Synthesis and binding properties of perylene-oligo-2′-deoxyribonucleotide conjugates

Article abstract of DOI:10.1016/S0040-4039(01)02010-X

Perylene has been covalently linked, via polymethylene tethers, to the 5′- and the 3′-ends of an oligopyrimidine sequence. The presence of the polycyclic ligand stabilizes the duplexes and the triplexes formed by the modified oligonucleotides and their single- and double-stranded DNA targets as compared to those formed with the parent unmodified oligonucleotide used as reference. Stabilization of the triplex is at its highest when perylene is linked to the 5′-end of the oligonucleotide via a nine-atom size linker. Stabilization of the duplexes is nearly equivalent whatever the position of the substitution (5′ or 3′) and the linker size used to tether both entities.

Full text of DOI:10.1016/S0040-4039(01)02010-X

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 42 (2001) 9005–9010
Synthesis and binding properties of
perylene-oligo-2%-deoxyribonucleotide conjugates
Ulysse Asseline* and Eric Cheng
Centre de Biophysique Mole´culaire, CNRS UPR 4301, affiliated with the University of Orle´ans and with INSERM,
Rue Charles Sadron, 45071 Orle´ans Cedex 02, France
Received 4 August 2001; accepted 23 October 2001
Abstract—Perylene has been covalently linked, via polymethylene tethers, to the 5%- and the 3%-ends of an oligopyrimidine
sequence. The presence of the polycyclic ligand stabilizes the duplexes and the triplexes formed by the modified oligonucleotides
and their single- and double-stranded DNA targets as compared to those formed with the parent unmodified oligonucleotide used
as reference. Stabilization of the triplex is at its highest when perylene is linked to the 5%-end of the oligonucleotide via a nine-atom
size linker. Stabilization of the duplexes is nearly equivalent whatever the position of the substitution (5% or 3%) and the linker size
used to tether both entities. © 2001 Elsevier Science Ltd. All rights reserved.
1. Introduction
A 3,4,9,10 di-anhydride perylene derivative has been
used to bridge the two pyrimidine strands of DNA in a
pyrimidine–purine–pyrimidine triplex.18,19 The planar
linker involving seven rings provides interactions with
all three base residues. To test the ability of the
perylene derivative involving five fused six-membered
rings to stabilize duplex and triplex structures, we
linked it to the 5%- and the 3%-ends of a pyrimidine
decamer. Linkers involving five or nine atom lengths
have been used to attach the dye to the 5%-end, while a
longer linker involving 11 atoms was used for linking to
the 3%-end of the oligonucleotide. Studies carried out
with oligonucleotide–acridine conjugates showed that
stabilization of triplex structures by a 3%-linked interca-
lator requires the use of a longer linker as compared to
that observed with a 5%-linked one.20 We report here the
synthesis and binding properties of these perylene-2%-
deoxyribonucleotide conjugates.
Due to the ability of oligonucleotides to specifically
regulate gene expression via ‘antisense’ and ‘antigene’
strategies, the development of analogues with improved
properties has been the focus of intense research.1,2 To
be efficient, modified oligonucleotides must efficiently
cross the cell membranes, be resistant to nuclease, and
specifically and strongly hybridize to their targets on
mRNA or double-stranded DNA.
Modifications developed to increase the affinity of
‘antisense’ and ‘antigene’ oligonucleotides for their
targets inside cells include that of oligonucleotides and
the covalent linking of ligands such as DNA minor
groove binders,3–5 polyamines,6,7 peptides,8 and interca-
lating agents.9–17 The double helix intercalating agents
involve molecules possessing a planar aromatic chro-
mophore with dimensions similar to those of a standard
Watson–Crick base pair corresponding to about three
fused six-membered rings. The search for triple helix
intercalating agents is based on the same principle with
the use of molecules corresponding to about four or
five six-membered rings able to interact with adjacent
base-triplets of the triple-stranded structure.
2. Synthesis
The synthesis of modified oligonucleotides was per-
formed as outlined in Schemes 1 and 2. The perylene-
linker derivatives 3–5 were obtained following a
two-step procedure adapted from the literature,21 as
described in Scheme 1. First, the preparation of the
aldehyde derivative 2 was achieved by reaction of the
perylene 1, in 1,2-dichloroethane, with SnCl4 (2 equiv.)
and then with 1,1-dichlorooxymethylether (1.2 equiv.)
followed by hydrolysis.22 Then, the synthesis of the
perylene-linker derivatives 3–5 was performed by reac-
Keywords: oligonucleotide–perylene conjugates; attachment to 5%-end;
attachment to 3%-end; absorption spectroscopy; Tm values.
* Corresponding author. Tel.: +33-2-38-25-55-97; fax: +33-2-38-63-
15-17; e-mail: asseline@cnrs-orleans.fr
0040-4039/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(01)02010-X

9006
U. Asseline, E. Cheng / Tetrahedron Letters 42 (2001) 9005–9010
H
iii
CH2-N-(CH2)8-NH2
i, ii
H
C
iv
O
H
CH2-N-(CH2)n-OH
1
2
4 = n = 2
5 = n = 6
v
vi, vii
viii,ix
H
O-CH2CH2CN
CH2-N-(CH2)nO-p-5'TTTTCTTTTC3'
_
CH2-N -(CH2)nO
H
P
N
8 = n = 2
9 = n = 6
6 = n = 2
7 = n = 6
Scheme 1. (i) CH2Cl2, SnCl4; (ii) 1,1-dichlorooxymethylether, H2O; (iii) H2N-(CH2)8-NH2, toluene, NaBH4; (iv) H2N-(CH2)n-OH
with n=2 or 6, toluene, NaBH4; (v) 2-cyanoethyldiisopropylchlorophosphoramidite, diisopropylethylamine, dichloromethane; (vi)
support bound trityl-off oligonucleotides; (vii) tetrazole; (viii) oxidation step; (ix) conc. NH4OH.
Scheme 2.
CPG-Long chain alkylamine; DMTrCl: 4,4%-dimethoxytritylchloride; (i) acetic anhydride, N-methyl-
imidazole; (iii) H+, conc. NH4OH.

U. Asseline, E. Cheng / Tetrahedron Letters 42 (2001) 9005–9010
9007
tion of the aldehyde 2 with 1,8-diaminooctane, 2-
aminoethanol or 6-aminohexanol (10 equiv.) in the
presence of paratoluene sulfonic acid (0.03 equiv.),
using toluene as a solvent, followed by NaBH4 (2
equiv.) treatment.23 Compounds 4 and 5 were phos-
phitylated by reaction of 2-cyanoethyl-diisopropyl-
chlorophosphoramidite (1 equiv.) in the presence of
diisopropylethylamine (2.5 equiv.) in dichloromethane
to give the phosphoramidites derivatives 6 and 7 with
85% yield after purification on a silica gel column using
ethyl acetate/hexane/NEt3, 50:50:4, v/v/v as eluent
(Rf6=0.74 and Rf4=0.10; Rf7=0.80, and Rf5=0.13) on
analytical TLC silica plates using the same eluent as
above.24,25
The mass values of the modified oligonucleotides 8 and
9 were confirmed by electrospray mass spectrometry
analysis (Table 1). In the case of the oligonucleotide–
perylene conjugate 14, the result of mass analysis indi-
cated a value superior by 42 Da to that expected. This
could be due to the acetylation of the secondary amino
function, present on the linker, during the capping steps
performed along the sequence assembly.28
The UV–vis spectra of the oligonucleotide–perylene
conjugates were recorded between u=220 and 520 nm
in 10 mM phosphate buffer, pH 7, containing 140 mM
KCl and 5 mM MgCl2. The spectra contain two
absorption bands in the visible region between u=350
and 520 nm (where only the perylene absorbs light)
with uvis
:446 and uvis :420 nm with a shoulder at
The modified support 13 was obtained following a
two-step procedure, as described in Scheme 2. First, the
modified support 1026 was reacted with the N4-benzoyl-
5%-O-(4,4%-dimethoxytrityl)-2%-deoxyribocytidine-3%-H-
phosphonate 1127 (24 equiv.) and pivaloyl chloride (75
equiv.) in pyridine/acetonitrile for 2 min to give the
modified support 12. Then after removal of the excess
reagent, the latter was reacted with the perylene deriva-
tive 3 (30 equiv.) in a pyridine/CCl4 (50:50, v/v) mixture
(2 mL) for 1 h to give the modified support 13. After
washing, the support was acetylated for 2 min, using
standard solutions, to block the unreacted hydroxyl
functions. Trityl assay indicated a loading of 30 mmol/g
of N4-benzoyl-5%-O-(4,4%-dimethoxytrityl)-2%-deoxyribo-
cytidine on the support 13. The yellow color of the
support indicated the presence of perylene.
u :395mnaxm. The other absorption band in the UV
range corresponds to the absorbance of the oligonucleo-
tide and the perylene with a uUV
:257 nm. The
max
UV–vis spectra of the oligonucleotide–perylene conju-
gate 9 recorded between u=220 and 520 nm in 10 mM
phosphate buffer, pH 7, containing 140 mM KCl and 5
mM MgCl2 is shown in Fig. 1.
Incorporation of the perylene to the 5%-end of the
oligonucleotide was performed manually by using 10
equiv. of the phosphoramidite derivatives 6 and 7 and a
coupling time of 10 min (Scheme 1). After removal of
the amidite excess, the oxidation step was performed
for 30 s by using a standard solution used on the
synthesizer. The synthesis of the oligonucleotide bear-
ing the perylene to the 3%-end was performed via the
phosphoramidite chemistry using the modified support
13 involving the perylene and the 3%-terminal nucleoside
(Scheme 2). The chain assembly was completed using a
standard coupling cycle with good efficiency as ascer-
tained by trityl measurements. The deprotection step
was performed by treatment at rt of the oligonucleo-
tide–perylene conjugates bound to the support with
a concentrated NH4OH solution overnight. After
removal of the ammonia, extraction of the organic
impurities and filtration, the crude oligonucleotide–
perylene conjugates 8, 9 and 14, were analyzed and
purified by reversed-phase chromatography. Retention
times are given in Table 1.
Figure 1. UV–vis spectra of the oligonucleotide–perylene con-
jugate 9 recorded between u=220 and 520 nm in 10 mM
phosphate buffer, pH 7, containing 140 mM KCl and 5 mM
MgCl2.
Table 1. Perylene-oligo-2%-deoxyribonucleotides conjugates
Oligonucleotides
Mass calculated
Mass observed
HPLC retention timea
8
9
14
3336.3
3392.4
3420.5
3337.2
3393.3
3462.1
27 min 3 s
31 min 13 s
44 min 20 s
a Analyses were performed on a reversed-phase RP 18 column (125×4 mm, Lichrospher 5 mM) from Merck, using a linear gradient of acetonitrile
(5 to 50% over 60 min) in 0.1 M triethylammonium acetate buffer, pH 7, with a flow rate of 1 mL/min and detection at 260 nm.

9008
U. Asseline, E. Cheng / Tetrahedron Letters 42 (2001) 9005–9010
3. Binding studies
the target duplex (around 61°C for all complexes) and
the transition with lower Tm to the dissociation of the
The experiments were performed, by absorption spec-
troscopy, in a 10 mM phosphate buffer, pH 6, contain-
ing 140 mM KCl and 5 mM MgCl2. We have used a
pH 6 buffer in order to facilitate the protonation of the
cytosines contained in the sequence. Thus, protonation
of the cytosines is a requirement to the formation of
two hoogsteen bonds between the cytosines and the
target guanines present on the purine strand of the
double-stranded DNA target. Pyrimidine sequences
containing cytosines form triple helices with weak sta-
bility at pH 7. Molar extinction coefficients of the
oligonucleotide–perylene conjugates 8, 9 and 14 were
determined by titration of conjugate solutions at 3°C
with a solution of single-stranded complementary
sequence 16. Molar extinction coefficients of the
unmodified oligonucleotides were determined according
to the literature.29 The mixing of the oligonucleotide–
perylene conjugates 8, 9 and 14 with either the single-
stranded 16 or the double-stranded 17/18 DNA targets
(see Fig. 2 for sequences) at 3°C induces a red shift of
the uvismax of the perylene spectra (:3 nm in the case of
compounds 8 and 9, and in the case of conjugate 14, 5
nm for the duplex and 4 nm for the triplex, respec-
tively). The spectral modifications reverse when the
temperature is increased to 50°C. Duplex and triplex
stabilities were determined by thermal denaturation.
Melting temperatures (Tms) and conditions used are
given in Table 2. One transition was observed in the
melting profile of each duplex, while two transitions
were observed in the melting of each triplex. The transi-
tion with the higher Tm corresponds to the melting of
third strand.
The duplexes formed by the oligonucleotide–perylene
conjugates 8:16, 9:16, and 14:16 are more stable than
the unmodified duplex 15:16 used as reference (Table
2). The presence of the perylene led to a Tm increase of
12.6°C when linked to the 5%-end of the oligonucleotide
(identical with both linkers) and a Tm increase of only
9.2°C when linked to the 3%-end of the oligonucleotide.
It must be noted that in the latter case the acetylation
of the linker could prevent the ligand from adopting the
better position for intercalation. The triple helices
formed by the oligonucleotide–ligand conjugates and
the double-stranded DNA target 8:17/18, 9:17/18, and
14:17/18 were also more stable than the unmodified
triple helix 15:17/18 used as reference. Once again when
the perylene was linked to the 5%-end of the oligonucle-
otide, the stabilization induced was nearly the same
with the two different linkers (DTm]15.4°C for conju-
gate 8 with short linker and DTm]16.4°C in the case of
conjugate 9 with a longer linker, respectively). When
the perylene was linked to the 3%-end of the oligonucle-
otide the stabilization observed was weaker (DTm]
9°C). This could be due to the acetylation of the linker
as mentioned above. Another explanation could be that
as in the case of oligonucleotide–acridine and oligonu-
cleotide–ethidium conjugates, weaker stabilization of
the triple helices was observed when the dye was cou-
pled to the 3%-end of the third strand as compared to
the stabilization observed when coupling was achieved
at the 5%-end.20 This question could be addressed by
using different linkers without an amino function to
prevent acetylation of the linker during the oligonucle-
otide chain assembly or post-synthetic coupling of the
ligand. The stabilization observed for the triple helices
is nearly equivalent to that provided by acridine20 and
naphthalene derivatives.17
4. Conclusions
This paper reports the synthesis and binding properties
of oligopyrimidine–perylene conjugates. Perylene was
covalently linked either to the 5%-end or the 3%-end of a
decamer. The covalent attachment of the perylene to
the 5%-end was performed via phosphoramidite chem-
istry using two linkers with different sizes. The coupling
of the perylene to the 3%-end of the oligonucleotide was
performed using a modified support involving the
perylene. The binding studies performed by absorption
spectroscopy showed that in any case the oligonucle-
otide–perylene conjugates formed more stable duplexes
and triplexes than did the unmodified oligonucleotide
used as reference.
Figure 2. Structures of the oligonucleotides used in the melt-
ing studies
Table 2. Melting temperatures for duplexes and triplexes
Duplexes
Tm (°C) (91°C) Triplexes
Tm (°C) (91°C)
8:16
9:16
14:16
15:16
34.4
34.4
31
8:17/18
9:17/18
14:17/18
15:17/18
29.4
30.4
23
Acknowledgements
21.8
514
The experiments were conducted with 1 mM oligonucleotide concen-
trations (each strand) in a 10 mM phosphate buffer, pH 6, containing
140 mM KCl and 5 mM MgCl2.
This work was supported by funds from the Agence
Nationale de Recherches sur le SIDA (ANRS). We

U. Asseline, E. Cheng / Tetrahedron Letters 42 (2001) 9005–9010
9009
thank H. Labbe´ (Centre de Biophysique Mole´culaire,
Orle´ans) for recording NMR spectra and C. Bure´ (Cen-
tre de Biophysique Mole´culaire, Orle´ans) for running
the electrospray mass spectrometry.
22. The perylene 1 (3 g, 11.88 mmol) was dissolved in 1,2-
dichloroethane (100 mL) placed in a three-necked round-
bottomed flask equipped with
a magnetic stirrer,
dropping funnel and nitrogen inlet tube with a bubbler.
The mixture was cooled to 5°C with an ice-water bath
and SnCl4 (2.85 mL, 23.34 mmol) was added in one
portion. Then, 1,1-dichlorooxymethylether (1.35 mL,
14.31 mmol) was added dropwise to the mixture over 1 h
and the temperature kept for 1 h at 55°C. The resulting
suspension was warmed to reflux for 2 h and further
stirred for 15 h. HCl was emitted during the warming
step. The reaction mixture was cooled to :10°C and
hydrolyzed by addition of cold water (100 mL). After 3 h
at room temperature the reaction mixture was extracted
with dichloromethane (150 mL). The organic phase was
washed with water (3×50 mL) dried over sodium sulfate,
and concentrated to dryness. The residue was purified by
silica gel chromatography using dichloromethane as elu-
ent to give the aldehyde derivative 2 (2.4 g, yield 75%,
Rf1=0.97 and Rf2=0.65 on analytical TLC silica plates
using CH2Cl2 as eluent). 1H NMR (CDCl3): l, 10.32–
10.30 (m, 1H, C(O)-H), 9.18–9.15 (m, 1H, Ar), 8.30–8.27
(m, 4H, Ar), 7.94–7.70 (m, 4H, Ar), 7.56–7.54 (m, 2H,
Ar). 13C NMR (CDCl3): l, 126.11, 127.65, 128.26,
129.70, 130.14, 133.20, 133.36, 134.25, 134.86, 135.27,
135.63, 136.01, 136.32, 136.94, 137.72, 140.09, 142.88,
143.44, 197.
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23. The aldehyde 2, 1,8-diaminooctane (or 2-aminoethanol or
6-aminohexanol) (10 equiv.), paratoluene sulfonic acid
(0.03 equiv.) and toluene were placed in a round-bot-
tomed flask equipped with a magnetic stirring bar and a
Dean–Stark trap. The reaction mixture was stirred at
reflux for 4 h and then cooled, diluted with ethanol (half
of the volume of toluene) and cooled to 0°C with an ice
bath. Solid NaBH4 (2 equiv.) was added to the stirred
mixture while maintaining the temperature below 15°C
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phase was washed with water, concentrated to dryness
and the residue purified on preparative TLC silica plates
using successively CH2Cl2/MeOH (80:20, v/v), CH2Cl2/
MeOH (50:50, v/v), and then CH2Cl2/MeOH/NH4OH
(50:50:5, v/v/v) as eluent for compound 3, and succes-
sively CH2Cl2/MeOH (95:5, v/v) and CH2Cl2/MeOH
(90:10, v/v) (three times) as eluent in the case of hydroxyl-
ated perylene-linker derivatives 4 and 5.
Compound 3. 1H NMR (DMSO, d6): l, 8.37–8.28 (m,
4H, Ar), 8.04–8.01 (m, 1H, Ar), 7.78–7.75 (m, 2H, Ar),
7.55–7.52 (m, 5H, Ar), 4.8 (s, 1H), 4.05–4.04 (m, 2H,
CH2-Ar), 2.60–2.57 (m, 2H, CH2), 2.49–2.43 (m, 2H,
CH2), 1.45–1.43 (m, 2H, CH2), 1.28–1.13 (m, 12H). Mass
analysis. ESI. Polarity positive. Calcd for C29H32N2: M+
H=408.5. Found: 409.4. Yield 50%.
17. Gianolio, D. A.; Segismundo, J. M.; McLaughlin, L. W.
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21. Bair, K. W.; Andrews, C. W.; Tuttle, R. L.; Knich, V. C.;
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1983–1990.
Compound 4. 1H NMR (CDCl3): l, 8.25–8.15 (m, 5H,
Ar), 7.97–7.95 (m, 1H, Ar), 7.71–7.69 (m, 2H, Ar), 7.57–
7.48 (m, 5H, Ar), 4.22 (s, 2H, CH2), 3.73 (t, 2H, J=5.5
Hz, CH2), 2.96 (t, 2H, J=6.5 Hz, CH2). Mass analysis.
ESI. Polarity positive. Calcd for C23H19NO: M=325.
Found: 326.1. Yield 65%.
Compound 5. 1H NMR (DMSO, d6): l, 8.22–8.13 (m,
5H, Ar), 7.94–7.92 (m, 1H, Ar), 7.69–7.68 (m, 2H, Ar),
7.67–7.66 (m, 2H, Ar), 7.55–7.48 (m, 5H, Ar), 4.16 (s,

9010
U. Asseline, E. Cheng / Tetrahedron Letters 42 (2001) 9005–9010
2H, CH2), 3.63 (t, 2H, J=6.5 Hz, CH2), 2.75 (t, 2 H, J=7 26. Asseline, U.; Thuong, N. T. Tetrahedron Lett. 1989, 19,
Hz, CH2), 1.58–1.39 (m, 4H, CH2), 1.38–1.37 (m, 4H,
CH2). 13C NMR (CDCl3): l, 31.67, 33.00, 35.75, 38.72,
55.47, 57.04, 66.83, 126.44, 126.76, 130.42, 132.76, 133.04,
133.75, 133.97, 134.34, 135.49, 136.83, 138.84, 140.42,
142.88. Mass analysis. ESI. Polarity positive. Calcd for
C27H27NO: M+H=381. Found: 382.3. Yield 68%.
2521–2524.
27. Froehler, B. C. Tetrahedron Lett. 1986, 27, 5575–5578.
28. Treatment of the perylene-linker derivative 5 with anhy-
dride acetic (10 equiv.) in anhydrous pyridine for 10 min
(corresponding to the cumulative capping time during the
sequence assembly) leads to the formation of two new
products identified by 1H NMR and electrospray mass
analysis as the mono- and the bis-acetylated derivatives.
This result confirmed that the oligonucleotide–perylene
conjugate 14 was probably acetylated.
24. Compound 6. 31P NMR [Ref. OP(OMe)3] (CDCl3): l ppm:
142.83. Mass analysis. ESI. Polarity positive. Calcd for
C32H36N3O2P: M=525.4. Found: 526.4.
25. Compound 7. 31P NMR [Ref. OP(OMe)3] (CDCl3): l ppm:
142.84. Mass analysis. ESI. Polarity positive. Calcd for
C36H44N3O2P: M=581.18. Found: 582.3.
29. Cantor, C. R.; Warshaw, M. M.; Shapiro, H. Biopolymers
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