Synthesis and properties of oligonucleotides with iodo-substituted aromatic aglycons: Investigation of possible halogen bonding base pairs

Article abstract of DOI:10.1021/jo701634t

(Chemical Equation Presented) Ab initio calculations of halogen bond energies of artificial base pairs constructed between iodinated aromatic nucleobase mimics and nitrogen-containing acceptor molecules such as pyridine and imidazole suggest that modified

Full text of DOI:10.1021/jo701634t

Synthesis and Properties of Oligonucleotides with Iodo-Substituted
Aromatic Aglycons: Investigation of Possible Halogen Bonding Base
Pairs
Ryuya Tawarada, Kohji Seio, and Mitsuo Sekine*
Department of Life Science, Tokyo Institute of Technology, 4259 Nagatsuta, Yokohama 226-8501, Japan,
and CREST, Japan Science and Technology Agency, 4259 Nagatsuta, Yokohama 226-8501, Japan
msekine@bio.titech.ac.jp
ReceiVed August 6, 2007
Ab initio calculations of halogen bond energies of artificial base pairs constructed between iodinated
aromatic nucleobase mimics and nitrogen-containing acceptor molecules such as pyridine and imidazole
suggest that modified base pairs are converted to optimized planar base pairs with weak ∆E values of
-0.19 to -3.93 kcal/mol. To evaluate the contribution of halogen bonding toward duplex stabilization
of such modified nucleobase mimics introduced into artificial base pairs, we synthesized three C-nucleoside
analogues 1-3 with several iodinated aromatic rings and an imidazole nucleoside derivative 4 and
incorporated them into oligodeoxynucleotides. Hybridization studies of modified oligodeoxynucleotides
incorporating iodoaromatic bases showed their unique universal base-like ability; however, no indication
of halogen bond formation was observed. A more sophisticated design is required for the development
of new base pairs stabilized by halogen bonding.
Introduction
reported new base pairs stabilized by hydrophobic interactions
between aromatic bases.8 In these studies of nucleosides with
aromatic bases, there are some examples of aromatic rings
modified by halogen atoms because of their inherent unique
properties. Seela and Kro¨shel reported that the bromo and iodo
groups enhanced base stacking on substitution into position 7
of 7-deazaguanine derivatives.9 5-Bromouracil has been used
instead of thymine because the bromo group affects the stacking
effect with the 5′-upstream and 3′-downstream bases.10 5-Fluo-
rouracil is known to exhibit strong antitumor activity.11 Other
examples of halogenated bases have also been reported for
diverse functions.12,13
Recently, many nucleoside derivatives having a functional
aromatic group as their aglycon have been reported.1-3 The
biological properties and base pairing abilities of these modified
bases upon incorporation into oligodeoxynucleotides have been
studied.4,5 For example, Shionoya et al. reported nucleotide
analogues as inhibitors of DNA polymerases using phenol- and
catechol-type modified bases.6 Wielckens et al. reported ribo-
sylated derivatives of benzamide as nicotinamide adenine
dinucleotide (NAD) analogues.7 Moreover, Romesberg et al.
* Corresponding author. Fax: +81-45-924-5772.
(1) Wu, Q.; Simons, C. Synthesis 2004, 1533-1553.
(2) Jiang, Y. L.; Stivers, J. T. Tetrahedron Lett. 2003, 44, 4051-4055.
(3) Kim, T. W.; Kool, E. T. Org. Lett. 2004, 6, 3949-3952.
(4) Hwang, G. T.; Romesberg, F. E. Nucleic Acids Res. 2006, 34, 2037-
2045.
(5) Morales, J. C.; Kool, E. T. Nat. Struct. Biol. 1998, 5, 950-954.
(6) Aketani, S.; Tanaka, K.; Yamamoto, K.; Ishihama, A.; Cao, H.;
Tengeiji, A.; Hiraoka, S.; Shiro, M.; Shionoya, M. J. Med. Chem. 2002,
45, 5594-5603.
(7) Krohn, K.; Heins, H.; Wielckens, K. J. Med. Chem. 1992, 35, 511-
517.
(8) Ogawa, A. K.; Wu, Y.; McMinn, D. L.; Liu, J.; Schultz, P. G.;
Romesberg, F. E. J. Am. Chem. Soc. 2000, 122, 3274-3287.
(9) Seela, F.; Kro¨shel, R. Nucleic Acids Res. 2003, 31, 7150-7158.
(10) Becher, G.; He, J.; Seela, F. HelV. Chim. Acta 2001, 84, 1048-
1065.
(11) Montgomery, J. A.; Hewson, K. J. Am. Chem. Soc. 1957, 79, 4559.
10.1021/jo701634t CCC: $40.75 © 2008 American Chemical Society
Published on Web 12/15/2007
J. Org. Chem. 2008, 73, 383-390
383

Tawarada et al.
TABLE 1. Theoretical Calculation of Energy (kcal/mol) of
Among halogens, the iodo group has a potentially interesting
property to form halogen bonds14 with other atoms having lone
electron pairs. The order of the strength of halogen bonding is
I > Br > Cl > F for the respective halogens. If such halogen
bonding can be achieved in DNA duplexes, it can be used
instead of hydrogen bonding for connecting different nucleobase
analogues in the development of new base pairing motifs. To
the best of our knowledge, this possibility has not yet been
investigated.
Halogen Bonding between Iodoaromatic Compounds and
N-Containing Donor Compoundsa
The iodo group when present in aromatic rings is reactive
toward various reagents such as Pd catalysts15 and butyl
lithium.16 Therefore, the synthesis of such modified nucleosides
having iodoaromatic rings and oligodeoxynucleotides incorpo-
rating them can be challenging. In this paper, we report that it
is theoretically possible to construct such base pairs using
halogen bonding and that oligodeoxynucleotides incorporating
iodoaromatic deoxynucleoside analogues can be successfully
synthesized. However, based on our results of hybridization
experiments with the halogenated oligodeoxynucleotides, it
seems that additional design modifications are required prior
to application of this new chemical bond for the synthesis of
base pairs with increased stability.
Results and Discussion
Theoretical Evaluation of Stability of Base Pairs of
Iodoaromatic Base Analogues and Heteroaromatic Base
Analogues Using Halogen Bonding. Halogen bonding has been
found in several crystallized organic compounds in naturally
occurring substances.17 A typical example is a weak but very
unique interaction between a halogen atom and a heteroaromatic
nitrogen atom. We focused on the possibility that this new
interaction might be used for stabilizing a base pair formed
between a set of artificially designed nucleobase analogues. As
an initial trial to evaluate this possibility, we calculated halogen
bonding energies of various possible combinations of iodoaro-
matic base donors and nitrogen-containing heteroaromatic
acceptors.
Because iodine is the 53rd element in the periodic table, we
could not use a high-level basis set, such as the 6-31G* level.
We had previously compared the results of the hydrogen bond
energies of several modified base pairs obtained using MO
calculations at the 3-21G* to 6-31G* levels. It was observed
that the relative stability remained unchanged. Therefore, in this
study, the theoretical calculation was performed at the HF/3-
21G* level in the gas phase considering basis set superposition
errors (BSSE). We also performed calculations considering the
effects of solvation using the conductor-like polarizable con-
tinuum (CPCM) model.18 These results are listed in Table 1
and Figure 2.
a ∆EBSSE ) ∆Eaq + (∆EBSSE - ∆Egas).
aq
gas
For calculation, the structures were initially arranged to keep
the two aromatic rings in a planar configuration. As a result,
after calculations, the halogen bonding structures of all com-
binations were found to be conversed, and the planarity of the
aromatic rings was maintained. The typical geometry of the
FIGURE 1. Halogen bonding between halogenated compounds and
acceptor molecules with heteroatoms (N, O, S, and Se).
halogen bond, the bond length, and the bond angle have been
reported in various literature.14 These studies revealed that the
optimal bond length was 3 Å and that the optimal bond angle
was 160-180°. In the case of the optimized structures shown
in Table 1 and Figure 2, the bond length and angle were close
to these optimal values.
(12) Hamm, M. L.; Rajguru, S.; Downs, A. M.; Cholera. R. J. Am. Chem.
Soc. 2005, 127, 12220-12221.
(13) Kim, T. W.; Kool, E. T. J. Org. Chem. 2005, 70, 2048-2053.
(14) Metrangolo, P.; Resnati, G. Encyclopedia of Supramolecular
Chemistry; Marcel Dekker, Inc.: New York, 2004; pp 628-635.
(15) Beletskaya, I. P.; Cheprakov, A. V. Chem. ReV. 2000, 100, 3009-
3066.
Gas phase calculations revealed that the halogen bonding
energies without BSSE correction (∆Egas in Table 1) varied from
-5.46 to -11.30 kcal/mol and that those with BSSE correction
(16) Clayden, J. Organolithiums: SelectiVity for Synthesis; Pergamon
Press: Oxford, 2002; pp 111-142.
(∆EBSSE in Table 1) varied from -1.98 to -6.63 kcal/mol.
gas
(17) Auffinger, P.; Hays, H. A.; Westhof, E.; Ho, P. S. Proc. Natl. Acad.
Sci. U.S.A. 2004, 101, 16789-16794.
These results suggested that the binding energies were overes-
timated by 3.46 kcal/mol (Table 1, entry 9) to 4.76 kcal/mol
(18) Barone, V.; Cossi, M. J. Phys. Chem. A 1998, 102, 1995-2001.
384 J. Org. Chem., Vol. 73, No. 2, 2008

Oligonucleotides with Iodo-Substituted Aglycons
FIGURE 3. Nucleoside derivatives with iodoaromatic rings (iPh, 2F,
and 3F), 4-iodoimidazole (iIm), pyridine-3-yl (Py),19 imidazol-1-yl ring
(Im),20 and phenyl (Ph)21 moieties.
components, as shown in Figure 3. We adopted Woski and
Wichai’s method for preparing the three C-glycosides 1-3.22
For the synthesis of 1, 1,3-diiodobenzene 5 was lithiated with
n-BuLi, and then silyl-protected 2-deoxyribonolactone 6 was
added to a solution of the 1-iodo-3-lithiobenzene intermediate.
The resulting hemiacetal was allowed to react in situ with excess
Et3SiH and BF3‚OEt2 to produce an inseparable anomeric
mixture (R/â ) 1:5.6) of 8 with a total yield of 32%. The free
C-nucleoside 9 (an anomeric mixture of 1) was obtained by
treatment of 8 with Et3N‚3HF and Et3N. Compound 10, obtained
by dimethoxytritylation of 9, could be easily isolated with an
overall yield of 57% by column chromatography. For preparing
oligodeoxynucleotides using standard â-cyanoethyl phosphora-
midite chemistry, the building block 11 was synthesized by
phosphitylation of 10 with 2-cyanoethyl N,N,N′,N′-tetra-isopro-
pylphosphordiamidite23 (Scheme 1).
Next, we attempted to synthesize the C-nucleoside 2 as an
anomeric mixture of 19 and its diastereomerically pure phos-
phoramidite derivative 21, as shown in Scheme 3. Schlosser
and Rausis reported that the treatment of 1,3-difluorobenzene
12 with s-BuLi led to selective 2-lithiation and that the lithiated
species 13 could react with iodine to give the iodinated
compound 1,3-difluoro-2-iodobenzene 14 quantitatively (Scheme
2).24
FIGURE 2. Optimized structures of gas phase calculations based on
data in Table 1.
(Table 1, entry 6), assuming that the BSSE corrections were
accurate. In the case of aqueous phase calculations, the energies
(∆Eaq) varied from -3.67 to -8.60 kcal/mol. Using the BSSEs
corresponding to the gas phase, ∆Eaq was further corrected to
result in the interaction energies in the aqueous phase ∆EBSSE
.
aq
The simplest example, as depicted in Table 1, entry 1, showed
the smallest ∆EBSSEaq value of -0.19 kcal/mol, and the halogen
bond formed between a tetra-fluoroiodophenyl derivative and
imidazole, as shown in Table 1, entry 8, resulted in the largest
∆EBSSEaq value of -3.93 kcal/mol. Because all of the interaction
energy values are consistently smaller than the canonical base
pairs, there is a possibility of destabilization of the double-
stranded oligonucleotides when halogen bonding base pairs are
incorporated. However, optimized structures, as shown in
Figure 2, indicate that these types of specific interactions are
possible.
In addition, by molecular modeling, we also confirmed that
the glycosyl bonds in these optimized base pair structures could
be superimposed over those in the natural base pairs, as shown
in the case of the superposition with an A-T base pair (see
Supporting Information). Further, we performed calculations of
partially fixed molecular mechanics for double-stranded DNA
containing the modified nucleosides iPh and Py (Figure 3) at
the central position of each single strand. The results showed
that iPh and Py maintained the halogen bonded base pair shown
in entry 1 of Figure 2 without a large distortion of the B-type
duplex structure (see Supporting Information). These data
suggest that there is a possibility of formation of such base pairs
in double-stranded oligonucleotides.
Synthesis of C- and N-Nucleosides Having Iodoaromatic
Aglycon and Their Phosphoramidite Building Blocks. We
synthesized several new deoxynucleoside derivatives 1-4 with
iodoaromatic nucleobase analogues. They include 3-iodophenyl
(iPh), 2,4-difluoro-3-iodophenyl (2F), 2,4,5-trifluoro-3-iodophe-
nyl (3F), and 4-iodoimidazol-1-yl (iIm) moieties as the aglycon
Therefore, we first attempted to synthesize the 1-(2-deoxy-
â-D-ribofuranosyl)-2,4-difluorobenzene derivative (18)25 as a key
intermediate to obtain 19. For the synthesis of 18, we attempted
to generate the organolithium compound by halogen-lithium
exchange of 1-bromo-2,4-difluorobenzene (15) with n-BuLi.
However, lithiation of the 3-H proton of 15 occurred simulta-
neously to give a mixture of the desired and undesired lithiated
species.
(19) Tanaka, K.; Yamada, Y.; Shionoya, M. J. Am. Chem. Soc. 2002,
124, 8802-8803.
(20) Durland, R. H.; Rao, T. S.; Bodepudi, V.; Seth, D. M.; Jayaraman,
K.; Revankar, G. R. Nucleic Acids Res. 1995, 23, 647-653.
(21) Matsuda, S.; Romesberg, F. E. J. Am. Chem. Soc. 2004, 126, 14419-
14427.
(22) Wichai, U.; Woski, S. A. Org. Lett. 1999, 1, 1173-1175.
(23) Barone, A. D.; Tang, J. Y.; Caruthers, M. H. Nucleic Acid Res.
1984, 12, 4051-4061.
(24) Rausis, T.; Schlosser, M. Eur. J. Org. Chem. 2002, 3351-3358.
(25) Lai, J. S.; Qu, J.; Kool, E. T. Angew. Chem., Int. Ed. 2003, 42,
5973-5977.
J. Org. Chem, Vol. 73, No. 2, 2008 385

Tawarada et al.
SCHEME 3. Synthesis of C-Nucleoside Derivatives 19
(Anomeric Mixture of 2), 26 (Anomeric Mixture of 3), and
Their Diastereomerically Pure Phosphoramidite Building
Blocks 21 and 28
SCHEME 1. Synthesis of C-Nucleoside 9 (Anomeric Mixture
of 1) and Its Diastereomerically Pure Phosphoramidite
Building Block 11
SCHEME 2. Lithiation of 1,3-Difluorobenzene
Synthesis of Nucleoside Analogue 4 with Iodoimidazole
Aglycon and Its Phosphoramidite Unit. The synthesis of 4
was carried out using a stereospecific glycosylation procedure.26
The reaction of the sodium salt of 4,5-diiodoimidazole (29)27
with 1-chloro-2-deoxy-3,5-di-O-p-toluoyl-R-D-erythro-pento-
furanose 30 in acetonitrile afforded the corresponding protected
nucleoside analogue 31 in 89% yield. Removal of the toluoyl
groups was accomplished by treatment with NaOMe in MeOH
to give the free nucleoside 32 in 90% yield, which was further
converted to the corresponding 5-O-dimethoxytrityl derivative
33 in 86% yield.
Previously, Lovely reported on the selective removal of iodine
atoms from 1-substituted 4,5-diiodoimidazole derivatives using
EtMgBr.28 We applied this reagent to the synthesis of the
monoiodinated derivative 34. It was found that treatment of 33
with 2.0 equiv of EtMgBr in THF followed by the addition of
Therefore, we chose an alternative way to synthesize 19.
Compound 15 was converted to the Grignard reagent 16, which
in turn was allowed to react with 6. Treatment of the resulting
hemiacetal 17 with Et3SiH and BF3‚OEt2 gave an anomeric
mixture of 18 in 25% yield (R/â ) 1:6.7). Although the yield
of the C-glycosylation product seemed rather low, it was
comparable to those obtained by similar C-glycosylation reac-
tions.24 The selective lithiation of 18 with s-BuLi followed by
reaction with I2 gave the iodinated product. Successive in situ
treatments of this product with Et3N‚3HF and Et3N resulted in
the desired nucleoside analogue 19 in 87% yield. The 5′-
hydroxyl group of 19 was protected with a 5′-dimethoxytrityl
(DMTr) group. At this stage, the desired â-isomer 20-â could
be easily separated from the R-isomer 20-R by silica gel column
chromatography. The R- and â-isomers were isolated in 9 and
81% yields, respectively. To incorporate the C-nucleoside
analogues 2 into oligodeoxynucleotides, the 3′-phosphoramidite
building block 21 was prepared in the usual manner in 92%
yield The phosphoramidite building block 28 of the 2,4,5-
trifluoro-3-iodophenyl derivatives 3 could be obtained by a series
of reactions starting from 22 via 23-26 and 27-â.
(26) Kazimierczuk, Z.; Cottam, H. B.; Revankar, G. R.; Robins, R. K.
J. Am. Chem. Soc. 1984, 106, 6379-6382.
(27) Garden, S. J.; Torres, J. C.; Melo, S. C.; Lima, A. S.; Pinto, A. C.;
Lima, E. L. S. Tetrahedron Lett. 2001, 42, 2089-2092.
(28) Lovely, C. J.; Du, H.; Dias, H. V. R. Heterocycles 2003, 60, 1-7.
386 J. Org. Chem., Vol. 73, No. 2, 2008

Oligonucleotides with Iodo-Substituted Aglycons
TABLE 2. Synthesis of Oligodeoxynucleotides with Iodoaromatic
Rings as Aglyconsa
SCHEME 4. Synthesis of Nucleoside Derivative 4 and Its
Phosphoramidite Building Block 35
d(GGACTAXACTGCG)
MALDI-TOF-mass
base X
isolated yield (%)
calculated
found
39
40
41
42
X ) iPh
2F
46
16
15
51
4049.6
4085.6
4103.6
4039.6
4049.8
4088.7
4108.7
4038.8
3F
iIm
d(CGCAGTYTAGTCC )
MALDI-TOF-mass
base Y
isolated yield (%)
calculated
found
45
46
47
48
Y ) iPh
2F
42
30
22
36
4000.6
4036.6
4054.6
3990.5
3997.8
4036.4
4055.9
3990.1
3F
iIm
a Oligodeoxynucleotides 45-48 and 39-42 were hybridized with
d(GGACTAXACTGCG) (36: X ) Py, 37: X ) Im, and 38: X ) Ph)
and d(CGCAGTYTAGTCC) (43: Y ) Py and 44: Y ) Im) containing
acceptor deoxynucleoside analogues, respectively, to measure Tm values.
water produced the desired product 34. To confirm the structure
of this product by NMR, it was detritylated with 80% acetic
acid to give the 3′,5′-O-free nucleoside 4 in 83% yield. All
1
resonance signal peaks of the H and 13C NMR spectra of 4
were unambiguously assigned by DEPT, COSY, and HMQC.
In the HMBC measurement, a cross-peak was observed between
the anomeric protons with the carbon on the dehalogenated
carbon. On the other hand, no cross-peak was observed between
the anomeric protons with the carbon on the iodinated carbon.
The usual phosphitylation of 34 gave the building block 35 in
15% yield.
FIGURE 4. Thermal stability of duplexes formed between modified
oligodeoxynucleotides with X and Y.
Synthesis of Modified Oligodeoxynucleotides. Oligodeoxy-
nucleotide 13-mers 39-42 5′-d(GGACTAXACTGCG)-3′ and
45-48 5′-d(CGCAGTYTAGTCC)-3′ incorporating the iodoaro-
matic nucleosides 1-4 at the central positions were synthesized
on a DNA/RNA synthesizer using standard â-cyanoethyl
phosphoramidite chemistry (Table 2). Each coupling yield was
more than 96%, as estimated by a DMTr cation assay. All
oligonucleotides were synthesized according to the DMTr-ON
procedure and purified by the use of C18 cartridge columns.
All oligonucleotides were characterized by MALDI-TOF mass
spectrometry, and their purities were checked by anion-exchange
HPLC (Table 2).
Interestingly, we found that minor peaks appeared as byprod-
ucts in the anion-exchange HPLC charts of 40, 41, 46, and 47.
MALDI-TOF mass spectrometry suggested that these peaks had
molecular weights corresponding to the products lacking the
iodo group. These results strongly implied the unexpected
removal of the iodine atom from the difluoro derivatives 40
and 46 and the trifluoro derivatives 41 and 47 during oligo-
nucleotide synthesis. In contrast to the results of the oligonucle-
otide synthesis, nucleoside analogues 19 and 26 were stable
under the conditions used for oligonucleotide synthesis such as
3% TFA in CH2Cl2, I2 in THF/pyridine/H2O, and concentrated
aqueous NH3. Moreover, these nucleosides did not react with 1
equiv of each of the T-phosphoramidite building block, triph-
enylphosphine, and triethyl phosphite in acetonitrile at room
temperature for 1 h. The reason for this dehalogenation is
unclear, and further studies are necessary.
Base Recognition Abilities of Iodoaromatic Bases in DNA
Duplexes. To analyze the thermal stability of the base pairs
that include the iodoaromatic bases in double-stranded oligode-
oxynucleotides, UV-melting experiments were performed. All
Tm values were obtained using 2 µM each oligonucleotide in
10 mM sodium phosphate buffer (pH 7.0), 100 mM NaCl, and
0.1 mM EDTA.
We measured the Tm values of oligodeoxynucleotide duplexes
containing a pyridine19 (Py) or imidazole20 (Im) residue as the
aglycon with oligodeoxynucleotides containing a simple iodo
donor deoxynucleoside analogue (iPh or iIm). As shown in
Figure 4, the duplexes containing iPh exhibited higher Tm values
than those containing iIm (see X/Y ) Py/iPh vs Py/iIm and
Im/iPh vs Im/iIm). When X/Y is reversed to Y/X, a similar
tendency was observed (see X/Y ) iPh/Py vs iIm/Py and iPh/
Im vs iIm/Im). Iodo donor bases, iPh, theoretically form the
weakest halogen bond with pyridine (Py) and the second weakest
halogen bond with imidazole (Im) (Table 1). Therefore, the
hybridization affinity of the oligodeoxynucleotides 40, 41, 46,
and 47 for incorporating 2F or 3F was examined to determine
J. Org. Chem, Vol. 73, No. 2, 2008 387

Tawarada et al.
oliogonucleotides incorporating these iodoaromatic nucleosides
were also synthesized. Interestingly, it was found that the iodine
atoms of oligodeoxynucleotides 40, 41, 46, and 47 were partly
removed when the oligonucleotides were synthesized by the
standard phosphoroamidite method; however, the reason for the
loss of the iodo group is unclear.
We compared the thermal stability of the modified oligode-
oxynucleotides with that of the unmodified oligodeoxynucle-
otides with the canonical bases. The results show that the Tm
value of X/Y ) G/iIm was higher than the other pairs with the
natural bases, thus suggesting the possibility of a base pair
shown in Figure 6.
In this model, iIm should be in a syn conformation (i.e., the
iodine atom should not face the side of the Watson-Crick base
pair but that of the 5′-hydroxyl group). Ab initio studies showed
that the energy of the syn conformation equals that of the anti
conformation (see Supporting Information). To the best of our
knowledge, this is the first published work on halogen bonded
base pairs in double-stranded DNA. On the basis of theoretical
and experimental data, our results imply that the halogen bond
interactions do not contribute as much to the increase in the
stability of the double-stranded DNA as the A-T pair. There
are several possibilities for this destabilization. In general,
halogen bonds have energies of 2-50 kcal/mol, which indicates
that a halogen bond could have an interaction energy comparable
to a canonical hydrogen bond, depending on the environment.14
The calculation of interaction energy of halogen bonded base
pairs suggested a much lower stability of halogen bonds in the
aqueous phase. This result gives a good account of destabiliza-
tion of double-stranded DNA incorporating the iodoaromatic
and halogen bond acceptor bases. In addition, there is a
possibility that the glycosyl bond of the iodoaromatic and
halogen bond acceptor nucleosides rotates so that the iodo group
and the nitrogen atoms move away from each other. Neverthe-
less, in the case of 2F and 3F, the iodo groups are expected to
face opposite strands because the fluorine atom at position 2 is
responsible for fixing the glycosyl bond in an anti conformation
similar to the carbonyl oxygens at position 2 of thymidine and
deoxycytidine. Therefore, a better design of the halogen bond
acceptor nucleosides, and possibly the iodoaromatic nucleotides,
can lead to the development of new artificial base pairs stabilized
by a halogen bond. Studies are currently underway to explore
this further.
FIGURE 5. Thermal stability of duplexes formed between modified
and unmodified oligodeoxynucleotides.
FIGURE 6. Possible hydrogen bond formation between iIm and G.
if they could act as iodo donors capable of forming more stable
halogen bonds with Py and Im. These results are also shown in
Figure 4.
For the halogen bond acceptors Py and Im, the highest Tm
value was observed when they were paired with 3F, and stability
decreased in the order of their pairing with 2F > iPh > iIm.
To further check if the halogen bond actually contributed to
the most stable base pair, we synthesized an oligodeoxynucle-
otide d(GGACTA[Ph]ACTGCG), 38, incorporating an unsub-
stituted phenyl (Ph) base instead of Py using the phosphora-
midite unit, as previously reported.21 The Tm value (42.3 °C)
of the duplex (X/Y ) Ph/3F) formed between 38 and
d(CGCAGT[3F]TAGTCC) 47 was essentially the same as that
(42.1 °C) of the duplex (X/Y ) Py/3F) formed between 36 and
47. This result implies that the contribution to stabilization of
the halogen bond was negligible and that more sophisticated
designs should be considered.
We also studied the hybridization affinity of 45 (Y ) iPh)
and 48 (Y ) iIm) for the complementary oligodeoxynucleotides
having naturally occurring nucleobases at the site opposite to
Y. For comparison, the data of non-natural base pairssiPh/Py,
iPh/Im, iIm/Py, and iIm/Imsare also shown in Figure 5. The
iodinated bases iPh and iIm considerably destabilized the DNA
duplexes as compared to their natural, fully matched duplex,
for which Tm ) 50.5 °C. Interestingly, the iIm moiety specif-
ically recognized guanine and not the other three natural bases,
as suggested by Tm ) 37.6 °C for X/Y ) G/iIm and Tm ) 34.1,
33.2, and 33.8 °C for the other pairssA/iIm, T/iIm, and C/iIm,
respectively. This specificity might be explained in terms of a
possible hydrogen bond between the 1-N nitrogen of the guanine
base and the 3-N nitrogen of iIm (Figure 6).
Experimental Section
3,5-O-[(1,1,3,3-Tetra-isopropyl)disiloxanediyl]-1,2-dideoxy-1-
(3-iodophenyl)-D-ribofuranose (8). To a solution of 1,3-diiodo-
benzene (5) (4.95 g, 15 mmol) in dry THF (30 mL) under argon at
-78 °C was added slowly n-BuLi (1.54 M in hexane, 9.74 mL,
15.0 mmol). The mixture was stirred at -78 °C for 30 min. The
mixure was added via a syringe to a solution of 3,5-O-[(1,1,3,3-
tetra-isopropyl)disiloxanediyl]-2-deoxy-D-ribono-1,4-lactone (6)
(3.74 g, 10.0 mmol) in dry THF (30 mL) at -78 °C. After 1 h, the
reaction mixture was quenched with saturated aq NH4Cl and
extracted with ethyl acetate. The combined organic layer was
washed with saturated aq NH4Cl, water, and brine. The organic
solution was dried over anhydrous Na2SO4 and concentrated under
reduced pressure. The resulting oil containing the initial product 7
was used without further purification in the next reaction.
Moreover, iPh formed duplexes with higher stabilities when
paired with synthetic bases Py and Im than when paired with
the natural bases. In addition, it should be noted that iPh
performs as a universal base because modified DNA duplexes
showed similar Tm values with the differences being within
3 °C.
A solution of the crude oil in CH2Cl2 (35 mL) under argon at
-78 °C was treated with Et3SiH (4.79 mL, 30.0 mol) and BF3‚
OEt2 (3.80 mL, 30.0 mmol). The resulting solution was stirred at
-78 °C for 6 h, and the reaction was quenched by the addition of
saturated aq NaHCO3 at -78 °C. The resulting mixture was
Conclusion
We synthesized a series of new C-deoxynucleoside derivatives
with iodinated aromatic ringssiPh, 2F, 3F, and iIm. Modified
388 J. Org. Chem., Vol. 73, No. 2, 2008

Oligonucleotides with Iodo-Substituted Aglycons
extracted with ethyl acetate. The combined organic layer was
washed with saturated aq NaHCO3 water and brine. The solution
was dried over anhydrous Na2SO4 and evaporated under reduced
pressure. The residue was purified by silica gel column chroma-
tography (C-200, 100 g, hexane/ethyl acetate, 0-1%) to give
anomeric mixture 8 (1.80 g, 32%, R/â ) 1:5.6). 1H NMR (CDCl3,
500 MHz) δ 7.73 (0.15H, s), 7.68 (0.85H, s), 7.60-7.58 (1H, m),
7.30-7.29 (1H, m), 7.07-7.04 (1H, m), 5.02 (0.85H, t, J ) 7.33
Hz), 4.96 (0.15H, dd, J ) 6.10, 9.77 Hz), 4.59-4.54 (0.15H, m),
4.52-4.48 (0.85H, m), 4.14-4.10 (0.85H, m), 4.07-4.03 (0.15H,
m), 3.93-3.86 (2H, m), 2.67-2.62 (0.15H, m), 2.39-2.34 (0.85H,
m), 2.12-2.00 (1H, m), 1.12-0.95 (28H, m); 13C NMR (CDCl3,
126 MHz, â-isomer) δ 144.8, 136.7, 134.9, 130.3, 125.3, 94.7, 86.7,
78.3, 73.2, 63.7, 43.3, 17.8, 17.7, 17.7, 17.5, 17.3, 17.3, 17.2, 13.7,
J ) 24.95 Hz), 85.9, 72.9 (d, J ) 2.88 Hz), 72.3, 63.1, 41.9, 17.8,
17.7, 17.6, 17.5, 17.3, 17.3, 17.2, 13.7, 13.6, 13.2, 12.8. ESI MS
+
calcd C23H38F2NaO4Si2 [M + Na]+ 495.2169, found 495.2170.
1,2-Dideoxy-1-(2,4-difluoro-3-iodophenyl)-D-ribofuranose (19).
To a solution of an anomeric mixture of 18 (460 mg, 0.97 mmol)
in dry THF (10 mL) was added dropwise via a syringe s-BuLi (0.95
M in hexane, 1.13 mL, 1.07 mmol) under argon at -78 °C. The
mixture was stirred at -78 °C for 2 h and then added via a syringe
to a solution of iodine (272 mg, 1.07 mmol) in dry THF (550 µL).
The mixture was warmed to room temperature and stirred for 1 h.
The reaction was quenched by the addition of saturated aq Na2-
SO3. The resulting mixture was extracted with ethyl acetate-H2O.
The organic extracts were combined, dried over anhydrous Na2-
SO4, and concentrated under reduced pressure. The resulting oil
was used without further purification. A solution of the residue in
THF (5 mL) was treated with Et3N (251 µL, 1.80 mmol) and Et3N‚
3HF (570 µL, 3.50 mmol) at room temperature. After stirring for
6 h, the mixture was extracted with ethyl acetate-H2O, and the
organic layer was washed with saturated aq NaHCO3 and brine.
The organic extract was dried over anhydrous Na2SO4 and
evaporated under reduced pressure. The residue was purified by
silica gel column chromatography (C-200, 30 g, CHCl3/MeOH,
0-2%) to give an anomeric mixture of 19 as a colorless oil (301
mg, 87%). 1H NMR (DMSO-d6, 500 MHz, â-isomer) δ 7.61 (1H,
dd, J ) 8.54, 15.13 Hz), 7.13 (1H, t, J ) 7.56 Hz), 5.20 (1H, dd,
J ) 5.61, 10.25 Hz), 5.12 (1H, d, J ) 3.90 Hz), 4.78 (1H, t, J )
5.86 Hz), 4.20-4.18 (1H, m), 3.81-3.74 (1H, m), 3.50-3.40 (2H,
m), 2.14-2.11 (1H, m), 1.80-1.74 (1H, m); 13C NMR (DMSO-
d6, 126 MHz, â-isomer) δ 161.2 (dd, J ) 6.72, 242.81 Hz), 158.7
(dd, J ) 6.72, 244.24 Hz), 128.6 (dd, J ) 3.84, 16.32 Hz), 111.5
(dd, J ) 2.88, 12.00 Hz), 87.7, 72.9, 72.4 (t, J ) 30.71 Hz), 72.2,
13.6, 13.2, 12.8. ESI MS calcd C23H43INO4Si2 [M + NH4]+
+
580.1770, found 580.2539.
1,2-Dideoxy-1-(3-iodophenyl)-D-ribofuranose (9). To a solution
of an R,â-anomeric mixture of 8 (1.68 g, 3.18 mmol) in THF (16
mL) were added Et3N (799 µL, 5.73 mmol) and Et3N‚3HF (1.81
mL, 11.2 mmol) at room temperature. After stirring for 1 h, the
mixture was extracted with CHCl3-H2O, and the organic layer was
washed with H2O. The organic solution was dried over anhydrous
Na2SO4 and evaporated under reduced pressure. The residue was
purified by silica gel column chromatography (C-200, 30 g, CHCl3/
MeOH, 0-3%) to give an anomeric mixture of 9 as a colorless oil
(951 mg, quant., R/â ) 1:5.6). 1H NMR (CDCl3, 500 MHz) δ 7.74
(0.15H, s), 7.70 (0.85H, s), 7.63-7.60 (1H, m), 7.33-7.29 (1H,
m), 7.10-7.05 (1H, m), 5.10 (0.85H, dd, J ) 5.62, 10.26 Hz), 5.38
(0.15H, t, J ) 7.81 Hz), 4.47-4.42 (1H, m), 4.07 (0.15H, dd, J )
4.88, 9.28 Hz), 4.02-4.00 (0.85H, m), 3.85-3.81 (1H, m), 3.78-
3.71 (1H, m), 2.70-2.65 (0.15H, m), 2.28-2.65 (0.85H, m), 2.04-
1.94 (3H, m); 13C NMR (CDCl3, 126 MHz, â-isomer) δ 143.7,
136.9, 135.0, 130.4, 125.4, 94.6, 87.5, 79.3, 73.6, 63.4, 43.9. ESI
+
62.2, 42.0. ESI MS calcd C11H11F2INaO3 [M + Na]+ 378.9613,
found 378.9607.
MS calcd. C11H13INaO3 [M + Na]+ 342.9802, found 342.9808.
1-[2-Deoxy-3,5-di-O-(4-toluoyl)-â-D-ribofuranose-1-yl]-4,5-di-
iodoimidazole (31). To a solution of 4,5-diiodoimidazole (29) (2.22
g, 6.95 mmol) in dry acetonitrile (70 mL) was added sodium hydride
(167 mg, 6.95 mmol). After the mixture had been stirred at room
temperature for 30 min, 1-chloro-2-deoxy-3,5-di-O-p-toluoyl-R-D-
erythro-pentofuranose (30) (500 mg × 4, 6.32 mmol) was added
in four portions at 15 min intervals. After a total 2 h of stirring,
the solvent was evaporated under reduced pressure. The residue
was diluted with ethyl acetate (200 mL). The organic solution was
washed with H2O (200 mL) and dried over anhydrous Na2SO4. The
solvent was evaporated under reduced pressure, and the residue
was purified by silica gel colum chromatography using a gradient
(0-12%) of CHCl3/hexane to give 31 (3.80 g, 89%) as a white
+
3,5-O-[(1,1,3,3-Tetra-isopropyl)disiloxanediyl]-1,2-dideoxy-1-
(2,4-difluorophenyl)-D-ribofuranose (18). Magnesium (384 mg,
15.8 mmol) and iodine (200 mg, 0.79 mmol) were added to dry
THF (15 mL) under argon at room temperature. 2,4-Difluorobro-
mobenzene (15) (1.89 mL, 15.8 mmol) was added dropwise via a
syringe to the mixture. The mixture was stirred for 1 h at room
temperature. After formation of the Grignard reagent 16 was
complete, 3,5-O-[(1,1,3,3-tetra-isopropyl)disiloxanediyl]-2-deoxy-
D-ribono-1,4-lactone (6) (2.00 g, 5.27 mmol) in dry THF (5 mL)
was added via a syringe to the mixture at 0 °C. After stirring for
2 h, the reaction mixture was quenched with saturated aq NH4Cl
and extracted with ethyl acetate. The combined organic layer was
washed with saturated aq NH4Cl, water, and brine. The organic
solution was dried over anhydrous Na2SO4 and concentrated under
reduced pressure. The resulting oil containing the hemiacetal 17
was used without further purification in the next reaction. A solution
of the crude oil in CH2Cl2 (15 mL) under argon at -78 °C was
treated with Et3SiH (2.53 mL, 15.8 mol) and BF3‚OEt2 (2.00 mL,
15.8 mmol). The resulting solution was stirred at -78 °C for 6 h,
and the reaction was quenched by the addition of saturated aq
NaHCO3 at -78 °C. The resulting mixture was extracted with ethyl
acetate. The combined organic layer was washed with saturated aq
NaHCO3, water, and brine. The solution was dried over anhydrous
Na2SO4 and evaporated under reduced pressure. The residue was
purified by silica gel column chromatography (C-200, 80 g, hexane/
ethyl acetate, 0-1%) to give an anomeric mixture of 18 (627 mg,
25%, R/â ) 1:6.7). 1H NMR (CDCl3, 500 MHz) δ 7.51-7.47 (1H,
m), 6.86-6.82 (1H, m), 6.79-6.75 (1H, m), 5.28 (0.86H, t, J )
7.32 Hz), 5.21 (0.14H, dd, J ) 6.09, 9.64 Hz), 4.59-4.54 (0.14H,
m), 4.09 (0.86H, dd, J ) 5.86, 13.43), 4.13-4.04 (1H, m), 3.95-
3.83 (2H, m, Râ-5H), 2.74-2.69 (0.14H, m), 2.46-2.41 (0.86H,
m), 2.07-2.02 (1H, m), 1.13-0.92 (28H, m); 13C NMR (CDCl3,
126 MHz, â-isomer) δ 162.5 (dd, J ) 12.48, 247.60 Hz), 159.8
(dd, J ) 12.48, 249.52 Hz), 128.3 (dd, J ) 6.72, 9.60 Hz), 126.0
(dd, J ) 3.84, 13.44 Hz), 111.3 (dd, J ) 3.84, 21.11 Hz), 103.8 (t,
1
solid. H NMR (CDCl3, 500 MHz) δ 7.95-7.85 (5H, m), 7.29-
7.23 (4H, m), 6.78 (1H, dd, J ) 5.62, 7.81 Hz), 5.64 (1H, m),
4.67, (2H, t, J ) 2.69, 3.42 Hz), 4.61 (1H, m), 2.81-2.80 (1H, m),
2.58-2.53 (1H, m), 2.44 (3H, s), 2.41 (3H, s); 13C NMR (CDCl3,
126 MHz) δ 166.3, 166.0, 144.8, 144.5, 139.1, 130.0, 129.7, 129.6,
129.5, 126.6, 126.4, 97.3, 89.6, 83.3, 79.8, 74.7, 63.9, 39.9, 21.9,
+
21.9. ESI MS calcd C24H23I2N2O5 [M + H]+ 672.9696, found
672.9682.
1-(2-Deoxy-â-D-ribofuranose-1-yl)-4,5-diiodoimidazole (32).
To a solution of 31 (3.79 g, 5.46 mmol) in MeOH (56 mL) was
added NaOMe (305 mg, 5.46 mmol). After this, the mixture was
stirred at room temperature for 1 h. The mixture was purified by
dry silica gel column chromatography (C-200, 80 g, methanol/
1
chloroform, 0-3%) to give 32 (2.21 g, 90%) as a white solid. H
NMR (DMSO-d6, 500 MHz) δ 8.14 (1H, s), 5.88 (1H, t, J ) 6.59
Hz), 5.34 (1H, d, J ) 4.15 Hz), 4.95 (1H, t, J ) 5.13 Hz), 4.30-
4.28 (1H, m), 3.83-4.82 (1H, m), 3.54-3.45 (2H, m), 2.41-2.27
(2H, m); 13C NMR (DMSO-d6, 126 MHz) δ 140.1, 97.2, 88.6, 88.0,
+
84.4, 70.3, 61.2, 40.3. ESI MS calcd C8H11I2N2O3 [M + H]+
436.8859, found 436.8845.
1-[2-Deoxy-5-O-(4,4′-dimethoxytrityl)-â-D-ribofuranose-1-yl]-
4,5-diiodoimidazole (33). The free nucleotide 32 (2.18 g, 5.00
mmol) was dried by coevaporation with dry pyridine (2 mL × 3)
J. Org. Chem, Vol. 73, No. 2, 2008 389

Tawarada et al.
and dissolved dry pyridine (50 mL). 4,4′-Dimethoxytrityl chloride
(1.86 g, 5.50 mmol) was added, and the mixture was stirred under
argon atmosphere at room temperature for 6 h. The reaction was
quenched by the addition of MeOH (5 mL), and the solvent was
evaporated under reduced pressure. The residue was diluted with
ethyl acetate (100 mL), and the organic solution was extracted with
saturated aq NaHCO3 (100 mL × 2). The organic layer was dried
by use of anhydrous Na2SO4 and evaporated under reduced pressure.
The residue was purified by silica gel column chromatography (C-
200, 50 g, methanol/chloroform, 0-2% with 1% triethylamine) to
(CDCl3, 126 MHz) δ 158.8, 144.5, 137.5, 135.7, 135.6, 130.7,
130.4, 128.2, 128.2, 127.2, 122.4, 133.4, 86.9, 86.2, 86.1, 82.8,
+
72.5, 63.9, 55.4, 41.7. ESI MS calcd C29H30IN2O5 [M + H]+
613.1200, found 613.1118.
Tm Measurement. Each oligonucleotide was dissolved in 10 mM
sodium phosphate (pH 7.0) containing 100 mM NaCl and 0.1 mM
EDTA so that the final concentration of each oligonucleotide
became 2 µM. The solutions were separated into quartz cells (10
mm) and incubated at 85 °C. After 10 min, the solutions were
cooled to 5 °C at 0.5 °C/min and then heated until the temperature
reached 85 °C at the same rate. During this annealing and melting,
the absorptions at 260 nm were recorded and used to draw UV
melting curves. The Tm values were calculated as the temperature
that gave maximum first derivatives of the UV melting curves. The
same experiment was repeated 4 times, and the average of the Tm
values is given in Figures 4 and 5. The oligonucleotide concentration
incorporating a modified nucleobase was determined on the
assumption that ꢀ260 is identical to that of thymidine.
1
give 33 as a foam (3.17 g, 86%). H NMR (CDCl3, 500 MHz) δ
7.77 (1H, s), 7.42-7.20 (9H, m), 6.84-6.82 (4H, m), 5.97 (1H, t,
J ) 6.35 Hz), 4.50 (1H, m), 3.79 (6H, s), 3.41-3.30 (2H, m), 2.53-
2.48 (1H, m), 2.39-2.34 (1H, m), 2.09 (1H, br); 13C NMR (CDCl3,
126 MHz) δ 158.8, 144.4, 139.3, 135.6, 135.6, 130.1, 130.1, 128.2,
128.2, 127.2, 113.4, 96.9, 89.2, 86.9, 86.1, 79.9, 72.2, 63.6, 55.4,
41.7. ESI MS calcd C29H29I2N2O5 [M + H]+ 739.0166, found
+
739.0226.
1-[2-Deoxy-5-O-(4,4′-dimethoxytrityl)-â-D-ribofuranose-1-yl]-
4-iodoimidazole (34). The 1.0 M EtMgBr in THF (1.0 mL, 1.0
mmol) was instilled into a solution of 33 (738 mg, 1.0 mmol) in
dry THF (10 mL) at -78 °C under argon atmosphere. After stirring
for 5 min, the mixture was warmed up to 0 °C and stirred for 5
min. A 1.0 M solution of EtMgBr in THF (1.0 mL, 1.0 mmol) was
added dropwise to the solution at 0 °C and stirred for 15 min. The
reaction mixture was quenched by the addition of H2O (3 mL),
and the residue was extracted with ethyl acetate and saturated aq
NaHCO3. The organic layer was dried with anhydrous Na2SO4 and
evaporated under reduced pressure. The residue was purified by
silica gel column chromatography (C-200, 15 g, methanol/
chloroform, 0-1% with 0.5% triethylamine) to give 34 as a foam
(334 mg, 56%). 1H NMR (CDCl3, 500 MHz) δ 7.50 (1H, s), 7.40-
7.22 (9H, m), 7.11 (1H, s), 6.85-6.83 (4H, m), 5.97 (1H, t, J )
6.59 Hz), 4.50 (1H, m), 4.05 (1H, dd, J ) 4.40, 8.06 Hz), 3.80
(6H, s), 3.39-3.26 (2H, m), 2.41 (2H, dd), 2.18 (1H, br); 13C NMR
Acknowledgment. We thank Drs. Akihiro Ohkubo and
Haruhiko Taguchi for helpful discussions. This work was
supported by a grant from CREST of the Japan Science and
Technology Agency and a Grant-in-Aid for Scientific Research
from the Ministry of Education, Culture, Sports, Science and
Technology, Japan. This work was supported in part by a grant
of the Genome Network Project from the Ministry of Education,
Culture, Sports, Science and Technology, Japan and by the
COE21 project.
1
Supporting Information Available: H, 13C, and 31P NMR data
of all new products and experimental procedures and characteriza-
tion of 4, 10, 11, 20-â, 21, 25, 26, 27-â, 28, and 35. This material
is available free of charge via the Internet at http://pubs.acs.org.
JO701634T
390 J. Org. Chem., Vol. 73, No. 2, 2008