7,5′-O-dibenzylinosines: Synthesis and studies on their conformational properties

Article abstract of DOI:10.1080/15257770701490696

Reaction of 2′,3′-O-isopropylidene inosine with benzyl bromide (1 h, rt) led to the 1,5′-O-dibenzylderivative 4, but by increasing the reaction time or the temperature, compound 4 is further transformed into the 1,7,5′-O-tribenzylinosine derivative 5. Sim

Full text of DOI:10.1080/15257770701490696

Nucleosides, Nucleotides, and Nucleic Acids, 26:695–699, 2007
Copyright ꢀ Taylor & Francis Group, LLC
C
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770701490696
7,5ꢀ-O-DIBENZYLINOSINES: SYNTHESIS AND STUDIES ON THEIR
CONFORMATIONAL PROPERTIES
Elena Casanova and Leire Aguado
Instituto de Qu´ımica Me´dica (CSIC),
2
Madrid, Spain
Mar´ıa-Luisa Jimeno
Centro de Qu´ımica Orga´nica Manuel Lora-Tamayo
2
(CSIC), Madrid, Spain
Federico Gago
Departamento de Farmacolog´ıa, Universidad de Alcala´,
2
Madrid, Spain.
Mar´ıa-Jose´ Camarasa and Mar´ıa-Jesu´ s Pe´rez-Pe´rez
Instituto de Qu´ımica
2
Me´dica (CSIC), Madrid, Spain
Reaction of 2ꢁ,3ꢁ-O-isopropylidene inosine with benzyl bromide (1 h, rt) led to the 1,5ꢁ-O-
2
dibenzylderivative 4, but by increasing the reaction time or the temperature, compound 4 is fur-
ther transformed into the 1,7,5ꢁ-O-tribenzylinosine derivative 5. Similarly, the 7-methyl-1,5ꢁ-O-
dibenzylderivative 6 has been synthesized from 4. The 1 H-NMR spectra of 5 and 6 showed peculiar
chemical shifts for geminal protons (H5ꢁ and H5ꢁꢁ of the ribose, and the CH2 of the benzyl groups).
Preliminary NMR studies have been performed, including NOESY experiments that point toward
the predominant existence of conformers that are stabilized by an electrostatic interaction between
the positively charged imidazole of the base moiety and the high electron density of the 5ꢁ-benzyl
substituent.
Keywords Inosine; benzylation; NMR studies; conformation; intramolecular
interactions
INTRODUCTION
The N7 position of guanine has been extensively studied due to cova-
lent bond formation at this site when double-stranded DNA is exposed to
electrophiles.[1] Moreover, 7-methylguanosine (1) (Figure 1) is present in
the mRNA 5ꢁ-cap structure that serves as a recognition site for numerous
enzymes.[2] The positive charge of the imidazole ring of the guanine base
E. Casanova acknowledges the Comunidad de Madrid, Spain, and the Fondo Social Europeo
(F.S.E.) for a predoctoral grant. L. Aguado acknowledges the Spanish MEC for a FPU grant. This work
has been supported by the Spanish MEC (SAF2003-07219-C02-01 and C02-02).
Address correspondence to Mar´ıa-Jesu´s Pe´rez-Pe´rez, Instituto de Qu´ımica Me´dica (CSIC), Madrid
28006, Spain. E-mail: mjperez@iqm.csic.es
695

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E. Casanova et al.
FIGURE 1
in both examples is essential for enzyme recognition. Therefore, model
compounds modified at position N7 of guanine have been subjected to
numerous studies to better understand the role of this site in biological in-
teractions. Interestingly, the N7 position of inosine has received much less
attention.
In this communication we report on the easy functionalization of posi-
tion N7 of inosine and on the preliminary conformational properties of the
resulting 5ꢁ-O-benzyl-7-susbstituted inosines of general formula 2.
RESULTS AND DISCUSSION
When performing the reaction between 2ꢁ,3ꢁ-O-isopropylidene ino-
sine (3) and benzyl bromide (Scheme 1), we found that the 1,5ꢁ-O-
dibenzylderivative 4 is initially obtained (1 h, rt). However, upon increasing
either the reaction time or the temperature, compound 4 is further trans-
1
formed into the 1,7,5ꢁ-O-tribenzylinosine derivative 5. The H-NMR spec-
trum of 5 showed peculiar chemical shifts for geminal protons (H5ꢁ and
H5ꢁꢁ of the ribose, and the CH2 of the benzyl groups). In order to evaluate
whether this characteristic spectrum is due to the simultaneous presence of
the benzyl groups at 5ꢁ and 7 positions, or to the positive charge of the imida-
zole ring, we synthesized the corresponding 7-methyl-1,5ꢁ-O-dibenzylinosine
(6) (Scheme 1) by reaction of 4 with MeI in DMF at 60◦C in the absence of
base, as recently reported for N1,N7-disubstituted purines.[3] Interestingly,
the 1H-NMR spectrum of the 7-methyl derivative 6 showed similar chemical
shifts for geminal protons to those of the 7-benzyl derivative 5.
Full structural assignments of compounds 5 and 6 have been performed
based on mono- and bidimensional experiments (gCOSY, gHSQC, and
gHMBC). Moreover, NOESY and ROESY experiments were carried out to
obtain information about the conformational properties. Since the spectra
of the 7-methyl derivative (6) are simpler than those of the 7-benzyl deriva-
tive (5), compound 6 was taken as a model compound, although the same
features are present in the spectra of compound 5. In the NOESY spectrum
of compound 6, particular attention has been paid to cross peaks relative to

7,5ꢁ-O-Dibenzylinosines: Synthesis and Studies
697
SCHEME 1 Reagents and conditions (a) 2.2 equiv BrBn, 3.3 equiv. Nah, DMF, 1 h rt; (b) longer reaction
time or heating; (c) 2.2 equiv. Mel, DMF, 24 h, 60◦C.
the H-8 of the base, and the o-proton of the Ph ring at 5ꢁ (Figures 2a and 2b,
respectively). On the basis of the available NMR restraints, the 3-D structure
of the molecule was constructed and energy minimization was run at the
AM1 level using HyperChemTM 7.52. The conformers 6A and 6B obtained
FIGURE 2 Selected peaks of the NOESY spectrum of 6. (a) interesting peaks between the H-8 of the
base and the sugar protons H1ꢁ (A) and H5ꢁꢁ (B) on one hand, and between the H-8 of the base and the
sugar protons H2ꢁ (Aꢁ) and H5ꢁ (Bꢁ). (b) interesting peaks between the o-proton of the Ph ring at 5ꢁ and
the sugar protons H2ꢁ (C) and H3ꢁ (D), and CH-Oꢁꢁ proton on one hand, and between the o-proton of
the Ph ring at 5ꢁ and CH-Oꢁ (Cꢁ); a strong NOE is observed between the o-proton of the Ph ring at 5ꢁ
and N7-CH3 (E).

698
E. Casanova et al.
FIGURE 3 Computed molecular models showing possible conformers of 6 compatible with the NOESY
cross peaks. Confomer 6A explains the cross peaks A, B, C, and D in Figure 2 while conformer 6B is
consistent with cross peaks Aꢁ, Bꢁ, Cꢁ, and D in Figure 2.
(Figure 3) are in agreement with the observed NOE effects. The strong NOE
between the o-proton of the Ph ring at 5ꢁ and the 7-CH3 could further sup-
port the spatial proximity of the aromatic ring at 5ꢁ and the imidazole ring
in both conformers.
These results allow us to propose the following conclusions:
1. In both cases (6A and 6B), the conformation of the sugar ring is O(4ꢁ)-
exo, probably due to the presence of the isopropylidene moiety, which is
in agreement with the reported X-ray structure of isopropylidene inosine
3.[4]
2. The glycosidic bond is predominantly anti based on the NOE effect be-
tween H8 and H5ꢁꢁ (conformer 6A), and the strong NOE between H8
and H2ꢁ (conformer 6B). This is in agreement with the preferred anti
conformation reported for 7-methyl guanosine.[2]
3. The proposed conformations for 6A and 6B are likely to result from the
stabilization brought about by the electrostatic interaction between the
positively charged base and the high electron density of the 5ꢁ-benzyl
moiety.
A more detailed study of the nucleoside conformation is in progress.
REFERENCES
1. Gates, K.S.; Nooner, T.; Dutta, S. Biologically relevant chemical reactions of N7-alkylguanine residues
in DNA. Chem. Res. Toxicology 2004, 17, 839–856.

7,5ꢁ-O-Dibenzylinosines: Synthesis and Studies
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2. Mikkola, S.; Zhang, Z.; Ma¨ki, E.; Lo¨nnberg, H. Preparation and properties of mRNA 5ꢁ-cap structure.
Current Organic Chem. 2005, 9, 999–1022.
3. Fu, H.; Lam, Y. Traceless solid-phase synthesis of N1,N7-disubstituted purines. J. Combinatorial Chem.
2005, 7, 734–738.
4. Mande, S.; Shamala, N.; Seshadri, T.P.; Viswamitra, M.A. Conformational variability in modified
nucleosides. Crystal and molecular structure of 2ꢁ,3ꢁ-O-isopropylidene inosine. Nucleosides and Nu-
cleotides. 1992, 11, 1103–1114.