Synthesis of Novel BicycloRibonucleosides; 2'-Amino- and 2'-Thio-LNA Monomeric Nucleosides

Full text of DOI:10.1021/jo9806658

6078
J . Org. Chem. 1998, 63, 6078-6079
Communications
Syn th esis of Novel Bicyclo[2.2.1]
Ribon u cleosid es: 2′-Am in o- a n d 2′-Th io-LNA
Mon om er ic Nu cleosid es
Sanjay K. Singh, Ravindra Kumar, and J esper Wengel*
Center for Synthetic Bioorganic Chemistry, Department of
Chemistry, Chemical Laboratory II, University of Copenhagen,
Universitetsparken 5, DK-2100 Copenhagen, Denmark
F igu r e 1. Structure and properties of LNA. ∆Tm ) change in
duplex melting temperatures per LNA monomer incorporated.
Base ) pyrimidine or purine nucleobases.
Received April 13, 1998
The immense potential of oligonucleotide analogues as
therapeutic agents or diagnostic molecules has stimulated
intensive research on nucleic acid mimics during the last
years.1 Conformationally restricted analogues have been
synthesized,2 and especially oligonucleotide 2′-fluoro N3′-
P5′-phosphoramidates have shown appealing characteristics.2a
We have recently introduced LNA (locked nucleic acids) as
a novel class of preorganized oligonucleotide analogues
showing very interesting properties (Figure 1).3,4 Parent
LNA monomeric nucleosides were in our initial approach
synthesized as indicated in Scheme 1.3 Condensation of 4-C-
(acetoxymethyl)-1,2-di-O-acetylfuranose 1 with silylated
nucleobases afforded nucleoside diols 2 after deacetylation.
LNA nucleosides 3 were subsequently obtained after mono-
tosylation, base-induced ring closure, and debenzylation.3,5
The characteristics of LNA appear to be related to the
fixed 3′-endo conformation of the monomeric LNA nucleo-
sides (Figure 2).6 One possible implication is that duplex
formation involving LNA is entropically favored. Stimulated
by LNA, a number of novel analogues can be identified as
attracting synthetic targets, among these the 2′-amino- and
2′-thio-LNA nucleosides 7 and 11. As the first step toward
the evaluation of the properties of 2′-amino- and 2′-thio-LNA
oligomers, e.g., the affinities toward complementary nucleic
acids, we have investigated the synthesis and conformational
behavior of compounds 7 and 11. Especially appealing
seems the possibility of utilizing the 2′-amino functionality
of nucleoside 7 as a structurally well-defined conjugation site
in LNAs.
Sch em e 1. Syn th esis of LNA Nu cleosid esa
a
Key: (a) (i) silylated base, TMS-triflate, (ii) deacetylation; (b) (i)
monotosylation, (ii) ring-closure, (iii) debenzylation. Base ) nucleobase.
F igu r e 2. Conformations of nucleic acid monomers in B-type
(generally DNA/DNA) duplexes and in A-type (generally DNA/
RNA and RNA/RNA) duplexes and of LNA nucleosides.
desired bicyclo[2.2.1] 2′-(benzylamino)-2′-deoxynucleoside 6a .
Analogously, the uracil nucleoside 5b using potassium
thioacetate in DMF gave the bicyclo[2.2.1] 2′-deoxy-2′-
thionucleoside 6b in 75% yield (deacetylation proceeds
during the reaction). The strategy of double nucleophilic
substitution reactions to obtain heteroatom analogues of
LNA nucleosides thus proved effective. Mechanistically,
these reactions are expected to proceed via 2,2′-anhydro
nucleoside intermediates,2d explaining the overall retention
of the configuration at the 2′-carbon atom. As the formation
of such intermediates requires intramolecular displacement
by an O-2 carbonyl in the base, this mechanistic suggestion
implies that the synthetic strategy will be applicable for
pyrimidine nucleosides only. The concomitant debenzylation
proved troublesome. Thus, catalytic hydrogenation (20% Pd-
(OH)2/C, H2) of 3′,5′-di-O-benzyl 2′-(benzylamino)-LNA nu-
cleoside 6a furnished a monobenzylated intermediate even
after prolonged reaction time (5 days) using a large excess
of catalyst. This intermediate could eventually be deben-
zylated using ammonium formate and 10% palladium on
carbon, giving the desired parent 2′-amino-LNA nucleoside
7 [1-(2-amino-2-deoxy-2-N,4-C-methylene-â-D-ribofuranosyl)-
thymine] in 54% yield from 6a . Alternatively, one-step
deprotection of 6a (affording 7 in 67% yield) was performed
with ammonium formate and 10% palladium on carbon.
4′-C-(Hydroxymethyl) nucleoside diols 4a and 4b3 were
converted into the di-O-tosylated nucleosides 5a and 5b in
80% and 78% yield, respectively, by reactions with p-
toluenesulfonyl chloride and 4-(N,N-dimethylamino)pyridine
(DMAP) in dichloromethane. Reaction of the thymine
nucleoside 5a in benzylamine afforded in 30% yield the
(1) (a) Herdewijn, P. Liebigs Ann. 1996, 1337. (b) Freier, S. M.; Altmann,
K.-H. Nucleic Acids Res. 1997, 25, 4429.
(2) (a) Schultz, D. G.; Gryaznov, S. M. Nucleic Acids Res. 1996, 24, 2966.
(b) Marquez, V. E.; Siddiqui, M. A.; Ezzitouni, A.; Russ, P.; Wang, J .;
Wagner, R. W.; Matteucci, M. D. J . Med. Chem. 1996, 39, 3739. (c) Bolli,
M,.; Litten, J . C.; Schu¨tz, R.; Leumann, C. J . Chem. Biol. 1996, 3, 197. (d)
Nielsen, P.; Pfundheller, H. M.; Olsen, C. E.; Wengel, J . J . Chem. Soc.,
Perkin Trans. 1 1997, 3423. (e) Steffens, R.; Leumann, C. J . J . Am. Chem.
Soc. 1997, 119, 11548.
(3) Singh, S. K.; Nielsen, P.; Koshkin, A. A.; Wengel, J . Chem. Commun.
1998, 455. (b) Koshkin, A. A.; Singh, S. K.; Nielsen, P.; Rajwanshi, V. K.;
Kumar, R.; Meldgaard, M.; Olsen, C. E.; Wengel, J . Tetrahedron 1998, 54,
3607.
(4) LNA is defined as an oligonucleotide (analogue) containing one or
more LNA nucleoside monomers.
(5) The LNA nucleosides containing uracil and cytosine nucleobases have
been synthesized independently by a linear approach. See: Obika, S.;
Nanbu, D.; Hari, Y.; Morio, K.; In, Y.; Ishida, T.; Imanishi, T. Tetrahedron
Lett. 1997, 38, 8735.
(6) Preorganization of LNA nucleosides into a 3′-endo-type conformation
has been shown by X-ray crystallography, see ref 5, and by NMR studies,
see ref 3.
S0022-3263(98)00665-3 CCC: $15.00 © 1998 American Chemical Society
Published on Web 08/08/1998

Communications
J . Org. Chem., Vol. 63, No. 18, 1998 6079
Sch em e 2a
Sch em e 3a
a
a
Key: (a) TsCl, DMAP, CH2Cl2; (b) BnNH2 (to give 6a ); KSAc, DMF
(to give 6b); (c) (i) 20% Pd(OH)2/C, H2, ethanol, (ii) 10% Pd/C,
ammonium formate, methanol (alternatively 10% Pd/C, ammonium
formate, methanol, one step) (to give 7).
Key: (a) 20% Pd(OH)2/C, H2, ethanol; (b) TIPDSCl2, anhydrous
pyridine; (c) KSAc, DMF; (d) TBAF, THF. [Si] ) 1,1,3,3-tetraisoprop-
yldisiloxane-1,3-diyl.
that 7 (2′-amino-LNA nucleoside, Figure 2, Y ) NH) and 11
(2′-thio-LNA nucleoside, Figure 2, Y ) S), like the parent
LNA nucleosides (Figure 2, Y ) O), exist in 3′-endo type
conformations. This conclusion is substantiated by the
strong and unusual NOEs observed between H-3′ and H-6
for compounds 6b and 7.10
Debenzylation of 2′-thio nucleoside 6b proved impossible in
our hands. Thus, probably because of catalyst poisoning,
catalytical hydrogenations (20% Pd(OH)2/C, H2, ethanol; 10%
Pd/C, H2, methanol; 10% Pd/C, 1,4-cyclohexadiene, metha-
nol) failed. Likewise, when a variety of other methods (BCl3,
dichloromethane, hexane; 20% Pd(OH)2/C, ammonium for-
mate, methanol; BBr3, dichloromethane; sodium, ethanol;
CrO3/CH3COOH; iodotrimethylsilane) were employed,7 ei-
ther no reaction or cleavage of the glycosidic bond was
observed (Scheme 2).
To obtain the desired 2′-thio nucleoside 11, the synthetic
strategy depicted in Scheme 3 was followed. Debenzylation
(49% yield, 20% Pd(OH)2/C, H2) of the di-O-tosylated uracil
nucleoside 5b to give 8 followed by 3′,5′-di-O-silylation in
97% yield using the bidentate reagent 1,3-dichloro-1,1,3,3-
tetraisopropyldisiloxane (TIPDSCl2) afforded the bicyclic
intermediate 9. Conversion of 9 into the tricyclic intermedi-
ate 10 was accomplished in 77% yield by reaction with
potassium thioacetate in DMF. Eventually, desilylation
using tetrabutylammonium fluoride (TBAF) in THF afforded
the desired 2′-thio-LNA nucleoside 11 [1-(2-deoxy-2-mer-
capto-2-S,4-C-methylene-â-D-ribofuranosyl)uracil] in 69%
yield.
On the basis of the results reported herein and earlier,3,5
the bicyclo[2.2.1] nucleoside skeleton appears very attractive
as a structural element of nucleic acid mimics containing
monomeric nucleosides in conformationally restricted 3′-
endo-type configurations. This has been clearly demon-
strated by the superior recognition of complementary nucleic
acids by parent LNA,3 and the conformational analyses
presented here for 2′-amino- and 2′-thio-LNA monomeric
nucleosides indicate that the corresponding 2′-amino- and
2′-thio-LNA oligonucleotides deserve further attention. In
our design of novel oligonucleotide analogues, we have been
rather conservative, focusing on pentofuranose analogues
containing phosphate esters at the natural 3′- and 5′-
positions. This strategy has been successful in the case of
LNA, and incorporation of the 2′-amino-LNA nucleoside 7
into oligonucleotides could represent a significant step
forward offering a potentially convenient conjugation site
in a preorganized nucleic acid mimic structurally resembling
RNA. The synthetic route devised in this report gives
convenient access to 2′-heteroatom substituted LNA pyri-
midine nucleosides and should in addition also be applicable
for synthesis of other bicyclic pyrimidine nucleoside ana-
logues.
The conformation of 2′-amino-LNA nucleoside 7 and 2′-
thio-LNA nucleoside 11 was evaluated from the coupling
constants of the 1H NMR spectra as described earlier for
other nucleosides.8 Importantly, the J 1′,2′ values for the
bicyclo[2.2.1] nucleosides 7 and 11 were 0 Hz,9 indicating
Ack n ow led gm en t. The Danish Natural Science Re-
search Council, The Danish International Development
Agency, and Exiqon A/S, Denmark, are thanked for finan-
cial support.
(7) (a) Beig, T.; Szeja, W. Synthesis 1985, 76. (b) Kutney, J . P.;
Abdurahman, N.; Gletsos, C.; Quesue, L. P.; Piers, E.; Vlattas, I. J . Am.
Chem. Soc. 1970, 92, 1727. (c) Bell, D. J .; Lorber, J . J . Chem. Soc. 1940,
453. (d) Angyal, S. J .; J ames, K. Carbohydr. Res. 1970, 12, 147. (e) Sakurai,
H.; Shirahata, A.; Sasaki, K.; Hosomi, A. Synthesis 1979, 740.
(8) (a) Altona, C.; Sundaralingam, M. J . Am. Chem. Soc. 1973, 95, 2333.
(b) Obika, S.; Morio, K., Nanbu, D.; Imanishi, T. Chem. Commun. 1997,
1643.
Su p p or tin g In for m a tion Ava ila ble: Supporting Information
Available: Experimental procedures, spectroscopic data for com-
pounds 4-11 and a copy of the 13C NMR spectrum of compound
11 (8 pages).
(9) 1H NMR data for 7: δH (DMSO-d6) 11.29 (br s, 1H, NH), 7.73 (d, 1H,
J ) 1.1 Hz, 6-H), 5.31 (s, 1H, 1′-H), 5.29 (d, 1H, J ) 3.7 Hz, 3′-OH), 5.13 (t,
1H, J ) 5.3 Hz, 5′-OH), 3.81 (br s, 1H, 3′-H), 3.69 (m, 2H, 5′-H), 3.23 (s, 1H,
2′-H), 2.88 (d, 1H, J ) 9.8 Hz, 1′′-Ha), 2.55 (d, 1H, J ) 9.8 Hz, 1′′-Hb), 1.77
(d, 3H, J ) 0.8 Hz, CH3). 1H NMR data for 11: δH (CD3OD) 8.19 (1H, d, J
) 8.1 Hz, 6-H), 5.77 (1H, s, 1′-H), 5.65 (1H, d, J ) 8.1 Hz, 5-H), 4.31 (1H,
d, J ) 2.1 Hz, 3′-H), 3.86 (2H, s, 5′-H), 3.53 (1H, d, J ) 2.2 Hz, 2′-H), 2.93
(1H, d, J ) 10.3 Hz, 1′′-Ha), 2.73 (1H, d, J ) 10.3 Hz, 1′′-Hb).
J O9806658
(10) For 6b: 9% NOE in H-6 (irradiation of H-3′). For 7: 11% NOE in
H-6 (irradiation of H-3′) and 6% NOE in H-3′ (irradiation of H-6).