Synthesis and properties of RNA analogues having amides as interuridine linkages at selected positions

Article abstract of DOI:10.1021/ja0360900

Oligoribonucleotide analogues having amide internucleoside linkages (AM1: 3′-CH₂CONH-5′ and AM2: 3′-CH ₂NHCO-5′) at selected positions have been synthesized and the thermal stability of duplexes formed by these analogues with complem

Full text of DOI:10.1021/ja0360900

Published on Web 09/06/2003
Synthesis and Properties of RNA Analogues Having Amides
as Interuridine Linkages at Selected Positions
Eriks Rozners,*,†,§ Dace Katkevica,†,‡ Erika Bizdena,‡ and Roger Stro¨mberg†
Contribution from DiVision of Organic and Bioorganic Chemistry, MBB, Scheele Laboratory,
Karolinska Institutet, S-17177 Stockholm, Sweden, and Faculty of Material Science and Applied
Chemistry, Riga Technical UniVersity, Azenes 14/24, LV 1048 Riga, LatVia
Received May 12, 2003; E-mail: e.rozners@neu.edu
Abstract: Oligoribonucleotide analogues having amide internucleoside linkages (AM1: 3′-CH2CONH-5′
and AM2: 3′-CH2NHCO-5′) at selected positions have been synthesized and the thermal stability of duplexes
formed by these analogues with complementary RNA fragments has been evaluated by UV melting
experiments. Two series of oligomers with either 2′-OH or 2′-OMe vicinal to the amide linkages were studied.
Monomeric synthons (3′ and 5′-C amines and carboxylic acids) were synthesized as follows: For synthesis
of the AM1 analogue, the known sequence of radical allylation followed by the cleavage of the double
bond was adopted. For synthesis of the AM2 analogue, novel routes via addition of nitromethane followed
by conversion of the nitro function to either amino or carboxyl groups were developed. Coupling of monomeric
amines and carboxylic acids followed by protecting group manipulation and phosphonylation gave dimeric
3′-hydrogenphosphonate building blocks for oligonucleotide synthesis. Monomeric model compounds having
3′-amide and 2′-OH or 2′-OMe groups were also prepared and their conformational equilibrium was
1
determined by H NMR. The AM1 and AM2 models showed equal preferences for the North conformers
(at 40 °C, 88-89% with 2′-OH, and 92-93% with 2′-OMe). At physiological salt concentration (0.1 M NaCl)
the duplexes between AM1 modified oligonucleotides and RNA had stability similar to unmodified RNA-
RNA duplexes (∆tm) -0.2 to +0.7 °C per modification). However, the AM2 modification resulted in
substantial stabilization of duplexes: ∆tm) +1 to +2.4 °C per modification compared to all RNA. A 2′-O-
methyl vicinal to the AM2 linkage further increased the duplex stability. Our results suggest that RNA
analogues having amide internucleoside bonds are very promising candidates for medicinal applications.
Introduction
ensure high nuclease stability of the modified oligonucleotide.
The stability of duplexes formed by modified oligonucleotide
and mRNA, however, is not so straightforward to foresee and
usually has to be thoroughly examined for each particular
dephospho linkage.
Of many nonionic oligodeoxynucleotide analogues screened
(for recent reviews, see ref 2) those having amide3-5 (3′-CH2-
CONH-5′ and 3′-CH2NHCO-5′), methylene(methylimino)6a,b
(3′-CH2N(CH3)O-5′), methylene(dimethylhydrazo)6c (3′-CH2N-
(CH3)N(CH3)-5′), formacetal7,8 (3′-OCH2O-5′) and thioform-
acetal8 (3′-SCH2O-5′) linkages give stable duplexes with
Antisense therapy with synthetic oligonucleotides is a promis-
ing alternative to conventional chemotherapy of genetic disor-
ders, cancer, and viral infections (such as HIV).1 Important
requirements for initial selection of potential antisense oligo-
nucleotides are high stability toward nuclease degradation and
high binding affinity to the intracellular target, usually a
messenger RNA. Oligonucleotide analogues with dephospho
internucleoside linkages could fulfill these requirements and are
suggested as potential second generation antisense compounds.2
The absence of the phosphodiester linkage must inherently
(3) For a review on amide substituted oligodeoxynucleotide analogues, see:
De Mesmaeker, A.; Waldner, A.; Lebreton, J.; Fritsch, V.; Wolf, R. M. In
Carbohydrate Modifications in Antisense Research; Sanghvi, Y. S., and
Cook, P. D., Eds.; ACS Symposium Series 580; American Chemical
Society: Washington, DC, 1994; pp 24-39.
(4) (a) Idziak, I.; Just, G.; Damha, M.; Giannaris, P. A. Tetrahedron Lett. 1993,
34, 5417-5420. (b) Lebreton, J.; Waldner, A.; Lesueur, C.; De Mesmaeker,
A. Synlett 1994, 137-140. (c) Mesmaeker, A.; Waldner, A.; Lebreton, J.;
Hoffmann, P.; Fritsch, V.; Wolf, R. M.; Freier, S. M. Angew. Chem., Int.
Ed. Engl. 1994, 33, 226-229. (d) De Mesmaeker, A.; Lesueur, C.; Bevierre,
M.-O.; Waldner, A.; Fritsch, V.; Wolf, R. M. Angew. Chem., Int. Ed. Engl.
1996, 35, 2790-2794. (e) De Mesmaeker, A.; Lebreton, J.; Jouanno, C.;
Fritsch, V.; Wolf, R. M.; Wendeborn, S. Synlett 1997, 1287-1290.
(5) Lebreton, J.; Waldner, A.; Fritsch, V.; Wolf, R. M.; De Mesmaeker, A.
Tetrahedron Lett. 1994, 35, 5225-5228.
† Karolinska Institutet.
‡ Riga Technical University.
§ Present address: Department of Chemistry and Chemical Biology,
Northeastern University, 360 Huntington Ave., Hurtig Hall, Boston, MA
02115. Tel: (617) 373-5826. Fax: (617) 373-8795.
(1) (a) Gewirtz, A. M.; Sokol, D. L.; Ratajczak, M. Z. Blood 1998, 92, 712-
736. (b) Antisense Drug Technology: Principles, Strategies, and Applica-
tions; Crooke, S. T., Ed.; Marcel Dekker: New York, 2001; p 929.
(2) (a) De Mesmaeker, A.; Ha¨ner, R.; Martin, P.; Moser, H. E. Acc. Chem.
Res. 1995, 28, 366-374. (b) De Mesmaeker, A.; Altman, K.-H.; Waldner,
A.; Wendeborn, S. Curr. Opin. Struct. Biol. 1995, 5, 343-355. (c) Sanghvi,
Y. S.; Cook, P. D. In Carbohydrate Modifications in Antisense Research;
Sanghvi, Y. S., and Cook, P. D., Eds.; ACS Symposium Series 580;
American Chemical Society: Washington, DC, 1994; pp 1-22. (d) Freier,
S. M., Altmann, K.-H. Nucleic Acids Res., 1997, 25, 4429-4443. (e)
Sanghvi, Y. S. In ComprehensiVe Natural Products Chemistry; Barton, D.
H. R., Nakanishi, K., and Meth-Cohn, O., Eds.; Elsevier:, 1999; pp 285-
311.
(6) (a) Vasseur, J.-J.; Debart, F.; Sanghvi, Y. S.; Cook, P. D. J. Am. Chem.
Soc. 1992, 114, 4006-4007. (b) Morvan, F.; Sanghvi, Y. S.; Perbost, M.;
Vasseur, J.-J.; Bellon, L. J. Am. Chem. Soc. 1996, 118, 255-256. (c)
Sanghvi, Y. S.; Vasseur, J.-J.; Debart, F.; Cook, P. D. Collect. Czech. Chem.
Commun. Special Issue 1993, 58, 158-162.
9
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J. AM. CHEM. SOC. 2003, 125, 12125-12136
12125

A R T I C L E S
Rozners et al.
complementary RNA. In general, duplex stability is enhanced
by modifications having restricted conformational freedom in
the middle of the backbone: by restricted rotation around amide
linkages and by other stereoelectronic effects.2,9 Both shifting
the amide bond by one atom along the backbone10 and changing
the backbone length11 significantly decreases the stability of the
duplex. The preference for North conformers in modified sugars
has also been correlated with an increased stability of the A-type
duplexes formed with RNA targets.12
Because RNA generally forms more stable duplexes than
DNA both with RNA and DNA targets, oligoribonucleotide
analogues having chemically and enzymatically stable inter-
nucleoside linkages may be better candidates also for therapy
at the gene level than their deoxy counterparts. For example, a
pentamer oligoribonucleotide (but not the corresponding deoxy-
oligoribonucleotide) hybridizes to a single-stranded DNA
template in the open complex formed with RNA polymerase.13a
Transcription inhibition with an oligoribonucleotide 2′-OMe
analogue has been demonstrated by this approach.13b Thus, RNA
analogues may be useful tools in both antisense applications
and emerging new gene therapy approaches.
Figure 1. Oligoribonucleotide duplexes studied, x denotes position of the
amide linkages, p denotes phosphodiesters.
guanidine18 (3′-NHC(dNH2+)NH-5′) and amide19 (3′-CH2-
CONH-5′) linkages have been synthesized, however, the thermal
stability of duplexes formed by these compounds has not been
reported.
There are only a few reports on RNA analogues having
dephospho linkages. Ribonucleoside dimers having thioform-
acetal14 and sulfide15 (3′-CH2CH2S-5′) linkages have been
prepared and incorporated in oligonucleotides otherwise con-
taining deoxynucleoside residues. Destabilization of such modi-
fied DNA-RNA duplexes was reported. The analysis of these
data, however, is complicated because the effect of internucleo-
side linkage is not separated from the effect of alternating sugar
composition: ribonucleoside dimers incorporated in otherwise
oligodeoxynucleotides.16 Uniformly modified oligoribonucleo-
tides with dimethylene sulfone linkages (3′-CH2SO2CH2-5′) have
been prepared, but the strong self-association of this analogue
apparently prevented hybridization with complementary RNA
and DNA.17 Uniformly modified riboadenosine pentamers with
We found that oligoribonucleotides where selected phos-
phodiester bonds were replaced by formacetal linkages had
increased affinity to the complementary RNA fragments as
compared to unmodified oligoribonucleotides.20 In contrast, the
formacetal modification in oligodeoxynucleotides is reported
to decrease the stability of both DNA-RNA and DNA-DNA
duplexes.8,9 Encouraged by these results, we extended our
studies to synthesize and investigate the properties of amide
linked oligoribonucleotide analogues. Amide analogues were
of particular interest because of: (1) potentially favorable
hybridization properties, as expected from results in the deoxy
series,3-5 (2) automated solid phase synthesis of such analogues
could be foreseen, similarly to peptide chemistry, (3) combining
one type of amide linkage (our AM1) with 2′-O-methyl groups
has been shown to increase affinity to RNA in mixed deoxyribo/
ribo oligonucleotides4d,e and (4) uniformly modified, amide
linked oligonucleotides could exhibit interesting properties,
similarly to peptide nucleic acids (PNA, for reviews, see refs
2b,21).
(7) (a) Matteucci, M. Tetrahedron Lett. 1990, 31, 2385-2388. (b) Quaedflieg,
P. J. L. M.; Pikkemaat, J. A.; van der Marel, G. A.; Kuyl-Yeheskiely, E.;
Altona, C.; van Boom, J. H. Recl. TraV. Chim. Pays-Bas 1993, 112, 15-
21.
(8) (a) Jones, R. J.; Lin, K.-Y.; Milligan, J. F.; Wadwani, S.; Matteucci, M. D.
J. Org. Chem. 1993, 58, 2983-2991. (b) Lin, K.-Y.; Pudlo, J. S.; Jones,
R. J.; Bischofberger, N.; Matteucci, M. D.; Froehler, B. C. Bioorg. Med.
Chem. Lett. 1994, 4, 1061-1064.
(9) De Mesmaeker, A.; Waldner, A.; Sanghvi, Y. S.; Lebreton, J. Bioorg. Med.
Chem. Lett. 1994, 4, 395-398.
In this paper, we report the synthesis of amide linked uridine-
uridine dimers (with either 2′-OH or 2′-OMe vicinal to the amide
linkages) and their incorporation in oligoribonucleotides (Figure
1). Novel synthetic routes toward 3′ and 5′-C one carbon
extended nucleoside homologues are reported. UV melting
experiments showed that both isomeric amides (AM1 and AM2,
see Figure 1) were well accommodated in RNA-RNA duplexes.
Whereas AM1 modified duplexes had an RNA affinity similar
to that of nonmodified oligoribonucleotides (FD), the AM2
modifications caused a large stabilization of up to over 2 °C
per modification. The different stabilities of AM1 and AM2
suggest that amide modified oligoribonucleotides are interesting
(10) (a) De Mesmaeker, A.; Lebreton, J.; Waldner, A.; Fritsch, V.; Wolf, R.
M.; Freier, S. M. Synlett 1993, 733-736. (b) Lebreton, J.; De Mesmaeker,
A.; Waldner, A.; Fritsch, V.; Wolf, R. M.; Freier, S. M. Tetrahedron Lett.
1993, 34, 6383-6386. (c) De Mesmaeker, A.; Lebreton, J.; Waldner, A.;
Fritsch, V.; Wolf, R. M. Bioorg. Med. Chem. Lett. 1994, 4, 873-878.
(11) Stork, G.; Zhang, C.; Gryaznov, S.; Schultz, R. Tetrahedron Lett. 1995,
36, 6387-6390. (b) De Mesmaeker, A.; Jouanno, C.; Wolf, R. M.;
Wendeborn, S. Bioorg. Med. Chem. Lett. 1997, 7, 447-452.
(12) Griffey, R. H.; Lesnik, E.; Freier, S.; Sanghvi, Y. S.; Teng, K.; Kawasaki,
A.; Wheeler, P.; Mohan, V.; Cook, P. D. In Carbohydrate Modifications
in Antisense Research; Sanghvi, Y. S., and Cook, P. D., Eds.; ACS
Symposium Series 580; American Chemical Society: Washington, DC,
1994; pp 212-224.
(13) (a) Perrin, D. M.; Mazumder, A.; Sadeghi, F.; Sigman, D. S. Biochemistry
1994, 33, 3848-3854. (b) Perrin, D. M.; Chen, C.-H. B.; Xu, Y.; Pearson,
L.; Sigman, D. S. J. Am. Chem. Soc. 1997, 119, 5746-5747.
(14) Cao, X.; Matteucci, M. D. Bioorg. Med. Chem. Lett. 1994, 4, 807-810.
(15) (a) Meng, B.; Kawai, S. H.; Wang, D.; Just, G.; Giannaris, P. A.; Damha,
M. J. Angew. Chem., Int. Ed. Engl. 1993, 32, 729-731. (b) Damha, M. J.;
Meng, B.; Wang, D.; Yannopoulos, C. G.; Just, G. Nucleic Acids Res. 1995,
23, 3967-3973.
(16) For a systematic study of thermal stability of mixed ribo-deoxyribo
sequences, see Hoheisel, J. D. Nucleic Acids Res., 1996, 24, 430-432.
(17) Richert, C.; Roughton, A. L.; Benner, S. A. J. Am. Chem. Soc. 1996, 118,
8, 4518-4531.
(18) Dempcy, R. O.; Luo, J.; Bruice, T. C. Proc. Natl. Acad. Sci. U.S.A. 1996,
93, 4326-4330. Kojima, N.; Bruice, T. C. Org. Lett. 2000, 3, 81-84.
Kojima, N.; Szabo, I. E.; Bruice, T. C. Tetrahedron 2002, 58, 867-879.
(19) Robins, M. J.; Doboszewski, B. D.; Nilsson, B. L.; Peterson, M. A.
Nucleosides, Nucleotides & Nucleic Acids 2000, 19, 69-86.
(20) Rozners, E.; Stro¨mberg, R. J. Org. Chem. 1997, 62, 1846-1850.
9
12126 J. AM. CHEM. SOC. VOL. 125, NO. 40, 2003

Synthesis and Properties of RNA Analogues
A R T I C L E S
Scheme 1. Synthesis of Monomers for AM1 Modificationa
resulted in somewhat lower yield of 5b (70% vs ca. 90%
reported in ref 29), but did not disturb further synthesis of the
dimer 14a. For synthesis of dimer 14b we employed the 2′,3′-
O-unprotected amine 5a, prepared using triphenylphosphine
reduction29b of 4a. However, the high polarity of 5a was
somewhat inconvenient.
In contrast, the preparation of building blocks for the 3′-CH2-
NHCO-5′ linkage (abbreviated as AM2, Figure 1) was prob-
lematic. For preparation of 3′-CH2NH2 in the deoxy series, De
Mesmaeker et al. used addition of styryltributyltin to 3′-C-
centered radicals of protected deoxyribonucleosides.5 In the ribo
series, however, we found that the reaction was difficult to
initiate and gave complex product mixtures. Synthesis via 3′-
deoxy-3′-methylene derivatives (Wittig addition followed by
hydroboration) was attempted but preliminary results gave poor
stereoselectivity. A high stereoselectivity in this reaction has
recently been reported but only for the ribonucleoside analogue
(not the 2′-OMe), because the bulky 2′-TBDMS group is
probably directing the stereoselectivity.30
a Reagents and conditions: (a) allyltributyltin, AIBN, 80 °C; (b) OsO4,
NaIO4; (c) NaClO2; for 5a (d) Ph3P, NH3; for 5b (e) acetyl chloride,
pyridine; (f) Bu3SnH, AIBN, 80 °C.
model systems for studies on factors that govern biopolymer
recognition. In particular, we suggest that the difference in
thermal stability is caused by different hydration of these
analogs. Furthermore, amide linked RNA might find potential
use as antisense compounds or as therapeutic ribozymes.
Of different one carbon homologization methods, the nitroal-
dol (Henry) reaction31 seemed most promising because of the
ease of carbon-carbon bond formation under relatively mild
conditions. Only a few but encouraging examples of such
reactions on carbohydrate derived ketones were known.32 In an
early report Rosenthal et al.32a described addition of nitro-
methane to 2′-keto xyloadenosine followed by hydrogenation
of the nitro function to give 2′-CH2NH2 lyxoadenosine. More
recently, Garg et al.32b reported addition of nitromethane to 3′-
keto ribothymidine. Importantly, reduction of the double bond
(NaBH4/EtOH) in 3′- nitromethylene derivative gave a 2.5:1
mixture of 3′,4′-trans and cis 3′C-CH2NO2 ribothymidines.32b
Our syntheses of amines 8a-d using the Henry reaction are
illustrated in Scheme 2. Addition of nitromethane32b to ap-
propriately protected 3′-keto nucleosides33 gave the 3′-C-
nitromethylene derivatives 6a-d in 70-80% yields. Reduction
of the double bond (NaBH4/EtOH, 0 °C) gave inseparable
mixtures of 7a-d and their 3′,4′-cis isomers in ratios of 6:1 for
7a,c and 15:1 for 7b,d.34 A brief investigation of the solvent
effect (THF, EtOH, EtOEt, CH2Cl2, ClCH2CH2Cl, for detail,
see Supporting Information) revealed that reduction in THF at
-78 °C gave the best results, diastereomer ratio (dr) better than
95:5. For 6c the use of (Bu)4NBH4 gave further significant
improvement, dr better than 98:2. In the 2′-OMe series, however,
the effects of solvent or reagent changes were insignificant: the
best result for 6d was dr 94:6 with NaBH4 in THF at -78 °C.
Results and Discussion
Syntheses of monomeric building blocks, carboxylic acids
3a,b and 13, and amines 5a,b and 8a-d are illustrated in
Schemes 1-2. For the 3′-CH2CONH-5′ linkage (abbreviated as
AM1, Figure 1), the key transformations previously used by De
Mesmaeker et al.3 were successfully employed (Scheme 1).
Free-radical allylation4b,10a,11a,22 of 3′-O-phenoxythiocarbonyl
derivatives 1a,b23 followed by cleavage of the double bond24
with OsO4 and NaIO4, and oxidation of the intermediate
4b,c,25
aldehyde with NaClO2
gave the carboxylic acids 3a and
3b in 38 and 29% overall yields, respectively (4 steps, from
2′,5′-protected nucleosides26). Consistent with results previously
reported in the deoxy series,4b,10a,11a,22 the radical allylation gave
preferably the 3′,4′-trans isomer shown in Scheme 1.27
Amines 5a,b were readily synthesized by reduction of the
known 5′-azido-5′-deoxyuridine28 (Scheme 1). During radical
reduction29a of 5′-azido-2′,3′-O-bis(acetyl)-5′-deoxyuridine 4b
(prepared by acetylation of 4a), we observed some transacet-
ylation yielding the N-acetyl byproduct. This side reaction
(21) (a) Nielsen, P. E.; Haaima, G. Chem. Soc. Rew. 1997, 73-78; Uhlmann,
E.; Peyman, A.; Breipohl, G.; Will, D. W. Angew. Chem., Int. Ed. Engl.
1998, 37, 2796-2823; Nielsen, P. E. Acc. Chem. Res. 1999, 32, 624-630.
(22) Chu, C. K.; Doboszewski, B.; Schmidt, W.; Ullas, G. V.; Van Roey, P. J.
Org. Chem. 1989, 54, 2767-2769.
(23) (a) Meier, C.; Huynh-Dinh, T. Synlett 1991, 227-228. (b) Robins. M. J.;
Wilson, J. S.; Hansske, F. J. Am. Chem. Soc. 1983, 105, 4059-4065.
(24) Sanghvi, Y. S.; Bharadwaj, R.; Debart, F.; De Mesmaeker, A. Synthesis
1994, 1163-1166.
(25) Dalcanale, E.; Montanari, F. J. Org. Chem. 1986, 51, 567-569.
(26) Syntheses of TBDMS protected uridine derivatives were done using
published procedures: Hakimelahi, G. H.; Proba, Z. A.; Ogilvie, K. K.
Can. J. Chem. 1982, 60, 1106-1113.
(27) Pure of 2a and 2b (3′,4′-trans isomers) were isolated after silica gel column
chromatography. The configuration at C3′ was confirmed by NOESY
spectra of the intermediate aldehydes. Informative NOEs were observed
between H1′ and 3′-CH2 and between H3′ and H6. Possible 3′,4′-cis isomers
(less than 10% as judged by TLC) were not isolated.
(28) (a) Hata, T.; Yamamoto, I.; Sekine, M. Chem. Lett. 1975, 977-980. (b)
Yamamoto, I.; Sekine, M.; Hata, T. J. Chem. Soc., Perkin Trans. 1 1978,
306-310.
(29) (a) Samano, M. C.; Robins, M. J. Tetrahedron Lett. 1991, 32, 6293-6296.
(b) Mungall, W. S.; Greene, G. L.; Heavner, G. A.; Letsinger, R. L. J.
Org. Chem. 1975, 40, 1659-1662.
(30) (a) Winqvist, A.; Stro¨mberg, R. Nucleosides, Nucleotides & Nucleic Acids,
2001, 1389-1392 (b) Winqvist, A.; Stro¨mberg, R. Eur. J. Org. Chem. 2001,
4305-4311.
(31) Rosini, G. In ComprehensiVe Organic Synthesis, Part 2, Addition to C-X
π Bonds; Trost, B.; Fleming, I., Eds.; Pergamon Press: Oxford, 1991; pp
329-340.
(32) (a) Rosenthal, A.; Sprinzl, M.; Baker, D. A. Tetrahedron Lett. 1970, 48,
4233-4236. (b) Garg, N.; Hossain, N.; Plavec, J.; Chattopadhyaya
Tetrahedron, 1994, 50, 4167-4178. (c) Albrecht, H. P.; Moffat, J. G.
Tetrahedron Lett. 1970, 1063-1066.
(33) (a) 3′-Keto derivatives were prepared from selectively protected nucleosides
(see ref 26) using the published oxidation procedures: (a) DMSO/acetic
anhydride or CrO3, see Hansske, F.; Madej, D.; Robins, M. J. Tetrahedron,
1984, 40, 125-135. (b) Dess-Martin 12-I-5 periodane oxidant, see Dess,
D. B.; Martin, J. C. J. Org. Chem. 1983, 48, 4155-4156. Samano, V.;
Robins, M. J. J. Org. Chem. 1990, 55, 5186-5188. For 5′-O-MMT
protected derivatives we preferred the Dess-Martin oxidation.
(34) Ratios determined by 1H NMR. The 3′,4′-trans configurations of the major
isomers were confirmed by NOESY spectra of 7c, d and 8a-d. Informative
NOEs were observed between H1′ and 3′-CH2 and between H3′ and H6.
As expected, no cross-peaks were detected for correlation between H1′
and H3′.
9
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A R T I C L E S
Rozners et al.
Scheme 2. Synthesis of Monomers for AM2 Modificationa
a one pot procedure (see Experimental Section). The double
bond in 6a-d was reduced with NaBH4 (4 equiv) in EtOH to
give 7a-d and successive addition of nickel boride (1.5 equiv
for 6a,b or 1 equiv. for 6c,d)38c and NaBH4 (10 equiv.) to the
same reaction mixture gave 8a-d in 40-50% overall yield from
6a-d. Silica gel column chromatography yielded isomerically
pure 8a, whereas 8b-d were obtained as still inseparable
mixtures of 3′,4′-cis and trans isomers (see above). When the
optimized conditions for the reduction of the double bond (see
above) were used in the one pot procedure (see the Experimental
Section) 8c was obtained with dr 98:2, as expected. For the
synthesis of AM2 linked dimers we used 8a and 8b prepared
using the NaBH4/NiB2 one pot procedure. Removal of the cis
isomer of 8b was achieved by chromatographic separation after
synthesis of 18b. In summary, addition of nitromethane to 3′-
keto nucleosides followed by successive reduction of double
bond and nitrofunction gave the required amines 8a-d in 20-
30% overall yields (5 steps, from the 2′,5′-protected nucleo-
sides).
Our initial attempts to prepare carboxylic acid 13 using
published methods for preparation of 5′-C one carbon homo-
logues of nucleosides also met with limited success.39 Instead,
we developed a novel route based again on the addition of
nitromethane followed by functional group interconversion
(Scheme 2). Oxidation of 2′,3′-O-(benzylidene)uridine 9 and
addition of nitromethane40 was done in the same reaction
mixture without isolation of the 5′-aldehyde. Acetylation of the
5′-OH under acidic conditions40b also conveniently cleaved the
benzylidene protection and acetylated the 2′ and 3′ hydroxyls.
Reductive removal of the 5′-O-acetyl group40b,41 followed by
transformation of the nitromethyl function42 gave the carboxylic
acid 13 in 27% overall yield (4 steps, from 2′,3′-O-(benzyli-
dene)uridine).
DCC and hydroxybenzotriazol (HOBt) mediated coupling43
of carboxylic acids 3a,b and 13 with amines 5a,b and 8a,b gave
the dimers 14a,b and 18a,b, respectively (Scheme 3). Selective
removal of the primary 5′-O-TBDMS group (limited hydrolysis
in 80% aqueous acetic acid), cleavage of the terminal 2′ and
3′-O-acetyl groups and protection of the 5′-OH as monometh-
oxytrityl (MMT) ether gave dimers 16a,b. Dimers 19a,b were
obtained after treatment of 18a,b with ammonia solution.
Synthesis of the dimeric H-phosphonate building blocks 17a,b
and 20a,b was achieved by one pot selective installation of the
2′-ortho-chlorobenzoyl (ClBz) group followed by 3′-O-phos-
phonylation as previously reported.20
a Reagents and conditions: (a) NaBH4 THF, -78 °C; (b) NiB2, NaBH4;
(c) DMSO, DCC, CHCl2COOH; (d) CH3NO2, NaOCH3; (e) Ac2O, HClO4,
0 °C; (f) NaBH4 ethanol-THF (1:1), 0 °C; (g) NaNO2, AcOH, DMSO, 40
°C.
The unwanted 3′,4′-cis isomers were separated after further
transformations.
Reduction of aliphatic nitro functions to give primary amines
is a well-known reaction in organic chemistry.35,36 However,
its application to nucleoside derivatives could be problematic
because of potential side reactions in the heterocyclic bases and
steric hindrance from protecting groups used. For nucleosides
only a few examples of successful catalytic hydrogenation of
nitro groups (in the presence of acetic acid) are reported.32a,c
We chose 7c for initial studies on the reduction of nitro group.
Catalytic hydrogenation (100 psi H2, EtOH, 20 °C, 48 h) using
36c
either 10% Pd/carbon,36a Raney nickel36b or PtO2 catalysts
gave no reaction. Hydrogenation using 10% Pd/carbon in the
presence of acetic acid led to cleavage of the 5′-O-TBDMS
group, whereas the nitrofunction remained intact. Loss of a
primary TBDMS protection during hydrogenation has been
previously observed by others.37 Reactions with LiAlH4,36d
sodium bis-(2-methoxyethoxy)aluminum hydride (Red-Al),36e
and NaBH4 in the presence of 10% Pd/carbon catalyst36f gave
complex mixtures containing also products from cleavage of
the glycosidic linkage. Reductions with hydrazine hydrate and
either nickel boride36g or Raney nickel36h as catalysts were slow.
At elevated temperatures or high hydrazine concentrations
complex mixtures were obtained.
(38) (a) Osby, J. O.; Ganem, B. Tetrahedron Lett. 1985, 26, 6413-6416. (b)
for a recent examples of this reaction, see Corey, E. J.; Zhang, F.-Y. Angew.
Chem., Int. Ed. Engl. 1999, 38, 1931-1934; Clive, D. L. J.; Bo, Y.; Tao,
Y.; Daigneault, S.; Wu, Y.-J.; Meignan, G. J. Am. Chem. Soc. 1998, 120,
10 332-10 349. (c) The use of less NaBH4 (5 equiv.) and nickel boride
catalyst (0.5 equiv) gave a mixture of hydroxylamine (isolated yield 50%,
for analytical data, see Supporting Information) and amine 8c (isolated yield
12%).
However, reduction with NaBH4 (10 equiv) and nickel
boride38 (1 equiv.) in ethanol gave 8c in ca. 50% isolated yield.
The reduction sequence from 6 to 8 was further developed into
(39) Carboxylic acid 13a (Scheme 2) was obtained from 1, 2; 5, 6-di-O-
(isopropylidene)glucose in a laborious multistep synthesis in 6% overall
yield. Carboxylic acid 13b was obtained from 2′,3′-O-(isopropylidene)-
uridine in 19% overall yield, however, the stability of the isopropylidene
protection complicated further operations. For synthetic details, see
Supporting Information. Synthesis via Wittig addition of 1,3-dithia-2-
cyclohexylidene triphenylphosphorane (see ref 5) failed because of instabil-
ity of 2′,3′-protections (benzylidene or benzoyl) under conditions required
to generate the carboxylic acid (HgCl2 in MeOH/H2O followed by NaOH
in H2O).
(40) (a) Kappler, F.; Hampton, A. J. Org. Chem. 1975, 40, 1378-1385. (b)
Mock, G. A.; Moffat, J. G. Nucleic Acids Res. 1982, 10, 6223-6234.
(41) Bachman, G. B.; Maleski, R. J. J. Org. Chem. 1972, 37, 2810-2814.
(42) Matt, C.; Wagner, A.; Mioskowski, C. J. Org. Chem. 1997, 62, 234-235.
(43) Rees, A. R.; Offord, R. E. Biochem. J. 1976, 159, 487-493.
(35) Larock, R. C. ComprehensiVe Organic Transformations. A Guide to
Functional Group Preparations; VCH Publishers: New York, 1989.
(36) (a) Poos, G. I.; Kleis, J.; Wittekind, R.; Rosenau, J. D. J. Org. Chem. 1961,
26, 4898-4904. (b) Bachman, G. B.; Hass, H. B.; Platau, G. O. J. Am.
Chem. Soc. 1954, 76, 3972-3974. (c) Secrist III, J. A.; Logue, M. V. J.
Org. Chem. 1972, 37, 335-336. (d) Nystrom, R. F.; Brown, W. G. J. Am.
Chem. Soc. 1948, 70, 3738-3740. (e) Butterick, J. R.; Unrau, A. M. J.
Chem. Soc. Chem. Commun. 1974, 307-308. (f) Petrini, M.; Ballini, R.;
Rosini, G. Synthesis 1987, 713-714. (g) Lloyd, D. H.; Nichols, D. E. J.
Org. Chem. 1986, 51, 4294-4295. (h) Podona, T.; Guardiola-Lemaitre,
B.; Caignard, D.-H.; Pfeiffer, B.; Renard, P.; Guillaumet, G. J. Med. Chem.
1994, 37, 1779-1793.
(37) Carreira, E. M.; Du Bois, J. J. Am. Chem. Soc. 1995, 117, 8106-8125.
9
12128 J. AM. CHEM. SOC. VOL. 125, NO. 40, 2003

Synthesis and Properties of RNA Analogues
A R T I C L E S
Table 1. Melting Temperatures of Oligonucleotide Duplexesa
Scheme 3. Synthesis of Dimeric Building Blocks Containing AM1
or AM2 Modificationsa
tm (oC)
tm (oC)
0.1 M NaCl
1 M NaCl
Mod 16
1
2
3
4
5
6
FD
AM1
AM2
55.9
70.3
55.7 (-0.1)
60.3 (+2.2)
57.4 (+0.8)
58.0 (+1.1)
59.4 (+1.8)
69.9 (-0.2)
72.7 (+1.2)
71.6 (+0.7)
70.9 (+0.3)
72.7 (+1.2)
FD+M b
AM1+M b
AM2+M b
Mod 15
7
8
9
10
11
12
FD
AM1
AM2
53.0
66.3
52.5 (-0.2)
57.0 (+1.3)
55.0 (+0.7)
54.4 (+0.5)
60.0 (+2.3)
64.1 (-0.8)
67.5 (+0.4)
68.7 (+0.8)
67.9 (+0.5)
71.2 (+1.6)
FD+M b
AM1+M b
AM2+M b
Mod 13
13
14
15
16
17
18
FD
AM1
AM2
13.3
30.4
17.2 (+0.7)
27.5 (+2.4)
19.1 (+1.0)
22.4 (+1.5)
31.6 (+3.1)
29.6 (-0.1)
38.1 (+1.3)
35.7 (+0.9)
32.1 (+0.3)
40.1 (+1.6)
FD+M b
AM1+M b
AM2+M b
a
∆tm per modification relative to the native RNA (FD) is given in
b
brackets, +M designates the 2-O-methyl series.
Table 2. Thermodynamic Parameters for the Formation of
Oligonucleotide Duplexes at 0.1 M NaCl and 1 M NaCl (Mod 13
only)
o
o
o
∆H
Τ∆S 310
∆G 310
kcal/mol
kcal/mol
kcal/mol
Mod 16 a
-139.7
-127.8
-154.1
-144.9
-127.6
-134.5
Mod 15 a
-130.1
-117.3
-123.8
-135.2
-116.7
-122.8
Mod 13 a
-76.8
-89.0
-90.3
-77.3
-82.1
-88.7
Mod 13 c
-99.5
-86.5
-96.6
-76.3
-65.4
-86.7
1
2
3
4
5
6
FD
AM1
AM2
-123.1
-111.8
-134.7
-127.2
-110.9
-116.8
-16.6
-16.1
-19.4
-17.7
-16.7
-17.7
FD+M b
AM1+M b
AM2+M b
a Reagents and conditions: (a) DCC, HOBt; (b) 80% AcOH, 50 °C; (c)
NH3/EtOH, 2:1; (d) MMTCl, pyridine; (e) o-Chlorobenzoyl chloride, 1.1
equiv., -78 °C; (f) PCl3, imidazole, NEt3, -78 °C; quenched with 2 M
triethylammonium bicarbonate (aqueous), pH 7.5.
7
8
9
10
11
12
FD
AM1
AM2
-115.5
-103.1
-107.6
-119.1
-101.8
-105.8
-14.6
-14.2
-16.1
-16.1
-14.8
-17.0
Three Model Oligoribonucleotides (Figure 1) having two
(Mod 16), three (Mod 15) and six (Mod 13) internucleoside
amide linkages (x) were prepared using dimers 17 or 20 and
standard H-phosphonate oligoribonucleotide synthesis proce-
dure.44 Reference oligoribonucleotides (unmodified and having
2′-O-Me groups vicinal to the phosphodiester linkages x) and
the complementary oligoribonucleotides were also synthesized
via the H-phosphonate route44 and the stabilities of the corre-
sponding duplexes (Figure 1) were characterized by UV melting
experiments.20 Experiments were done at low (0.1 M NaCl) and
high (1 M NaCl) salt concentrations; melting temperatures are
collected in Table 1 and thermodynamic data in Table 2. For
each model sequence two series of oligomers were studied: (1)
in the ribo series AM1 and AM2, were x (Figure 1) represents
the corresponding amide linkage, were compared to unmodified
phosphodiester oligoribonucleotide FD, and (2) in the corre-
sponding 2′-O-methyl series AM1+M and AM2+M were
compared to FD+M (in all series only the 2′-hydroxyls vicinal
to x were methylated).
FD+M b
AM1+M b
AM2+M b
13
14
15
16
17
18
FD
AM1
AM2
-74.6
-86.5
-84.5
-73.5
-77.6
-81.7
-2.3
-2.6
-5.7
-3.8
-4.5
-7.0
FD+M b
AM1+M b
AM2+M b
19
20
21
22
23
24
FD
AM1
AM2
-93.0
-80.0
-87.7
-68.1
-57.8
-77.2
-6.5
-6.5
-8.9
-8.2
-7.6
-9.5
FD+M b
AM1+M b
AM2+M b
a
b
c
0.1 M NaCl, +M designates the 2-O-methyl series, 1 M NaCl
controls: ∆tm) -0.2 to +0.7 in the ribo series and, ∆tm) -0.2
to +0.5 in the 2′-O-methyl series. This result correlates well
with previous observations in the deoxy series.4 In contrast, the
AM2 modification gave substantially higher duplex stabiliza-
tion: ∆tm) +1.3 to +2.4 in the ribo series and, ∆tm) +1.0 to
+2.1 in the 2′-OMe series. This result is approaching the greatest
positive effects observed when a phosphodiester bond in an
At low salt concentration (0.1 M NaCl, Table 1) the AM1
modified duplexes showed a stability similar to that of the
(44) (a) Westman, E.; Sigurdsson, S.; Stawinski, J.; Stro¨mberg, R. Nucleic Acids
Sym Ser. 1994, 31, 25. (b) Stro¨mberg, R.; Stawinski, J. Current Protocols
in Nucleic Acid Chemistry, unit 3.4; John Wiley & Sons: New York, 2000.
(c) Sarkar, M.; Sigurdsson, S.; Tomac, S.; Sen, S.; Rozners, E.; Sjo¨berg,
B. M.; Stro¨mberg, R.; Gra¨slund, A. Biochemistry 1996, 35, 4678-4688.
9
J. AM. CHEM. SOC. VOL. 125, NO. 40, 2003 12129

A R T I C L E S
Rozners et al.
Scheme 4 . Synthesis of Model Compounds for Conformational
25b were 46 and 54% North, respectively. These results are
also in good agreement with conformational studies on meth-
ylene(methylimino)48 and methylene(dimethylhydrazo)12 linked
dimers. Because both amides caused the same conformational
preferences of the sugar residues in the model compounds, the
conformational preorganization induced by the 3′-CH2 modifica-
tion cannot be responsible for the observed differences in
stability of AM1 and AM2 modified RNA duplexes. Before other
interactions (see below) are better understood, direct correlation
between stability of modified oligonucleotide duplexes and
conformational preference of the corresponding nucleoside
models is best used with caution.
Studiesa
Analysis of the thermodynamic data (Table 2) of the two
different amide modifications suggests that in general AM1 is
more entropically favored, whereas the AM2 modification is
more enthalpically favored. This suggests that conformational
preorganization could be a dominating factor with AM1 and
that some additional bonding interaction, e.g., direct or water
mediated H-bonding, stabilizes the AM2 duplexes. The thermal
stability of modified nucleic acid duplexes should depend on
many factors hydrogen bonding, preferred sugar conformation,
hydration, electrostatic and steric factors, hydrophobic interac-
tions, etc. Substitution of neutral dephosphono linkages for
phosphodiesters is a radical change that should interfere with
almost all of these factors. The example of the isomeric amides
AM1 and AM2, studied herein, is of particular interest: although
many factors should be affected similarly, the net difference
between these modifications is surprisingly large. In the deoxy
series, molecular mechanics and molecular dynamics studies
on both modifications give similar geometry and UV melting
experiments give similar thermal stability.4,5 Our results showed
a very similar preference for North conformers in the different
model compounds. For both AM1 and AM2 modified duplexes
we found a similar gain in stability due to reduction of
electrostatic repulsion (see above). Still, the AM2 modified
duplexes are much more stable than AM1, the difference being
as large as 1 to 1.5 °C per modification.
To gain more insight into possible reasons for the greater
stability of the AM2 modification, we studied the dependence
of melting temperatures of Mod 13 on the concentration of
different types of salts. At high concentrations (>1M) salts exert
specific effects on the stability of biopolymer structures through
indirect interaction with the surrounding aqueous solvent.
According to the Hofmeister series,49 kosmotropes (polar water
structure-makers, e.g., sodium acetate) stabilize whereas chao-
tropes (water-structure breakers, e.g., sodium perchlorate)
destabilize the native conformation of biopolymers. Sodium
chloride has little effect on water structure. The comparison of
AM1 (triangles) and AM2 (circles) melting points in the presence
of added sodium chloride (empty markers) or sodium acetate
(filled markers) is shown in Figure 2. According to the
Hofmeister series,49 Mod 13 was expected to be more stable in
sodium acetate than in sodium chloride solution. This was indeed
observed for the unmodified RNA (data not shown) and the
AM1 modified oligonucleotide (Figure 2, filled vs empty
triangles). In contrast, Mod 13 with AM2 modifications did not
a
Reagents and conditions: (a) DCC, HOBt, ethylamine; (b) tetrabu-
tylammonium fluoride; (c) propionic anhydride.
oligonucleotide is replaced with a dephospho linkage2,45 and is
in contrast to the deoxy series where similar thermal stabilities
of AM1 and AM2 modified duplexes were observed.
4,5
At high salt concentration (1 M NaCl, Table 1) where
electrostatic repulsion is greatly reduced, generally higher tm
values were observed. At 1 M NaCl the AM1 modified duplexes
showed a slight destabilization (∆tm) -0.8 to -0.1) compared
to the controls, whereas the stabilization caused by the AM2
modification remained positive (∆tm) +0.4 to +1.3), although
less so than at lower salt concentration. The difference in ∆tm
per modification compared to native RNA between high and
low salt conditions (∆∆tm) 0.1 to 1.2 for AM1 and ∆∆tm)
0.4 to 1.4 for AM2) suggests that a substantial part of the gain
in duplex stability at physiological salt concentration (0.1 M)
is due to reduced electrostatic repulsion when replacing charged
phosphodiester linkages with neutral amides. However, this is
not responsible for all of the stabilization, especially in the case
of the AM2 modification.
Substitution of 3′-CH2 for 3′-O should shift the conforma-
tional equilibrium of modified sugars toward North46 which in
turn has been correlated with increased stability of the A-type
duplexes formed by modified oligonucleotides with RNA
targets.12 To evaluate the changes in sugar conformation caused
by the amide linkages we synthesized monomeric nucleoside
models 23a,b and 24a,b (Scheme 4) and studied their confor-
mational equilibrium by H NMR.47
1
Amide modified models showed a greatly increased prefer-
ence for North conformation: percentage of North conformers
was for the 2′-OH models 23a 88%, 24a 89% and for the 2′-
OMe models 23b 92%, 24b 93% (for full experimental data,
see Supporting Information). In comparison, the equilibrium
positions of the corresponding phosphodiester models 25a and
(45) Cationic guanidine and S-methylthiourea modified DNA forms extremely
stable 2: 1 complexes with natural DNA, see (a) Linkletter, B. A.; Szabo,
I. E.; Bruice, T. C. Nucleic Acids Res. 2001, 29, 2370-2376. (b) Linkletter,
B. A.; Szabo, I. E.; Bruice, T. C. J. Am. Chem. Soc. 1999, 121, 3888-
3896. (c) Arya, D. P.; Bruice, T. C. J. Am. Chem. Soc. 1998, 120, 12 419-
12 427.
(46) (a) Plavec, J.; Garg, N.; Chattopadhyaya, J. J. Chem. Soc. Chem. Commun.
1993, 1011-1014. (b) For a discussion on the stereoelectronic effects in
nucleosides and analogues, see also Plavec, J.; Tong, W.; Chattopadhyaya,
J. J. Am. Chem. Soc. 1993, 115, 9734-9746.
(48) Bhat, B.; Swayze, E. E.; Wheeler, P.; Dimock, S.; Perbost, M.; Sanghvi,
Y. S. J. Org. Chem. 1996, 61, 8186-8199.
(49) (a) Cacace, M. G.; Landau, E. M.; Ramsden, J. J. Quart. ReV. Biophysics
1997, 30, 241-277. (b) Collins, K. D.; Washabaugh, M. W. Quart. ReV.
Biophysics 1985, 18, 323-422.
(47) Altona, C.; Sundaralingam, M. J. Am. Chem. Soc. 1973, 95, 2333-2344.
9
12130 J. AM. CHEM. SOC. VOL. 125, NO. 40, 2003

Synthesis and Properties of RNA Analogues
A R T I C L E S
stability of RNA-RNA duplexes compared to DNA-DNA
duplexes both entropically, by conformational preorganization
of the ribose and enthalpically, by improvement of hydration.50
The water mediated hydrogen bond network observed in crystal
structures of A-RNA duplexes is suggested to play a crucial
role in thermal stability.51 Thus, we can suggest that the changes
in hydration pattern of modified RNA-RNA duplexes are more
favorable for AM2 than for AM1. The enthalpical stabilization
of AM2 modified duplexes (when compared to AM1) supports
this hypothesis. It could be that the amide linkage of the AM2
modification interacts with the hydration network through
hydrogen bonding, thereby stabilizing the duplex, but more
structural data is needed to see if this is a viable hypothesis.
Structural regularity, as in Mod 13 having alternating amide
and phosphodiester linkages, could be favorable for formation
of well-defined hydration pattern. This would also explain why
Mod 13 showed generally higher ∆tm values (Table 1). A similar
increase in stability when the number of periodic modifications
are increased has also been observed by others.6c
At this stage, alternative explanations for the greater stability
of the AM2 modified duplexes are also feasible. Previous
molecular dynamics studies in the deoxy series suggest that both
AM1 and AM2 modified DNA-RNA duplexes adopt similar
A-DNA like conformation having trans amide linkages.4c,d,5
Although less likely, we cannot without structural data rule out
the possibility that the 2′-oxygen in our models causes the
alternative cis amide conformation in either AM1 or AM2 leading
to different thermal stabilities of the modified RNA-RNA
duplexes. We can also not rule out the possibility that the
observed effects are either sequence specific or caused by the
alternating nature of the phosphodiester and amide linkages.
Analysis of high salt concentration experiments on Mod 13 may
also be complicated by a possible duplex-triplex equilibrium.
It has been shown that short oligo(rU)-oligo(rA) form duplexes
at low salt concentration, whereas increasing the salt concentra-
tion stabilizes triple helical structures.52 In our melting experi-
ments with Model 13 we always observed single thermal
transitions under all salt concentrations (0.01 to 5 M). However,
duplex to single strands and triplex to duplex transitions in short
oligonucleotides could overlap.52 Therefore, we cannot rule out
the possibility that increasing salt concentration causes AM1
and/or AM2 modified Model 13 to adopt different higher order
structures leading to different behavior in the high salt experi-
ments. Whatever the cause, the higher thermal stability of AM2
modified sequences in all models is nevertheless unambiguous.
Figure 2. Dependence of melting points for AM1 (triangles) and AM2
(circles) modified Mod 13 on the concentration of sodium chloride (empty
markers) and sodium acetate (filled markers). A Mod 13 in the 2′-OH series;
B Mod 13 in the 2′-OMe series.
display a similar behavior, being more stable in sodium chloride
solutions in the 2′-OH series (Figure 2A, filled vs empty circles).
In the 2′-O-methyl series AM2 also displayed an unusual
behavior: the slight stabilization in sodium acetate vs sodium
chloride disappeared as the concentration of salts increased
beyond 3 M (Figure 2B, filled vs empty circles). Addition of
sodium perchlorate (data not shown) strongly destabilized all
sequences with no significant differences between AM1 and
AM2. These results suggested a substantial difference in the
water structure surrounding the duplexes having AM1 and AM2
modifications.
Conclusions
Although there are many different sides to developing
antisense and antigene compounds, favorable hybridization and
stability toward nucleases are of major importance. The amide
modifications studied herein are well tolerated in RNA-RNA
duplexes and are potential candidates for application in oligo-
nucleotide therapeutics: either as 3′- and 5′-end modifications
in a “gapmer” antisense approach, or as uniformly modified
oligonucleotides in alternative procedures (for recent example,
(50) (a) Egli, M. Angew. Chem., Int. Ed. Engl. 1996, 35, 1894-1909. (b) For
a recent review on DNA hydration, see Berman, H. M. Curr. Opin. Struct.
Biol. 1994, 4, 345-350.
(51) (a) Portmann, S.; Usman, N.; Egli, M. Biochemistry 1995, 34, 7569-7575.
(b) Egli, M.; Portmann, S.; Usman, N. Biochemistry 1996, 35, 8489-8494.
(52) Porschke, D. Biopolymers 1971, 10, 1989-2013.
Although more structural data have to be obtained to disclose
the origin of the difference in behavior of duplexes containing
AM1 and AM2 modified oligoribonucleotides, some suggestions
can be made. The 2′-OH contributes to the higher thermal
9
J. AM. CHEM. SOC. VOL. 125, NO. 40, 2003 12131

A R T I C L E S
Rozners et al.
methylsilyl)-2-O-methyl-3-O-phenylthiocarbonyl-â-d-pentofuranosyl]-
uracyl 1b (prepared from 5′-O-(tert-butyldimethylsilyl)-2′-O-methyl-
uridine26 following the published procedures23a) as described above for
see ref 13). The excellent hybridization properties of AM2
modified oligoribonucleotides (∆tm ) +1.0 to +2.4) make this
analogue particularly interesting for further studies. This is to
our knowledge the largest positive effect observed with nonionic
oligonucleotide dephospho analogues studied so far. For com-
parison with other studies it should be noted that these ∆tm
values are relative to an oligoribonucleotide reference as opposed
to an oligodeoxyribonucleotide, which if used as reference would
result in a higher ∆tm for the amide and OH or OMe
modification as a whole.
Although some explanations can be suggested, the difference
between AM1 and AM2 modified duplexes is still puzzling and
further structural studies are of great interest. We therefore
believe that this should be carefully investigated in future
structural studies that must include model systems with uni-
formly modified amide linked RNA. Efforts toward this goal
are well underway and our current synthetic efforts focus on
synthesis of oligoribonucleotides having all phosphodiesters
replaced with amides.53
1
2a. Yield: 70%, Rf) 0.52 (Solvent D), H NMR (CDCl3) δ: 8.47 (s,
1H, NH), 8.24 (d, J ) 8.0 Hz, 1H, H6), 5.86 (s, 1H, H1′), 5.78-5.65
(m, 1H, CH2dCH), 5.63 (dd, JNH-H5 ) 2.2 Hz, 1H, H5), 5.13-5.02
(m, 2H, CH2dCH), 4.15 (m, JH5′-H5′′ ) 12.1 Hz, 1H, H5′), 4.00 (m,
1H, H4′), 3.76-3.70 (m, 2H, H5′, H2′), 3.57 (s, 3H, OCH3), 2.40-
2.20 (m, 2H, CH2dCH-CH2, H3′), 2.01 (m, 1H, CH2dCH-CH2), 0.93
(s, 9H, CH3), 0.11 (s, 6H, SiCH3). 13C NMR (CDCl3) δ: 164.22 (C4),
150.54 (C2), 140.69 (C6), 135.81 (CH2)CH), 117.00 (CH2dCH),
101.41 (C5), 88.48, 86.22, 85.54 (C1′, C4′, C2′), 61.23 (C5′), 58.37
(OCH3), 39.19 (C3′), 28.61 (CH2dCH-CH2), 26.05 (CH3), 18.60
(quaternary C in t-Bu), -5.25, -5.39 (SiCH3).
1-[2,5-O-bis(tert-Butyldimethylsilyl)-3-C-carboxymethyl-â-d-pento-
furanosyl]-uracyl (3a) Compound 2a (3.48 g, 7 mmol) and 4-methyl-
morpholine N-oxide (1.04 g, 7.7 mmol) were dissolved in dioxane (35
mL). A solution of OsO4 (3.5 mL, 250 mg in 25 mL water, 0.14 mmol)
was added, the reaction mixture was protected from light and stirred
for 3 h (TLC, Solvent D). The mixture was diluted with CHCl3 (200
mL) and extracted with saturated NaHCO3 (aqueous) (2 × 200 mL).
The organic layer was separated, dried over Na2SO4, evaporated and
redissolved in dioxane (35 mL). A solution of NaIO4 (1.65 g in 5 mL,
7.7 mmol) was added dropwise and the mixture was stirred for 3-4 h
(TLC Solvent D). The mixture was diluted with CHCl3 (200 mL) and
extracted with saturated NaHCO3 (aqueous) (2 × 200 mL). The organic
layer was separated, dried over Na2SO4, evaporated and the residue
was purified by silica gel column chromatography (0-40% of ethyl
acetate in toluene). Yield: 2.87 g, 82%, Rf) 0.24 (Solvent D), 0.28
Experimental Section
General Methods. Pyridine, acetonitrile, and toluene (pa) were dried
over 3 Å molecular sieves. Methylenechloride was dried over 4 Å
molecular sieves. Triethylamine and DMSO were dried by refluxing
with CaH2 overnight followed by distillation. Pivaloyl chloride and PCl3
were distilled. All other reagents and solvents were used as purchased.
Triethylammonium bicarbonate buffer (pH ca. 7.5) was prepared by
passing CO2 (g) through a mixture of triethylamine and water until
saturation. TLC was performed on Merck silica gel 60 F254 precoated
plates using solvents A (CHCl3/methanol, 9:1, v/v), B (CHCl3/methanol,
4:1, v/v), C (toluene/ethyl acetate, 1:4, v/v), D (CHCl3/methanol, 19:
1, v/v), E (toluene/ethyl acetate, 4:1, v/v). Silica gel (35-70 µm) from
Amicon Europe was used for column chromatography and the columns
were run in the flash mode. Chloroform was passed through basic Al2O3
prior to use. NMR spectra were recorded on a JEOL GSX-270
1
(Solvent E), H NMR (CDCl3) δ: 9.79 (s, 1H, COH), 9.68 (s, 1H,
NH), 8.15 (d, J ) 8.4 Hz, 1H, H6), 5.73 (s, 1H, H1′), 5.66 (dd, JNH-H5
) 1.6 Hz, 1H, H5), 4.44 (d, JH2′-H3′ ) 4.4 Hz, 1H, H2′), 4.04 (m,
JH3′-H4′ ) 9.9 Hz, 1H, H4′), 4.13 and 3.69 (ABX, JH5′-H5′′ ) 11.9 Hz,
JH4′-H5′ ) < 1 Hz, 2H, H5′), 2.82 and 2.42 (ABX, J ) 18.3, 9.5 and
4.2 Hz, 2H, CH2COH), 2.61 (m, 1H, H3′), 0.92, 0.90 (2s, 18H, CH3),
0.24, 0.11, 0.10, 0.05 (4s, 12H, SiCH3). 13C NMR (CDCl3) δ: 199.66
(COH), 163.94 (C4), 150.56 (C2), 140.39 (C6), 101.51 (C5), 91.42
(C1′), 84.72 (C4′), 77.66 (C2′), 61.41 (C5′), 39.40 (CH2COH), 35.05
(C3′), 25.99 (CH3), 18.51, 18.19 (quaternary C in t-Bu), -4.26, -5.39,
-5.45 (SiCH3).
This material (2.25 g, 4.5 mmol) was dissolved in a mixture of
DMSO (10 mL) and tert-butyl alcohol (20 mL) and a solution of NaH2-
PO4 × H2O (0.21 g in 2 mL of water, 1.35 mmol) were added. A
solution of NaClO2 (0.72 g in 6 mL of water, 6.3 mmol) was added
during 2 h under stirring and cooling (ice bath) and the mixture was
further stirred for 4 h at 20 °C (TLC Solvent D). The mixture was
diluted with CHCl3 (200 mL) and extracted with saturated NaCl (3 ×
200 mL, containing 0.5 mL of acetic acid). The organic layer was
separated, dried over Na2SO4, evaporated and the residue was purified
by silica gel column chromatography (0-60% of ethyl acetate in toluene
containing 0.1% of acetic acid). Yield: 2.87 g, 82%, Rf ) 0.20 (Solvent
D). 1H NMR (CDCl3) δ: 10.14 (s, 1H, NH), 8.23 (d, J ) 8.4 Hz, 1H,
H6), 5.71 (s, 1H, H1′), 5.68 (d, 1H, H5), 4.44 (d, JH2′-H3′ ) 4.4 Hz,
1H, H2′), 4.04 (m, JH3′-H4′ ) 9.9 Hz, 1H, H4′), 4.14 and 3.72 (ABX,
JH5′-H5′′ ) 11.2 Hz, JH4′-H5′ ) < 1 Hz, 2H, H5′), 2.68 and 2.28 (ABX,
J ) 16.5 and <1 Hz, 2H, CH2COOH), 2.51 (m, 1H, H3′), 0.92, 0.90
(2s, 18H, CH3), 0.23, 0.11, 0.10, 0.08 (4s, 12H, SiCH3). 13C NMR
(CDCl3) δ: 176.81 (COOH), 164.76 (C4), 150.62 (C2), 140.98 (C6),
101.38 (C5), 91.63 (C1′), 84.63 (C4′), 77.82 (C2′), 61.31 (C5′), 37.01
(C3′), 29.23 (CH2COOH), 25.98 (CH3), 18.55, 18.20 (quaternary C in
t-Bu), -4.28, -5.42, -5.53 (SiCH3).
1
spectrometer at 25 °C. Signals were assigned by H-1H and 13C-1H
COSY experiments.
1-[3-C-Allyl-2,5-O-bis(tert-butyldimethylsilyl)-â-D-pentofurano-
syl]-uracyl (2a). 1-[2,5-O-bis(tert-butyldimethylsilyl)-3-O-phenyl-
thiocarbonyl-â-d-pentofuranosyl]-uracyl 1a (6.10 g, 10 mmol, prepared
from 2′,5′-O-bis(tert-butyldimethylsilyl)uridine26 following the pub-
lished procedures23a) was dissolved in dry toluene, tributylallyltin (12.5
mL, 40 mmol) was added and the solution was degassed by passing
through dry nitrogen gas (ca. 30 min). AIBN (0.82 g, 5 mmol) was
added, and the mixture was heated at 80 °C for 2 h. Another portion
of AIBN (0.41 g, 2.5 mmol) was added and the mixture heated at 80
°C for 4 h. The mixture was cooled to 20 °C and purified by silica gel
column chromatography (0-30% of ethyl acetate in toluene). Yield:
3.76 g, 75%, Rf ) 0.36 (Solvent D), 0.38 (Solvent E), 1H NMR (CDCl3)
δ: 9.85 (s, 1H, NH), 8.26 (d, J ) 8.0 Hz, 1H, H6), 5.86-5.67 (m, 1H,
CH2dCH), 5.66 (s, 1H, H1′), 5.60 (dd, JNH-H5 ) 1.8 Hz, 1H, H5),
5.12-5.02 (m, 2H, CH2dCH), 4.26 (d, JH2′-H3′ ) 3.6 Hz, 1H, H2′),
4.06 (m, JH3′-H4′ ) 9.9 Hz, 1H, H4′), 4.17 and 3.74 (ABX, JH5′-H5′′
)
12.1 Hz, JH4′-H5′ ) 1.5 and < 1 Hz, 2H, H5′), 2.34 and 2.02 (2m, 2H,
CH2dCH-CH2), 2.14 (m, 1H, H3′), 0.93 (s, 18H, CH3), 0.28, 0.13,
0.12, 0.11 (4s, 12H, SiCH3). 13C NMR (CDCl3) δ: 164.26 (C4), 150.61
(C2), 140.79 (C6), 135.80 (CH2)CH), 116.72 (CH2dCH), 101.10 (C5),
91.42 (C1′), 85.29 (C4′), 77.98 (C2′), 63.49 (C5′), 40.07 (C3′), 28.63
(CH2dCH-CH2), 26.02 (CH3), 18.54, 18.27 (quaternary C in t-Bu),
-4.00, -5.34, -5.42 (SiCH3).
1-[5-O-(tert-Butyldimethylsilyl)-3-C-carboxymethyl-2-O-methyl-
â-d-pentofuranosyl]-uracyl (3b) was prepared from 2b using the same
procedures as for 3a. Intermediate aldehyde: yield: 70%, Rf ) 0.28
(Solvent D), 0.45 (Solvent A), 1H NMR (CDCl3) δ: 9.86 (s, 1H, NH),
9.78 (s, 1H, COH), 8.12 (d, J ) 8.1 Hz, 1H, H6), 5.91 (s, 1H, H1′),
1-[3-C-Allyl-5-O-(tert-butyldimethylsilyl)-2-O-methyl-â-d-pento-
furanosyl]-uracyl (2b) was synthesized from 1-[5-O-(tert-butyldi-
(53) Rozners, E.; Liu, Y. Org. Lett. 2003, 5, 181-184.
9
12132 J. AM. CHEM. SOC. VOL. 125, NO. 40, 2003

Synthesis and Properties of RNA Analogues
A R T I C L E S
5.65 (d, 1H, H5), 4.11 and 3.67 (ABX, JH5′-H5′′ ) 12.1 Hz, JH4′-H5′
)
5.61 (d, 1H, H5), 4.39 (d, J ) 4.0 Hz, 1H, H2′), 4.13 (m, 2H, H4′,
H5′), 3.74 (m, 1H, H5′), 2.73 and 2.97 (ABX, J ) 12.2, 8.4, 5.1 Hz,
2H, CH2N), 2.23 (m, 1H, H3′), 0.92, 0.91 (2s, 18H, CH3), 0.25-0.11
(4s, 12H, SiCH3). 13C NMR (CDCl3/CD3OD, 19:1) δ: 164.03 (C4),
150.60 (C2), 140.62 (C6), 101.28 (C5), 91.30 (C1′), 83.85 (C4′), 77.17
(C2′), 62.33 (C5′), 43.74 (C3′), 37.61 (CH2N), 26.07, 25.88 (CH3),
18.58, 18.39 (quaternary C in t-Bu), -4.15, -4.58, -5.39 (SiCH3).
HRMS calcd for C22H43O5N3Si2+Na 508.2639, found 508.2653.
8d yield 53% (dr 85:15), Rf ) 0.12 (Solvent A), 1H NMR (CDCl3/
CD3OD, 19:1) δ: 8.19 (d, J ) 8.1 Hz, 1H, H6), 5.90 (s, 1H, H1′),
5.61 (d, 1H, H5), 4.16-4.06 (m, 2H, H4′, H5′), 3.90 (d, J ) 4.8 Hz,
1H, H2′), 3.73 (m, 1H, H5′), 3.60 (s, 3H, OCH3), 2.97 and 2.76 (2m,
2H, CH2N), 2.35 (m, 1H, H3′), 0.93 (s, 9H, CH3), 0.18 (s, 6H, SiCH3).
13C NMR (CDCl3/CD3OD, 19:1) δ: 164.30 (C4), 150.54 (C2), 140.47
(C6), 101.43 (C5), 88.13, 86.05, 83.71 (C1′, C4′, C2′), 61.61 (C5′),
58.13 (OCH3), 42.21 (C3′), 37.00 (CH2N), 25.99 (CH3), 18.55
(quaternary C in t-Bu), -5.41, -5.48 (SiCH3). HRMS calcd for
C17H31O5N3Si+Na 408.1931, found 408.1928.
< 1 Hz, 2H, H5′), 3.98 (m, JH3′-H4′ ) 10.8 Hz, 1H, H4′), 3.93 (d,
JH2′-H3′ ) 5.1 Hz, 1H, H2′), 3.50 (s, 3H, OCH3), 2.81 and 2.39 (ABX,
J ) 17.8, 9.7 and 3.8 Hz, 2H, CH2COH), 2.65 (m, 1H, H3′), 0.90 (s,
9H, CH3), 0.09 (s, 6H, SiCH3).
Carboxylic acid 3b: yield 86%, Rf ) 0.15 (Solvent D), 0.66 (Solvent
B). 1H NMR (CDCl3/CD3OD, 19:1) δ: 8.16 (d, J ) 8.1 Hz, 1H, H6),
5.85 (s, 1H, H1′), 5.60 (d, 1H, H5), 4.10 and 3.66 (2m, 2H, H5′), 3.92
(m, 2H, H2′, H4′), 3.51 (s, 3H, OCH3), 2.57 (m, 2H, CH2COOH, H3′),
2.20 (m, 1H, CH2COOH), 0.88 (s, 9H, CH3), 0.08, 0.07 (2s, 6H, SiCH3).
13C NMR (CDCl3/CD3OD, 19:1) δ: 174.14 (COOH), 164.26 (C4),
150.32 (C2), 140.58 (C6), 101.32 (C5), 88.58 (C1′), 85.89 (C4′), 84.75
(C2′), 60.96 (C5′), 58.34 (OCH3), 35.97 (C3′), 28.63 (CH2COOH),
25.91 (CH3), 18.49 (quaternary C in t-Bu), -5.50 (SiCH3).
Amines 5 were prepared from 5′-azido-5′-deoxyuridine28 by reduc-
tion with triphenylphosphine29b (5a, 81%) or via acylation with acetyl
chloride in CH2Cl2/pyridine, 9:1 and radical reduction with tributyl-
stannane29a (5b, 61%, two steps).
5b 1H NMR (CDCl3/CD3OD, 4:1) δ: 7.49 (d, J ) 8.1 Hz, 1H, H6),
5.76 (d, JH1′-H2′ ) 5.1 Hz, 1H, H1′), 5.65 (d, 1H, H5), 5.34 (t, JH2′-H3′
) 5.8 Hz, 1H, H2′), 5.22 (t, JH3′-H4′ ) 5.9 Hz, 1H, H3′), 3.98 (m, 1H,
H4′), 2.94 and 2.84 (ABX, JH5′-H5′′ ) 14.2 Hz, JH4′-H5′ ) 3.5 and 5.3
Hz, 2H, H5′), 2.01, 2.00 (2s, 6H, Ac). 13C NMR (CDCl3/CD3OD, 4:1)
δ: 170.11, 169.97 (CdO), 163.97 (C4), 150.43 (C2), 141.22 (C6),
102.93 (C5), 88.87 (C1′), 82.55 (C4′), 73.01, 70.28 (C2′, C3′), 42.23
(C5′) 20.34, 20.26 (CH3 in Ac).
Compound 6c (205 mg, 0.4 mmol) was dissolved in anhydrous THF
(5 mL). The mixture was cooled on an acetone-dry ice bath (-78 °C)
(Bu)4NBH4 (616 mg, 2.4 mmol) was added and the mixture was stirred
at -78 °C for 5 h. The mixture was treated with NaBH4/Ni2B (1 equiv.),
stirred at 20 °C overnight, worked up and purified as described above
to give 8c, yield 101 mg (49%), dr 98:2.
2′,3′-O-Benzylidene-5′-C-(nitromethyl)uridine (10). 2′,3′-O-(Ben-
zylidene)uridine54 9 (1.99 g, 6 mmol) was dried by evaporation of added
dry toluene (50 mL) and dry acetonitrile (50 mL) and then dissolved
in dry DMSO (15 mL). DCC (3.71 g, 18 mmol) and dichloroacetic
acid (0.24 mL) were added and the mixture was stirred for 18 h at 20
°C. Nitromethane (30 mL), methanol (12 mL) and NaOCH3 (9 mL,
30% in methanol) were mixed, stirred for 10 min at 20 °C and added
to the reaction mixture. After stirring for 2 h at 20 °C the mixture was
neutralized and the excess DCC was hydrolyzed by a careful addition
(cooling on ice) of oxalic acid dihydrate (7.9 g, 63 mmol) in methanol
(25 mL). The mixture was stirred for 30 min at 0 °C, filtered, the
precipitate was washed with cold methanol (10 mL), and the filtrate
was evaporated. The residue was dissolved in CHCl3 (200 mL) and
extracted with saturated NaHCO3/saturated NaCl (aqueous) (1:1, 150
mL). Organic layer was separated, dried over Na2SO4, evaporated and
the residue was purified by silica gel column chromatography (0-5%
of methanol in CHCl3, two purifications were necessary to remove
nonnucleosidic contamination completely). Yield 1.21 g, 51% Rf ) 0.24
(Solvent A), 1H NMR (major diastereomer, CDCl3) δ: 7.52-7.36 (m,
5H, Ar), 7.27 (d, J ) 8.1 Hz, 1H, H6), 6.03 (s, 1H, benzylidene), 5.73
(d, 1H, H5), 5.59 (d, J ) 1.8 Hz, 1H, H1′), 5.29 (m, 1H, H3′), 5.14
General Procedure for Synthesis of Amines 8. Ni2B catalyst38 was
prepared as follows: NiCl2-6H2O (238 mg, 1 mmol) was dissolved in
anhydrous ethanol (10 mL), NaBH4 (113 mg, 3 mmol) was added and
the mixture was stirred at 0 °C for 30 min. Compounds 6a-d (1 mmol,
prepared as described in ref 32) were dissolved in cold (0 °C) anhydrous
ethanol (15 mL), NaBH4 (151 mg, 4 mmol) was added and the mixture
was stirred at 0 °C for 2 h. A solution of freshly prepared Ni2B catalyst
(1.5 equiv. for 6a,b or 1 equiv. for 6c,d) was then added. After 45 min
another portion of NaBH4 (375 mg, 10 mmol) was added to the reaction
mixture. After stirring at 20 °C for 8 h the mixture was diluted with
CHCl3 (100 mL) and extracted first with 10% citric acid (aqueous)
(50 mL) and then with saturated NaHCO3 (aqueous) (50 mL). Organic
layer was separated, dried over Na2SO4, evaporated and the residue
was purified by silica gel column chromatography (0-8% of methanol
in CHCl3).
1
8a yield 40%, Rf ) 0.35 (Solvent A), H NMR (CDCl3/CD3OD,
19:1) δ: 8.19 (d, J ) 8.2 Hz, 1H, H6), 7.46-7.27, 6.88 (m, 14H,
MMT), 5.72 (s, 1H, H1′), 5.29 (d, 1H, H5), 4.43 (d, J ) 4.2 Hz, 1H,
H2′), 4.14 (m, 1H, H4′), 3.82 (s, 3H, OCH3), 3.72 and 3.35 (ABX, J
) 11.5, 2.0, 2.9 Hz, 2H, H5′), 2.89 and 2.50 (ABX, J ) 12.8, 8.6, 5.0
Hz, 2H, CH2N), 2.37 (m, 1H, H3′), 0.92 (s, 9H, CH3), 0.27, 0.18 (2s,
6H, SiCH3). 13C NMR (CDCl3/CD3OD, 19:1) δ: 164.09 (C4), 150.60
(C2), 158.98, 143.98, 143.82, 134.77, 130.71, 128.63, 128.20, 127.44,
113.47 (MMT), 140.58 (C6), 101.47 (C5), 91.61 (C1′), 82.64 (C4′),
77.37 (C2′), 62.15 (C5′), 55.42 (OCH3), 44.77 (C3′), 37.26 (CH2N),
25.91 (CH3), 18.24 (quaternary C in t-Bu), -4.01, -5.36 (SiCH3).
HRMS calcd for C36H45O6N3Si+Na 666.2975, found 666.2989.
(m, 1H, H2′), 4.62-4.41 (m, 3H, CHCH2NO2), 4.08 (m, 1H, H4′). 13
C
NMR (major diastereomer, CDCl3) δ: 163.98 (C4), 150.98 (C2),
135.63, 130.17, 128.65, 126.82, 104.28 (benzylidene), 143.87 (C6),
103.28 (C5), 96.80 (C1′), 85.72 (C4′), 83.61 (C2′), 81.85 (C3′), 78.45
(CH2NO2), 68.61 (C5′).
2′,3′,5′-O-Triacetyl-5′-C-(nitromethyl)uridine (11). Compound 10
(0.99 g, 2.5 mmol) was dissolved in cold (0 °C) acetic anhydride (15
mL), HClO4 (0.24 mL) was added and the mixture was stirred for 1 h
at 0 °C. The mixture was diluted with CHCl3 (100 mL), saturated
NaHCO3 (aqueous) (50 mL) and saturated NaCl (aqueous) (50 mL)
were added, and the mixture was stirred for 30 min at 20 °C. Organic
layer was extracted with saturated NaHCO3/saturated NaCl (aqueous)
(1:1, 100 mL), separated, dried over Na2SO4, evaporated, coevaporated
with toluene (2 × 100 mL), and the residue was purified by silica gel
column chromatography (0-6% of methanol in CHCl3). Yield 0.97 g,
90% Rf ) 0.42 (Solvent A), 1H NMR (major diastereomer, CDCl3) δ:
9.90 (s, 1H, NH), 7.21 (d, J ) 8.1 Hz, 1H, H6), 5.85-5.75 (m, 3H,
H1′, H5, H5′), 5.60-5.52 (m, 2H, H2′, H3′), 4.89-4.62 (m, 2H, CH2-
1
8b yield 47% (dr 94:6), Rf ) 0.10 (Solvent A), H NMR (CDCl3/
CD3OD, 19:1) δ: 8.19 (d, J ) 8.1 Hz, 1H, H6), 7.44-7.28, 6.86 (m,
14H, MMT), 5.92 (s, 1H, H1′), 5.25 (d, 1H, H5), 4.08 (m, 1H, H4′),
3.93 (d, J ) 4.2 Hz, 1H, H2′), 3.80 (s, 3H, OCH3 in MMT), 3.68 and
3.30 (ABX, J ) 11.3, 1.5, 2.5 Hz, 2H, H5′), 3.59 (s, 3H, OCH3), 2.91
(m, 1H, CH2N), 2.55 (m, 2H, CH2N, H3′). 13C NMR (CDCl3/CD3OD,
19:1) δ: 164.22 (C4), 150.51 (C2), 158.90, 143.85, 143.68, 134.67,
130.60, 128.52, 128.14, 127.41, 113.41 (MMT), 140.42 (C6), 101.70
(C5), 88.29, 85.78, 82.60 (C1′, C4′, C2′), 61.50 (C5′), 58.10, 55.35
(OCH3), 43.37 (C3′), 36.84 (CH2N). HRMS calcd for C31H33O6N3+Na
566.2267, found 566.2288.
(54) Prepared using the procedure described for 2′,3′-O-(p-anisylidene)uridine:
Smith, M.; Rammler, D. H.; Goldberg, I. H.; Khorana, H. G. J. Am. Chem.
Soc. 1976, 84, 430-440.
8c yield 55% (dr 85:15), Rf ) 0.25 (Solvent A), 1H NMR (CDCl3/
CD3OD, 19:1) δ: 8.17 (d, J ) 8.1 Hz, 1H, H6), 5.68 (s, 1H, H1′),
9
J. AM. CHEM. SOC. VOL. 125, NO. 40, 2003 12133

A R T I C L E S
Rozners et al.
NO2), 4.27 (t, 1H, H4′), 2.12, 2.09 (2s 9H, acetyl). 13C NMR (major
diastereomer, CDCl3) δ: 170.02, 169.86, 169.67 (CdO), 163.51 (C4),
150.32 (C2), 142.28 (C6), 103.46 (C5), 92.42 (C1′), 80.52 (C4′), 73.99
(CH2NO2), 72.45, 70.30 (C3′, C2′), 69.03 (C5′), 20.76, 20.57 (CH3).
2′,3′-O-Diacetyl-5′-deoxy-5′-C-(nitromethyl)uridine (12). Com-
pound 11 (1.08 g, 2.5 mmol) was dried by evaporation of added dry
acetonitrile (30 mL) and then dissolved in cold (0 °C) absolute ethanol/
THF (1:1, 20 mL). NaBH4 (0.19 g 5 mmol) was added in small portions
over 10 min and the mixture was stirred for 45 min at 0 °C. The reaction
mixture was neutralized with acetic acid, diluted with CHCl3 (100 mL),
and extracted with saturated NaHCO3/saturated NaCl (aqueous) (1:1,
100 mL). The organic layer was separated, dried over Na2SO4,
evaporated, and the residue was purified by silica gel column chro-
matography (0-5% of methanol in CHCl3). Yield 0.82 g, 88%, Rf )
0.42 (Solvent A), 1H NMR (CDCl3) δ: 7.20 (d, J ) 8.1 Hz, 1H, H6),
5.74 (d, 1H, H5), 5.59 (d, JH1′-H2′ ) 4.0 Hz, 1H, H1′), 5.47 (dd, JH2′-H3′
) 6.3 Hz, 1H, H2′), 5.24 (t, 1H, H3′), 4.53 (t, 2H, CH2NO2), 4.12 (m,
1H, H4′), 2.55-2.32 (m, 2H, H5′), 2.08, 2.07 (2s 6H, acetyl). 13C NMR
(CDCl3) δ: 170.07, 169.96 (CdO), 163.57 (C4), 150.24 (C2), 141.77
(C6), 103.27 (C5), 91.48 (C1′), 78.44 (C4′), 72.97 (C2′), 72.48 (C3′),
71.54 (CH2NO2), 29.96 (C5′), 20.52 (CH3).
14b a modified procedure was used: amine 5a was added in dry
DMF (10 mL), after stirring for 4 h, the solvent was evaporated (no
aqueous workup) and the residue was purified by silica gel column
chromatography (2-10% of methanol in CHCl3), yield 0.35 g, 47%,
1
Rf ) 0.45 (Solvent B), H NMR (CDCl3/CD3OD, 4:1)55 δ: 8.09 (d, J
) 8.6 Hz, 2H, H6), 7.36 (d, J ) 8.0 Hz, 2H, H6*), 5.76 (s, 1H, H1′),
5.61 (d, 1H, H5*), 5.52 (d, 1H, H5), 5.46 (d, JH1′-H2′ ) 4.0 Hz, H1′*),
4.20 (m, 1H, H2′*), 4.03-3.87 (m, 4H, H3′*, H4′, H4′*, H5′), 3.77
(d, JH2′-H3′ ) 5.2 Hz 1H, H2′), 3.62-3.53 (m, 2H, H5′′, H5′*), 3.40
(s, 3H, OCH3), 3.34-3.24 (m, 1H, H5′′*), 2.56 (m, 1H, H3′), 2.37,
2.08 (2m, 2H, CH2CO), 0.82 (s, 9H, t-Bu), 0.01 (s, 6H, SiCH3). 13C
NMR (CDCl3/CD3OD, 4:1)55 δ: 172.03 (CdO), 164.48, 164.24 (C4),
150.77, 150.48 (C2), 142.19 (C6*), 140.55 (C6), 102.29 (C5*), 101.18
(C5), 93.12 (C1′*), 88.12 (C1′), 85.67 (C2′), 84.78, 82.49 (C4′, C4′*),
73.18 (C2′*), 70.59 (C3′*), 61.09 (C5′), 57.88 (OCH3), 40.75 (C5′*),
36.32 (C3′), 30.60 (CH2CO), 25.74 (CH3), 18.32 (quaternary C in t-Bu),
-5.72 (SiCH3).
18a 50-100% of ethyl acetate in toluene, yield 0.85 g, 86%, Rf )
0.21 (Solvent D), 0.40 (Solvent C), 1H NMR (CDCl3)55 δ: 7.99 (d, J
) 8.2 Hz, 1H, H6), 7.39-7.14 (m, 13H, MMT, H6*), 6.83 (m, 2H,
MMT), 5.72 (d, JH1′-H2′ ) 2.0 Hz, 1H, H1′), 5.66 (d, JH1′-H2′ ) 4.6
Hz, 1H, H1′*), 5.59-5.53 (m, 2H, H2′*, H5*), 5.31 (t, 1H, H3′*),
5.18 (d, 1H, H5), 4.38 (m, 2H, H4′*, H2′), 4.18 (m, 1H, H4′), 3.77 (s,
3H, OCH3), 3.63-3.36 (m, 3H, H5′, CH2NH), 3.15 (m, 1H, CH2NH),
2.59-2.44 (m, 3H, H5′*, H3′), 2.06 (2s, 6H, CH3CO), 0.86 (s, 9H,
t-Bu), 0.15, 0.07 (2s, 6H, SiCH3). 13C NMR (CDCl3)55 δ: 170.09,
169.95, 169.84 (CdO), 163.79, 163.44 (C4), 150.63 (C2), 142.44 (C6*),
140.50 (C6), 158.98, 143.85, 143.66, 134.58, 130.66, 128.58, 128.20,
127.50, 113.47 (MMT), 103.07 (C5*), 101.88 (C5), 91.50 (C1′*), 91.01
(C1′), 87.53 (MMT), 82.34 (C4′), 79.26, 77.45 (C2′, C4′*), 72.93
(C3′*), 72.80 (C2′*), 62.96 (C5′), 55.37 (OCH3), 41.86 (C3′), 38.91
(C5′*), 36.10 (CH2NH), 25.83 (CH3), 20.56 (CH3CO), 18.10 (quaternary
C in t-Bu), -4.41, -5.38 (SiCH3). HRMS calcd for C50H59O14N5Si+Na
1004.3726, found 1004.3714.
Carboxylic acid 13. Compound 12 (0.80 g, 2.15 mmol) was dried
by evaporation of added dry acetonitrile (30 mL) and then dissolved
in dry DMSO (5 mL). NaNO2 (0.69 g, 10 mmol) and acetic acid (1.8
mL, 33 mmol) were added and the mixture was stirred at 40 °C for 30
h. Water (5 mL) was added, pH was adjusted to ca. 4.5 with 1 M HCl,
and the mixture was partitioned between CH2Cl2 (100 mL) and saturated
NaCl (aqueous) (50 mL). The water layer was extracted with CH2Cl2
(4 × 50 mL). Combined organic layers were separated, dried over Na2-
SO4, evaporated and the residue was purified by silica gel column
chromatography (0-10% of methanol in CHCl3). Yield 0.51 g, 66%
Rf ) 0.20 (Solvent B), 1H NMR (CDCl3) δ: 7.44 (d, J ) 8.1 Hz, 1H,
H6), 5.95 (d, JH1′-H2′ ) 5.9 Hz, 1H, H1′), 5.74 (d, 1H, H5), 5.45-5.31
(m, 2H, H2′, H3′), 4.31 (m, 1H, H4′), 2.80 (d, J ) 5.1 Hz, 2H, H5′),
2.07, 2.05 (2s, 6H, CH3). 13C NMR (CDCl3) δ: 172.95, 170.06, 169.87
(CdO), 163.71 (C4), 150.47 (C2), 140.98 (C6), 103.47 (C5), 87.53
(C1′), 79.01, 72.77, 72.23 (C4′, C3′, C2′), 36.59 (C5′), 20.72, 20.59
(CH3).
18b 0-10% of methanol in CHCl3, yield 0.77 g, 87%, Rf ) 0.18
(Solvent D), 0.45 (Solvent C), 1H NMR (CDCl3)55 δ: 8.07 (d, J ) 8.1
Hz, 1H, H6), 7.41-7.18 (m, 13H, MMT, H6*), 6.83 (m, 2H, MMT),
5.83 (s, 1H, H1′), 5.65-5.55 (m, 3H, H1′*, H2′*, H5*), 5.32 (t, 1H,
H3′*), 5.19 (d, 1H, H5), 4.45 (m, 1H, H4′*), 4.10 (m, 1H H4′), 3.86
(m, 1H, H2′), 3.76 (s, 3H, OCH3), 3.60-3.20 (m, 4H, H5′, CH2NH),
3.50 (s, 3H, 2′-OCH3), 2.71-2.53 (m, 3H, H5′*, H3′), 2.06, 2.05 (2s,
6H, CH3CO). 13C NMR (CDCl3)55 δ: 170.04, 169.50, 169.40 (CdO),
164.03, 163.49 (C4), 150.86, 150.51 (C2), 142.39 (C6*), 140.47 (C6),
158.90, 143.95, 143.68, 134.62, 130.65, 128.55, 128.20, 127.41, 113.47
(MMT), 103.21 (C5*), 101.70 (C5), 91.34 (C1′*), 88.56 (C1′), 87.43
(MMT), 85.95 (C2′), 82.52 (C4′), 79.63 (C4′*), 72.83 (C3′*, C2′*),
61.82 (C5′), 58.24 (2′-OCH3), 55.38 (OCH3), 40.56 (C3′), 39.00
(C5′*), 34.98 (CH2NH), 20.63, 20.54 (CH3CO). HRMS calcd for
C45H47O14N5+Na 904.3017, found 904.3018.
Selective Cleavage of 5′-O-(tert-Butyldimethylsilyl) Protection.
Dimers 14a and 14b were dissolved in 80% acetic acid (aqueous) (20
mL/mmol) and the solutions were heated at 50 °C for 3 h. The solvent
was evaporated and the residue was dried by coevaporating with
absolute ethanol (2 × 20 mL). Dimer 14a′ was purified by silica gel
column chromatography (0-15% of methanol in CHCl3), yield 59%,
Rf) 0.42 (Solvent A). Later fractions gave fully desilylated dimer 34%,
Rf ) 0.17 (Solvent A). Crude 15b was used in subsequent steps.
General Procedure for Synthesis of Amide Linked Dinucleosides.
Carboxylic acid (1 mmol) and 1-hydroxybenzotriazole (0.135 g 1 mmol)
were dried by evaporation of added dry acetonitrile (2 × 30 mL) and
were then dissolved in dry CH2Cl2 (10 mL). DCC (0.213 g, 1 mmol)
was added, and the mixture was stirred at 20 °C for 30 min. The mixture
was filtered, and the required amine (1 mmol) and triethylamine (0.14
mL, 1.5 mmol) were added. The mixture was stirred at 20 °C for 4 h
(TLC, Solvents B and C), diluted with CH2Cl2 (40 mL) and extracted
with saturated NaHCO3 (aqueous) (2 × 50 mL). The organic layer was
separated, dried over Na2SO4, evaporated, and purified by silica gel
column chromatography using the solvent systems specified below.
14a 50-100% of ethyl acetate in toluene, yield 0.56 g, 68%, Rf )
0.22 (Solvent D), 0.29 (Solvent C), 1H NMR (CDCl3)55 δ: 9.97, 9.74
(2s, 2H, NH), 8.17 (d, J ) 8.1 Hz, 2H, H6), 7.33 (d, J ) 8.1 Hz, 2H,
H6*), 5.81 (m, 2H, H5*, H1′), 5.68 (m, 2H, H5, H1′*), 5.59 (t, 1H,
H2′*), 5.36 (t, 1H, H3′*), 4.51 (m, 1H, H2′), 4.25 (m, 1H, H4′*), 4.06
(m, 2H, H4′, H5′), 3.79 (m, 2H, H5′′, H5′*), 3.52 (m, 1H, H5′′*), 2.68
(m, 2H, H3′, CH2CO), 2.26 (m, 1H, CH2CO), 2.15, 2.13 (2s, 6H, CH3-
CO), 0.97, 0.92 (2s, 18H, t-Bu), 0.20, 0.15, 0.09 (3s, 12H, SiCH3). 13
C
NMR (CDCl3)55 δ: 171.59, 170.00, 169.84 (CdO), 163.92, 163.49
(C4), 150.70, 150.35 (C2), 142.30 (C6*), 140.71 (C6), 103.06 (C5*),
101.68 (C5), 91.82 (C1′*), 90.36 (C1′), 84.60 (C4′), 80.66 (C4′*), 77.47
(C2′), 72.79 (C2′*), 70.82 (C3′*), 62.74 (C5′), 40.42 (C5′*), 37.98 (C3′),
31.55 (CH2CO), 26.01, 25.85 (CH3), 20.53 (CH3CO), 18.50, 18.09
(quaternary C in t-Bu), -4.55, -5.36, -5.42, -5.50 (SiCH3). HRMS
calcd for C36H57O13N5Si2 823.3491, found 823.3508.
Cleavage of Acetyl Protections. Dimers 14a′, 18a, and 18b were
dissolved in 32% NH3/ethanol, 2:1 (20 mL/mmol) and kept at 20 °C
for 6 h. The solvent was evaporated. Crude 15a was dissolved in water
(20 mL/mmol), freeze-dried and used in subsequent steps. 18a and 18b
were dried by evaporation of added absolute ethanol (2 × 20 mL) and
purified by silica gel column chromatography: 19a 0-12% of methanol
in CHCl3, yield 85%, Rf ) 0.38 (Solvent A); 19b 5-15% of methanol
in CHCl3, yield 69%, Rf ) 0.66 (Solvent B).
(55) * indicates resonances from protons and carbons in 5′-yl unit of the dimer.
9
12134 J. AM. CHEM. SOC. VOL. 125, NO. 40, 2003

Synthesis and Properties of RNA Analogues
A R T I C L E S
1
5′-O-Monometoxytritylation. Dimers 15a and 15b were reacted
with 4-monomethoxytrityl chloride (1.1 equiv) according to the standard
procedure,56 and the products were purified by silica gel column
chromatography (0-10% of methanol in CHCl3): 16a, yield 74% (43%,
three steps from 14a), Rf ) 0.29 (Solvent A); 19b yield 60%, two steps
from 14b, Rf ) 0.63 (Solvent B).
20b yield 75%, Rf ) 0.27 (Solvent B), H NMR (CDCl3/CD3OD,
9:1)55 δ: 8.13 (d, J ) 8.1 Hz, 1H, H6), 7.95 (d, J ) 7.7 Hz, 1H,
o-ClBz), 7.65 (d, J ) 8.1 Hz, 1H, H6*), 7.47-7.21 (m, 15H, Ar), 6.87
(m, 2H, MMT), 6.86 (d, J ) 635 Hz, 1H, PH), 6.04 (bd, 1H, H1′*),
5.88 (s, 1H, H1′), 5.57 (d, 2H, H5*, H2′*), 5.21 (d, 1H, H5), 4.90 (p,
1H, H3′*), 4.58 (m, 1H, H4′*), 4.12 (m, 1H, H4′), 3.92 (m, 1H, H2′),
3.79 (s, 3H, OCH3), 3.62-3.37 (m, 4H, CH2NH, H5′), 3.56 (s, 3H,
2′-OCH3), 2.90 (q, J ) 7.4 Hz, 6H, NCH2), 2.86-2.70 (m, 3H, H3′,
H5′*), 1.19 (t, 9H, CH3). 13C NMR (CDCl3/CD3OD, 9:1)55 δ: 170.07
(CdO in amide), 164.14, 163.78 (C4, C4*, CdO in o-ClBz), 150.64,
150.46 (C2, C2*), 158.82, 144.03, 143.71, (MMT) 141.39, 140.47 (C6,
C6*), 134.67, 134.11, 133.30, 132.27, 131.14, 130.63, 128.79, 128.52,
128.12 127.28, 126.90, 113.41 (MMT, o-ClBz), 103.00, 101.51 (C5,
C5*), 88.78, 88.51 (C1′, C1′*), 87.29 (MMT), 85.49 (C2′), 82.52 (C4′),
80.90 (C4′*), 74.83 (C2′*), 72.51 (C3′*), 61.72 (C5′), 58.13 (2′-OCH3),
55.32 (OCH3), 45.74 (NCH2), 40.46 (C3′), 38.59 (C5′*), 34.76 (CH2-
NH), 8.92 (CH3). HRMS calcd for C48H47O15N5ClP+Na 1022.2395,
found 1022.2433.
Synthesis of H-Phosphonates 17a,b and 20a,b was done as
previously reported.20
1
17a yield 69%, Rf ) 0.24 (Solvent B), H NMR (CDCl3/CD3OD,
9:1)55 δ: 8.11 (d, J ) 8.1 Hz, 1H, H6), 7.91 (d, J ) 7.7 Hz, 1H,
o-ClBz), 7.43-7.23 (m, 16H, Ar, H6*), 6.85 (m, 2H, MMT), 6.82 (d,
J ) 640 Hz, 1H, PH), 5.89 (bs, 1H, H1′*), 5.73 (s, 1H, H1′), 5.69 (d,
J ) 8.1 Hz, 1H, H5*), 5.57 (m, 1H, H2′*), 5.21 (d, 1H, H5), 4.91 (p,
1H, H3′*), 4.49 (m, 1H, H2′), 4.23 (m, 1H, H4′*), 4.08 (m, 1H, H4′),
3.78 (s, 3H, OCH3), 3.63-3.55 (m, 3H, H5′*, H5′), 3.34 (m, 1H, H5′′),
2.97 (q, J ) 7.3 Hz, 6H, NCH2), 2.73 (m, 1H, H3′), 2.49 and 2.07
(2m, 2H, CH2CO), 1.24 (t, 9H, CH3), 0.86 (s, 9H, t-Bu), 0.19, 0.06
(2s, 6H, SiCH3). 13C NMR (CDCl3/CD3OD, 9:1)55 δ: 171.33 (CdO in
amide), 164.21, 164.13, 163.64 (C4, C4*, CdO in o-ClBz), 150.50,
150.42 (C2, C2*), 158.73, 143.87, 143.68, (MMT) 141.57, 140.76 (C6,
C6*), 134.64, 133.99, 133.34, 132.08, 131.10, 130.57, 128.49, 128.00
127.25, 126.84, 113.30 (MMT, o-ClBz), 102.99, 101.32 (C5, C5*),
91.23 (C1′), 89.69 (C1′*), 87.29 (MMT), 83.32 (C4′), 81.89 (C4′*),
77.39 (C2′), 74.55 (C2′*), 71.26 (C3′*), 62.01 (C5′), 55.24 (OCH3),
45.77 (NCH2), 40.67 (C5′*), 38.32 (C3′), 30.57 (CH2CO), 25.80 (t-
Bu), 18.00 (quaternary C in t-Bu), 8.67 (CH3), -4.53, -5.53 (SiCH3).
HRMS calcd for C53H59O15N5ClSiP 1099.3203, found 1099.3279.
Oligonucleotides were synthesized, purified and analyzed as previ-
ously reported.20 MALDI-TOF MS and enzymatic degradation followed
by RP HPLC analysis data are included in Supporting Information
(Table 4). Dimers 17a,b and 20a,b were used under standard coupling
conditions. Oligonucleotides bearing 2′-O-TBDMS protections were
deprotected as follows: after removal of the acyl protections and
cleavage of the oligomer from polymeric support (32% NH3/EtOH 3:1,
for 8 h at 20 °C) the ammonia solution was lyophilized, the residue
was dissolved in neat triethylamine trihydrofluoride57 (0.3 mL, Aldrich)
and kept overnight at 20 °C. Water (1 mL) was added, the aqueous
phase was extracted with ethyl acetate (4 × 1 mL), and lyophilized.
Further purification and analysis were done as reported.20
1
17b yield 66%, Rf ) 0.25 (Solvent B), H NMR (CDCl3/CD3OD,
9:1)55 δ: 8.14 (d, J ) 8.4 Hz, 1H, H6), 7.92 (d, J ) 7.7 Hz, 1H,
o-ClBz), 7.51-7.22 (m, 16H, Ar, H6*), 6.86 (m, 2H, MMT), 6.84 (d,
J ) 636 Hz, 1H, PH), 6.00 (d, JH1′-H2′ ) 5.1 Hz, 1H, H1′*), 5.87 (s,
1H, H1′), 5.74 (d, J ) 8.4 Hz, 1H, H5*), 5.52 (t, 1H, H2′*), 5.27 (d,
1H, H5), 4.88 (p, 1H, H3′*), 4.27 (m, 1H, H4′*), 4.04 (m, 1H, H4′),
3.91 (m 1H, H2′), 3.80 (s, 3H, OCH3), 3.80-3.29 (m, 4H, H5′*, H5′),
3.52 (s, 3H, 2′-OCH3), 2.71 (q, J ) 7.4 Hz, 7H, NCH2, H3′), 2.43 and
2.04 (2m, 2H, CH2CO), 1.11 (t, 9H, CH3). 13C NMR (CDCl3/CD3OD,
9:1)55 δ: 171.68 (CdO in amide), 164.24, 163.62 (C4, C4*, CdO in
o-ClBz), 150.65, 150.30 (C2, C2*), 158.76, 143.87, 143.68, (MMT)
141.19, 140.46 (C6, C6*), 134.71, 134.06, 133.35, 132.11, 131.14,
130.57, 128.63, 128.49, 128.06 127.25, 126.84, 113.33 (MMT, o-ClBz),
103.17, 101.44 (C5, C5*), 89.03, 88.63 (C1′, C1′*), 87.28 (MMT),
85.76 (C2′), 83.82 (C4′), 82.25 (C4′*), 74.57 (C2′*), 71.31 (C3′*), 61.33
(C5′), 58.06 (2′-OCH3), 55.28 (OCH3), 45.85 (NCH2), 40.39 (C5′*),
37.69 (C3′), 30.80 (CH2CO), 9.99 (CH3).
Thermal Melting and Hybridization Thermodynamics. Absor-
bance vs temperature profiles were measured at 260 nm on a Varian
Cary 3 spectrophotometer in buffers 10 mM sodium phosphate (pH
7.2), 0.1 mM EDTA, 2 µM of each oligonucleotide (analogue and
complementary RNA) and various concentrations of added sodium salts
(chloride, acetate, perchlorate). Extinction coefficients were calculated
from the nearest-neighbor approximation.58 The temperature was
increased at a rate of 0.2 °C per minute (control runs at a rate of 0.1
°C per minute gave essentially the same results) and data points were
collected every 0.1 °C. A thermostatable multicell (2 × 6) block was
used to simultaneously monitor up to five samples, the sixth cell was
used for internal temperature control. At temperatures below 15 °C
the sample compartment was flushed with dry nitrogen gas. The melting
curves for all models uniformly showed single thermal transitions with
a well-defined lower and upper baseline over all experimental conditions
(for Model 13 0.01 to 5 M Na+) allowing us to fit the data to a two-
state model. The melting temperatures and thermodynamic parameters
(Tables 1 and 2) were obtained using Varian Cary software, Version
2.5. The experimental absorbance vs temperature curves were converted
into fractions of strands remaining hybridized (R) vs temperature curve
by fitting the melting profile to a two-state transition model, with
linearly sloping lower and upper baselines. The tm’s were obtained
directly from the temperature at R ) 0.5. The thermodynamic
parameters were determined from van’t Hoff plot (ln K vs 1/T) with
(-∆H/R) as the slope and (∆S/R) as the intercept. Values of K
(equilibrium constant) were determined at each temperature using
equation K ) R /(Ct/n)n-1 Rn) where Ct is the total strand concentration
and n is the molecularity of the reaction. Reported values are the average
of at least three experiments.
1
20a yield 56%, Rf ) 0.26 (Solvent B), H NMR (CDCl3/CD3OD,
9:1)55 δ: 8.07 (d, J ) 8.1 Hz, 1H, H6), 7.93 (d, J ) 7.7 Hz, 1H,
o-ClBz), 7.42-7.21 (m, 16H, Ar, H6*), 6.85 (m, 2H, MMT), 6.79 (d,
J ) 641 Hz, 1H, PH), 5.89 (bs, 1H, H1′), 5.73 (s, 1H, H1′*), 5.61 (m,
1H, H2′*), 5.55 (d, J ) 8.1 Hz, 1H, H5*), 5.16 (d, 1H, H5), 4.90 (p,
1H, H3′*), 4.45 (m, 2H, H2′, H4′*), 4.18 (m, 1H, H4′), 3.78 (s, 3H,
OCH3), 3.63-3.37 (m, 3H, H5′, CH2NH), 3.15 (m, 1H, CH2NH), 2.97
(q, J ) 7.3 Hz, 6H, NCH2), 2.74-2.58 (m, 3H, H3′, H5′*), 1.19 (t,
9H, CH3), 0.86 (s, 9H, t-Bu), 0.17, 0.09 (2s, 6H, SiCH3). 13C NMR
(CDCl3/CD3OD, 9:1)55 δ: 170.61 (CdO in amide), 164.32, 164.08,
163.81 (C4, C4*, CdO in o-ClBz), 150.62, 150.48 (C2, C2*), 158.85,
143.95, 143.60, (MMT) 142.30, 140.66 (C6, C6*), 134.59, 134.05,
133.38, 132.22, 131.14, 130.60, 128.49, 128.09, 127.36, 126.90, 113.38
(MMT, o-ClBz), 102.81, 101.57 (C5, C5*), 90.91 (C1′), 90.30 (C1′*),
87.40 (MMT), 82.25 (C4′), 80.17 (C4′*), 77.04 (C2′), 74.83 (C2′*),
72.67 (C3′*), 62.61 (C5′), 55.27 (OCH3), 45.72 (NCH2), 41.54 (C3′),
38.59 (C5′*), 35.76 (CH2NH), 25.75 (t-Bu), 18.01 (quaternary C in
t-Bu), 8.51 (CH3), -4.52, -5.47 (SiCH3). HRMS calcd for C53H59O15N5-
ClPSi+2Na 1144.2920, found 1144.2915.
Synthesis and Conformational Analysis of Monomeric Models
23a,b and 24a,b. Carboxylic acids 3a,b were coupled with ethylamine
(57) (a) Gasparutto, D.; Livache, T.; Bazin, H.; Duplaa, A.-M.; Guy, A.; Khorin,
A.; Molko, D.; Roget, A.; Teoule, R. Nucleic Acids Res. 1992, 20, 5159-
5166. (b) Westman, E.; Stro¨mberg, R. Nucleic Acids Res. 1994, 22, 2430-
2431.
(56) Connolly, B. A. In Oligonucleotides and Analogues: A Practical Aproach;
Eckstein, F., Ed.; IRL Press: Oxford, 1991; pp 161-162.
(58) Puglisi, J. D.; Tinoco, I., Jr. Methods in Enzymology 1989, 180, 304-324.
9
J. AM. CHEM. SOC. VOL. 125, NO. 40, 2003 12135

A R T I C L E S
Rozners et al.
using the HOBt/DCC procedure outlined above. Amines 8c,d were
reacted with propionic anhydride (for full procedures, see Supporting
Information). The TBDMS groups were removed in 80% acetic acid
(aqueous) at 50 °C for 24 h. Preparation of compounds 25a,b has been
previously reported.20 Purification of the products (RP HPLC) and NMR
experiments were done as previously reported.20 For experimental
coupling constants, see Tables 5-8 in the Supporting Information. The
equilibrium between North and South conformers was estimated using
4.11 (m, 1H, H4′), 3.96 and 3.73 (ABX, J H5′-H5′′ ) 13.1 Hz, 2H, H5′),
3.43 and 3.31 (ABX, J ) 8.1, 6.0 and 14.0 Hz, 2H, NCH2), 2.41-2.30
(m, 1H, H3′), 2.23 (q, J ) 7.6 Hz, 2H, CH2CO), 1.06 (t, 3H, CH3).
24b 1H NMR (D2O, 270 MHz, 40 °C) δ: 7.97 (d, J ) 8.0 Hz, 1H,
H6), 5.90 (d, 1H, H1′), 5.83 (d, 1H, H5), 4.09 (m, 1H, H4′), 4.03 (m,
1H, H2′), 3.96 and 3.73 (ABX, J H5′-H5′′ ) 13.0 Hz, 2H, H5′), 3.51 (s,
3H, OCH3), 3.44 and 3.27 (ABX, J ) 8.2, 5.9 and 13.9 Hz, 2H, NCH2),
2.49-2.38 (m, 1H, H3′), 2.22 (q, J ) 7.7 Hz, 2H, CH2CO), 1.08 (t,
3H, CH3).
a straightforward approximation- South (%)) (3JH1′-H2′ /(3JH1′-H2′
+
3JH3′-H4′)) × 100.59 To ensure that the results obtained in D2O are
representative for water buffers, control NMR experiments were also
done in buffers used in UV melting studies containing only 10% D2O
(both at 1.0 and 0.1 M NaCl and at 40, 60 and, 80 °C). No significant
differences were observed in these experiments (typically deviations
within (0.3 Hz).
Acknowledgment. This study was supported by the Swedish
Natural Science Research Council, the Swedish Research
Council for Engineering Sciences and the Swedish Royal
Academy of Sciences. We thank Dr. J. Johansson (Karolinska
Institutet) for help with MALDI-TOF MS analysis, the Nordic
Council of Ministers and the Swedish Institute for studentships
to D.K., and the Wenner-Gren Center Foundation for a
postdoctoral fellowship to E.R.
23a 1H NMR (D2O, 270 MHz, 40 °C) δ: 7.96 (d, J ) 8.1 Hz, 1H,
H6), 5.83 (d, 1H, H5), 5.77 (d, 1H, H1′), 4.38 (m, 1H, H2′), 4.13-
4.06 (m, 1H, H4′), 3.92 and 3.72 (ABX, JH5′-H5′′ ) 13.0 Hz, 2H, H5′),
3.16 (m, 2H, NCH2), 2.51-2.29 (m, 3H, CH2CO, H3′), 1.07 (t, J )
7.5 Hz, 3H, CH3).
23b 1H NMR (D2O, 270 MHz, 40 °C) δ: 7.99 (d, J ) 8.2 Hz, 1H,
H6), 5.90 (d, 1H, H1′), 5.84 (d, 1H, H5), 4.08-4.02 (m, 1H, H4′),
3.96 (m, 1H, H2′), 3.90 and 3.72 (ABX, JH5′-H5′′ ) 13.3 Hz, 2H, H5′),
3.49 (s, 3H, OCH3), 3.17 (m, 2H, NCH2), 2.56-2.28 (m, 3H, CH2CO,
H3′), 1.08 (t, J ) 7.3 Hz, 3H, CH3).
Supporting Information Available: Experimental procedures
and spectral data (1
H and 13C NMR) not included in Experi-
mental Section, Tables with experimentally obtained spin-spin
coupling constants for model compounds 23a,b and 24a,b. This
material is available free of charge via the Internet at
http://pubs.acs.org.
24a 1H NMR (D2O, 270 MHz, 40 °C) δ: 7.94 (d, J ) 8.1 Hz, 1H,
H6), 5.82 (d, 1H, H5), 5.77 (d, 1H, H1′), 4.39 (m, 1H, H2′), 4.18-
(59) Altona, C.; Sundaralingam, M. J. Am. Chem. Soc. 1973 95, 2333-2344.
JA0360900
9
12136 J. AM. CHEM. SOC. VOL. 125, NO. 40, 2003