Synthesis and DNA cleavage of 2′-O-amino-linked metalloporphyrin-oligonucleotide conjugates

Article abstract of DOI:10.1039/b004431h

A new general method for an easy 2′-O-modification of nucleosides is reported. We describe the preparation of modified 19-mer oligonucleotides carrying an aminoalkyl linker at the 2′-position of cytidine residues and the covalent attachment of an artifici

Full text of DOI:10.1039/b004431h

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Synthesis and DNA cleavage of 2Ј-O-amino-linked
metalloporphyrin–oligonucleotide conjugates
Igor Dubey,† Geneviève Pratviel and Bernard Meunier*
1
Laboratoire de Chimie de Coordination du CNRS, 205 route de Narbonne,
31077 Toulouse cedex 4, France
Received (in Cambridge, UK) 2nd June 2000, Accepted 31st July 2000
Published on the Web 31st August 2000
A new general method for an easy 2Ј-O-modification of nucleosides is reported. We describe the preparation
of modified 19-mer oligonucleotides carrying an aminoalkyl linker at the 2Ј-position of cytidine residues and the
covalent attachment of an artificial chemical nuclease, a manganese() tris(N-methylpyridinium-4-yl)porphyrin
moiety, onto the functionalized linker. The positions of attachment of the manganese cationic porphyrin within the
19-mer vector sequence and the length of the tether were selected in order to have the DNA-cleaver entity close to an
(AT)3-sequence in the duplex region formed by the 19-mer oligonucleotide vector and a single-stranded DNA target.
The cleavage pattern of the target DNA by these new metalloporphyrin–oligonucleotide conjugates was compared
with that obtained with the 19-mer oligonucleotide conjugate carrying the metalloporphyrin at the 5Ј-end.
The DNA degradation was mainly due to guanine oxidation in
the single-stranded region of the target DNA, together with
direct breaks due to C-5Ј hydroxylations of the sugar in the
AT-rich regions located in the double-stranded region of the
conjugate/target hybrid.10,11
Introduction
Diaquamanganese()
meso-tetrakis(N-methylpyridinium-4-
yl)porphyrin (Mn-TMPyP) is an efficient chemical nuclease
when associated to KHSO5, an oxygen-atom donor.1 The mech-
anism of DNA cleavage involves the formation of a high-valent
metal-oxo species2–4 able to hydroxylate CH bonds of deoxy-
ribose units of DNA2–5 or to abstract electrons from the guan-
ine bases.6,7 The reactivity of oxo-Mn-TMPyP (sugar versus
guanine oxidation) depends on the sequence of the DNA
target. Mn-TMPyP has a high affinity for the minor groove of
AT-rich regions of double-stranded B-DNA. Within that site,
the oxidized form of Mn-TMPyP, namely oxo-Mn-TMPyP,
mediates direct DNA breaks which are located on the 3Ј-side
of each sequence consisting of three consecutive AT base
pairs or (AT)3-site.5 These direct single-strand breaks are due
to C-5Ј hydroxylation of the deoxyribose moiety of the
adjacent nucleoside on the 3Ј-side of the (AT)3-site. The
resulting products are fragments of DNA ending with a 3Ј-
phosphate and a 5Ј-aldehyde respectively. Further reduction
of the 5Ј-aldehyde into a 5Ј-OH function by NaBH4 trans-
formed the products of the oxidative cleavage into products
of DNA hydrolysis.8 Thus the interaction of Mn-TMPyP
with its high (AT)3-affinity site can afford a way to mediate
the ‘pseudo-hydrolysis’ of the adjacent phosphodiester bond.
When no (AT)3-site is available on the target B-DNA, as well
as in single-stranded DNA, the reactivity of the activated
metalloporphyrin is shifted towards the oxidation of the
guanines.6,7
In order to target this metalloporphyrin-based DNA-
cleaving agent onto a specific region of DNA, an Mn-tris(methyl-
pyridiniumyl) motif (Fig. 1) was covalently attached to an
oligonucleotide (ODN) as a vector. The first generation of
metalloporphyrin–ODN conjugates consisted of compounds
with the metalloporphyrin moiety attached at the 5Ј-end of the
ODN vector.9–13 When these manganese cationic porphyrin–
ODN conjugates were annealed with a long single-stranded
DNA, the single-stranded target was damaged over several
bases in the vicinity of the metalloporphyrin location, i.e. at the
junction between the single- and the double-stranded DNA.
A large number of redox-active metallocomplexes, including
Fe-EDTA, Fe-porphyrins, Cu-phenanthroline, Fe-bleomycin,
Mn-porphyrins, metal-chelating peptides, etc. have been
attached to oligonucleotides to produce sequence-specific
chemical nucleases.14,15 Among them, the cationic manganese
tris(methylpyridiniumyl) motif is one of the most efficient arti-
ficial nucleases when attached at the 5Ј-end of an ODN.9–13 This
efficiency was rationalized in terms of closer interaction
between the cationic nuclease and DNA.13 However, in all these
different cases, the ODN vector allowed the targeting of a
selected sequence within large single- or double-stranded DNA
targets but the cleavage pattern of these redox metal-based
nuclease–ODN conjugates usually diffused over several nucleo-
tide residues.16–19 This is due to the fact that these chemical
nucleases attached onto the ODN vector did not possess any
special affinity or any strong specific binding site within the
target DNA. We addressed the question whether it would be
possible to build a metalloporphyrin–ODN conjugate which
would allow us to take advantage of the proper high affinity
of the Mn-tris(methylpyridiniumyl) motif for an (AT)3-site. A
metalloporphyrin–ODN conjugate capable of directing the
metalloporphyrin to a single (AT)3-site within the target DNA
sequence and capable of promoting ‘pseudo-hydrolysis’ within
this (AT)3-site would represent a truly artificial restriction
nuclease. Such a conjugate would promote the cleavage of a
single phosphodiester bond on a strand of DNA and the result-
ing DNA fragments could be processed further. To favor the
fitting of the metalloporphyrin with its favorite (AT)3-binding
site, the metalloporphyrin residue must be directed towards the
minor groove of the duplex formed by the vector and its com-
plementary target sequence. This could be done via an attach-
ment to an oligonucleotide at a position oriented into the minor
groove, i.e. at one 2Ј-position of sugar residue. By considering
the structure of B-DNA, the functionalization of oligonucleo-
tides by incorporating 2Ј-modified nucleosides seems to be the
most convenient approach to target a DNA cleaver within the
minor groove. Furthermore, as the 2Ј-position of deoxyribose
in the duplex is just in the middle of the minor groove, the
† Present address: Institute of Molecular Biology and Genetics,
National Academy of Sciences, Kiev, Ukraine.
3088
J. Chem. Soc., Perkin Trans. 1, 2000, 3088–3095
This journal is © The Royal Society of Chemistry 2000
DOI: 10.1039/b004431h

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Fig. 1 Structures of the metalloporphyrin–oligonucleotide conjugates. The metalloporphyrin has two water molecules as axial ligands and thus an
additional positive charge.
2Ј-linker groups should not sterically disturb formation of
the duplex.
mercaptan in the presence of NaH, but the yield was only
27%.29 Adenosine was also alkylated to prepare a series of
common 2Ј-O-alkyl nucleoside derivatives for the incorporation
into oligonucleotides.30 However, selective alkylation reactions
were only reported for non-protected adenosine. A more
general way for the incorporation of a mercaptoalkyl linker
into 2Ј-position of nucleosides was proposed by Douglas
et al.31 The multi-step synthesis was based on the alkylation of
3Ј,5Ј-O-silyl-protected nucleosides with ethyl bromoacetate in
the presence of strong base with subsequent conversion of the
2Ј-O-substituent into a 2Ј-O-mercaptoethyl group. Neverthe-
less, O-alkylation of nucleosides is not a general and convenient
approach, since a strong base like NaH must be used in the
reaction, and is hardly compatible with several protecting
groups.
In this report we propose a new general method for an easy
2Ј-O-modification of nucleosides. We describe the preparation
of modified 19-mer-ODNs carrying an aminoalkyl linker at the
2Ј-position of cytidine residues and the covalent attachment of
a metalloporphyrin moiety onto this functionalized linker. The
site of conjugation of the manganese cationic porphyrin within
the 19-mer vector sequence was selected taking into account the
linker length so that the tethered nuclease residue would be
close to an (AT)3-site in the duplex region formed by the 19-
mer-ODN vector and a single-stranded target. The cleavage of
the target DNA by these new metalloporphyrin–ODN conju-
gates was compared with that of the 19-mer-ODN conjugate
carrying the metalloporphyrin at the 5Ј-end.
There are many reports on the preparation of 2Ј-O-modified
nucleosides and oligonucleotides. The 2Ј-O-alkyl (especially
methyl, as well as butyl, allyl, etc.) DNA analogs have been
widely used in molecular biology.20–22 Corresponding nucleo-
sides are usually synthesized by alkylation of the 2Ј-hydroxy
group of 3Ј,5Ј-protected nucleosides in the presence of sodium
hydride. Oligonucleotides carrying at the 2Ј-position an inter-
calator group like anthraquinone,23,24 anthracene25 or pyrene26
were obtained using 2Ј-O-modified nucleotide phosphor-
amidites. These latter derivatives were synthesized via alkyl-
ation of the 2Ј-hydroxy group of suitably protected nucleosides
with the 2-(bromomethyl)anthraquinone, 2-(chloromethyl)-
anthracene or 1-(chloromethyl)pyrene, respectively. As these
alkylation reagents were benzyl halides, the alkylation reaction
proceeded rather easily. Oligonucleotides having a dansyl fluor-
escence label at the 2Ј position have been also synthesized. The
key step in the preparation of the 2Ј-modified phosphoramidite
was the reaction of 2Ј-amino-2Ј-deoxyuridine with dansyl
chloride.27 There are, however, only a few examples of a general
method for introducing into oligonucleotides a 2Ј-linker suit-
able for the conjugation of a variety of different compounds.
Manoharan et al. reported the synthesis of a 2Ј-O-(amino-
alkyl)adenosine phosphoramidite. The synthetic method was
based on the preferential 2Ј-O-alkylation (<5% of 3Ј-isomer) of
non-protected adenosine with an alkyl halide in the presence
of sodium hydride. Insertion of the corresponding phosphor-
amidite into oligonucleotides with subsequent deprotection
yielded 2Ј-aminoalkylated oligomers which were then modified
with fluorescein and other reporter groups.28 A 2Ј-thiol tether
was incorporated into oligonucleotides in a similar way, by
2Ј-O-alkylation of adenosine with S-protected 6-bromohexyl
Results and discussion
We assayed the DNA-cleavage activity of the metalloporphyrin–
19-mer-oligonucleotide conjugates onto the single-stranded 35-
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pared by monoacylation of hexamethylenediamine with ethyl
trifluoroacetate. The nucleoside 3 bearing N-protected amino-
alkyl linker was purified with the same gradient. Another
possible way to transform 2 into 3, by treating the imidazolide
first with the diamine followed by protection of the end amino
group with ethyl trifluoroacetate, was found to be less efficient,
because of the formation of nucleoside dimer at the first step.
The NMR spectrum of 3 clearly showed the disappearance of
imidazole protons and the presence of NH and methylene
group signals. The silyl protecting group was then removed by
rapid treatment with tetrabutylammonium fluoride (TBAF) in
tetrahydrofuran (THF), and 2Ј-O-modified cytidine derivative
4 was recrystallized from aq. ethanol. This nucleoside was
5Ј-O-tritylated by the standard method to give the protected
nucleoside 5. The latter was phosphitylated in dichloromethane.
This reaction was relatively slow, because of the presence of the
2Ј-substituent, and was achieved in 1.5 h. Two diastereomers
of the resulting phosphoramidite 6 were formed (total yield
74%) which could be separated by silica gel chromatography.
The ‘fast’ isomer was eluted with CHCl3–EtOAc–triethylamine
(TEA) 7:2:1, whereas the ‘slow’ diastereomer was eluted
with the same eluting system but in the proportions 45:45:10.
Purity of diastereomers was >90% as was shown by 31P NMR.
1
The structure of all reaction products was confirmed by H
NMR spectroscopy and FAB-MS. The overall yield from
cytidine was 22%. Although in the present work we have
prepared only the 2Ј-modified cytidine reagent, this method
is quite general and can be extended to other nucleosides.
Fig. 2 Structure of the duplex formed by hybridization of the metallo-
porphyrin–19-mer ODN conjugates with the 35-mer single-stranded
DNA target. The two (AT)3-sites are shown by boxes. Mn-Por stands
for the covalent attachment of a metalloporphyrin moiety. Each 19-mer
vector is linked to one metalloporphyrin unit, but the four different
possibilities of linkage are presented in this Figure. The arrows show
the sites of cleavage of the 35-mer ODN by C-5Ј hydroxylation of
sugar. The 35-mer ODN was 5Ј-end-labelled.
Synthesis of manganese(III) tris(N-methylpyridinium-4-yl)-
porphyrin–oligonucleotide conjugates
Cytidine carrying a 2Ј-O-aminoalkyl linker (6, Scheme 1) was
then introduced in the sequence of the 19-mer ODN vector by
phosphoramidite automated solid-phase synthesis. One modi-
fied cytidine residue was introduced in the sequence where it
replaced a standard cytidine at position C5, C6 or C11 (see
sequence of the 19-mer in Fig. 2). The coupling time for amidite
reagent 6 (0.1 M in acetonitrile) was increased to 2 min. The
coupling yield of 2Ј-modified amidite at C5, C6 and C11 posi-
tions was 95% when estimated by the trityl method (as in ref.
28) but appeared much lower, 65%, when calculated from the
HPLC analysis of the crude product of the ODN synthesis. The
lower yield of the coupling of the cytidine monomer carrying a
2Ј-O-aminoalkyl linker compared with that of an unmodified
cytidine amidite was probably due to the steric effect of the 2Ј-
O-alkyl substituent. The major impurity was an oligonucleotide
corresponding to a stop at the nucleotide before the 2Ј-modified
amidite (14-mer, 13-mer and 8-mer for the C5-, C6- and C11-
modified ODNs at retention times 33, 34 and 17 min, respect-
ively, compared with 39 min for the full-length ODN). The
trifluoroacetyl-blocked amine linker arm was fully deprotected
during the ammonia cleavage and deblocking steps of oligo-
nucleotide synthesis. The coupling of the metalloporphyrin
moiety was performed as previously described11,32 by formation
of an amide bond between the activated ester of the metallo-
porphyrin and the alkylamine function of the various 2Ј-O-
aminoalkyl ODNs. The Mn–C5, Mn–C6 and Mn–C11 19-mer
conjugates were obtained in very good yield (70%), with respect
to the starting ODN, after HPLC purification on an anion-
exchange column. The conjugated molecule with the metallo-
porphyrin at the 5Ј-end of the ODN sequence (Mn–5Ј 19-mer)
was included for comparison. The structures of the metallo-
porphyrin–ODN conjugates are shown in Fig. 1 and Fig. 2.
mer-ODN, 5Ј-CAGAGGAGAGCAAGAAATGGAGCCAG-
TAGATCCTA-3Ј which is complementary to the 19-mer vector
sequence from the G6 to the C24 positions. The numbering of
the nucleosides on ODNs starts at the 5Ј-side of the sequence.
The oligonucleotide sequence of the 19-mer-ODN was thus
5Ј-GGCTCCATTTCTTGCTCTC-3Ј. The 2Ј-O-modified
cytidines were positioned either at the C5, C6 or the C11 posi-
tions of the 19-mer-ODN. The resulting metalloporphyrin–
ODN conjugates were C5–Mn 19-mer, C6–Mn 19-mer or C11–
Mn 19-mer, respectively (Figs. 1 and 2). These new metallo-
porphyrin-ODN conjugates were compared to the conjugate
carrying the metalloporphyrin at the 5Ј-end of 19-mer
ODN (Mn- 5Ј 19-mer) (see also Figs. 1 and 2 for structure). The
metalloporphyrin residue linked to the 2Ј-O-position of C5 or
C6 in 19-mer-vector is forced to sit in the minor groove in the
direction of the (AT)3-site of the vector–target duplex. The
metalloporphyrin tethered to the C-11 residue is oriented
within the minor groove in the opposite direction with respect
to the (AT)3-sites.
Synthesis of modified phosphoramidite precursor
The synthesis of a cytidine phosphoramidite modified with 2Ј-
aminoalkyl linker group (reagent 6) is presented in Scheme 1.
The 3Ј,5Ј-tetraisopropyldisiloxane (TPDS)-protected N-benz-
oylcytidine 1 was first treated with 1,1Ј-carbonyldiimidazole
(CDI), affording nucleoside imidazolide 2. It should be noted
that starting nucleoside and product 2 have close Rf-values (0.81
and 0.77 in chloroform–methanol 9:1, respectively), and there-
fore the control of reaction by TLC was rather difficult; this
was also the case for the transformation of 2 into 3. Imidazolide
2 was purified by silica gel chromatography with a gradient
of methanol (0–2.5%) in dichloromethane. Imidazolide 2 was
treated with N-(trifluoroacetyl)hexamethylenediamine, pre-
Cleavage of the 35-mer single-stranded DNA by the Mn–5Ј 19-
mer conjugate compared with that by the intra-strand, Mn–C5,
Mn–C6 and Mn–C11 19-mer conjugates
Comparative DNA-cleavage experiments were performed with
the 5Ј-[32P]labelled 35-mer-ODN at a final concentration of
3090
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Scheme 1 Synthetic scheme for the preparation of 2Ј-O-modified phosphoramidite 6. Reagents, conditions and yields: (i) CDI (1.5 eq.), CH2Cl2, 2 h
(80%); (ii) H2N(CH2)6NHCOCF3 (2 eq.), CH2Cl2, 1.5 h (83%); (iii) TBAF (2.1 eq. in THF), 20 min (84%); (iv) DMTrCl (1.25 eq.), Py, 4 h (84%); (v)
Pri2NP(Cl)OCH2CH2CN (2 eq.), DIPEA (4 eq.), CH2Cl2, 1.5 h (74%).
100 nM which was incubated with 100 nM of metallo-
porphyrin–oligonucleotide conjugates with an excess of random
double-stranded herring testes DNA (0.2 mM base pairs) in
50 mM phosphate buffer (pH 7) and 100 mM NaCl. The results
are reported in Fig. 3. The cleavage reaction was initiated by
the addition of KHSO5. After 1 h at 0 ЊC the reaction was
stopped by the addition of an excess of HEPES‡ buffer which
reacts with KHSO5. The final concentration of KHSO5 was
0.1 mM (Fig. 3, lanes 1–8) or 0.01 mM (Fig. 3, lanes 9–16).
Direct DNA damage is illustrated through lanes 1–4 and lanes
9–12, with piperidine-labile breaks (shown as pip in Fig. 3) in
lanes 5–8 and lanes 13–16 for 0.1 and 0.01 mM KHSO5 concen-
trations, respectively. Each set of four lanes corresponded to the
comparative analysis of the four different metalloporphyrin–
ODN conjugates. The assays with the Mn–5Ј, Mn–C5, Mn–C6
and Mn–C11 19-mer conjugates were loaded from left to
right. The DNA fragments were identified according to the
Maxam–Gilbert sequencing reaction (not shown). All these
bands corresponded to the migration of 3Ј-phosphate-ending
fragments.
In the presence of 0.1 mM of KHSO5, extensive cleavage of
the 35-mer target DNA by the Mn–5Ј 19-mer conjugate con-
sisted of a smear appearing in the G20–G26 region of the
sequence and two individual bands at G19 and T18 before
piperidine treatment (Fig. 3, lane 1).10,11 The G19 and T18 cleav-
age bands are due to the C-5Ј hydroxylation of the G19 or T18
sugar residues for the metalloporphyrin interacting in the minor
groove of the (A16A17A18)-site and the (A15A16T17)-site respect-
ively. Extensive cleavage mediated by the Mn-5Ј 19-mer conju-
gated molecule could be estimated by the very low intensity of
the full-length material at the top of the gel (Fig. 3, lane 1). The
smear was probably due to an altered mobility of the full-length
DNA strand carrying multiple oxidative damage (several oxid-
ized G’s on the same strand under drastic oxidative conditions
and probable partial lability of these lesions during electro-
phoresis). Upon piperidine treatment, the smear and a part of
the full-length material were transformed into cleavage bands
observed only at G residues (Fig. 3, lanes 5 and 13). At a low
concentration of KHSO5, lane 13 of Fig. 3, G26 was the main
alkali-labile site. At higher concentrations of KHSO5, lane 5,
extensive oxidation of the 35-mer led to cleavage fragments
mainly located at G19 residue. The labelled DNA probably
included also some alkali-labile G-lesions located higher up on
the gel and which were not visualized because only the shortest
fragment with respect to the labelled 5Ј-end was observed in
this case. The metalloporphyrin entity of the Mn–5Ј 19-mer,
attached onto the terminal G of the 19-mer vector, first reacted
with the most accessible guanines in the single-stranded region
of the 19-/35-mer duplex (G22, G26). Upon drastic oxidation
conditions (0.1 mM KHSO5, Fig. 3, lane 5), the activated
metalloporphyrin was able to oxidize guanine bases in the
double-stranded region or could reach also the (AT)3-site where
direct strand breaks were mediated by hydroxylation at C-5Ј
of the deoxyribose on the G19 and T18 residues. The reaction of
the oxo-metalloporphyrin within the (AT)3-sites occurred only
after a significant degradation of the guanine bases, namely
at a late stage of the reaction. This may be due to a partial
dehybridization of the 19-/35-mer duplex in the region where
guanines have been oxidized. The ratio of the reactivity of
the oxo-metalloporphyrin towards G residues compared with
one of the (AT)3-sites was highly in favor of the oxidation at
G bases.
In order to optimize the metalloporphyrin interaction in the
minor groove of the (AT)3-sites and to lower the G-oxidation,
the metalloporphyrin entity was then positioned internally
within the 19-mer sequence by its covalent attachment onto
oligonucleotides containing a 2Ј-O-modified cytidine. The Mn–
C5 19-mer conjugate mediated direct cleavage at G19 and T18
(Fig. 3, lane 2) and alkali-labile lesion at the G19 residue (Fig. 3,
lane 6). The difference in intensity of the G19 band in lane 6
‡ HEPES, 2-[NЈ-(2-hydroxyethyl)piperazin-N-yl]ethanesulfonic acid.
J. Chem. Soc., Perkin Trans. 1, 2000, 3088–3095
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stranded region of the target DNA, but probably only with G19.
The overall efficiency of the Mn–C5 19-mer was lower than that
of the terminal metalloporphyrin, as can be seen by com-
parison of the level of intact full-length material at the top of
lanes 13 and 14 of Fig. 3 for the Mn-5Ј and the Mn–C5 19-mer
conjugates, respectively.
The Mn–C6 19-mer conjugate did not produce any smear
before piperidine treatment and mediated direct breaks located
downward on the gel, at T18 [(A15A16A17)-site] and A17
[(G14A15A16)-site] (Fig. 3, lane 3) at an even earlier stage of
the reaction than with the C5–Mn 19-mer ODN conjugate
(compare the intensity of the full length DNA in lanes 2 and 3
or 6 and 7 in Fig. 3). The Mn–C6 19-mer conjugate, as well as
the Mn–C5 19-mer one, showed that the metalloporphyrin
reacted within the minor groove of the duplex DNA as a
primary event of DNA degradation. No direct cleavage at the
G19 residue was observed. The G19 band was only revealed as an
alkali-labile site, indicating a guanine-oxidation mechanism
(Fig. 3, lanes 3 and 7). In the case of the Mn–C6 19-mer conju-
gate, no cleavage was observed in the single-stranded region
(Fig. 3, lane 7). The metalloporphyrin now faced the G19 resi-
due on the 35-mer sequence and was able to mediate oxidation
of this guanine (whereas the previous Mn–C5 19-mer conjugate
did not oxidize the complementary G20 base). The efficiency of
the Mn–C6 19-mer was much lower than the two previously
described conjugates. At 0.1 mM of KHSO5 only 50% of
the full length DNA was damaged after piperidine treatment
(Fig. 3, lane 7) compared with a total degradation observed in
lanes 5 and 6 of Fig. 3.
The Mn–C11 19-mer conjugate did not find any (AT)3-site to
interact with since it was directed to the other side with respect
to the AT-rich region of the duplex. The metalloporphyrin was
facing the G14 residue on the target DNA. This compound
allowed us to evaluate the reactivity of an internally located
metalloporphyrin onto the guanine that is directly comple-
mentary to the modified cytidine residue. As shown in Fig. 3,
lanes 4, 8 and lanes 12, 16, a weak oxidation of the G14 residue
was evidenced only at high concentration of KHSO5 (less than
10% of cleavage).
Concerning the direct cleavage within (AT)3-sites by the met-
alloporphyrin conjugates tethered by 2Ј-linkers at different sites
within the sequence of the 19-mer vector, the results can be
summarized as follows. When the metalloporphyrin is attached
in front of G20 (Mn–C5 19-mer) direct damage at G19, (A16-
A17T18)-site, and T18, (A15A16A17)-site, was observed, whereas
when the metalloporphyrin was attached in front of G19 (Mn–
C6 19-mer) direct damage at T18, (A15A16A17)-site, and A17,
(G14A15A16)-secondary site, was observed, no interaction with
the upper box [G19 band for interaction within the (A15A16A17)-
site] being possible. Thus the oxo-metalloporphyrin oxidized a
CH-5Ј bond of the nucleotide adjacent to the complementary
base in the 5Ј-direction on the target DNA. The metallo-
porphyrin clearly interacted with the (AT)3-site that is separated
by one nucleotide unit in the direction on the 5Ј-end of the
target sequence and this reactivity appeared as a primary event
of DNA damage. However, the intra-strand tethered metallo-
porphyrins did not reach the high reactivity of the free metallo-
porphyrin, Mn-TMPyP, for the (AT)3-sites.5,8 With the 2Ј-linked
metalloporphyrin conjugates, it has been possible to favor the
approach of the metalloporphyrin entity to an (AT)3-site, but
without being able to tune the exact binding mode in order to
hydroxylate one of the CH bonds at C-5Ј. These hydroxylation
reactions are O-atom-transfer reactions and the high-valent
metal-oxo species should approach very close to the H-atom
that will be abstracted. Otherwise the oxo-manganese por-
phyrin behaves like a strong two-electron oxidant and abstracts
electrons from the nearest guanine residues. This balance
between CH-hydroxylation versus guanine oxidation is also
dependent on the strength of the binding affinity of the cleaver
within double-stranded DNA.
Fig. 3 Polyacrylamide gel analysis of the oxidative damage of the 5Ј-
[32P]labelled 35-mer ODN by the Mn–5Ј, Mn–C5, Mn–C6 and Mn–C11
19-mer ODN conjugates in the presence of 0.1 (lanes 1 to 8) or 0.01 mM
(lanes 9 to 16) of KHSO5. The cleavage of the 35-mer target was
monitored before (lanes 1 to 4 and 9 to 12) or after (lanes 5 to 8 and 13
to 16) piperidine treatment. Each set of four lanes corresponded to the
comparative analysis of the four metalloporphyrin–ODN conjugates.
The Mn–5Ј, Mn–C5, Mn–C6 and Mn–C11 19-mer ODN conjugates
assays were loaded from left to right. The bands of DNA fragments
were identified according to the Maxam–Gilbert sequencing reaction.
See Experimental for further details.
compared with that of lane 2 was indicative of the guanine
oxidation mechanism versus the C-5Ј-hydroxylation mechanism
on that residue. The metalloporphyrin covalently bound at C5
of the 19-mer vector is now facing the G20 residue of the 35-mer
target DNA (Fig. 2). One can note that the direct cleavage of
the metalloporphyrin within the (A16A17T18)-site (G19, direct
cleavage) and the (A15A16A17)-site (T18, direct cleavage)
occurred at an earlier stage of DNA degradation indicating
thus a better interaction of the C5–Mn 19-mer metallo-
porphyrin moiety within the minor groove. After piperidine
treatment (Fig. 3, lanes 5 and 6) these two conjugates showed
the same pattern of cleavage but the internal metalloporphyrin
(Mn–C5 19-mer) probably mediated a more specific cleavage
since comparison of lanes 13 and 14 of Fig. 3 clearly indicated
that the intra-strand porphyrin did not react so well, at lower
concentrations of KHSO5, with the guanines of the single-
3092
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The reactivity towards guanine bases was found to account
for the major mechanism of oxidative damage of the target
DNA for the four tested metalloporphyrin–ODN conjugates.
The guanines in a single-stranded region or belonging to a GG
sequence in the double-stranded region of the vector/target
hybrid were particularly reactive. The sensitivity of the
guanines located in single-stranded regions was illustrated by
the reaction of Mn–5Ј 19-mer ODN conjugate at low concen-
trations of KHSO5 (Fig. 3 lane 13), while the three other com-
pounds were almost inactive under the same conditions. The
easy GG oxidation was evidenced by the high efficiency of the
Mn–C5 19-mer conjugate (lane 6, Fig. 3). The accessibility of
the G bases in the single-stranded region was probably respon-
sible for the high efficiency of DNA degradation of 5Ј-end-
modified conjugates (lane 13 of Fig. 3), and the low oxidation
potential of a 5Ј-G of a GG sequence33 explains the higher
efficiency of the Mn–C5 compared with the Mn–C6 or Mn–
C11 19-mer conjugates. Concerning the intra-strand-located
metalloporphyrins (Mn–C5, Mn–C6 and Mn–C11 19-mer ODN
conjugates), the Mn–C6 derivative was able to oxidize the com-
plementary paired G19 on the target DNA, while the Mn–C11
derivative reacted with the complementary G14 but to a much
lower extent. The difference in oxidation of these two guanines
may be explained by the higher oxidation potential of the
isolated G14 compared with G19 (G19 is the 5Ј-G of a GG
sequence).33 The Mn–C5 19-mer conjugate did not show any
cleavage band corresponding to oxidation of the comple-
mentary G20 but exhibited the highest reactivity towards the G19
residue (one nucleoside shift in the 5Ј-direction of target). This
result may be explained by a better positioning of the tethered
metalloporphyrin towards a guanine shifted by one nucleotide
unit (in the 5Ј-direction of the target DNA), as was observed in
the case of the reaction within the (AT)3-sites, due to the fact
that the metalloporphyrin was forced to be oriented towards the
5Ј-direction of the target DNA. However, the total absence of
G20 damage in the case of the Mn–C5 19-mer conjugate might
indicate the existence of transfer of oxidative damage from the
G20 to the G19 base.34–36
1H and 31P NMR, respectively. Mass spectrometry analyses
were performed on a Nermag R1010 apparatus [FABϩ/
m-nitrobenzyl alcohol (MNBA) matrix] and a Perkin-Elmer
SCIEX API 365 ESI-MS spectrometer (negative mode). TLC
was performed on Alugram Sil G/UV254 plates (Macherey-
Nagel) using CHCl3–MeOH 9:1 (system A) and CHCl3–
EtOAc–TEA 45:45:10 (system B) as developers. The oligo-
nucleotides were synthesized on an Expedite 8900 PerSeptive
Biosystems instrument using commercially available reagents.
Oligonucleotides were analyzed by HPLC using an anion-
exchange column (Protein Pak DEAE 8 HR from Waters, 8
cm × 1 cm) eluted in a gradient mode (solvent A, 25 mM Tris/
HCl, pH 8.5; solvent B, A ϩ 1 M NaCl) from 20 to 40% of
solvent B in 1 h, flow rate 0.7 ml minϪ1. Detection was at 260
and/or 468 nm (diode array detector from Kontron).
Synthesis of modified phosphoramidite precursor 6
4-N-Benzoyl-3Ј,5Ј-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-
diyl)cytidine 1. Starting N-benzoylcytidine was prepared by the
reaction of cytidine with benzoic anhydride in boiling ethanol
(yield 91%), as per Watanabe and Fox.37 It was silylated with
1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane in pyridine to give
1 (yield 72%), as described previously.38
4-N-Benzoyl-2Ј-O-(imidazol-1-ylcarbonyl)-3Ј,5Ј-O-(tetraiso-
propyldisiloxane-1,3-diyl)cytidine 2. The 3Ј,5Ј-O-tetraisopropyl-
disiloxanediyl-N-benzoylcytidine 1 (783 mg, 1.33 mmol) was
evaporated twice with anhydrous acetonitrile (2 × 15 ml), and
dissolved in 10 ml of dry dichloromethane. CDI was added (1.5
eq., 2 mmol, 324 mg) and the reaction mixture was stirred at
room temperature for 2 h. Mixture was directly applied to a
silica column and product was isolated (gradient 0–2.5%
MeOH in CH2Cl2). Corresponding fractions were evaporated
1
under vacuum to give a white solid foam (726 mg, 80%); H
NMR (CDCl3) δ 8.84 [br s, 1 H, NH (Cyt)], 8.22 (d, 1 H, H-6,
J6,5 7.5 Hz), 8.16 (s, 1 H, imidazole), 7.89 (d, 2 H, Bz, J 7.1 Hz),
7.50–7.65 [m, 4 H, 3 H (Bz) ϩ H-5], 7.44 (s, 1 H, imidazole),
7.09 (s, 1 H, imidazole), 6.06 (s, 1 H, H-1Ј), 5.63 (d, 1 H, H-2Ј,
J 4.6 Hz), 4.48 (m, 1 H, H-3Ј), 4.25–4.40 (m, 1 H, H-4Ј), 3.95–
4.15 (m, 2 H, H2-5Ј), 0.85–1.15 (m, 28 H, 4 × Pri); FAB-MS m/z
684 [(M ϩ Hϩ)], 572 [(M Ϫ OCOIm)ϩ], 469 [(M Ϫ B)ϩ], 357
[(M Ϫ B Ϫ OCOIm)ϩ]; Rf 0.77 (system A).
Conclusions
The currently reported synthesis of a 2Ј-amino-linked cytidine
allows site-selective positioning of a DNA cleaver and can be
extended to the 2Ј-labelling of oligonucleotides with various
molecules. The length and the chemical structure of linker can
be selected to optimize interaction with the minor groove. The
illustration of the variation in cleavage of DNA by different
metalloporphyrin–oligonucleotide conjugates showed that the
efficiency of the degradation of the target DNA, due to the
binding of the 2Ј-linked-metalloporphyrin within the minor
groove of (AT)3-sites, was not significantly increased to favor
the C-5Ј-hydroxylation of sugar units by the oxo-metallo-
porphyrin and to completely avoid neighbouring G-oxidation.
4-N-Benzoyl-3Ј,5Ј-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-
diyl)-2Ј-O-{[6-(trifluoroacetamido)hexyl]carbamoyl}cytidine 3.
Imidazolide 2 (720 mg, 1.05 mmol) was stirred in 40 ml of
dichloromethane with 445 mg of N-trifluoroacetyl-1,6-diamino-
hexane (2.1 mmol, 2 eq.) for 1.5 h. The latter was prepared by
monoacylation of hexamethylenediamine with ethyl trifluoro-
acetate.39 The reaction mixture was applied to a silica column
and product was isolated using a gradient of methanol (0–3%)
in dichloromethane. Fractions containing product were evapor-
1
ated in vacuo to give a white solid foam (721 mg, 83%); H
NMR (DMSO-d6) δ 11.47 [s, 1 H, NH (Cyt)], 9.52 (t, 1 H,
NHCOCF3, J 5.3 Hz), 8.27 (d, 1 H, H-6, J6,5 7.5 Hz), 8.12
(d, 2 H, Bz, J 7.1 Hz), 7.50–7.80 [m, 5 H, 3 H (Bz) ϩ
H-5 ϩ NHCOO], 5.88 (s, 1 H, H-1Ј), 5.44 (d, 1 H, H-2Ј, J 5.1
Hz), 4.50–4.55 (m, 1 H, H-3Ј), 4.30–4.40 (m, 1 H, H-4Ј), 4.05–
4.15 (m, 2 H, H2-5Ј), 3.25–3.35 (m, 2 H, CH2N), 2.95–3.20 (m,
2 H, CH2N), 1.50–1.70 (m, 4 H, 2 × CH2 internal), 1.30–1.50
(m, 4 H, 2 × CH2 internal), 1.05–1.25 (m, 28 H, 4 × Pri); FAB-
MS m/z 850 [(M ϩ Na)ϩ], 828 [(M ϩ H)ϩ], 613 [(M Ϫ B)ϩ],
357 [(M Ϫ B Ϫ OCONHalkyl)ϩ]; Rf 0.75 (system A).
Experimental
General methods
Cytidine, 1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane, CDI,
1,6-diaminohexane, TBAF, 4,4Ј-dimethoxytrityl chloride
(DMTrCl), diisopropylethylamine (DIPEA), 2-cyanoethyl
N,N-diisopropylchlorophosphoramidite,
oxytris(dimethylamino)phosphonium
(BOP) and 1-hydroxybenzotriazole (HOBT) were purchased
from Aldrich, ethyl trifluoroacetate from Lancaster. Anhydrous
solvents were from Merck. Other commercially available
reagents and all other solvents were purchased from standard
chemical suppliers and used without further purification.
NMR spectra were recorded on a Bruker AM 250 spec-
trometer at 250 MHz for 1H NMR or 101 MHz for 31P NMR.
External standards were tetramethylsilane and 85% H3PO4 for
benzotriazol-1-yl-
hexafluorophosphate
4-N-Benzoyl-2Ј-O-{[(6-trifluoroacetamido)hexyl]carbamoyl}-
cytidine 4. The 3Ј,5Ј-O-silyl-protected nucleoside derivative 3
(715 mg, 0.86 mmol) was dissolved in 5 ml of THF. A solution
of TBAF (2.1 eq., 1.81 mmol, 474 mg) in 2 ml of THF was
added and the slightly yellow solution was kept at room
J. Chem. Soc., Perkin Trans. 1, 2000, 3088–3095
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temperature for 20 min. The product was precipitated by the
addition of 50 ml of water. Precipitate was filtered off, and
washed with water several times. Product was recrystallized
refrigerator under nitrogen. Separation of diastereomers of
phosphoramidite 6 by silica gel chromatography was possible;
a ‘fast’ isomer was eluted with CHCl3–EtOAc–TEA 7:2:1,
whereas a ‘slow’ diastereomer eluted with the same system
in the proportions 45:45:10. Purity of individual isomers was
>90%, as was shown by 31P NMR. (a) ‘Fast’ isomer: Rf
1
from aq. ethanol to give 422 mg of a white powder (84%); H
NMR (DMSO-d6) δ 11.42 [s, 1 H, NH (Cyt)], 9.51 (br t, 1 H,
NHCOCF3, J 5.0 Hz), 8.57 (d, 1 H, H-6, J6,5 7.5 Hz), 8.13 (d,
2 H, Bz, J 7.2 Hz), 7.60–7.85 (m, 3 H, Bz), 7.49 (d, 1 H, H-5),
7.43 (t, 1 H, NHCOO, J 5.6 Hz), 6.11 (d, 1 H, H-1Ј, J1Ј,2Ј 4.0
Hz), 5.58 (d, 1 H, 3Ј-OH, J 5.3 Hz), 5.37 (m, 1 H, H-2Ј), 5.20 (t,
1 H, 5Ј-OH, J 4.6 Hz), 4.33 (m, 1 H, H-3Ј), 4.00–4.10 (m, 1 H,
H-4Ј), 3.70–3.95 (m, 2 H, H2-5Ј), 3.27 (m, 2 H, CH2N), 3.07 (m,
2 H, CH2N), 1.50–1.70 (m, 4 H, 2 × CH2 internal), 1.30–1.45
(m, 4 H, 2 × CH2 internal); FAB-MS m/z 608 [(M ϩ Na)ϩ], 586
[(M ϩ H)ϩ], 371 [(M Ϫ B)ϩ], 330 [(M Ϫ OCONHalkyl)ϩ], 216
(BH2ϩ); Rf 0.34 (system A); mp 209–211 ЊC (Calc. for C25H30-
F3N5O8ؒH2O: C, 49.75; H, 5.35; N, 11.60. Found: C, 50.00; H,
5.02; N, 11.50%).
1
0.68 (system B); H NMR (DMSO-d6) δ 6.10 (d, 1 H, H-1Ј,
J 4.1 Hz), 5.48 (m, 1 H, H-3Ј); 31P NMR (DMSO-d6) δ 150.2.
1
(b) ‘Slow’ isomer: Rf 0.53 (system B); H NMR (DMSO-d6)
δ 6.14 (d, 1 H, H-1Ј, J 4.6 Hz), 5.51 (m, 1 H, H-3Ј); 31P
NMR (DMSO-d6)
δ 149.6; FAB-MS of both isomers,
m/z 1110 [(M ϩ Na)ϩ], 1088 [(M ϩ H)ϩ], 987 [(M Ϫ Pri2N)ϩ],
683 [(M Ϫ Pri2N Ϫ DMTr)ϩ], 566 {[(M Ϫ Pri2NP(OH)OCH2-
CH2CN Ϫ DMTr]ϩ}.
Oligonucleotides
The oligonucleotides (19- and 35-mer ODNs) were synthesized
by solid-phase β-cyanoethyl phosphoramidite chemistry. The
35-mer ODN was purified by 20% polyacrylamide gel electro-
phoresis and desalted on a Sep-pak cartridge from Waters. The
19-mer ODNs containing a modified cytidine with an amino-
alkyl linker at the C5, C6 or C11 position were obtained by direct
incorporation of one cytidine amidite precursor 6 in the
sequence of ODN. The coupling time for amidite reagent 6 (0.1
M in acetonitrile) was increased to 2 min. The HPLC analysis
retention times of the 2Ј-O-aminoalkyl-modified ODNs were
38, 39 and 39 min for the C5-, C6- and C11-modified ODNs,
respectively. Detection was at 260 nm. The 2Ј-O-aminoalkyl-
linker-modified ODNs were purified by HPLC under the same
conditions as described for analysis and were desalted on
Sep-pak cartridges before the coupling reaction with the
metalloporphyrin entity. Concentrations of the ODNs were
deduced from UV-absorbance measurements at 260 nm taking
ε-values from the literature.40
4-N-Benzoyl-5Ј-O-(4,4Ј-dimethoxytrityl)-2Ј-O-{[6-(trifluoro-
acetamido)hexyl]carbamoyl}cytidine 5. The 2Ј-modified nucleo-
side 4 (415 mg, 0.71 mmol) was evaporated with dry pyridine
(2 × 10 ml), dissolved in 10 ml of the same solvent, and
DMTrCl (1.25 eq., 0.88 mmol, 298 mg) was added. After 4 h at
room temperature, the reaction mixture was poured into 5% aq.
NaHCO3 (50 ml), and extracted with dichloromethane (3 × 25
ml). Extract was washed successively with aq. NaHCO3 (25 ml)
and water (25 ml), dried over sodium sulfate, and evaporated in
vacuo. Excess of pyridine was removed by coevaporation with
toluene (2 × 10 ml). Product was isolated by silica gel column
chromatography using a gradient of methanol (0–4%) in
dichloromethane. Fractions containing tritylated product were
evaporated in vacuo to give a slightly yellowish solid foam (528
mg, 84%); 1H NMR (DMSO-d6) δ 11.44 [s, 1 H, NH (Cyt)], 9.52
(br t, 1 H, NHCOCF3, J 5.2 Hz), 8.43 (d, 1 H, H-6, J6,5 7.5 Hz),
8.12 (d, 2 H, Bz, J 7.1 Hz), 7.0–7.8 [m, 18 H, 3 H (Bz) ϩ
H-5 ϩ NHCOO ϩ 13 H (DMTr)], 6.06 (d, 1 H, H-1Ј, J 2.6 Hz),
5.68 (d, 1 H, 3Ј-OH, J 5.7 Hz), 5.30 (m, 1 H, H-2Ј), 4.55 (m,
1 H, H-3Ј), 4.15 (m, 1 H, H-4Ј), 3.87 (s, 6 H, 2 × OCH3), 3.4–3.6
(H-5Ј, overlapping with water signal), 3.27 (m, 2 H, CH2N),
3.09 (m, 2 H, CH2N), 1.50–1.70 (m, 4 H, 2 × CH2 internal),
1.30–1.45 (m, 4 H, 2 × CH2 internal); FAB-MS m/z 910 [(M ϩ
Na)ϩ], 888 [(M ϩ H)ϩ], 584 [(M Ϫ B)ϩ], 330 [(M Ϫ DMTr)ϩ],
303 (DMTrϩ); Rf 0.69 (system A).
Synthesis of manganese(III) tris(N-methylpyridinium-4-yl)-
porphyrin–oligonucleotide conjugates
The manganese() 5-[(4-(carboxybutoxy)phenyl]-10,15,20-
tris(N-methylpyridinium-4-yl)porphyrin precursor bearing a
carboxylate group was prepared as described.41 The metallo-
porphyrin–ODN conjugate at the 5Ј-end was prepared as
described.11 The coupling reaction between the purified C5, C6
and C11 2Ј-O-aminoalkyl-modified ODNs and the metallo-
porphyrin involved the activation of a carboxylic function of a
metalloporphyrin precursor by BOP and HOBT as a first step.
The coupling reaction was performed according to the method
of Duarte et al.32 and was analyzed by HPLC using an anion-
exchange column as described above. Detection of the products
was at 260 and 468 nm simultaneously. The yield of the coup-
ling reaction was around 90%. The impurities consisted of
either unchanged starting 2Ј-O-aminoalkyl-modified ODN or
one ODN product containing two metalloporphyrin moieties.
The retention times were 27, 28 and 27 min for the C5-,
C6- and C11-modified conjugates, respectively. The conjugates
were purified by HPLC using the same conditions as described
for the analyses. Yields were approximatively 70% with respect
to the starting 2Ј-O-aminoalkyl ODN (after purification and
desalting of the conjugate); λmax (H2O) 262 and 468 nm, calcu-
4-N-Benzoyl-5Ј-O-(4,4Ј-dimethoxytrityl)-2Ј-O-{[6-(trifluoro-
acetamido)hexyl]carbamoyl}cytidin-3Ј-yl 2-cyanoethyl N,N-
diisopropylphosphoramidite 6. The 5Ј-O-tritylated nucleoside 5
(523 mg, 0.59 mmol) was evaporated twice with dry dichloro-
methane and dried under vacuum over P2O5 for 2 h. It was
dissolved in 4 ml of dry CH2Cl2, and 4 eq. of DIPEA were
added (2.36 mmol, 408 µl). The mixture, under N2, was
cooled (ice-bath) before the phosphitylating reagent, 2-cyano-
ethyl N,N-diisopropylchlorophosphoramidite (2 eq., 1.18
mmol, 263 µl), was added dropwise by syringe over a period of
5 min with stirring. The mixture was stirred for 10 min at ice-
bath, then the bath was removed, and stirring was continued at
room temperature. During 1.5 h, 200 µl of methanol was added.
The mixture was diluted 10 min later with 25 ml of dichloro-
methane, washed successively with saturated aq. NaHCO3
(2 × 15 ml) and saturated aq. NaCl (15 ml), dried over Na2SO4,
and evaporated under vacuum. The residue was dissolved in
CHCl3–TEA (90:10) and applied to a silica column equilib-
rated with the same solvent. The column was washed with two
volumes of this eluent, and then product was eluted with
CHCl3–EtOAc–TEA (45:45:10). Corresponding fractions
were evaporated under vacuum. The amidite was precipitated
from chloroform into hexane, filtered, washed with hexane,
and dried under vacuum over phosphorus pentaoxide to give
a white powder (474 mg, 74%). The product was stored in a
lated ε260 = 190 000 MϪ1 cmϪ1 and ε468 = 130 000 MϪ1 cmϪ1
,
observed visible–UV ratio on the diode array detector spectra
of the conjugate HPLC peak was 0.7; ESI-MS (negative mode)
m/z 951.5 [(M Ϫ 7H)7Ϫ], 1110.0 [(M Ϫ 6H)6Ϫ], 1332.0 [(M Ϫ
5H)5Ϫ], 1665.5 [(M Ϫ 4H)4Ϫ], 2221.0 [(M Ϫ 3H)3Ϫ] compared
to calculated 951.25, 1109.95, 1332.15, 1665.43 and 2220.9.
The calculated neutral mass of the three metalloporphyrin–
ODN conjugates was 4155.8 Da. Concentration of metallo-
porphyrin–ODN conjugate solutions was determined by UV
absorbance at 260 nm as for single-stranded ODNs.
3094
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10 B. Mestre, G. Pratviel and B. Meunier, Bioconjugate Chem., 1995, 6,
Cleavage of the 35-mer oligonucleotide
466.
Labelling at the 5Ј-end of the 35-mer ODN was achieved by
treatment with [γ-32P]ATP from Amersham and T4 poly-
nucleotide kinase from Gibco-BRL using the standard
procedure. DNA-cleavage reactions (final reaction volume = 15
µl) were performed with 5Ј-[32P]labelled 35-mer ODN target
(final concentration 100 nM; 20 × 103 cpm) and the correspond-
ing metalloporphyrin–ODN conjugates (final concentration
100 nM) in the presence of an excess of random double-
stranded herring testes DNA (0.4 mM in nucleotides) in 100
mM NaCl, 50 mM phosphate buffer (pH 7). Annealing of the
conjugates with complementary 35-mer was achieved by heat-
ing at 90 ЊC for 1 min followed by slow cooling to 25 ЊC. DNA-
cleavage reactions were initiated by adding 1 µl of a freshly
prepared solution of KHSO5 (final concentration 0.01 or 0.1
mM). The DNA-cleavage reaction lasted 1 h at 0 ЊC and was
stopped by the addition of 1 µl of 1 M HEPES buffer pH 8. The
DNA material was precipitated overnight at Ϫ20 ЊC after addi-
tion of 100 µl of 0.3 M sodium acetate buffer (pH 5.2) contain-
ing 0.1 mg mlϪ1 yeast tRNA and 200 µl of cold ethanol.
After centrifugation (10 min; 4 ЊC; 12 × 103 rpm), the DNA
pellet was washed with cold 90% ethanol, dried under vacuum
(Speedvac), dissolved in 100 µl of 1 M piperidine and incu-
bated at 90 ЊC for 40 min. Samples were then evaporated to
dryness, the DNA pellet was washed/dried twice with water to
remove excess of piperidine, and dissolved in formamide with
marker dyes. DNA fragments were analyzed by electrophoresis
on denaturing (7 M urea) 20% polyacrylamide gel (3 h at
2300 V).
11 B. Mestre, A. Jacobs, G. Pratviel and B. Meunier, Biochemistry,
1996, 35, 9140.
12 P. Bigey, G. Pratviel and B. Meunier, Nucleic Acids Res., 1995, 23,
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Acknowledgements
Igor Dubey is indebted to the Université Paul Sabatier
(Toulouse) for financial support. Dr Henri-Louis Plaisancié
from Isoprim (Toulouse) is acknowledged for the preparation of
2Ј-amino-linked ODNs. We are grateful to Dr Béatrice Mestre
for providing a sample of the metalloporphyrin precursor.
32 V. Duarte, G. Pratviel, B. Meunier, M. Berton, S. Sixou and
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