Application of BODIPY-trimethylmelamine conjugate for DNA crosslinking in vitro

Article abstract of DOI:10.1134/S1068162011020038

The conjugate of the fluorescent dye 4,4,-difluoro-1,3,5,7-tetramethyl-4- bora-3a,4a-diaza-s-indasten-8-propionic acid (BODIPY) with N²,N ⁴,N⁶-trimethylmelamine was obtained. This compound was shown to generate covalent crosslinks between DNA strands in vitro in the presence of formaldehyde.

Full text of DOI:10.1134/S1068162011020038

ISSN 1068ꢀ1620, Russian Journal of Bioorganic Chemistry, 2011, Vol. 37, No. 2, pp. 249–253. © Pleiades Publishing, Ltd., 2011.
Original Russian Text © V.A. Efimov, S.V. Fedunin, O.G. Chakhmakhcheva, 2011, published in Bioorganicheskaya Khimiya, 2011, Vol. 37, No. 2, pp. 278–283.
LETTER
TO THE EDITOR
Application
of BODIPYꢀTrimethylmelamine Conjugate
for DNA Crosslinking in Vitro
V. A. Efimov†, S. V. Fedunin, and O. G. Chakhmakhcheva1
Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences,
ul. MiklukhoꢀMaklaya 16/10, Moscow, 117997 Russia
Received September 1, 2010; in final form, November 3, 2010
Abstract—The conjugate of the fluorescent dye 4,4,ꢀdifluoroꢀ1,3,5,7ꢀtetramethylꢀ4ꢀboraꢀ3
ꢀindastenꢀ8ꢀpropionic acid (BODIPY) with N2 N4 N6ꢀtrimethylmelamine was obtained. This comꢀ
pound was shown to generate covalent crosslinks between DNA strands in vitro in the presence of formꢀ
aldehyde.
a,4aꢀdiazaꢀ
s
,
,
Keywords
BODIPY
: DNA, interstrand crosslinking reagents, hexamethylmelamine, trimethylmelamine, trimelamol,
DOI: 10.1134/S1068162011020038
† 1
Crosslinks between the strands of nucleic acids result of oxidation by cytochrom Pꢀ450, giving
Nꢀ
(NA) in living cells can be induced by both exterꢀ
nal chemical agents and endogenous compounds.
All of them, being of different chemical nature,
function, as a rule, as alkylating of arylating
agents. Crosslinking reagents are currently used as
antitumor preparations and tools for the investigaꢀ
tion of the biological behavior of crosslinked NA
duplexes [1].
(hydroxymethyl) intermediates (Ia) (Fig. 1a) [3]
and, after dehydration, forms the active iminium
ion (Ib). This compound is a potential electrophile
and, apparently, an active species responsible for the
alkylation of DNA.
Clinical trials of HMM have revealed a number
of deficiencies with the peroral use caused by the
difference in the formation of active Nꢀhydroxymeꢀ
Some
two DNA strands. In particular, hexamethꢀ
ylmelamine (HMM, ) exhibits pronounced antituꢀ
mor activity (Fig. 1a); it is efficient against metaꢀ
static forms of breast cancer, lymphoma, lung canꢀ
cer, and some other forms of cancer [2]. The exact
mechanism of its action is still unknown. It is
assumed that HMM is activated in an organism as a
sꢀtriazine derivatives are able to crosslink
thyl metabolites in different patients’ bodies [4].
The bioactive form of HMM, i.e., 2,4,6ꢀ
tris[(hydroxymethyl)methylamino]ꢀ1,3,5ꢀtriazine
(trimelamol, TM, (II), Fig. 1b), was suggested as an
alternative to HMM for intravenous administration
I
[3, 5]. TM is easily synthesized from N2 N4 N6ꢀtriꢀ
, ,
methylmelamine (TMM, (III)) by its treatment
with formaldehyde [6, 7] (Scheme 1). TM is well
soluble in water, does not require activation by oxiꢀ
dation, and acts like HMM as an efficient reagent to
crosslink DNA. Unlike HMM, however, TM is
unstable in aqueous solutions; its halfꢀlife is about
200 min at pH 7.4 and room temperature.
Abbreviations: ISC, interstrand crosslink of DNA; BODIPY,
4,4,ꢀdifluoroꢀ1,3,5,7ꢀtetramethylꢀ4ꢀboraꢀ3a,4aꢀdiazaꢀsꢀindastenꢀ
8ꢀpropionic acid; HMM – hexamethylmelamine; TM, triꢀ
2
4
6
melamol; TMM,
N ,N ,N ꢀtrimethylmelamine; TMMB,TMMꢀ
BODIPY conjugate.
Deceased.
Corresponding author: phone: +7 (495) 336ꢀ5911; fax: +7 (495)
330ꢀ6738; eꢀmail: eva@mx.ibch.ru.
†
1
249

250
EFIMOV et al.
OH
R
R
H
N
N
NH
N
N
N
R
R
OH
NaHCO /H O/EtOH
2
3
+ CH2O
N
N
R
N
N
R
HN
N
HO
(
(
III) R = –CH3
II) R = –CH3
Scheme 1. Synthesis of TM from TMM. Reaction was carried out with 4ꢀfold molar excess of formaldehyde
in an ethanol–water mixture (4 : 1) containing 0.25 M NaHCO at 25°C for 24 h. Yield of TM was 54%.
3
The mechanism of TM’s action and preferred suggests, instead of the covalent addition of this
reagent to DNA strands, crosslinks of the strands due
to the action of formaldehyde formed after deformilaꢀ
tion of TM [8].
binding sites in DNA have not been exactly deterꢀ
mined. A mechanism based on acidic elimination of
water with the formation of a reactive electrophilic ion
(IIa) was postulated (Fig. 1b). Another proposed
The possibility of the occurrence of one or the other
mechanism of ISC formation under the action of TM process was studied. It was shown that, in the presence
(a)
+
N
DNA
N
N
N
N
OH
N
–H O
2
oxidation
N
N
N
N
N
N
N
N
N
N
N
N
(I
)
(Ia
)
(Ib)
N
DNA
N
DNA
N
N
DNA
–H O
2
N
N
N
N
N
N
DNA
DNA
+
N
N
N
N
N
N
N
OH
N
N
(b)
OH
OH
OH
DNA
DNA
N
N
N
N
N
N
N
N
N
+
OH
N
N
N
N
N
N
N
N
HO
HO
HO
OH
DNA
OH
DNA
N
N
N
N
N
N
N
N
N
N
N
+
N
DNA
DNA
Fig. 1. Oxidative activation of hexamethylmelamine (a) and hypothetical mechanism of crosslinking of strands in DNA
duplexes (b).
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APPLICATION OF BODIPYꢀTRIMETHYLMELAMINE CONJUGATE
251
'
'
(
(
(
(
(
VIII)
'
'
IX
)
'
'
X
)
'
'
XI
)
'
'
XII)
Fig. 2. Duplexes used for crosslinking with TM, TMM, and TMMB.
of thiophenol, TM forms only trisubstituted thioester sible to measure [8]. It is assumed that, after intercalaꢀ
product. At the same time, neither deformilation
adducts or bisthiophenol products linked via a methylꢀ
ene bridge (bisthiophenol products) were revealed [5].
These facts do not support the second mechanism. Furꢀ
ther, linear 5'ꢀ32Pꢀlabeled plasmid DNA pBR322 was
analyzed by electrophoresis in agarose gel after its crossꢀ
liking under TM action. The analysis was based on the
fact that crosslinks should be revealed by the presence in
the denaturing gel of spots corresponding to doubleꢀ
stranded DNA. It was shown that crosslinks began to
form at TM concentrations of about 2.5–5.0 pM, and
the extent of their formation increased with the TM
concentration. Such a level of crosslinking activity is
comparable with the activity of alkylating agents on the
basis of nitrogen mustards [5]. Another indirect confirꢀ
mation of the mechanism in Fig. 1b was the fact that the
efficiency of crosslinking increased with a reduction of
pH from 7.0, and its rate at pH 4.0 increased by a factor
of 10 over that at neutral pH. At the same time, the
treatment of the linear form of pBR322 with formaldeꢀ
hyde at various concentrations did not lead to the forꢀ
mation of the crosslinks in DNA. Thus, these in vitro
results showed that formaldehyde itself did not lead to
the crosslinking of DNA [5].
tion of TM into DNA and alkylation of one of the
strands with the first iminium group of TM, the other
activated iminium group almost immediately alkylates
the second DNA strand, being in close proximity to it;
in this case, presumably a small change in the conforꢀ
mation of the duplex occurs [8].
We compared the efficiency of the formation of
crosslinks in DNA in vitro under the action of a numꢀ
ber of intercalating crosslinking reagents. To study
the mechanism of the crosslinking with TM action,
we investigated the interaction of doubleꢀstranded
DNA with TM. Synthetic DNA duplexes (30 bp)
were used as DNA fragments. The crosslinking was
carried out in solutions containing oligonucleotide
duplexes (20–30
µ
M) and TM (50–500 M) preꢀ
µ
pared from TMM (Scheme 1 [7, 9]) in 0.03 M triethaꢀ
nolamineꢀHCl (pH 5.0) buffer for 3–5 h at room temꢀ
perature. The efficiency of the crosslinking was evaluꢀ
ated by gelꢀelectrophoresis in denaturing PAAG
according to Jackson et al. [8]. Crosslinked duplexes
had lower electrophoretic mobility than singleꢀ
stranded oligonucleotides. It was shown that the yield
of crosslinked duplexes increased with increasing TM
concentration, not exceeding, however, 10%. The
increase of the reaction time did not significantly
influence the yield, apparently due to degradation of
TM in water, while the second addition of the
crosslinking agent 3 h after the beginning of the reacꢀ
tion led to a certain enhancement of the yield of
Some authors have previously hypothesized that
alkylation of both DNA strands with ISC formation
occurred very quickly one after the other, almost
simultaneously. The alkylation of the second strand
(usually limiting the rate of crosslinking in the case of
many reagents) occurs so quickly that its rate is imposꢀ crosslinked DNA.
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 37
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252
EFIMOV et al.
To increase the crosslinking yield, we used a mixꢀ
ture of trimethylmelamine (III) (50–500 M) and
formaldehyde (150–1500 M). TMM was synthesized
NH2
H2N
μ
(CH2)6
μ
according to Ramurthy and Miller [7]. Addition of
formaldehyde to TMM resulted in the formation of
TM in situ. This approach led to an increase in
crosslinked DNA by a factor of almost 5. In control
experiments, doubleꢀstranded DNA was treated under
the same conditions with either formaldehyde or
TMM. The formation of ISC in DNA was not
observed in either case.
i
Cl
N
NHTr
H2N
+
N
N
(CH2)6
Cl
Cl
OH
ii
O
N
For further evidence of the presence of the triꢀ
melamol residue in the crosslinked DNA, we syntheꢀ
sized the TMM derivative where one of the CH3
groups was substituted by the fluorescent label.
BODIPY, one of the most sensitive neutral dyes, was
chosen as the fluorescent label to be conjugated with
TMM. BODIPY was linked with TMM through the
Cl
N
H
N
N
NHTr
(CH2)6
⊕
N
ᮎ
N
B
Cl
F
F
iii
(V)
flexible hexamethylenediamine linker, giving 2ꢀ
N
ꢀ(6ꢀ
HN
H
N
v
N
NHTr
aminohexyl)aminoꢀ4,6ꢀdiꢀNꢀmethylaminoꢀ
ine (IV) (Scheme 2).
sꢀtriazꢀ
(CH2)6
O
N
N
N
ꢀoxysuccinimide ester of BODIPY (VI) was also
synthesized. For this purpose, 4,4,ꢀdifluoroꢀ1,3,5,7ꢀtetꢀ
ramethylꢀ4ꢀboraꢀ ,4 ꢀdiazaꢀ ꢀindastenꢀ8ꢀpropionic
acid ( ) synthesized according to [10] was treated with
ꢀhydroxysuccinimide and DCC. The interaction of
O N
O
NH
iv
O
3a
a
s
V
HN
N
H
N
N
NH2
ester (VI) and compound (IV) resulted in a fluorescent
(CH2)6
⊕
N
TMM derivative containing the BODIPY residue
ᮎ
N
N
N
(TMMB, (VII)) (λmax 499 nm,
ε
~ 8
×
104 cm–1 M–1,
B
F
F
NH
λex 500 nm, λem 506 nm). The structures of (VII) and all
intermediate compounds were confirmed by NMR specꢀ
troscopy and mass spectrometry.
(
IV
)
(
VI
)
Compound (VII) was used for crosslinking of the
DNA duplexes (Fig. 2) in the presence and the absence
of formaldehyde. The introduction of TMMB into
doubleꢀstranded DNA was studied by HPLC and elecꢀ
trophoresis in PAAG (Fig. 3). The results unambiguꢀ
ously showed that the fluorescent label was incorpoꢀ
rated in the crosslinked DNA. The treatment of DNA
with only TMMB without formaldehyde did not lead to
DNA crosslinking. TMMB was not attached to singleꢀ
stranded DNA under our conditions. Thus, additional
confirmation was obtained that the triazine residue was
involved in the structure of crosslinked DNA.
vi
⊕
H
N
ᮎB
N
H
N
F
N
HN
N
(CH2)6
F
O
N
N
NH
(
VII)
Scheme 2. Synthesis of conjugate of 4,4,ꢀdifluoroꢀ1,3,5,7ꢀ
tetramethylꢀ4ꢀboraꢀ3 ,4 ꢀdiazaꢀ ꢀindastenꢀ8ꢀpropionic
acid with trimelamine derivative containing aminoalkyl
spacer. , TrCl/DBU/CH Cl , 0°C, 30 min (98% yield); ii
DIPEA/CH CN, 0°C, 30 min; iii, CH NH /H O, 160°C,
It was previously hypothesized that TM causes ISC
preferably at GC sites [5], which was confirmed by
experiments on the thermal denaturation of DNA
crosslinked with TM. It was also assumed that an even
distribution of GCꢀ and ATꢀreach sequences in close
proximity to the TM binding site can influence the
crosslinking [7]. Preliminary experiments on deterꢀ
mining TM binding sites in DNA showed that
duplexes (XI) and (XII), containing no GC sites, were
not crosslinked with TM (or TMMB with formaldeꢀ
hyde). At the same time, duplexes containing these
sites were crosslinked with these reagents. Experiꢀ
a
a
s
i
,
2
2
3
3
2
2
6 h; iv, 80% CH COOH, 100°C, 1 h (98% yield of (IV)); v,
3
DCC/CH Cl , 25°C, 10 min (95% yield of (VI)); vi, CH Cl ,
2
2
2
2
1
25°C, 20 min, (72% yield of (VII)); H NMR for (VII):
(2 H, s, BODIPY, 3 , 6 , 5.90 (1 H, br. s, C(O)ꢀN ), 5.15
(3 H, br. s, ꢀtriazine, N ), 3.1–3.25 (4 H, m, ꢀtriazine,
NHꢀC ), BODIPY, C ), 3.19 (2 H, k, 6.5 Hz, C(O)NHꢀ
), 2.48 (6 H, s,
δ
6.02
H
H
H
s
H
s
H
H
J
2
2
C
H ), 2.88 (6 H, br. s, sꢀtriazine, NHꢀCH
2
3
BODIPY, 1,7ꢀCH , 2.40 (8 H, m, BODIPY, 3,5ꢀC
H ,
3
3
BODIPY, CH =CH ), 1.54–1.29 (8 H, m,
NHCH ꢀ(C
sꢀtriazine,
2
2
+
H ) ). Massꢀspectrum (ESI): 556 (M ).
2
2 4
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APPLICATION OF BODIPYꢀTRIMETHYLMELAMINE CONJUGATE
253
GC or CG sites under treatment with TM and its
derivatives and the study of structural features of the
formed DNA adducts are ongoing.
(a)
(b)
3
1
2
3
4
1
2
4
5
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