Intramolecular cation-π interactions as the driving force to restrict the conformation of certain nucleosides

Article abstract of DOI:10.1021/jo902677s

"Chemical Equation Presented" Despite the well-established importance of intermolecular cation-π interactions in molecular recognition, intramolecular cation-jr interactions have been less studied. Here we describe how the simultaneous presence of an arom

Full text of DOI:10.1021/jo902677s

pubs.acs.org/joc
Intramolecular Cation-π Interactions As the Driving Force To Restrict
the Conformation of Certain Nucleosides
Elena Casanova,† Eva-Marı
a Priego,† Marıa-Luisa Jimeno,‡ Leire Aguado,† Ana Negri,§
´ ´
†
a-Jose Camarasa, and Marı
,†
Federico Gago,§ Marı
´
´
ꢀ
ꢀ
ꢀ
ꢀ
a-Jesus Perez-Perez*
†
‡
Instituto de Quımica Medica (CSIC), Juan de la Cierva 3, E-28006 Madrid, Spain, Centro de Quımica
´
ꢀ
´
Orgaꢀnica Lora-Tamayo (CSIC), Juan de la Cierva 3, E-28006 Madrid, Spain, and §Departamento de
´
ꢀ
Farmacologıa, Universidad de Alcala, Alcala de Henares, E-28871 Madrid, Spain
ꢀ
mjperez@iqm.csic.es
Received December 17, 2009
Despite the well-established importance of intermolecular cation-π interactions in molecular
recognition, intramolecular cation-π interactions have been less studied. Here we describe how
the simultaneous presence of an aromatic ring at the 50-position of an inosine derivative and a
positively charged imidazolium ring in the purine base drive the conformation of the nucleoside
toward a very major conformer in solution that is stabilized by an intramolecular cation-π
interaction. Therefore, the cation-π interaction between imidazolium ions and aromatic rings can
also be proposed in the design of small molecules where this type of interaction is desirable. The
imidazolium ion can be obtained by a simple acidification of the pH of the media. So a simple change
in pH can shift the conformational equilibrium from a random to a restricted conformation stabilized
by an intramolecular cation-π interaction. Thus the here described nucleosides can be considered as
a new class of pH-dependent conformationally switchable molecules.
Introduction
the nucleoside field is the 50-cap structure in mRNA
(m7GpppN) that consists of a 7-methylguanosine (m7G)
that is linked via a 50-50 triphosphate bridge (ppp) to the
50-end of the transcribed RNA where N represents the
initiating nucleotide of the encoded sequence (Figure 1).6
The presence of the 50-cap structure in mRNAs is specifically
recognized by a number of enzymes involved in mRNA
translation (including the eukaryotic initiation factor eIF4E,7,8
the CBC nuclear cap-binding complex,9 the vaccinia virus
Cation-π interactions in proteins take place between
positively charged groups and aromatic residues1-3 and they
have been recognized as an important component of non-
covalent binding forces in biological systems.4,5 The most
relevant interactions occur between the side chain of aro-
matic amino acids (Phe, Trp, Tyr) and metal or organic ions,
including, in the latter group, the imidazolium ring of a
protonated histidine. A particular example of organic ion in
(6) Mikkola, S.; Salomaki, S.; Zhang, Z.; Maki, E.; Lonnberg, H. Curr.
Org. Chem. 2005, 9, 999–1022.
* To whom correspondence should be addressed. Phone: 34 91 2587579.
Fax: 34 91 5644853.
(1) Dougherty, D. A. Science 1996, 271, 163–168.
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281–287.
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2003, 42, 1210–1250.
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Sonenberg, N.; Wagner, G. Nat. Struct. Biol. 1997, 4, 717–724.
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Published on Web 03/01/2010
DOI: 10.1021/jo902677s
r
2010 American Chemical Society

Casanova et al.
JOCArticle
started within our research program on purine nucleosides.23
While performing the synthesis of 1-benzyl-50-O-benzyl-
20,30-O-isopropylideneinosine23 (1, Figure 2a), the desired
compound was obtained together with a novel derivative
identified as 1,7-dibenzyl-50-O-benzyl-20,30-O-isopropylide-
neinosine bromide24 (2, Figure 2b).
The 1H NMR spectra of both compounds in CDCl3
showed significant differences particularly affecting in com-
pound 2 the splitting of signals corresponding to geminal
protons (the CH2 of the benzyl groups at the 50-position of
the sugar and at the N1 of the purine; the protons at the 50
position of the sugar H50 and H500) and the “upfield” shift of
the aromatic protons of the benzyl group at the 50-position
(Figure 2b). Therefore we decided to investigate how
the introduction of other substituents at position N7 in the
inosine or other benzyl groups at the 50-position of the
nucleoside affected the appearance of these “fingerprint”
FIGURE 1. 50-Cap structure of mRNA.
methyltransferase VP3910,11) or in RNA transcription (as in
polymerase basic protein (PB2) of the influenza virus12). The
structural insights provided by both X-ray crystallography
and NMR spectroscopy within some of the 50-capped com-
plexes and their target enzymes reveal that the positi-
vely charged 7-methylguanine establishes a sandwich-type
cation-π interaction with the side chain of aromatic residues
such as Trp, Tyr, or Phe.13-15 Complexation of 7mGTP with
the eIF4E factor, with a dissociation constant in the nM
range, represents an excellent example of the importance of
cation-π interactions in molecular recognition.8
Cation-π interactions are also finding an increasingly
important role in guest-host recognition and supramole-
cular chemistry.16-19 Quite recently, intramolecular cation-π
interactions, and in particular pyridinium-π interactions, are
being successfully applied in organic synthesis20 or to study
isomer populations.21 Most reported cases of intramolecular
pyridinium-π interactions include some restrictions in the
conformation of the substrate to favor the occurrence of
stacking interactions, although there are some very recent
examples that overcome this limitation.22
1
signals in the H NMR spectra. This was followed by a
1
detailed conformational study in solution by means of H
NMR techniques and complemented with computational
studies. With the information collected, we hypothesized
that the conformational restrictions in this series of nucleo-
sides required the simultaneous presence of a positive charge
at the imidazole of the purine ring and an aromatic sub-
stituent at the 50-position of the sugar. Therefore, the con-
formational freedom of the dibenzyl derivative 1 should be
driven to a more restricted conformation by a simple change
in the pH of the medium through protonation at N7 of the
purine, and this was shown to be the case, as reported in
the final section of this paper.
Results and Discussion
Synthesis and NMR Experiments. The tribenzyl derivative
2 (Scheme 1) can be obtained in 56% yield in a single step by
reaction of 20,30-O-isopropylideneinosine (3) with an excess
of benzyl bromide in the presence of NaH at room tempera-
ture for 1 h followed by heating at 80 °C for an additional
hour. The synthesis of the N7-methyl derivative 4 was
achieved by treatment of 1-benzyl-50-O-benzyl-20,30-O-iso-
propylideneinosine (1)23 with methyl iodide in DMF at 60 °C
for 24 h and in the absence of base, as recently reported for
other purine bases.25
Here we report on a series of inosine derivatives whose
conformational freedom is constrained in solution, as shown
by 1H NMR, and for which we propose that the driving force
for this behavior is an intramolecular interaction between the
imidazolium of the purine base and the phenyl ring of the
benzyl substituent at the 50-position of the sugar. This study
On the other hand, treatment of 20,30-O-isopropylidenei-
nosine (3) with benzyl bromide and DBU26 in acetonitrile at
rt afforded 1-benzyl-20,30-O-isopropylideneinosine (5) in
91% yield (Scheme 2). Reaction of 5 with p-methylbenzyl
bromide in the presence of NaH in DMF at -20 °C for
30 min afforded the 50-O-(4-methylbenzyl) derivative 6 in
63% yield. Further treatment of 6 with methyl iodide in
DMF at 60 °C for 24 h yielded the N7-methyl nucleoside 7.
The structure of the N7-substituted inosines 2, 4, and 7 was
determined by 1H NMR, 13C NMR, gCOSY, gHMBC, and
gHSQC experiments. The 1H NMR spectra of compounds 4
and 7 showed the same pattern as for 2, with similar splitting
for the signals corresponding to the methylene groups and
the upfield chemical shift for the aromatic protons of the
(10) Hodel, A. E.; Gershon, P. D.; Quiocho, F. A. Mol. Cell 1998, 1, 443–
447.
(11) Lockless, S. W.; Cheng, H.-T.; Hodel, A. E.; Quiocho, F. A.;
Gershon, P. D. Biochemistry 1998, 37, 8564–8574.
(12) Guilligay, D.; Tarendeau, F.; Resa-Infante, P.; Coloma, R.; Crepin,
T.; Sehr, P.; Lewis, J.; Ruigrok, R. W. H.; Ortin, J.; Hart, D. J.; Cusack, S.
Nat. Struct. Mol. Biol. 2008, 15, 500–506.
(13) Hu, G.; Gershon, P. D.; Hodel, A. E.; Quiocho, F. A. Proc. Natl.
Acad. Sci. U.S.A. 1999, 96, 7149–7154.
(14) Quiocho, F. A.; Hu, G.; Gershon, P. D. Curr. Opin. Struct. Biol.
2000, 10, 78–86.
(15) Fechter, P.; Brownlee, G. G. J. Gen. Virol. 2005, 86, 1239–1249.
(16) Heemstra, J. M.; Moore, J. S. Chem. Commun. 2004, 1480–1481.
(17) Fokkens, M.; Jasper, C.; Schrader, T.; Koziol, F.; Ochsenfeld, C.;
Polkowska, J.; Lobert, M.; Kahlert, B.; Klarner, F.-G. Chem.;Eur. J. 2005,
11, 477–494.
(18) Salorinne, K.; Tero, T. R.; Riikonen, K.; Nissinen, M. Org. Biomol.
Chem. 2009, 7, 4211–4217.
(19) Schneider, H. J. Angew. Chem., Int. Ed. 2009, 48, 3924–3977.
(20) Yamada, S. Org. Biomol. Chem. 2007, 5, 2903–2912.
(21) Lavieri, S.; Zoltewicz, J. A. J. Org. Chem. 2001, 66, 7227–7230.
(22) Richter, I.; Minari, J.; Axe, P.; Lowe, J. P.; James, T. D.; Sakurai, K.;
Bull, S. D.; Fossey, J. S. Chem. Commun. 2008, 1082–1084.
(24) Casanova, E.; Aguado, L.; Jimeno, M. L.; Gago, F.; Camarasa,
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M. J.; Perez-Perez, M. J. Nucleosides, Nucleotides Nucleic Acids 2007, 26,
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Camarasa, M. J.; Balzarini, J.; Perez-Perez, M. J. J. Med. Chem. 2006, 49,
5562–5570.
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2003, 46, 1878–1885.
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FIGURE 2. 1H NMR spectra of compounds 1 and 2 in CDCl3.
SCHEME 1. Synthesis of 1-Benzyl-50-O-benzyl-7-methyl-20,30-O-isopropylideneinosine Iodide (4)
benzyl group at the 50-position (Figure 3 and Figure S1 in the
Supporting Information). Thus, as a representative example,
the 1H NMR spectrum of 4 (Figure 3) showed two different
signals for H-50 and H-500 protons (two doublets at δ 3.70 and
δ 4.69 ppm, respectively). Also, the signals corresponding to
the methylene unit of the benzyl group at the 50 position
appearedas two doublets (δ 4.47 and 4.72 ppm), and the same
pattern was observed for the benzylic protons at N1 (doublets
at 5.12 and 5.24 ppm). Moreover, the aromatic protons of the
benzyl at 50 were upfield shifted and appeared as three distinct
sets of signals at δ 6.85, 6.96, and 7.02 ppm for the para,
ortho, and meta protons, respectively. Finally, the H-8 from
the inosine moiety was markedly downfield shifted.
The 1H NMR spectrum of compound 4 was recorded
at two different concentrations (10 mg/0.55 mL or 0.5 mg/
0.55 mL) but no differences were observed. In addition,
changing the solvent from CDCl3 to the more polar DMSO-
d6 did not significantly modify the chemical shifts. Thus, these
results pointed toward an intramolecular stabilization. The
1H NMR spectrum of 4 in CDCl3 was also registered at
variable temperature (from 45 to -50 °C) (Figure S2 in the
Supporting Information). By increasing the temperature,
there was no evidence of coalescence, while by lowering the
temperature, there was no splitting of signals, exclusively
the upfield shift of some signals was observed (i.e, H8
and the aromatic protons at the 50-position, in particular the
1976 J. Org. Chem. Vol. 75, No. 6, 2010

Casanova et al.
JOCArticle
FIGURE 3. 1H NMR spectrum of 4 in CDCl3.
SCHEME 2. Synthesis of 1-Benzyl-7-methyl-50-O-(4-methylbenzyl)-20,30-O-isopropylideneinosine Iodide (7)
proton at the para-position). This suggests the existence of a
vastly populated conformer in a CDCl3 solution.
The 2D-NOESY and 2D-ROESY experiments carried
out with compound 7, with a 4-methylbenzyl group at the
50-position, perfectly matched with the correlations des-
cribed above for compound 4 (Figure S3 in the Supporting
Information). In this case, 1D-NOESY experiments were
performed by selective irradiation of the signal at 4.06 ppm,
assigned to the methyl at N7 of the purine, and the signal at
2.20 ppm, which corresponds to the methyl at the position
para of the benzyl group at 50 (Figure S4 in the Supporting
Information). From the irradiation of the signal at 4.06 ppm,
NOE signals were observed for the ortho and meta protons
of the benzyl group at 50, pointing again to a close proximity
between the imidazole of the base and the substituent at 50.
The irradiation of the signal at 2.20 ppm showed NOEs with
the ortho and meta protons of the same substituent as
expected, but also NOEs with the benzylic protons at N1,
that should only be possible if the aromatic substituent at 50,
and in particular its para position, is located over the sugar
and pointing toward the N1 of the base.
2D-NOESY and 2D-ROESY experiments provide valuable
information about the proximity of distal substituents. The
most significant correlation signals corresponding to com-
pound 4 are shown in Figure 4. Special attention should be
paid to the strong cross-peak between H8 and H500 (Figure 4a)
indicating a close proximity between both. The correlations
observed between the sugar protons H50 and H500 and the
benzylic protons also at this position are very illustrative: H500
shows a strong NOE only with one of the protons at the
benzylic position, while H50 shows NOE exclusively with the
geminal proton also at the benzylic position. In addition, H50
showed correlations with H30 andwithH40 of the furanose ring,
while H500 showed a cross-peak only with H40, suggesting there
was no free rotation around the C40-C50 bond. Thus, these
correlations point toward the lack of conformational freedom
at this part of the molecule (Figure 4b). Some other peaks of
medium or low intensity between the aromatic protons at the
50-position and the methyl at N7 or the H20 of the sugar
(Figure 4c) may suggest that the aromatic ring at the 50-position
is frequently located over the sugar plane and quite close to the
imidazole of the purine ring.
NMR Conformational Analysis. The conformation of
nucleosides can be defined by three structural parameters:
the puckering of the furanose ring, the conformation of the
exocyclic bond C40-C50 (defined by the torsion angle γ), and
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FIGURE 4. Correlations peaks from the 2D-NOESY and 2D-ROESY experiments for compound 4.
TABLE 1. Comparison of the Pseudorotational Parameters for Com-
pound 4 Based on Experimental and Calculated Values
NMR dataa AMBER datab 3J exptl (Hz)
θH1 -C1 -C2 -H2 c (deg)
110.4
-9.7
-96.0
252 (OT4)
33
109.1
-13.0
-89.9
234 (4E)
30
1.3
5.4
0
0
0
0
0
θH2 -C2 -C3 -H3 c (deg)
0
0
0
0
θH3 -C3 -C4 -H4 c (deg)
P furanose ringd
τme (deg)
0
0
0
0
aObtained from PSEUROT analysis. bObtained after geometry opti-
mization with HyperChem 7.5. cDihedral angle. dPseudorotational
angle (P), the corresponding conformation in the pseudorotational
circuit is represented in parentheses. eMaximum out-of-plane pucker.
the conformation of the glycosidic bond (defined by the
torsion angle χ).27,28 The puckering of the furanose ring in
solution can be determined experimentally by 1H NMR from
vicinal proton-proton coupling constants using the concept
of pseudorotation,29-31 in which the conformation of the
sugar ring is described by two parameters: the phase angle of
pseudorotation (P) and the puckering amplitude (νm). These
pseudorotational parameters have been calculated from the
FIGURE 5. Two different views of the 3D structure obtained for
compound 4 after geometry optimization.
gaucheþ rotamer, where the dihedral angles H40-C40-
C50-H50 and H40-C40-C50-H500 adopt values of þ60°
and -60°, respectively. This is in agreement with the cross-
peaks observed in Figure 4.
Concerning the conformation of the glycosidic bond, this
was determined from the cross-peaks observed for the signals
corresponding to protons H2 and H8 of the purine in the
NOESY/ROESY experiments. Thus, while H8 showed
cross-peaks with some protons on the β-face of the furanose
(H500 and H20), the H2 of the purine gave NOEs exclusively
with the benzylic and aromatic protons of the substituent at
N1, while no correlations were detected with any protons
from the furanose. These data point to a major anti con-
formation for the glycosidic bond.
On the basis of these data, a preliminary 3D structure for 4
was constructed and subjected to geometry optimization by
means of molecular mechanics by using the AMBER force
field as implemented in HyperChem 7.5.34 As a result, a
conformer was obtained in which the benzyl group at the
50-position faces the purine base (Figure 5). Comparison of
three experimental interprotonic coupling constants, 3JH1 ,H2
0
0
,
JH2 ,H3 , JH3 ,H4 , using the software PSEUROT-2.32 Thus, the
experimental data for compound 4 (Table 1) strongly support
the presence of a major conformer in solution with calculated
values for P and νm of 252° and 33°, respectively. These values
comply with a twist-type conformation OT4.
The conformation around the exocyclic C40-C50 bond, in
terms of three rotamers population, has been calculated from
3
3
0
0
0
0
3
3
0
0
0
00
the experimental JH4 ,H5 and JH4 ,H5 coupling constants
by using the appropriately parametrized Karplus equation.33
Results obtained pointed to a population of 90% of the
ꢀ
(27) Marquez, V. E.; Ezzitouni, A.; Russ, P.; Siddiqui, M. A.; Ford, H.,
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2344.
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(32) Deleew, F. A. A. M.; Vanbeuzekom, A. A.; Altona, C. J. Comput.
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1980, 36, 2783–2792.
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I. W.: Ontario, Canada, 2005.
1978 J. Org. Chem. Vol. 75, No. 6, 2010

Casanova et al.
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FIGURE 6. 1H NMR spectrum of 8 in CDCl3.
the angle values in this minimized conformer with those
determined experimentally on the basis of the interprotonic
coupling constants was in excellent agreement (Table 1).
Moreover this conformer accounts for the correlations seen
in the NOESY/ROESY experiments.
tonated species 1 (Figure S7 in the Supporting Information).
Thus the conformation of the dibenzyl derivative 1 switches
from a random to a restricted conformation in the presence of
TFA while the addition of base to the latest restores the random
conformation.
Can the Conformation of 50-O-Benzylinosines Be Driven by
a Change in pH?. From the experiments described above, it
could be argued that the simultaneous presence of a benzyl
group at the 50-position of the nucleoside and a positive
charge at the N7 of the imidazole ring lead to very significant
conformational restrictions such that the benzyl group is
located over the furanose ring and stacking with the posi-
tively charged base. Therefore an intramolecular cation-π
interaction could be the driving force for these conforma-
tional restrictions. If this is the case, a simple protonation of
the N7 of the purine in the dibenzyl derivative 1 could also
favor this type of interaction and result in a conformationally
restricted nucleoside in solution. In this respect, it has been
recently reported that purine nucleosides, when treated with
TFA, are thermodynamically protonated at N7.35 Thus,
when a solution of the dibenzyl derivative 1 in CDCl3 was
acidified by addition of TFA (10 equiv), the 1H NMR
spectrum drastically changed. The new species (8) showed
the “fingerprint” signals already observed for the other N7-
substituted compounds, affecting in particular the signals
corresponding to CH2O50, NCH2, H50, and H500 and aro-
matic signals (Figure 6). Although in our hands these
Molecular Dynamics Simulations. To assess whether a
molecular mechanics force field can account for these con-
formational preferences in solution, the behavior of the
major conformers determined for compounds 4 and 8 on
the basis of the NMR data was simulated by using molecular
dynamics (MD). The dibenzyl derivative 1 was also included
in these studies for comparative purposes. (See the Support-
ing Information for details.)
The NMR-determined structures for compounds 4 and 8
were used as the starting structures for the simulation. The
torsional restraints compatible with the experimental values
for 3JH-H were kept for 10 ns and this period was followed by
90 ns of unrestrained simulations. For the dibenzyl deriva-
tive 1, and since no hints of conformational restrictions had
been observed in the 1H NMR spectrum, the simulation was
performed unrestrained for the total time of 100 ns. All
simulations were carried out in an explicit solvent, that is, in a
CHCl3 box at 300 K and 1 atm. The AMBER force field was
employed as implemented in the Amber 8.0 release.36-38
Different conformational parameters were monitored as a
function of time for the three compounds by using the
PTRAJ module in Amber 8.0,38 including some dihedral
angles and distances between relevant atoms belonging to
distal substituents.
1
differences in the H NMR spectrum were already highly
indicative of similar conformational restrictions to those
observed for 2, 4, or 7, compound 8 was subjected to the
same battery of NMR experiments, conformational deter-
minations, and geometry optimization already enumerated
for the N7-methyl derivative (Figures S5 and S6, and Table
S1 in the Supporting Information). The results obtained with
compound 8 pointed again to an intramolecular stabilization
between the aromatic ring at 50 and the imidazolium ion of
the purine base.
Regarding the conformation around the C40-C50 bond in
compounds 4 and 8, MD simulations monitoringthedihedral
angle H40-C40-C50-H500 confirmed the gaucheþ confor-
mation, with an average value of -66° for both compounds,
as shown in Figure 7 for compound 8. These values are in
excellent agreement with the experimental values determined
from the NMR studies that indicated between 90% and 93%
of the conformers with such a dihedral angle value.
In addition, and based on a referee’s suggestion, it was
important to figure out if a new change in the pH toward
neutral values would restore the conformation of the
dibenzyl derivative to a random conformation. By adding
a base as diisopropylethylamine to a solution of the proto-
nated species 8in CDCl3, the1H NMR spectrum changed again
and became almost identical with the spectrum of the unpro-
To determine the relative spatial disposition of the sub-
stituent at the 50 position and the purine base, the distance
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J. Org. Chem. Vol. 75, No. 6, 2010 1979

Casanova et al.
JOCArticle
has been investigated by NMR techniques and molecular
modeling, including molecular dynamics simulations. These
compounds behave as conformationally restricted nucleo-
sides and we propose that the driving force for this restriction
is the stabilization through an intramolecular cation-π
interaction between the aromatic ring at the 50-position of
the ribose and the positively charged imidazolium ring in the
purine base. To the best of our knowledge there are no
previous examples involving imidazolium ions in intramole-
cular cation-π interactions in small molecules. Thus, the
cation-π interaction between imidazolium ions and aro-
matic rings can be proposed in the design of small molecules
involved in guest-host interactions or organic synthesis.
The imidazolium ion required for the above-described
interaction can be obtained by an acidification of the pH
of the medium. Thus, by a simple change in pH the con-
formation of the nucleoside switches from a random to a
restricted conformation, as shown for compound 8. There-
fore, these inosine derivatives can be considered as a new
class of pH-dependent conformationally switchable mole-
cules.
FIGURE 7. MD simulations. H40-C40-C50-H500 dihedral angle
(deg) for compound 8.
Experimental Section
1,7-Dibenzyl-50-O-benzyl-20,30-O-isopropylideneinosine Bro-
mide (2). To a solution of 20,30-O-isopropylideninosine (3, 200
mg, 0.63 mmol) in dry DMF (4 mL) was added NaH (60% in
mineral oil, 81 mg, 2.10 mmol). The reaction was stirred at room
temperature for 15 min. Then, benzyl bromide (0.24 mL, 2.03
mmol) was added and stirring was continued for 1 h at room
temperature and 1 h at 80 °C. Volatiles were removed, and the
residue was purified by flash column chromatography (CH2Cl2:
MeOH, 100:1) to yield 2 (235 mg, 56%) as a white solid. Mp
(CH2Cl2:MeOH) 82-84 °C; 1H NMR (500 MHz, CDCl3) δ
1.43, 1.63 (s, 6H, C(CH3)2), 3.64 (dd, J = 10.8, 2.2 Hz, 1H, H-
50), 4.33 (d, J = 11.2 Hz, 1H, CH2O), 4.66 (d, J = 11.2 Hz, 1H,
CH2O), 4.75 (dd, J = 10.9, 1.8 Hz, 1H, H-500), 4.85 (m, 1H, H-
40), 4.94 (d, J = 5.8 Hz, 1H, H-30), 5.11 (d, J = 14.3 Hz, 1H,
CH2N-1), 5.25 (d, J = 14.3 Hz, 1H, CH2N-1), 5.41 (dd, J = 5.7,
1.4 Hz, 1H, H-20), 5.54 (d, J = 13.6 Hz, 1H, CH2N-7), 6.01 (d,
J = 1.4 Hz, 1H, H-10), 6.39 (d, J = 13.6 Hz, 1H, CH2N-7), 6.83
(m, 1H, p-CH-OBn), 6.96 (m, 4H, OBn), 7.25-7.33 (m, 3H, 7-
NBn), 7.41-7.49 (m, 5H, 1-NBn), 7.84-7.87 (m, 2H, o-CH-7-
NBn), 8.24 (s, 1H, H-2), 10.96 (s, 1H, H-8); 13C NMR (125
MHz, CDCl3) δ 25.2 (CH3), 26.9 (CH3), 50.3 (CH2N-1), 51.9
(CH2N-7), 70.2 (C-50), 72.25 (CH2O), 82.8 (C-30), 85.9 (C-20),
88.7 (C-40), 96.6 (C-10), 113.8 (C(CH3)2), 114.2 (C-5), 127.0,
127.5, 127.9, 134.0 (7-NBn), 129.0, 129.2, 129.3, 134.0 (1-NBn),
129.0, 129.2, 129.7, 137.5 (OBn), 139.6 (C-8), 144.3 (C-4), 150.6
(C-2), 151.7 (C-6); MS (ES, positive mode) m/z 579 [M]þ. Anal.
FIGURE 8. MD simulations. Distances (expressed in A) between
the ipso carbon of the aromatic ring at 50 and the N7 of the purine
base from the MD trajectories calculated for 1 (black), 4 (magenta,)
and 8 (blue) in a CHCl3 solution.
between the ipso carbon of the aromatic ring at 50 and the N7 of
the purine base was monitored. As shown in Figure 8, this
distance for 1 (black line) showed great variations, with values
oscillating between 4 and 10 A, as expected for a conformation-
ally unrestrained compound. However, this distance remained
almost constant for 4 (displayed in magenta) and 8 (displayed in
blue) along the whole simulation, with average values of 4.3 and
3.8 A, respectively, in agreement with the lack of conformational
freedom of both substituents and as an indication of the
proximity between the inosine base and the aromatic ring at
the 50 position. Since the only structural difference between
compounds 1 and 8 is the presence of a proton at N7, leading to
the transformation of the purine imidazole into an imidazolium
ion, it can be proposed that the conformational restrictions
observed for the protonated compound 8 are due to an intra-
molecular cation-π interaction between the aromatic substitu-
ent at 50 and the imidazolium ion in the purine base. It should be
noticed that the distance of 3.8 A monitored in the MD
calculations closely resembles the reported intermolecular dis-
tances between the cationic 7-methylG of the 50-cap and the side
chains of Trp102B and Trp56B in the cap binding protein
eIF4E8 and other systems involving cation-π interactions.4
Calcd for C34H35BrN4O5 H2O: C, 60.27; H, 5.50; N, 8.27.
3
Found: C, 59.89; H, 5.13; N, 8.58.
1-Benzyl-50-O-benzyl-7-methyl-20,30-O-isopropylideneinosine
Iodide (4). To a solution of 1-benzyl-50-O-benzyl-20,30-O-isopro-
pylideneinosine (1, 87 mg, 0.18 mmol) in dry DMF (1.5 mL) was
added methyl iodide (22 μL, 0.35 mmol). The reaction was
stirred at 60 °C overnight. Volatiles were removed, and the
residue was purified by CCTLC in the Chromatotron (CH2Cl2:
MeOH, 20:1) to yield 4 (76 mg, 69%) as a yellow solid. Mp
(CH2Cl2:MeOH) 186-188 °C; 1H NMR (500 MHz, CDCl3) δ
1.43, 1.59 (s, 6H, C(CH3)2), 3.70 (dd, J = 10.8, 1.8 Hz, 1H, H-
50), 4.12 (s, 3H, CH3-N7), 4.47 (d, J = 11.1 Hz, 1H, CH2O), 4.69
(dd, J = 10.8, 2.0 Hz, 1H, H-500), 4.72 (d, J = 11.1 Hz, 1H,
CH2O), 4.83 (m, 1H, H-40), 4.98 (d, J = 5.8 Hz, 1H, H-30), 5.12
(d, J = 14.4 Hz, 1H, CH2N-1), 5.24 (d, J = 14.4 Hz, 1H, CH2-
N-1), 5.43 (dd, J = 5.8, 1.4 Hz, 1H, H-20), 6.11 (d, J = 1.4 Hz,
Conclusions
The conformational behavior of a series of N7-substituted
inosines with an aromatic ring at the 50-position of the ribose
1980 J. Org. Chem. Vol. 75, No. 6, 2010

Casanova et al.
JOCArticle
1H, H-10), 6.85 (m, 1 H, p-CH-OBn), 6.96 (m, 2H, m-CH-OBn),
7.02 (m, 2H, o-CH-OBn), 7.39-7.46 (m, 5H, NBn), 8.31 (s, 1H,
H-2), 10.31 (s, 1H, H-8); 13C NMR (125 MHz, CDCl3) δ 25.2
(CH3), 26.9 (CH3), 36.8 (CH3-N7), 50.3 (CH2N-1), 70.5 (C-50),
72.4 (CH2O), 82.7 (C-30), 86.1 (C-20), 88.7 (C-40), 96.4 (C-10),
113.9 (C(CH3)2), 115.2 (C-5), 126.9, 127.6, 128.0, 137.5 (OBn),
128.9, 129.3, 129.3, 133.8 (NBn), 139.3 (C-8), 144.1 (C-4), 150.5
(C-2), 151.8 (C-6); MS (ES, positive mode) m/z 503 [M]þ. Anal.
Calcd for C28H31IN4O5: C, 53.34; H, 4.96; N, 8.89. Found: C,
53.18; H, 5.02; N, 8.87.
dry DMF (1.0 mL) was added methyl iodide (25 μL, 0.40 mmol).
The reaction was stirred at 60 °C overnight. Volatiles were
removed, and the residue was purified by CCTLC in the
Chromatotron (CH2Cl2:MeOH, 10:1) to yield 7 (50 mg, 49%)
1
as a yellow solid. Mp (CH2Cl2:MeOH) 99-101 °C; H NMR
(500 MHz, CDCl3) δ 1.43, 1.58 (s, 6H, C(CH3)2), 2.20 (s, 3H,
CH3-Ph), 3.70 (dd, J = 10.9, 2.0 Hz, 1H, H-50), 4.06 (s, 3H, CH3-
N7), 4.48 (d, J = 11.0 Hz, 1H, CH2O), 4.61 (dd, J = 10.9, 2.2
Hz, 1H, H-500), 4.69 (d, J = 11.1 Hz, 1H, CH2O), 4.81 (m, 1H,
H-40), 4.99 (d, J = 5.9 Hz, 1H, H-30), 5.13 (d, J = 14.5 Hz, 1H,
CH2N-1), 5.29 (d, J = 14.5 Hz, 1H, CH2N-1), 5.45 (dd, J = 1.4,
5.8 Hz, 1H, H-20), 6.13 (d, J = 1.0 Hz, 1H, H-10), 6.90 (m, 2H,
m-CH-OBn), 6.96 (m, 2H, o-CH-OBn), 7.33-7.45 (m, 5H,
NBn), 8.29 (s, 1H, H-2), 10.31 (s, 1H, H-8); 13C NMR (125
MHz, CDCl3) δ 21.0 (CH3-Ph), 25.2 (CH3), 26.8 (CH3), 36.7
(CH3-N7), 50.0 (CH2N-1), 70.4 (C-50), 72.4 (CH2O), 82.6 (C-30),
86.1 (C-20), 88.7 (C-40), 96.1 (C-10), 113.8 (C(CH3)2), 115.1 (C-5),
127.0, 128.7, 134.7, 137.9 (OBn), 128.5, 129.1, 129.3, 134.2
(NBn), 139.3 (C-8), 144.21 (C-4), 150.7 (C-2), 151.8 (C-6); MS
(ES, positive mode) m/z 517 [M]þ. Anal. Calcd for C29H33-
1-Benzyl-20,30-O-isopropylideneinosine (5).39,40. To a solution
of 20,30-O-isopropylideninosine (3, 500 mg, 1.62 mmol) in
CH3CN (10 mL) were added benzyl bromide (0.17 mL, 1.41
mmol) and DBU (208 μL, 2.14 mmol). The reaction was stirred
at room temperature for 30 min. Volatiles were removed, and
the residue was purified by flash column chromatography
(AcOEt:MeOH, 10:1) to yield 5 (585 mg, 91%) as an amorphous
solid. 1H NMR (300 MHz, CDCl3) δ 1.36, 1.64 (s, 6H, C(CH3)2),
3.78 (m, 1H, H-50), 3.93 (m, 1H, H-500), 4.49 (m, 1H, H-40),
5.03-5.13 (m, 3H, H-30, H-20, OH), 5.16 (m, 2H, CH2N), 5.84
(d, J = 4.2 Hz, 1H, H-10), 7.35 (m, 5H, Ph), 7.87 (s, 1H, H-8),
8.04 (s, 1H, H-2); MS (ES, positive mode) m/z 399 [M þ 1]þ.
1-Benzyl-50-O-(4-methylbenzyl)-20,30-O-isopropylideneinosine
(6). To a solution of 5 (150 mg, 0.38 mmol) in dry DMF (2.5 mL)
were added NaH (60% in mineral oil, 50 mg, 1.24 mmol) and
4-methybenzyl bromide (63 mg, 0.34 mmol). The reaction was
stirred at -20 °C for 30 min, and then it was neutralized by the
addition of AcOH. Volatiles were removed, and the residue was
purified by CCTLC in the Chromatotron (CH2Cl2:MeOH,
20:1) to yield 6 (120 mg, 63%) as an amorphous solid. 1H
NMR (300 MHz, CDCl3) δ 1.35, 1.59 (s, 6H, C(CH3)2), 2.32
(s, 3H, CH3-Ph), 3.57 (dd, J = 10.5, 3.8 Hz, 1H, H-50), 3.64 (dd,
J = 10.5, 3.1 Hz, 1H, H-500), 4.42 (d, J = 11.7 Hz, 1H, CH2O),
4.46 (d, J = 11.7 Hz, 1H, CH2O), 4.52 (m, 1H, H-40), 4.89 (dd,
J = 6.1, 2.2 Hz, 1H, H-30), 5.12 (dd, J = 6.1, 2.7 Hz, 1H, H-20),
5.22 (d, J = 14.7, 1H, CH2N-1), 5.27 (d, J = 14.7, 1H, CH2N-1),
6.10 (d, J = 2.7 Hz, 1H, H-10), 7.10-7.13 (m, 4H, OBn),
7.29-7.35 (m, 5H, NBn), 7.98 (s, 1H, H-8), 8.02 (s, 1H, H-2);
13C NMR (125 MHz, CDCl3) δ 21.1 (CH3-Ph), 25.3 (CH3), 27.2
(CH3), 49.1 (CH2N-1), 69.9 (C-50), 73.5 (CH2O), 81.8 (C-30),
85.3 (C-20), 85.8 (C-40), 91.5 (C-10), 114.1 (C(CH3)2), 124.8 (C-5),
128.0, 129.1, 134.0, 137.7 (OBn), 128.2, 128.3, 129.0, 135.9
(NBn), 138.5 (C-8), 146.9 (C-4), 147.1 (C-2), 156.4 (C-6); MS
(ES, positive mode) m/z 503 [M þ 1] þ. Anal. Calcd for
C28H30N4O5: C, 66.92; H, 6.02; N, 11.15. Found: C, 66.80; H,
6.14; N, 11.09.
IN4O5 H2O: C, 52.57; H, 5.32; N, 8.46. Found: C, 52.36; H,
3
5.10; N, 8.65.
Treatment of 1 with TFA. 1-Benzyl-50-O-benzyl-20,30-O-iso-
propylideneinosine (1, 7.0 mg, 0.014 mmol) was dissolved in
CDCl3 (0.55 mL) and introduced in a NMR tube. A H 1D
1
NMR spectrum was recorded at 298 K. Then, TFA (10.6 μL,
0.143 mmol) was added and a new 1H-1D NMR spectrum was
recorded that corresponds to compound 8. 1H NMR (500 MHz,
CDCl3) δ 1.46, 1.64 (s, 6H, C(CH3)2), 3.69 (dd, J = 10.9, 1.5 Hz,
1H, H-50), 3.91 (dd, J = 10.9, 1.7 Hz, 1H, H-500), 4.24 (d, J =
10.8 Hz, 1H, CH2O), 4.38 (d, J = 10.8 Hz, 1H, CH2O), 4.86 (m,
1H, H-40), 4.94 (d, J = 5.7 Hz, 1H, H-30), 5.17 (d, J = 14.4 Hz,
1H, CH2N-1), 5,28 (d, J = 14.4 Hz, 1H, CH2N-1), 5.29 (d, J =
1.4 Hz, 1H, H-20), 6.16 (d, J = 1.4 Hz, 1H, H-10), 6.89-7.04
(m, 5, OBn), 7.36-7.46 (m, 5H, NBn), 8.26 (s, 1H, H-2), 9.03 (s,
1H, H-8).
Acknowledgment. This research was supported by grants
from the Spanish CICYT [SAF2006-12713-C02] and the
Comunidad de Madrid [BIPEDD-CM, S-BIO/0214/2006].
E.C. thanks the Comunidad de Madrid and the Fondo Social
Europeo (FSE) for a predoctoral fellowship. E-M.P. has a
CSIC contract from the I3P programme financed by the
ꢀ
FSE. L.A. thanks the Spanish Ministerio de Educacion y
Ciencia for a FPU predoctoral fellowship.
1-Benzyl-7-methyl-50-O-(4-methylbenzyl)-20,30-O-isopropyli-
deneinosine Iodide (7). To a solution of 6 (100 mg, 0.20 mmol) in
Supporting Information Available: General chemical pro-
cedures and conditions for NMR studies and molecular dyna-
mics simulations are provided, Figures S1-S7 and Table S1,
and spectra of compounds 2 and 4-8. This material is available
free of charge via the Internet at http://pubs.acs.org.
(39) Lichtenthaler, F. W.; Kitahara, K.; Riess, W. Nucleic Acids Res.,
Spec. Publ. 1978, 4, s115-s118.
(40) Montgomery, J. A.; Hewson, K.; Clayton, S. J.; Thomas, H. J.
J. Org. Chem. 1966, 31, 2202–2210.
J. Org. Chem. Vol. 75, No. 6, 2010 1981