Synthesis, incorporation into triplex-forming oligonucleotide, and binding properties of a novel 2'-deoxy-C-nucleoside featuring a 6-(thiazolyl-5)benzimidazole nucleobase.

Article abstract of DOI:10.1021/ol026609h

[reaction: see text] 6-(Thiazolyl-5)benzimidazole (B(t)()) was designed as a novel nucleobase for the specific recognition of an inverted A.T base pair in a triple helix motif. It was successfully incorporated into an 18-mer triplex-forming oligonucleotid

Full text of DOI:10.1021/ol026609h

ORGANIC
LETTERS
2002
Vol. 4, No. 24
4209-4212
Synthesis, Incorporation into
Triplex-Forming Oligonucleotide, and
Binding Properties of a Novel
2′-Deoxy-C-Nucleoside Featuring a
6-(Thiazolyl-5)benzimidazole Nucleobase
Dominique Guianvarc’h,† Jean-Louis Fourrey,† Rosalie Maurisse,‡
,‡
,†,§
Jian-Sheng Sun,* and Rachid Benhida*
Institut de Chimie des Substances Naturelles, CNRS, 1 aVenue de la Terrasse,
91198 Gif-sur-YVette, France, Laboratoire de Biophysique, UR 565 INSERM UMR
8646 CNRS, Muse´um National d’Histoire Naturelle, 43 rue CuVier 75231 Paris Cedex
05, France, and Laboratoire de Chimie Bioorganique, UMR 6001 CNRS,
UniVersite´ de Nice-Sophia Antipolis, Parc Valrose, 06108 Nice Cedex 2, France
benhida@unice.fr
Received July 25, 2002
ABSTRACT
t
6-(Thiazolyl-5)benzimidazole (B ) was designed as a novel nucleobase for the specific recognition of an inverted A‚T base pair in a triple helix
motif. It was successfully incorporated into an 18-mer triplex-forming oligonucleotide (TFO) using the 2′-deoxy-C-nucleoside phosphoramidite
16. The triple helix binding properties of the modified TFO were examined by means of thermal denaturation experiments targeting an
oligopyrimidine‚oligopurine 26-mer DNA duplex containing an A‚T base pair inversion.
Pyrimidine oligonucleotides can interact, in a parallel
orientation, with the oligopurine strand of an oligopyrimidine‚
oligopurine DNA duplex through the formation of T‚AxT
and C‚GxC+ triplets.1 This recognition process occurs in the
major groove of the double stranded DNA (ds-DNA) and
involves specific Hoogsteen hydrogen bonds between pyri-
midines of the triplex forming-oligonucleotide (TFO) and
the Watson-Crick purine bases of its cognate ds-DNA
duplex.2 Accordingly, TFOs have attracted considerable
interest because of their potential applications in gene
expression modulation and gene targeting technologies.3
Unfortunately, when a single A‚T or G‚C base pair interrupts
an oligopyrimidine‚oligopurine ds-DNA target, one observes
a strong triplex destabilization.4 Hence, to date, the recogni-
tion of mixed purine/pyrimidine sequences remains a chal-
lenge. In the past few years, many TFO chemical modifi-
(2) (a) Le Doan, T.; Perrouault, L.; Praseuth, D.; Habhoub, N.; Decout,
J. L.; Thuong, N. T.; Lhomme, J.; He´le`ne, C. Nucleic Acids Res. 1987, 15,
7749-7760. (b) Moser, H. E.; Dervan, P. B. Science 1987, 238, 645-650.
For reviews, see: (c) Thuong, N. T.; He´le`ne, C. Angew. Chem., Int. Ed.
Engl. 1993, 32, 666-690. (d) Frank-Kamenetskii, M. D.; Mirkin, S. M.
Annu. ReV. Biochem. 1995, 64, 65-95. (e) Neidle, S. Anti-Cancer Drug
Des. 1997, 12, 433-442.
(3) (a) Maher, J. L., III. Cancer InVest. 1996, 14, 66-82. (b) Giovan-
nangeli, C.; He´le`ne, C. Antisense Nucleic Acid Drug DeV. 1997, 7, 413-
421. (c) Vasquez, K. M.; Wilson, J. H. Trends Biochem. Sci. 1998, 23,
4-9. (d) Praseuth, D. Guieysse-Peugeot, A. L.; He´le`ne, C. Biochim. Biophys.
Acta 1999, 1489, 181-206.
† Institut de Chimie des Substances Naturelles.
‡ Laboratoire de Biophysique.
(4) (a) Mergny, J. L.; Sun, J. S.; Rouge´e, M.; Garestier, T.; Barcelo, F.;
Chomilier, J.; He´le`ne, C. Biochemistry 1991, 30, 9791-9798. (b) Greenberg,
W. A.; Dervan, P. B. J. Am. Chem. Soc. 1995, 117, 5016-5022. (c) Best,
G. C.; Dervan, P. B. J. Am. Chem. Soc. 1995, 117, 1187-1193.
§ Laboratoire de Chimie Bioorganique.
(1) The symbols ‚ and x indicate Watson-Crick and Hoogsteen hydrogen
bonds, respectively.
10.1021/ol026609h CCC: $22.00 © 2002 American Chemical Society
Published on Web 10/31/2002

cations were reported to overcome this sequence limitation.5
The most promising approach consists of designing new
modified bases able to form hydrogen bond contacts on both
bases of the A‚T or G‚C Watson-Crick inverted base pairs
in the major groove.6 We have recently reported on the
selective recognition of A‚T base pair in ds-DNA by
pyrimidine-motif TFO containing an unnatural nucleoside
S.7 This new base analogue consists of two unfused aromatic
rings (3-aminophenyl-thiazole) linked to a 2-deoxyribose unit
by an acetamide motif. This interesting result led us to design
a nucleobase analogue featuring a thiazolyl-benzimidazole
system (Bt), to form selective hydrogen bonds with an
inverted A‚T base pair, as shown in Figure 1.
an aldol-type condensation between the 2-lithiated derivative
of 5 and 6 with a suitably protected 2-deoxyribose appeared
to be the most appropriate and straightforward procedure
toward the preparation of B- and Bt-derived 2′-deoxy-C-
nucleosides. The key steps in this synthesis consisted of a
Stille-type coupling, for the biaryl aglycone Bt formation
(Scheme 1), and an aryl-aldol type condensation followed
Scheme 1a
a Reagents and conditions: (i) Boc2O, pyridine (100%); (ii)
K2CO3, MeI, DMF (95%); (iii) LDA, THF, -78 °C then Bu3SnCl
(>95%); (iv) NaH, DMF, ClSO2NMe2 (80%); (v) H2, Pd/C, MeOH/
THF (1/1) (>95%); (vi) TMSI, NaNO2, TEBAC, CH3CN (73%);
(vii) Pd(PPh3)4, DMF, 60 °C (95%). *Compound 6 was obtained
from benzimidazole following conditions (iv).
Figure 1. The proposed interactions between nucleobase Bt and
the inverted A‚T base pair.
Bt was designed on the basis of molecular modeling studies
suggesting that a more rigid modified base would better
stabilize triplex formation. Here we report on the synthesis
of the new nucleobase Bt together with its incorporation into
a TFO whose recognition properties have been thoroughly
examined. To incorporate Bt into a TFO we needed to
synthesize, as suggested by modelization, a 2′-deoxy-C-
nucleoside derivative of Bt in which the deoxyribose unit is
connected to the C2-position of the thiazolyl-benzimidazole
aglycone Bt (Figure 1). A 2′-deoxy-C-nucleoside, in which
the aglycone moiety is reduced to a single benzimidazole
ring B (Figure 1), was also synthesized as a control.
by a regioselective isopropylidene cleavage-ring closure
(Schemes 2 and 3).
In this study, the N,N-dimethylsulfamoyl and tert-butoxy-
carbonyl protecting groups were used for the respective
protection of the benzimidazole and aminothiazole rings of
Bt, and they were found to be compatible with oligonucle-
otide solid-phase synthesis.
First, the biaryl nucleobase Bt was obtained, according to
our previously described procedure,9 by a palladium-
catalyzed Stille cross-coupling reaction between derivative
2 and 5(6)-iodobenzimidazole 4 in 95% yield (Scheme 1).10
Compound 2 was obtained from 2-aminothiazole 1 in three
steps (>90% overall yield), which consisted of (i) standard
A large number of synthetic approaches to a variety of
C-nucleosides have been reported.8 In our case, the use of
(5) Reviews: (a) Sun, J. S.; He´le`ne, C. Curr. Opin. Struct. Biol. 1993,
3, 345-356. (b) Doronina, S. O.; Behr, J. P. Chem. Soc. ReV. 1997, 26,
63-71. (c) Gowers, D. M.; Fox, K. R. Nucleic Acids Res. 1999, 27, 1569-
1577 and references therein.
(8) For reviews on the chemistry, biochemistry, and synthesis of
C-nucleosides analogues, see: (a) Buchanan, J. G. Prog. Chem. Org. Nat.
Prod. 1983, 44, 243-299. (b) Hacksell, U.; Daves, G. D., Jr. Prog. Med.
Chem. 1985, 22, 1-65. (c) Postema, M. H. D. Tetrahedron 1992, 40, 8545-
8599. (d) Watanabe, K. A. Chemistry of Nucleosides and Nucleotides;
Townsend, L. B., Ed.; Plenum Press: New York, 1994; Vol. 3, pp 421-
535. (e) Jaramillo, C.; Knapp, S. Synthesis 1994, 1-20. (f) Chaudhuri, N.
C.; Ren, R. X. F.; Kool, E. T. Synlett 1997, 341-347. (g) Togo, H.; He,
W.; Waki, Y.; Yokoyama, M. Synlett 1998, 700-716.
(9) Benhida, R.; Lecubin, F.; Fourrey, J.-L.; Rivas, L.; Quintero, L.
Tetrahedron Lett. 1999, 40, 5701-5703.
(10) (a) Stille, J. K. J. Am. Chem. Soc. 1979, 4992-4998. (b) Stille, J.
K. Angew. Chem., Int. Ed. Engl. 1986, 25, 508-524.
(6) (a) Griffin, L. C.; Kiessling, L. L.; Beal, P. A.; Gillespie, P.; Dervan,
P. B. J. Am. Chem. Soc. 1992, 114, 7976-7982. (b) Koshlap, K. M.;
Gillespie, P.; Dervan, P. B.; Feigon, J. J. Am. Chem. Soc. 1993, 115, 7908-
7909. (c) Huang, C. Y.; Cushman, C. D.; Miller, P. S. J. Org. Chem. 1993,
58, 5048-5049. (d) Huang, C. Y.; Miller, P. S. J. Am. Chem. Soc. 1993,
15, 10456-10457. (e) Huang, C. Y.; Bi, G.; Miller, P. S. Nucleic Acids
Res. 1996, 24, 2606-2613. (f) Lehmann, T. E.; Greenberg, W. A.; Liberles,
D. A.; Wada, C. K.; Dervan, P. B. HelV. Chim. Acta 1997, 80, 2002-
2022. (g) Obika, S.; Hari, Y.; Sekiguchi, M.; Imanishi, T. Angew. Chem.,
Int. Ed. 2001, 40, 2079-2081.
(7) Guianvarc’h, D.; Benhida, R.; Fourrey, J.-L.; Maurisse, R.; Sun, J.
S. J. Chem. Soc., Chem. Commun. 2001, 1814-1815.
4210
Org. Lett., Vol. 4, No. 24, 2002

treatment with Boc2O, (ii) methylation of the resulting
carbamate, and (iii) stanylation at the thiazole C-5 position
using LDA in THF, then trapping of the intermediate with
tributyltin chloride. The 5(6)-iodo regioisomers 411 derived
from 5(6)-nitro-benzimidazole 3: (i) benzimidazole protec-
tion (ClSO2NMe2/NaH/DMF), (ii) catalytic hydrogenolysis
of the nitro function, and finally (iii) transformation of the
obtained amine to the target iodo-derivatives 4 (NaNO2/
TMSI)12 in 73% yield (Scheme 1).
SnCl2 treatment of 1′-O-mesyl derivatives 10S and 11S in
methylene chloride at room temperature resulted in a clean
conversion to the cyclized product 12 and 13, respectively,
(60% yield) following a cleavage-ring closure process
(Scheme 3).
Scheme 3a
Next, and as shown in Scheme 2, the protected Bt and
benzimidazole B were lithiated by LDA in THF at -50 °C
Scheme 2a
a Reagents and conditions: (i) SnCl2‚2H2O, CH2Cl2 (58-62%);
(ii) Bu4NF, THF (85-87%); (iii) DMTCl, Et3N, DMF (70-88%);
(iv) ClP(OCH2CH2CN)(iPr)2, Hunig’s base, CH2Cl2 (63-73%).
a Reagents and conditions: (i) LDA, THF, -78 °C then 7 (59-
61%); (ii) MsCl, Et3N, DMAP (88-90%).
Under these mild conditions, the S-isomers of 10 and 11
led to the cyclized products 12(â) and 13(â), respectively,
whereas the R-isomers afforded the corresponding R-anomers
(12(R) and 13(R), respectively, see Supporting Information).
In view of these data, it appears that pentitols 10 and 11
cyclize, under SnCl2 treatment, according to an SN2 process.16
The â-anomeric configuration of 12 and 13 was assigned
by 2D-NOESY experiments that showed, as expected for a
â-configuration, a clear NOE correlation between H1′ and
H4′.17 The 5′-TBDPS group cleavage leading to the target
B- and Bt-derived 2′-deoxy-C-nucleosides 14 and 15 was
achieved by treatment of 12 and 13 with Bu4NF in THF.
Finally, the preparation of the phosphoramidite building
blocks 16 and 17, which were used for TFO synthesis, was
accomplished by successive 5′-dimethoxytritylation and 3′-
phosphitylation of nucleosides 14 and 15, respectively.
to give the corresponding 2-lithio derivatives in quantitative
yield.13 These 2-lithiated heterocycles were allowed to react
with the known deoxyribose precursor 714 to afford the
corresponding pentitol derivatives 8 and 9 in 60% yield as
a mixture of R/S diastereoisomers in 7/3 ratio, respectively,
1
as determined by H NMR. The diastereoisomers of each
compounds 8 and 9 were then easily separated by silica gel
flash chromatography.15 Mesylation of 8 and 9 with meth-
anesulfonyl chloride afforded the 1-O-mesyl derivatives 10
(88%) and 11 (90%), respectively. To keep the required N,N-
dimethylsulfamoyl and tert-butoxycarbonyl protecting groups
on the aglycone moiety for oligonucleotide solid-phase
synthesis, we needed to use mild conditions to eliminate the
isopropylidene group of compounds 10 and 11. Interestingly,
The phosphoramidites 16 and 17 were then incorporated
into 18-mer TFOs (III) at the internal position Z (Figure 2)
using automated oligonucleotide synthesis. The protected
oligonucleotides thus obtained were purified by reverse phase
HPLC, and the protecting DMT, SO2NMe2 and Boc groups
(11) The iodo derivative 4 was obtained as a mixture of 5- and
6-regioisomers in nearly 40/60 ratio, respectively. Both regioisomers will
give the same oligonucleotide after cleavage of the sulfamoyl protecting
1
group. To facilitate the interpretation of the H and 13C NMR spectra, we
only used the 6-isomer in the next steps of our synthesis.
(12) Lee, J. G.; Cha, H. T. Tetrahedron Lett. 1992, 33, 3167-3168.
(13) We have verified that this lithiation occurred exclusively and
quantitatively at the C-2 position of the benzimidazole moiety by trapping
the lithio derivatives with hexachloroethane.
(14) Joos, P. E.; Esmans, E. L.; Domisse, R. A.; De Bruyn, A.; Balzarini,
J. M.; De Clercq, E. D. HelV. Chim. Acta 1992, 75, 1613-1620.
(15) The R and S configurations of compounds 8 and 9 were determined
on the basis of the anomeric stereochemistry of their cyclized products 12
and 13, respectively (NOESY experiments).
(16) Under more drastic conditions (TFA, H2O, 60 °C),14 the cyclization
occurred but with (i) concomitant cleavage of all the protecting TBDPS,
SO2NMe2 and Boc groups and (ii) epimerization of the C-1′ stereocenter,
which suggested a partial SN2-type mechanism for the outcome of this
reaction.
(17) In the same way, we observed for the other anomers (12(R) and
13(R) obtained from 10R and 11R, respectively) clear NOE H1′-H3′ and
H1′-H5′ correlations, in accordance with an R configuration.
Org. Lett., Vol. 4, No. 24, 2002
4211

To determine the specificity of the nucleobase Bt, the 18-
mer TFO (III, Z ) Bt) was also screened against duplexes
containing the three other possible base pairs (X‚Y ) T‚A,
C‚G and G‚C) (Table 1). It was observed that (i) the
Table 1. Melting Temperature of All Combinations of Base
Triplets at the X‚YxZ Site (Tm ( 1 °C)
Z
X‚Y
T
C
G
Bt
T‚A
C‚G
A‚T
G‚C
51
40
33
38
31
50
33
35
31
31
45
35
37
38
43
41
nucleobase Bt can moderately discriminate a Pu‚Py from a
Py‚Pu base pair (A‚TxBt and G‚CxBt vs T‚AxBt and
C‚GxBt) and (ii) that Bt provides a +3 °C triplex stabilization
in the case of a G‚C interruption compared to the best base
triplet made of the natural bases G‚CxT. The moderate
discrimination of nucleobase Bt for all four base pairs argues
for a non-sequence-specific interaction, i.e, third strand base
stacking/intercalation.
In conclusion, we have synthesized a novel 2′-deoxy-C-
nucleoside analogue featuring a highly functionnalized
aglycone moiety (Bt) and shown the N,N-dimethylsulfamoyl
protecting group to be compatible with oligonucleotide
synthesis. The new nucleobase was successfully incorporated
into a TFO using automated oligonucleotide synthesis. The
triplexes featuring an A‚TxBt triplet or the most stable natural
triplet A‚TxG were shown to be of comparable stability. In
view of these results, Bt represents an interesting starting
point for the synthesis of other nucleobases, for structure-
affinity relationship studies which is currently in progress.
Figure 2. Sequence of the studied triplexes I‚IIxIII (X‚Y ) A‚T
and Z ) G, Bt, or B) and melting temperature curves. Conditions:
I‚II (1.2 µM), III (1.5 µM) in 10 mM cacodylate buffer (pH 6)
containing 100 mM NaCl, 10 mM MgCl2, and 0.5 mM spermine.
were cleanly cleaved by treatment of the oligonucleotides
with aqueous TFA solution (10%). To our knowledge, this
is the first example of oligonucleotides synthesis in which
N,N-dimethylsulfamoyl-protected nucleobases were involved.
The triple helix binding properties of these TFOs were
examined by means of thermal denaturation experiments with
the oligopyrimidine‚oligopurine 26-mer ds-DNA (I‚II) con-
taining an A‚T interruption (X‚Y ) A‚T) (Figure 2).
We observed that the Tm value obtained for the triplex
containing an A‚TxBt triplet (Tm ) 43 °C) is very close to
that obtained with G, which is known to form the most stable
natural triplet (A‚TxG, Tm ) 45 °C). However, as it is well-
known that G involves one hydrogen bond with T in
A‚TxG triplet (NH2(G)-O4 (T)),5c our Bt nucleobase most
likely does not involve three hydrogen bonds as illustrated
in Figure 1. Interestingly, the replacement of Bt by the
benzimidazole nucleobase B caused a dramatic destabiliza-
tion of the triplex (∆Tm ) -9 °C from A‚TxBt to A‚TxB)
(Figure 2). This result shows clearly the implication of the
methylaminothiazole moiety of Bt in the recognition process,
which has not been yet elucidated.
Acknowledgment. The authors thank M. Thomas for
oligonucleotides synthesis, Dr. J.-C. Blais for characterization
of oligonucleotides, and CNRS for a doctoral fellowship
(BDI) to D.G.
Supporting Information Available: Experimental pro-
cedures and spectroscopic characterization (1H, 13C, 31P (for
phosphoramidites) NMR and MS) of all new compounds.
Also automated oligonucleotides synthesis, melting temper-
ature experiments, and MALDI-TOF/MS characterization of
oligonucleotides. This material is available free of charge
via the Internet at http://pubs.acs.org.
OL026609H
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Org. Lett., Vol. 4, No. 24, 2002