMR (100 MHz,
CD3COCD3): 118.6 (s), 124.9 (s), 129.01 (d), 136.88
(d), 137.96 (d), 149.64 (s).
2-(Hydroxyethyl)benzenediazonium tetrafluobo- DNA was also exposed to control solutions of the diaz-
rate (9); yellow crystals (0.30 g, 62%); mp 118–120°C; onium salt with none of these additives and to each of
ν
max (cm−1): 2924, 2507, 2264, 1603, 1497, 1067, 756; the additives separately with no diazonium salt added.
1H NMR (400 MHz, CD3COCD3): 7.9 (1 H, dd, J 8.3, After incubation for 2 h, 5 µl of a gel-loading dye solu-
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 34 No. 4 2008

SYNTHESIS AND DNA-CLEAVING ACTIVITY
497
14. Diple, A, Michejda, C.J, and Weisburger, E.K, in Metab-
olism of Chemical Carcinogenes Part 1, Grunberg, D.
and Goff, S., Eds., New York: Academic, 1987.
tion [48] (containing 0.1% Bromophenol Blue,
150 mM EDTA, 1% SDS in 2 mM Tris, 1 mM acetate,
pH 8) was added into each well before loading (in dark)
onto 1% Agarose gel containing an aqueous Ethidium
Bromide solution (10 mg/ml). The gel was electro-
phoresed for 2 h at 120 V in 1 × TAE (40 mM Tris ace-
tate, 1 mM EDTA, pH 8.2) buffer. Incubations with are-
nediazonium salts were carried out for 2 h using the fol-
lowing final concentrations: 31.53 nM DNA, 0.375 mM
arenediazonium salt, and 0.775 mM copper chloride.
DNA in gel was analyzed with BioRad Gel Doc XR
Gel-Documentation system. The amount of supercoiled
form I DNA (sc-DNA), the relaxed open circular form
II DNA (oc-DNA), and linear form III DNA (l-DNA)
were quantified by the intensity of the bands with
Quantity One programme (version 4.5.2, BioRad Co.).
15. Gali, C., Chem. Rev., 1988, vol. 88, pp. 765–792.
16. Chin, A., Hung, M.-H., and Stock, L.M., J.Org. Chem,
1981, vol. 46, pp. 2203–2207.
17. Hung, M.-H. and Stock, L.M., J. Org. Chem., 1982,
vol. 47, pp. 448–453.
18. Gannett, P.M., Powell, J.H., Rao, R., Shi, X., Lawson, T.,
Kolar, K., and Toth, B., Chem. Res. Toxicol., 1999,
vol. 12, pp. 297–304.
19. Stiborova, M., Asfaw, B., Frei, E., Schemeiser, H.H., and
Weiessler, M., Chem. Res. Toxicol., 1995, vol. 8,
pp. 489–498.
20. Berh, J.P., J. Chem. Soc. Chem. Commun., 1989,
pp. 101–103.
The percentage of net DNA cleavage was calculated
by the following equation: [(Form II)s + (Form III)s] –
[(Form II)c + (Form III)c]. The subscripts ‘s’ and ‘c’
refer to as the samples and controls, respectively [51].
21. Griffiths, J. and Murphy, J.A., J. Chem. Soc. Chem.
Commun., 1992, pp. 24–26.
22. Kato, T., Kojima, K., and Hiramoto, K., Mutat. Res.,
1992, vol. 268, pp. 105–114.
23. Hazlewood, C., Davies, M.J., Gilbert, B.C., and
Packer, J.E., J. Chem. Soc., Perkin Trans. 2, 1995,
pp. 2167–2174.
ACKNOWLEDGMENTS
24. Hiramoto, K., Kaku, M., Kato, T., and Kikugawa, K.,
˙
We thank Dr. Ebru I nce Yılmaz for assistance with
Chem. Biol. Interact., 1995, vol. 94, pp. 21–36.
plasmid DNA purification, and Generous financial sup-
port by Dicle University Research Foundation
(DÜAPK, Project numbers: 02-FF-10 and 03-FF-63).
˙
25. Kizil, M., Yilmaz, E.I., Pirinccioglu, N., and
Aytekin, C., Turk J. Chem., 2003, vol. 27, pp. 539–544.
26. Gannett, P.M., Ye, J., Ding, M., Powell, J., Zhang, Y.,
Darian, E., and Daft, J., Chem. Res. Toxicol., 2000,
vol. 10, pp. 1020–1027.
27. Zollinger, H., Diazo Chemistry: Aromatic and Heteroar-
omatic Compounds, Vol. 1, New York: Wiley, 1994.
28. Bravo Diaz, C. and Gonzalez, Romero E., Curr. Topics
Colloid Interface Sci., 2001, vol. 4, p. 58.
29. Quintero, B. Cabeza, M.C., Martinez, Puentedura, M.I.,
Gutierrez, P., Martinez, De Ias Parras, P.J., Llopis, L.,
and Zarzuelo, A., Ars Pharm., 2003, vol. 44, pp. 239–
255.
REFERENCES
1. Preussman, R., Ivankovic, S., Landschuttz, C., Gimmy, J.,
Flohr, E., and Grisebach, U., Z. Krebsforsch., 1974,
vol. 81, pp. 285–310.
2. Gold, B. and Salmasi, S., Cancer Lett., 1982, vol. 15,
pp. 289–300.
3. Ames, B.N., Magaw, R., and Gold, L.S., Science, 1987,
vol. 236, pp. 271–280.
4. Kikugawa, K., Kato, T., and Takeda, Y., Mutat. Res.,
1987, vol. 172, pp. 35–43.
30. Dornberger, K., Ihn, W., Schade, W., Tresselt, D.,
Zureck, A., and Radics, I., Tetrahedron Lett., 1986,
vol. 27, pp. 559–560.
5. Kikugawa, K. and Kato, T., Food Chem. Toxicol., 1988,
vol. 26, pp. 209–214.
31. Kikugawa, K.., Kato, T., and Kojima, K., Mutat. Res.,
6. Ohsima, H., Furihata, C., Matsushima, T., and Bartsch, H.,
1992, vol. 268, pp. 65–75.
Food Chem. Toxicol., 1989, vol. 27, pp. 193–203.
32. Toth, B., Patil, K., Taylor, J., Stessman, C., and Gannett, P.,
7. Lawson, T., Gannett, P.M., Yau, W.M., Dalal, N.S., and
Toth, B., J. Agric. Food Chem., 1995, vol. 43, pp. 2627–
2635.
8. Stiborova, M., Hansikova, H., Schmeiser, H.H., and
Frei, E., Gen. Physiol. Biophys, 1997, vol. 16,
pp. 285–300.
In Vivo, 1989, vol. 3, pp. 301–306.
33. Levenberg, B., Biochim. Biophys. Acta, 1962, vol. 63,
pp. 212–214.
34. Stiborova, M., Asfaw, B., Anzenbacher, P., Leseticky, L.,
and Hodek, P., Cancer Lett., 1988, vol. 40, pp. 319–326.
9. Toth, B. and Gannett, P., Mycopathologia, 1993,
35. Stiborova, M., Asfaw, B., Anzenbacher, P., and Hodek, P.,
vol. 124, pp. 73–77.
Cancer Lett., 1988, vol. 40, pp. 327–333.
10. Toth, B., Patil, K., Erickson, J., and Gannett, P., In Vivo,
36. Toth, B., Patil, K., and Jae, H., Cancer Res., 1981,
1998, vol. 12, pp. 379–382.
vol. 41, pp. 2444–2449.
11. Toth, B., Patil, K., Erickson, J., and Gannett, P., In Vivo,
37. Toth, B., Nagel, D., and Ross, A., Br. J. Cancer, 1982,
1999, vol. 13, pp. 125–128.
vol. 46, pp. 417–422.
12. Toth, B., In Vivo, 2000, vol. 14, pp. 299–319.
38. Gannett, P.M., Lawson, T., Miller, M., Thakkar, D.D.,
Lord, J.W., Yau, W.-M., and Toth, B., Chem. Biol. Inter-
act., 1996, vol. 101, pp. 149–164.
13. Koepke, S.R., Kroeger-Koepke, M.B., and Michejda, C.,
Chem. Res. Toxicol., 1990, vol. 3, pp. 17–20.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 34 No. 4 2008

498
ÇEKEN, KÍZÍL
39. Pratviel, G., Bernadou, J., and Meunier, B., Angew. 55. Wenthold, P.G. and Squries, R.R., J. Am. Chem. Soc.,
Chem. Int. Ed. Eng., 1995, vol. 34, pp. 746–769.
1994, vol. 116, pp. 6401–6412.
56. Bunnett, J.F. andYijima, J., J. Org. Chem., 1977, vol. 42,
40. Arya, D.P. and Jebaratnam, D.J., Tetrahedron Lett.,
pp. 639–643.
1995, vol. 25, pp. 4369–4372.
57. Zollinger, H., Diazo Chemistry, vol. 1, Aromatic and
Heteroaromatic Compounds, Weinheim: VCH Publish-
ers, 1994.
41. Mohler, D.L., Coonce, J.G., and Predecki, D., Bioorg.
Med. Chem. Lett., 2003, vol. 13, pp. 1377–1379.
42. Stubbe, J. and Kozarich, J.W., Chem. Rev., 1987, vol. 87,
58. Griffiths, J. and Cox, R., Dyes Pigm., 2000, vol. 47,
pp. 1107–1136.
p. 65.
43. Steenken, S., Chem. Rev., 1989, vol. 89, pp. 503–520.
44. Hazlewood, C. and Davies, M.J., J. Chem. Soc. Perkin
Trans., 1995, pp. 895–901.
45. Carter, K.N., Taverner, T., Schieser, C.H., and Green-
berg, M.M., J. Org. Chem., 2000, vol. 65, pp. 8375–
8378.
46. Quintero, B., Cabeza, M.C., Martinez, M.I., Gutierrez, P.,
and Martinez, P.J., Can. J. Chem., 2003, vol. 81, pp. 832–
839.
59. Williams, W.J., Handbook of Anion Determination, Lon-
don: Butterworths, 1979.
60. Ambroz, H.B., Kemp, T.J., and Przybtniak, G.K., J.
Photo Chem. Photobiol. A, 1997, vol. 108, pp. 149–153.
61. Bravo-Diaz, C., Romsted, M., Harbowy, M., Romero-
Nieto, M.E., and Gonzalez-Romero, E., J. Phys. Org.
Chem., 1999, vol. 12, pp. 130–140.
62. Pazo Llorente, R., Sarabia Rodriguez, M.J., Bravo-Diaz, C.,
and Gonzalez-Romero, E., Int. J. Chem. Kinet., 1999,
vol. 31, pp. 73–82.
47. Dill, J.D., Schleyer, P.V.R., and People, J.A., J. Am.
Chem. Soc., 1977, vol. 99, pp. 5361–5378.
63. Canning, P.S.J., McCrudden, K., Maskill, H., and Sex-
48. Liu, J.W., Beelman, R.B., Lineback, D.R., and Speroni, J.J.,
ton, B., Chem. Commun., 1998, pp. 1971–1972.
J. Fd. Sci, 1982, vol. 47, pp. 1542–1544.
64. Canning, P.S.J., McCrudden, K., Maskill, H., and Sex-
ton, B., J. Chem. Soc., Perkin Trans. 2, 1999, pp. 2735–
2740.
65. Gannett, P.M., Powell, J.H., Rao, R., Shi, X., Lawson, T.,
Kolar, C., and Toth, B., Chem. Res. Toxicol., 1999,
vol. 12, pp. 297–304.
66. Ando, W, in Photochemistry of the Diazonium and Diazo
Groups, in The Chemistry of Functional Groups, Sal,
Patai., Ed., Wiley, 1978.
49. Friederic, U., Fischer, B., Lüthy, J., Hann, D., Schlatter, C.,
and Würgler, E., Z. Lebensm. Unters. Forsch, 1986,
vol. 183, pp. 85–89.
50. Galli, C., Chem. Rev., 1998, vol. 88, pp. 765–792.
51. Hiramoto, K., Kaku, M., Sueyoshi, A., Fujise, M.., and
Kikugawa, K., Chem. Res. Toxico., 1995, vol. 8,
pp. 356–362.
52. Arizmendi, L.E., Heidbrink, J.L., Guler, L.P., and Kent-
tamaa, H.I., J. Am. Chem. Soc., 2003, vol. 125, pp. 2272–
2281.
53. Li, R.M., Smith, R.L., and Kenttamaa, H.I., J. Am.
Chem. Soc., 1996, vol. 118, pp. 5056–5061.
54. Wenthold, P.G. and Squries, R.R., J. Mass Spectrom. Ion
Processes, 1998, vol. 175, pp. 215–224.
67. Zhou, C.,Yang,Y., and Jong, A.Y., BioTechniques, 1990,
vol. 8, pp. 172–173.
68. Sambrook, J., Fritsch, E.F., and Maniatis, T., Molecular
Cloning: A LaboratoryManual, 2nd ed., Cold Spring
Harbor, NY: Cold Spring Harbor Lab. Press, 1989,
pp. 209–286.
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