Synthesis of (2-deoxy-α- and -β-D-erythro-pentofuranosyl)(thymin-1-yl)alkanes and their incorporation into oligodeoxyribonucleotides. Effect of nucleobase-sugar linker flexibility on the formation of DNA-DNA and DNA-RNA hybrids

Article abstract of DOI:10.1021/jo961548k

On the basis of modeling studies, the (2-deoxy-α- and β-D-erythro-pentofuranosyl) (thymin-1-yl) alkanes 1a,b and 2a,b were selected as potential conformational probes for altDNA oligonucleotides. A straightforward approach to the synthesis of 1a,b and 2a,b from commercial 2-deoxy-D-ribose (3) and 1-O-methyl-2-deoxy-3,5-di-0-p-toluoyl-D-erythro-pentofuranose (13), respectively, was developed. These nucleoside analogues were converted to the phosphoramidite derivatives 27a,b-30a,b and incorporated into oligonucleotide 31 at predetermined sites and defined internucleotidic motifs. The insertion of 1a,b according to either a (3′ → 5′)- or a (3′ → 3′)-internucleotidic polarity produced oligonucleotides exhibiting a slightly higher affinity for their complementary unmodified DNA sequence than for the corresponding RNA sequence (Table 3). Conversely, the incorporation of 2a into 31 according to a (3′ → 3′)-orientation generated, for the first time, an altDNA oligonucleotide displaying a greater affinity for its complementary unmodified RNA sequence (ΔT_m = 6°C) than for the corresponding DNA sequence (ΔT_m = 10°C). This hybrid was, however, thermodynamically less stable than the duplex having unmodified α-2′-deoxythymidine similarly incorporated into 31 (ΔΔT_m = 3°C).

Full text of DOI:10.1021/jo961548k

J . Org. Chem. 1996, 61, 8617-8626
8617
Syn th esis of (2-Deoxy-r- a n d
-â-D-er yth r o-p en tofu r a n osyl)(th ym in -1-yl)a lk a n es a n d Th eir
In cor p or a tion in to Oligod eoxyr ibon u cleotid es. Effect of
Nu cleoba se-Su ga r Lin k er F lexibility on th e F or m a tion of
DNA-DNA a n d DNA-RNA Hybr id s
J ila H. Boal, Andrzej Wilk, Carlo L. Scremin, Glenn N. Gray,1 Lawrence R. Phillips,1 and
Serge L. Beaucage*
Division of Hematologic Products, Center for Biologics Evaluation and Research,
Food and Drug Administration, 8800 Rockville Pike, Bethesda, Maryland 20892, and Laboratory of Drug
Discovery Research and Development, Developmental Therapeutics Program, National Cancer Institute,
Frederick, Maryland 21701
Received August 8, 1996X
On the basis of modeling studies, the (2-deoxy-R- and â-D-erythro-pentofuranosyl) (thymin-1-yl)
alkanes 1a ,b and 2a ,b were selected as potential conformational probes for altDNA oligonucleotides.
A straightforward approach to the synthesis of 1a ,b and 2a ,b from commercial 2-deoxy-D-ribose
(3) and 1-O-methyl-2-deoxy-3,5-di-O-p-toluoyl-D-erythro-pentofuranose (13), respectively, was
developed. These nucleoside analogues were converted to the phosphoramidite derivatives 27a ,b-
30a ,b and incorporated into oligonucleotide 31 at predetermined sites and defined internucleotidic
motifs. The insertion of 1a ,b according to either a (3′ f 5′)- or a (3′ f 3′)-internucleotidic polarity
produced oligonucleotides exhibiting a slightly higher affinity for their complementary unmodified
DNA sequence than for the corresponding RNA sequence (Table 3). Conversely, the incorporation
of 2a into 31 according to a (3′ f 3′)-orientation generated, for the first time, an altDNA
oligonucleotide displaying a greater affinity for its complementary unmodified RNA sequence (∆Tm
) 6 °C) than for the corresponding DNA sequence (∆Tm ) 10 °C). This hybrid was, however,
thermodynamically less stable than the duplex having unmodified R-2′-deoxythymidine similarly
incorporated into 31 (∆∆Tm ) 3 °C).
Our efforts toward designing oligonucleotide analogues
that possess achiral phosphorus centers and offer sub-
stantial resistance to nucleases have led to the develop-
ment of R,â-oligodeoxyribonucleotides with alternating
(3′ f 3′)- and (5′ f 5′)-internucleotidic phosphodiester
linkages (altDNA).2 These oligonucleotide analogues
have not only shown superior resistance to nucleases but
also have generated relatively stable complexes with
complementary DNA or RNA sequences.2,3 Thermody-
namically, altDNA-DNA hybrids are only slightly less
stable than unmodified DNA-DNA duplexes (∆Tm/
modification ) 1.1 °C).2a By comparison, altDNA-RNA
complexes are considerably less stable than unmodified
DNA-RNA duplexes (∆Tm/modification ) 2.2 °C).2a The
reduced thermal stability of altDNA-RNA complexes is
thought to result from the unnatural arrangement of the
internucleotidic phosphodiester motifs of altDNA oligo-
mers that could affect base-pair formation more exten-
sively in A-type than in B-type helices.4 In this context,
it can be argued that providing additional flexibility to
altDNA nucleobases by the insertion of a methylene or
ethylene tether between each carbohydrate and nucleo-
base of R-nucleotidic residues would allow complemen-
tary nucleobases to better align and form stable Watson-
Crick base-pairs. We modeled the modified nucleosides
1a and 1b in either the parallel or the antiparallel
orientation into an A-type DNA-RNA helix and a B-type
DNA-DNA duplex and discovered two energetically
preferred B-type helices (see Figure 1, top and middle
sections).
The torsion angle ø1 (C1′-C6′-C7′-N1) of the ethylene
tether for either 1a or 1b, which allowed for optimal base-
pairing within B-helices, was 180°. Similarly modeled,
the less flexible nucleoside analogue 2a led to an A-type
helix that was energetically comparable to that of an
unmodified DNA-RNA duplex (see Figure 1, bottom
section). Given the favorable structural features of these
models, we now report the detailed synthesis and char-
acterization of the nucleoside analogues 1a ,b and 2a ,b5
and their incorporation into oligonucleotides according
to defined internucleotidic motifs. Thermal stability of
hybrids generated upon annealing these oligodeoxyribo-
nucleotide analogues with complementary unmodified
DNA and RNA oligomers will also be reported.
* To whom correspondence should be addressed. Tel.: (301) 496-
3378. Fax: (301) 480-3256. E-mail: beaucage@helix.nih.gov.
X Abstract published in Advance ACS Abstracts, November 15, 1996.
(1) National Cancer Institute at Frederick.
(2) (a) Koga, M.; Wilk, A.; Moore, M. F.; Scremin, C. L.; Zhou, L.;
Beaucage, S. L. J . Org. Chem. 1995, 60, 1520-1530. (b) Koga, M.;
Moore, M. F.; Beaucage, S. L. J . Org. Chem. 1991, 56, 3757-3759.
(3) Debart, F.; Tosquellas, G.; Rayner, B.; Imbach, J .-L. Bioorg. Med.
Chem. Lett. 1994, 4, 1041-1046.
(4) (a) Scremin, C. L.; Boal, J . H.; Wilk, A.; Phillips, L. R.; Zhou, L.;
Beaucage, S. L. Tetrahedron Lett. 1995, 36, 8953-8956. (b) Scremin,
C. L.; Boal, J . H.; Wilk, A.; Phillips, L. R.; Beaucage, S. L. Bioorg. Med.
Chem. Lett. 1996, 6, 207-212.
S0022-3263(96)01548-4
This article not subject to U.S. Copyright. Published 1996 by the American Chemical Society

8618 J . Org. Chem., Vol. 61, No. 24, 1996
Boal et al.
F igu r e 1. Top: Stereoview of an energy-minimized B-type DNA-DNA helix composed of the oligodeoxyribonucleotide
5′-d(GCGTTN*TTGCG) and its complementary unmodified DNA sequence. N* is 1a linked through (3′ f 3′)- and (5′ f 5′)-
phosphodiester linkages (shown as broader lines). Middle: Same as above but boldface N* is 1b inserted according to natural (3′
f 5′)-internucleotidic polarity. Bottom: Stereoview of an energy-minimized A-type DNA-RNA helix composed of the above oligomer
and its complementary unmodified RNA sequence. Boldface N* is 2a linked through (3′ f 3′)- and (5′ f 5′)-phosphodiester linkages.
Resu lts a n d Discu ssion
phosphonate produced the (2-deoxyribofuranosyl)aceto-
nitrile derivatives 5a ,b in almost quantitative yields as
nearly an equimolar mixture of R- and â-epimers.7 The
facile separation of 5a and 5b by silica gel chromatog-
raphy allowed isolation of each epimer in gram quanti-
ties. Reduction of 5a and 5b to the corresponding
amines, however, presented difficulties. Treatment of
either 5a or 5b with lithium aluminum hydride (LAH)
in THF produced a complex mixture of reaction products.
A straightforward approach to the preparation of 1a ,b
from 2-deoxy-D-ribose is described in Scheme 1. The 5-O-
trityl-2-deoxyribofuranoside (4) was prepared in 60%
yield from the reaction of 2-deoxy-D-ribose (3) with
triphenylmethyl chloride under conditions adapted from
the procedure of Kam and Oppenheimer.6 A Wittig-
Horner condensation of 4 with diethyl (cyanomethyl)-
(5) Preliminary data pertaining to the preparation of 1a ,b and 2a ,b
have been reported earlier by us.4 During the edition of this manu-
script, an alternate synthesis of 2b and its incorporation into oligo-
nucleotides have appeared; see: Hossain, N.; Hendrix, C.; Lescrinier,
E.; Van Aerschot, A.; Busson, R.; De Clercq, E.; Herdewijn, P. Bioorg.
Med. Chem. Lett. 1996, 6, 1465-1468.
(6) Kam, B. L.; Oppenheimer, N. J . Carbohydr. Res. 1979, 69, 308-
310.
(7) Syntheses of (ribofuranosyl)acetonitrile derivatives from the
corresponding ribofuranoses and either (cyanomethyl)triphenylphos-
phorane or diethyl (cyanomethyl)phosphonate have been reported.29

Formation of DNA-DNA and DNA-RNA Hybrids
J . Org. Chem., Vol. 61, No. 24, 1996 8619
Sch em e 1a ,b
sumed to be responsible for the decreased yields of amine
products obtained in these reduction reactions.8 How-
ever, addition of aluminum chloride to LAH enables
complete reduction of nitriles, even those with active
R-hydrogens, without release of hydrogen.10 Incidentally,
the reaction of aluminum chloride with LAH is known
to generate aluminum hydride,11 which is also effective
in the reduction of nitriles with active R-hydrogens.9
Thus, the reduction of 5a ,b with aluminum hydride
in THF gave highly pure (2-deoxyribofuranosyl)amino-
ethanes 6a ,b;12 thin-layer chromatography (TLC) analy-
sis of the corresponding peracetylated derivatives indi-
cated a nearly quantitative reaction. The preparation
of aluminum hydride according to the method of Finholt
et al.13 can, however, be hazardous and must be carried
out in a drybox under an inert atmosphere. Conse-
quently, the use of other acidic reducing reagents was
investigated. As a result, the commercial borane-
dimethyl sulfide complex was found to be especially
effective in the conversion of 5a ,b to 6a ,b; the reduction
proceeded smoothly to provide yields exceeding 95%
within 1 h in refluxing THF. The aminoethylated gly-
cosides 6a ,b, were characterized as their diacetate de-
rivatives 7a ,b. Without further purification, 6a ,b were
reacted with trimethylsilyl chloride and then condensed
with 3-methoxy-2-methylacryloyl isocyanate, a reagent
generated in situ from the parent acryloyl chloride and
silver cyanate.14 The resulting crude acryloylurea de-
rivatives 8a ,b were treated with 10% trifluoroacetic acid
in dichloromethane for 2 h at ambient temperature.
After workup and purification of the reaction products
by silica gel chromatography, the acryloylurea derivatives
9a ,b were isolated in ca. 40% yield (based on 5a ,b).
Cyclization of 9a ,b to give 1a ,b in high yield (ca. 90%)
was accomplished in concentrated ammonium hydroxide
at 50 °C for 12 h in a pressure vessel.
A similar approach to the preparation of the R- and
â-2′-deoxyribonucleoside analogues 2a ,b is outlined in
Scheme 2. Although syntheses of “homouridine” (10, 11),
“homocytidine” (12), and analogous D-ribitol derivatives
have been reported,15,16 the preparation of 2a ,b has not
been described.5
a
Conditions: (i) TrCl/DMAP/C5H5N, 50 °C, 6 h; (ii) (EtO)2P-
(O)CH2CN/NaH/THF, 5 °C, 6 h; (iii) silica gel chromatography;
(iv) (CH3)2S‚BH3/THF/reflux, 40 min; (v) Ac2O/C5H5N, 25 °C; (vi)
TMS-Cl/Et3N/THF, 25 °C, 2 h; (vii) CH3OCHC(CH3)CONCO/Et3N/
C6H6, 25 °C, 3 h; (viii) 10% TFA/CH2Cl2, 25 °C, 2 h; (ix) concd
NH4OH, 50 °C, 12 h. bKey: Et, ethyl; Tr, triphenylmethyl; Ac,
acetyl; TMS, trimethylsilyl; TFA, trifluoroacetic acid.
Thus, commercial 1-O-methyl-2-deoxy-3,5-di-O-p-tolu-
oyl-D-erythro-pentofuranoside (13) was converted by the
(9) Brown, H. C.; Yoon, N.-M. J . Am. Chem. Soc. 1966, 88, 1464-
1472.
Consistent with these findings is the reduction of â-ox-
ypropionitriles with LAH in THF, which produces sig-
nificant cleavage at the ether linkage.8 Moreover, the
reduction of aliphatic nitriles with LAH proceeds with
evolution of hydrogen resulting from an attack of the
reagent on the active hydrogens adjacent to the nitrile
function.8,9 Such hydrogen abstraction has been pre-
(10) Nystrom, R. F. J . Am. Chem. Soc. 1955, 77, 2544-2545.
(11) (a)Brown, H. C.; Choi, Y.-M.; Narasimhan, S. Synthesis 1981,
605-606. (b) Lane, C. F. Aldrichim. Acta 1975, 8, 20-23.
(12) Zhou, L.; Beaucage, S. L. Unpublished results.
(13) Finholt, A. E.; Bond, J r., A. C.; Schlesinger, H. I. J . Am. Chem.
Soc. 1947, 69, 1199-1203.
(14) Shealy, Y. F.; O’Dell, C. A.; Thorpe, M. C. J . Heterocycl. Chem.
1981, 18, 383-389.
(15) Bobek, M.; Farkas, J . Collect. Czech. Chem. Commun. 1969,
34, 1684-1689.
(8) (a) Soffer, L. M.; Parotta, E. W. J . Am. Chem. Soc. 1954, 76,
3580-3583. (b) Soffer, L. M.; Katz, M. J . Am. Chem. Soc. 1956, 78,
1705-1709.
(16) (a) Cleophax, J .; Defaye, J .; Gero, S. D. Bull. Soc. Chim. Fr.
1967, 104-107. (b) Defaye, J .; Reyners, T. Bull. Soc. Chim. Biol. 1968,
50, 1625-1635.

8620 J . Org. Chem., Vol. 61, No. 24, 1996
Boal et al.
Sch em e 2a ,b
F igu r e 2. Structure displaying the proximity of H-1′ to
H-2′and H-6′′ in the case of 1a and 2a (top) and H-1′ to H-2′′,
H-4′, and H-6′ in the case of 1b and 2b (bottom) according to
NOESY experiments performed at 300 MHz in D2O.
Treatment of 16a ,b with 3-methoxy-2-methylacryloyl
isocyanate gave the acryloylurea derivatives 17a ,b in ca.
80% yield. Acid-catalyzed cyclization15 of purified 17a ,b
produced the thymine nucleoside analogues 18a ,b in ca.
90% yield. Saponification of 18a ,b with aqueous potas-
sium hydroxide quantitatively produced 2a ,b. The nu-
cleoside analogues 1a ,b and 2a ,b were characterized by
1
high-resolution H- and 13C-NMR spectroscopies; spectral
data are presented in Tables 1 and 2, respectively. 1H-
NMR chemical shift assignments were based on the
results of COSY experiments,19 and coupling constants
were determined from fine splitting patterns of reso-
nances at 500 MHz. The structure of the R-deoxyribo-
nucleosides 1a and 2a is clearly differentiated from that
of the â-deoxyribonucleosides 1b and 2b on the basis of
their respective NOESY spectra at 300 MHz. The
presence of the corresponding crosspeaks confirms the
proximity of H-1′ to H-2′ and H-6′′ in the case of 1a and
2a and the proximity of H-1′ to H-2′′, H-4′, and H-6′ in
the case of 1b and 2b (see Figure 2).20
a
Conditions: (i) HCl (g)/AcOH; (ii) Et2AlCN/toluene/THF, 25
°C, 7 h; (iii) silica gel chromatography; (iv) BH3/THF 25 °C, 1 h;
(v) HCl (g)/CH3OH; (vi) CH3OCHC(CH3)CONCO/Et3N/C6H6, 25 °C,
16 h; (vii) AcOH/concd HCl (10:1 v/v), 25 °C, 16 h; (viii) KOH in
EtOH-H2O (2:1) and, then AG 50W-X12 (H+). bKey: p-Tol,
p-toluoyl; Ac, acetyl; Et, ethyl.
method of Hoffer17 to the halogenated glycoside 14.
Condensation of 14 with diethylaluminum cyanide under
the conditions reported by Iyer et al.18a produced the
cyanoglycosides 15a ,b as an almost equimolar mixture
of R- and â-anomers in yields greater than 95%. Like
5a and 5b, these anomers were easily separated by silica
gel chromatography and chemoselectively reduced by
diborane in THF to generate the aminomethylated gly-
cosides 16a and 16b in isolated yields of ca. 85%.
1
Table 1 shows the similarities between the H-NMR
chemical shifts of H-5′ and H-5′′ and those reported for
unmodified R- and â-thymidine.21 On the basis of previ-
ously reported chemical shifts for H-5′ and H-5′′ of
unmodified â-deoxyribonucleosides,21,22 H-5′ signals of
(19) 1H-COSY spectra of 1a ,b and 2a ,b are available as Supporting
Information.
(20) 1H-NOESY spectra of 1a ,b and 2a ,b are shown as Supporting
Information. H-2′ and H-2′′ are defined according to: Wood, D. J .;
Hruska, F. E.; Ogilvie, K. K. Can. J . Chem. 1974, 52, 3353-3366.
(21) Cadet, J .; Ta¨ıeb, C.; Remin, M.; Niemczura, W. P.; Hruska, F.
E. Biochim. Biophys. Acta 1980, 608, 435-445.
(22) Ritchie, R. G. S.; Perlin, A. S. Carbohydr. Res. 1977, 55, 121-
128.
(17) Hoffer, M. Chem. Ber. 1960, 93, 2777-2781.
(18) (a) Iyer, R. P.; Phillips, L. R.; Egan, W. Synth. Commun. 1991,
21, 2053-2063. The synthesis of 15a ,b has also been achieved by
treatment of 14 with sodium cyanide in 1,2-dimethoxyethane.18b (b)
Kolb, A.; Huynh Dinh, T.; Igolen, J . Bull. Soc. Chim. Fr. 1973, 3447-
3448.

Formation of DNA-DNA and DNA-RNA Hybrids
J . Org. Chem., Vol. 61, No. 24, 1996 8621
Ta ble 1. 500 MHz 1H-NMR Sp ectr a l Da ta for th e Deoxyr ibon u cleosid e An a logu es 1a ,b a n d 2a ,b in D2Oa ,b
H-1′
(J 1′-2′
(J 1′-2′′
(J 1′-6′
H-6′
(J 6′-1′
(J 6′-6′′
(J 6′-7′
H-6′′
(J 6′′-1′
(J 6′′-6′
(J 6′′-7′
)
)
)
)
H-2′
(J 2′-1′
(J 2′-2′′
(J 2′-3′
H-2′′
(J 2′′-1′
(J 2′′-2′
(J 2′′-3′
H-3′
(J 3′-2′
(J 3′-2′′
(J 3′-4′
H-4′
(J 4′-3′
(J 4′-5′
)
)
)
)
)
)
)
H-7′
H-7′′
)
)
)
)
)
)
)
)
)
)
)
)
H-5′
(J 5-4′
(J 5′-5′′
H-5′′
(J 5′′-4′
(J 5′′-5′
(J 7′-6′
)
(J 7′′-6′)
)
)
)
)
(J 7′-6′′
(J 7′-7′′
)
)
(J 7′′-6′′
(J 7′′-7′
)
)
H-6
(J 6-CH
CH3
(J CH )
3-6
(J 1′-6′′
(J 4′-5′′
(J 6′-7′′
(J 6′′-7′′
)
)
3
1a
1b
2a
2b
4.16
(6.9)
(7.5)
(4.8)
(7.8)
4.17
(10.2)
(5.6)
(2.3)
(7.9)
4.36
(7.8)
(5.5)
(3.5)
(7.9)
4.36
(9.7)
(5.9)
(2.9)
(7.3)
2.47
(6.9)
(13.3)
(6.9)
1.70
(7.5)
(13.3)
(6.0)
4.30
(6.9)
(6.0)
(5.0)
3.93
(5.0)
(3.7)
(6.3)
3.70
(3.7)
(12.1)
3.61
(6.3)
(12.1)
1.99
(4.8)
(14.1)
(7.1)
(7.8)
1.95
(2.3)
(13.8)
(6.7)
(8.2)
3.84
2.05
(7.8)
(14.1)
(7.8)
(6.1)
1.89
(7.9)
(13.8)
(8.1)
(5.9)
3.89
3.83
(7.1)
(7.8)
3.90
(7.8)
(6.1)
7.54
(1.0)
1.89
(1.0)
(14.8)
(14.8)
1.81
(10.2)
(13.5)
(6.3)
2.00
(5.6)
(13.5)
(2.0)
4.25
(6.3)
(2.0)
(2.7)
3.83
(2.7)
(4.3)
(5.8)
3.62
(4.3)
(12.0)
3.55
(5.8)
(12.0)
3.77
(6.7)
(8.1)
3.86
(8.2)
(5.9)
7.48
(1.2)
1.84
(1.2)
(14.0)
(14.0)
2.36
(7.6)
(13.9)
(6.9)
1.69
(5.5)
(13.6)
(4.7)
4.24
(6.9)
(4.7)
(4.1)
3.90
(4.1)
(4.0)
(5.5)
3.58
(4.0)
(12.2)
3.51
(5.5)
(12.2)
7.43
(1.1)
1.81
(1.1)
(3.4)
(14.5)
(7.8)
(14.5)
1.81
(9.8)
(13.6)
(5.8)
1.99
(5.9)
(13.7)
(2.2)
4.24
(5.6)
(2.3)
(2.6)
3.84
(2.4)
(4.6)
(5.5)
3.53
(4.6)
(12.1)
3.47
(5.6)
(12.1)
3.95
(2.9)
(14.6)
3.79
(7.3)
(14.6)
7.41
(1.1)
1.81
(1.1)
a
Chemical shifts are given in parts per million relative to the sodium salt of 3-(trimethylsilyl)propionic acid as an internal standard
b
(0 ppm). J couplings are in parentheses and expressed in Hz.
Ta ble 2. 125 MHz 13C-NMR Ch em ica l Sh ifts of th e Deoxyr ibon u cleosid e An a logu es 1a ,b a n d 2a ,b in D2Oa
C-2
C-4
C-5
C-6
C-1′
C-2′
C-3′
C-4′
C-5′
C-6′
C-7′
CH3
1a
1b
2a
2b
154.7
154.7
154.8
155.0
169.3
169.4
169.2
169.4
113.1
113.2
112.8
113.0
145.7
145.6
146.2
146.2
78.2
78.7
79.0
79.2
41.8
42.1
39.0
39.1
74.3
75.0
74.2
74.8
87.2
89.1
87.9
89.4
63.7
64.6
63.9
64.5
36.5
36.2
54.3
53.9
48.4
48.4
13.7
13.8
13.8
13.7
a
Proton-decoupled chemical shifts are listed in parts per million relative to the resonance of the middle line of CDCl3 (77.0 ppm) as an
external standard.
1a ,b and 2a ,b are tentatively assigned downfield relative
to those of H-5′′. According to coupling constant argu-
ments, chemical shifts for H-6′, H-6′′, H-7′, and H-7′′
listed in Table 1 are also tentatively assigned. Specifi-
cally, H-6′ signals for 1b and 2b are downfield to those
of H-6′′. Conversely, H-6′ signals for 1a and 2a are
upfield of those of H-6′′, and signals for H-7′ are also
assigned upfield of those of H-7′′ for both 1a and 1b.
Incidentally, 13C-NMR chemical shift assignments for
in isolated yields ranging from 90 to 95%. These 5′-O-
silylated nucleoside analogues were reacted with DMTr-
Cl in pyridine to give the fully protected nucleosides
23a ,b and 24a ,b. Without further purification, 23a ,b
and 24a ,b were desilylated by treatment with tetra-n-
butylammonium fluoride in THF to provide 25a ,b and
26a ,b in isolated yields of 68-80%. Phosphitylation of
19a,b, 20a,b, 25a,b, and 26a,b by 2-cyanoethyl N,N,N′,N′-
tetraisopropylphosphorodiamidite and catalytic amounts
of N,N-diisopropylammonium tetrazolide in anhydrous
dichloromethane25 generated the deoxyribonucleoside
phosphoramidites 27-30a ,b in yields exceeding 88%.
These phosphoramidites were characterized by 31P-NMR
spectroscopy (see Experimental Section).
Each of the modified phosphoramidites 27-28a ,b and
29-30a ,b was incorporated by routine solid-phase oli-
gonucleotide synthesis into 5′-d(TCGACACCCAATN*C-
N*GAAAATGGA) (31) at selected sites (N*) according to
native (3′ f 5′)- or inverted (3′ f 3′)-internucleotidic
polarity, respectively (Table 3). Additional DNA se-
quences carrying unmodified â-2′-deoxythymidine (â-dT)
and R-2′-deoxythymidine (R-dT) inserted in both inter-
nucleotidic orientations at the indicated sites within 31
have been synthesized to generate meaningful control
experiments. These modified oligonucleotides were an-
nealed to either a complementary unmodified DNA or an
RNA oligomer. The stability of each complex was deter-
mined and its thermal denaturation temperature (Tm)
reported in Table 3. To better assess the thermodynamic
stability of the hybrids, mismatches were created
1
1a ,b and 2a ,b (Table 2) are supported by H-13C-COSY
data.23
The incorporation of 1a ,b and 2a ,b into oligodeoxyri-
bonucleotides according to defined internucleotidic motifs
was achieved by standard phosphoramidite chemistry.24
Scheme 3 illustrates the conversion of 1a ,b and 2a ,b to
the phosphoramidites 27-30a ,b. Specifically, the con-
densation of 1a ,b and 2a ,b with 4,4′-dimethoxytrityl
chloride (DMTr-Cl) in pyridine generated the 5′-O-
protected nucleosides 19a ,b and 20a ,b, in 75-85% yield.
Unlike the synthesis of 19a ,b and 20a ,b the preparation
of the 3′-O-protected nucleosides 25a ,b and 26a ,b re-
quired additional steps. Typically, 1a ,b and 2a ,b were
silylated, regioselectively, at the 5′-hydroxy function upon
reaction with tert-butyldimethylchlorosilane in the pres-
ence of imidazole in dry DMF to produce 21a ,b and 22a ,b
(23) 1H-13C-COSY spectra of of 1a ,b and 2a ,b are provided as
Supporting Information.
(24) (a) Beaucage, S. L. In Methods in Molecular Biology, Vol. 20:
Protocols for Oligonucleotides and Analogs; Agrawal, S., Ed.; Humana
Press: Totowa, 1993; pp 33-61. (b) Beaucage, S. L.; Caruthers, M. H.
In Bioorganic Chemistry: Nucleic Acids; Hecht, S. M., Ed.; Oxford
University Press: New York, 1996; pp 36-74. (c) Beaucage, S. L.; Iyer,
R. P. Tetrahedron 1992, 48, 2223-2311.
(25) Barone, A. D.; Tang, J .-Y; Caruthers, M. H. Nucl. Acids Res.
1984, 12, 4051-4061.

8622 J . Org. Chem., Vol. 61, No. 24, 1996
Boal et al.
Ta ble 3. Th er m a l Den a tu r a tion Tem p er a tu r es of
Sch em e 3a ,b
DNA-DNA a n d DNA-RNA Hybr id s Com p osed of
5′-d (TCGACACCCAATN*CN*GAAAATGGA) (31) a n d
5′-d (TCCATTTTCAGAATTGGGTGTCGA) or
5′-r (UCCAUUUUCAGAAUUGGGUGUCGA)
DNA-DNA
hybrids
DNA-RNA
hybrids
a
b
N*
orientation Tm (°C) ∆Tm (°C) Tm (°C) ∆Tm (°C)
â-d T
R-d T
â-d A
1a
1b
2a
(3′ f 5′)
(3′ f 5′)
(3′ f 5′)
(3′ f 5′)
(3′ f 5′)
(3′ f 5′)
(3′ f 5′)
(3′ f 3′)
(3′ f 3′)
(3′ f 3′)
(3′ f 3′)
(3′ f 3′)
(3′ f 3′)
68
60
57
59
63
57
58
55
66
61
58
58
56
61
60
50
52
54
49
52
50
58
52
50
55
50
8
11
9
1
11
9
7
12
9
11
3
9
11
6
11
5
11
10
13
2
2b
â-d T
R-d T
1a
1b
2a
7
10
10
12
2b
a
Thermal denaturation of DNA-DNA and DNA-RNA hybrids
was performed in PBS buffer (pH 7.2) and monitored at 260 nm.
The concentration of each oligomer was ca. 2.5 µM. ∆Tm is the
b
difference between the Tm of either modified DNA-DNA or DNA-
RNA complexes and the Tm of the corresponding unmodified
duplex. The variability between repeated Tm measurements was
ca. 1 °C.
studies (vide supra), it is reasonable to assume that the
increased number of conformers causes an entropic
destabilization of the hybrid.
As expected, the (3′ f 3′)-incorporation of 1b into 31
produced DNA-DNA and DNA-RNA hybrids that were
less stable than the parent unmodified duplexes (∆Tm
)
10 and 11 °C, respectively). The thermal stability of
these complexes is comparable to either those resulting
from the (3′ f 3′)-incorporation of â-dT into 31 (∆Tm
)
13 °C for the DNA-DNA complex and 11 °C for the
DNA-RNA hybrid, Table 3) or those having mismatches
created by the (3′ f 5′)-insertion of â-dA in 31 (∆Tm
)
11 °C for both DNA-DNA and DNA-RNA complexes).
The (3′ f 5′)-incorporation of 1a or an unmodified R-dT
into 31 led to the formation of DNA-DNA hybrids
displaying similar thermal stabilities (Tm ) 59 or 60 °C,
respectively). Surprisingly, the (3′ f 3′)-incorporation
of 1a into 31 only slightly improves the stability of the
DNA-DNA hybrid (Tm ) 61 °C) relative to that obtained
with the (3′ f 5′)-insertion of 1a (Tm ) 59 °C). This
stabilization (∆Tm ) 2 °C) is small when compared to that
generated by the (3′ f 3′)-incorporation of unmodified
R-dT into 31 (∆Tm ) 6 °C) under similar conditions. It
must also be noted that the incorporation of 1a and 1b
into 31 in either orientation did not provide a higher
affinity for the complementary RNA oligomer. For
example, the (3′ f 3′)-incorporation of 1a into 31 gener-
ated an alt-motif that showed a higher affinity for its
DNA complement (∆Tm ) 7 °C) than for its RNA
complementary sequence (∆Tm ) 9 °C, Table 3). These
data are consistent with the physicochemical properties
of altDNA oligomers studied earlier.2
The incorporation of 2a and 2b into 31 according to
either orientation, and â-dA in the native (3′ f 5′)-
internucleotidic polarity, produced DNA-DNA hybrids
of similar thermal stabilities (Tm ) 56-58 °C). Thus,
the incorporation of 2a or 2b into a DNA-DNA hybrid
is equivalent to the incorporation of a mismatch into an
unmodified DNA-DNA duplex. However, the (3′ f 3′)-
incorporation of 2a into 31 generated a significantly
greater affinity for the complementary RNA oligomer
a
Conditions: (i) DMTrCl/DMAP/C5H5N, 25 °C; (ii) TBDMSCl/
imidazole/DMF, 25 °C, 1 h; (iii) 1.0 M n-Bu4NF in THF, 25 °C, 2
h; (iv) (iPr2N)2POCH2CH2CN/cat. DIAT/CH2Cl2, 25 °C, 4 h. Key:
b
DMTr, 4,4′-dimethoxytrityl; DMAP, 4-(dimethylamino)pyridine;
TBDMS, tert-butyldimethylsilyl; Bu, butyl; DIAT, N,N-diisopro-
pylammonium salt of 1H-tetrazole; CE, 2-cyanoethyl; iPr, (1-
methyl)ethyl; Thy, thymin-1-yl.
at the indicated sites within 31 (Table 3) by insertion of
â-2′-deoxyadenosine (â-dA) through natural (3′ f 5′)-
internucleotidic linkages.
Modeling studies predicted that the incorporation of
1a ,b into oligodeoxyribonucleotides would produce ener-
getically preferred B-type helices (vide supra). Experi-
ments showed that insertion of 1b in 31, according to
natural (3′ f 5′)-internucleotidic orientation, led to the
formation of a DNA-DNA complex (Tm ) 63 °C) that is
thermodynamically more stable than the corresponding
DNA-RNA hybrid (Tm ) 54 °C, Table 3) but less stable
than the parent unmodified DNA-DNA or DNA-RNA
duplex (Tm ) 68 or 61 °C, respectively). Despite the fact
that the preferred torsion angle ø1 (C1′-C6′-C7′-N1) of
the ethylene tether in 1b is 180° according to modeling

Formation of DNA-DNA and DNA-RNA Hybrids
J . Org. Chem., Vol. 61, No. 24, 1996 8623
1a ,b or 2a ,b inserted according to defined internucleotidic
motifs, served in the construction of B- or A-type helices for
DNA-DNA or DNA-RNA duplexes, respectively. Sixteen
preliminary structures were relaxed by initial minimization,
and short simulations (10 ps) were performed in vacuum on
constrained helices. Trajectories of lowest energy were se-
lected and subjected to the Adopted Basis Newton-Raphson
minimization method to achieve a 10-4 kcal/mol energy gradi-
ent. Stereoviews of the most energetically favored structures
are shown in Figure 1.
(∆Tm ) 6 °C) than for the corresponding DNA comple-
ment (∆Tm ) 10 °C). This is the first time that an
oligodeoxyribonucleotide with an alt-motif demonstrates
a higher affinity for RNA than for DNA complementary
sequences. These data are also in agreement with the
predictions that emerged from modeling studies (see
Figure 1, bottom section). It must, however, be pointed
out that the (3′ f 3′)-incorporation of R-dT into 31 still
leads to a DNA-RNA hybrid that is more stable (Tm
)
2-Deoxy-5-O-tr ityl-D-er yth r o-p en tofu r a n ose (4). The
synthesis of 4 was achieved by modification of a previous
procedure.6 Typically, 2-deoxy-D-ribose (3) (30.0 g, 0.22 mol)
was dried by coevaporation with dry pyridine (3 × 30 mL) and
then dissolved in the same anhydrous solvent (250 mL).
Triphenylmethyl chloride (73.6 g, 26.4 mol) and a catalytic
amount of 4-(dimethylamino)pyridine (200 mg) were added to
the solution. After the solution was stirred at 50 °C for 6 h,
methanol (10 mL) was added. The resulting dark orange
solution was stirred for 30 min at room temperature prior to
evaporation of the solvents under reduced pressure. To ensure
the removal of any remaining pyridine, the gumlike material
was coevaporated with ethanol-toluene (1:4) (2 × 50 mL). The
residue was then dissolved in methylene chloride (600 mL)
and washed with water (3 × 300 mL). The collected organic
phases were dried over anhydrous sodium sulfate and evapo-
rated to dryness under vacuum. The crude product 4 was
purified by silica gel chromatography, first using methylene
chloride and, then, an increasing gradient of methanol (1-
4%) in methylene chloride as eluents. 2-Deoxy-5-O-trityl-D-
erythro-pentofuranose was isolated as a colorless amorphous
material in 60% yield (49.6 g, 0.13 mol). 1H-NMR spectral data
agreed with those reported by Kam and Oppenheimer.6
(2-Deoxy-5-O-tr ityl-r- a n d -â-D-er yth r o-p en tofu r a n o-
syl)a ceton itr ile (5a a n d 5b). Diethyl (cyanomethyl)phos-
phonate (66.3 mL, 72.6 g, 0.41 mol) was added dropwise over
50 min to a stirred ice-cold suspension of sodium hydride (8.6
g, 0.36 mol) in dry THF (280 mL) under a stream of argon.
After the evolution of hydrogen subsided, a solution of 4 (44.0
g, 0.12 mol) in dry THF (300 mL) was added dropwise over 30
min. The reaction mixture was stirred at 0 °C under an inert
atmosphere, and reaction progress was monitored by TLC until
5a and 5b were formed in a nearly equimolar ratio (6 h).
Volatiles were then removed by the use of a rotary evaporator.
The residue was dissolved in ether, washed with water, dried
over magnesium sulfate, and finally evaporated to dryness.
The mixture of epimers (5a and 5b) was separated by silica
gel chromatography using a gradient of ethyl acetate in
hexane. The epimer 5b eluted from the silica gel column with
20% ethyl acetate in hexane, whereas elution of 5a required
25-30% ethyl acetate in hexane. The glycoside 5a (12.01 g,
30.1 mmol) was isolated as a white solid. 1H- and 13C-NMR
data pertaining to 5a have been reported (ref 4a). FAB-
HRMS: calcd for C26H25NO3 (M•+) 399.1834, found 399.1833.
The glycosylated acetonitrile derivative 5b (8.08 g, 20.2 mmol)
was also isolated as a white solid. 1H-NMR (300 MHz,
CDCl3): δ 1.95 (ddd, J ) 13.1, 9.2, 6.2 Hz, 1H), 2.08 (ddd, J )
13.1, 5.7, 2.6 Hz, 1H), 2.62 (dd, J ) 16.7, 5.4 Hz, 1H), 2.66
(dd, J ) 16.7, 5.5 Hz, 1H), 3.17 (dd, J ) 9.9, 5.9 Hz, 1H), 3.30
(dd, J ) 9.9, 4.7 Hz, 1H), 3.92 (ddd, J ) 5.9, 4.7, 3.0 Hz, 1H),
4.38 (m, 1H), 4.40 (m, 1H), 7.25 (t, J ) 7.8 Hz, 3H), 7.31 (t, J
) 7.8 Hz, 6H), 7.44 (d, J ) 7.8 Hz, 6H). 13C-NMR (75 MHz,
CDCl3): δ 23.9, 40.0, 64.3, 73.4, 74.1, 86.3, 86.9, 116.9, 127.2,
58 °C) than that obtained from the (3′ f 3′)-integration
of 2a into 31 (Tm ) 55 °C). Thus, the nucleobase
flexibility of 2a presumably contributed to entropic
factors that adversely affected the thermostability of the
modified DNA-RNA hybrid to a greater extent than that
observed with the parent unmodified R-nucleoside under
identical conditions.
Exp er im en ta l Section
Ma ter ia ls a n d Meth od s. Common chemicals and solvents
including those that are anhydrous were purchased from
commercial sources and used without further purification.
2-Deoxy-D-ribose (3) and 1-O-methyl-2-deoxy-3,5-di-O-p-tolu-
oyl-D-erythro-pentofuranoside (13) were obtained from Sigma
and used as received. Triphenylmethyl chloride, 4,4′-dimethoxy-
trityl chloride, 4-(dimethylamino)pyridine, diethyl (cyano-
methyl)phosphonate, sodium hydride, 1.0 M borane-tetrahy-
drofuran complex in THF, 1.0 M diethylaluminum cyanide in
toluene, 2.0 M borane-methyl sulfide complex in THF, tri-
methylsilyl chloride, methyl methacrylate, sodium hydrogen
sulfate, silver cyanate, tert-butyldimethylchlorosilane, imida-
zole, 1.0 M tetra-n-butylammonium fluoride in THF, (2-
cyanoethyl)-N,N,N′,N′-tetraisopropyl phosphordiamidite, di-
isopropylamine, and 1H-tetrazole were purchased from Aldrich.
Trifluoroacetic acid (peptide synthesis grade) and basic ion-
exchange resin Amberlite IRA-93 (free base) were obtained
from Applied Biosystems and Sigma, respectively. 3-Methoxy-
2-methylacryloyl chloride was prepared according to the
method of Shaw and Warrener.26
Chromatography on silica gel columns was performed using
Merck silica gel 60 (230-400 mesh), whereas analytical thin-
layer chromatography (TLC) was conducted on 2.5 cm × 7.5
cm glass plates coated with a 0.25 mm thick layer of silica gel
60 F254 (Whatman). Reported melting points are uncorrected.
Optical rotations (R) of 1a and 1b were measured at 589 nm
in methanol at 25 °C. Specific rotations ([R]) were calculated
according to the formula [R] ) R/(lc), where R is the observed
rotation, l is the sample cell length in dm, and c is the sample
concentration in g/mL.
NMR spectra were recorded at either 7.05 T (300 MHz for
1H) or 11.75 T (500 MHz for 1H). Proton-decoupled 31P- and
1H-NMR spectra were obtained using deuterated solvents;
unless otherwise indicated, tetramethylsilane (TMS) was used
1
as internal reference for H-NMR spectra and 85% phosphoric
acid in deuterium oxide as an external reference for 31P-NMR
spectra. Proton-decoupled 13C-NMR spectra were recorded in
either CDCl3 or D2O. When indicated, the middle line of the
resonances observed for CDCl3 (77.00 ppm) was used as an
internal or external reference. Chemical shifts δ are reported
in parts per million (ppm). 1H-NOESY (mixing time τm ) 1 s)
and 1H-COSY experiments were performed at 300 MHz in
D2O.
127.9, 128.6, 143.7. FAB-HRMS: calcd for C26H25NO3 (M•+
399.1834, found 399.1835.
)
Low-resolution FAB mass spectra were acquired from
samples dissolved in
a 3-nitrobenzyl alcohol matrix and
A mixture of both epimers (9.26 g, 23.2 mmol) was also
isolated along with pure 5a and 5b. Thus, the overall yield
for the preparation of 5a and 5b was 61%.
bombarded with 8 keV fast xenon atoms. Accurate mass scans
covering a spectral region that included the molecular species
was performed at a resolution of ca. 10 000; a mass calibration
standard consisting of cesium iodide:rubidium iodide:sodium
iodide with molar ratios of 1:1:1 was added to the sample prior
to analysis.
1-(2-Deoxy-5-O-tr ityl-r- a n d -â-D-er yth r o-p en tofu r a n o-
syl)-2-a m in oeth a n e (6a a n d 6b). (2-Deoxy-5-O-trityl-R-D-
erythro-pentofuranosyl)acetonitrile (5a , 9.5 g, 23.8 mmol) was
dried by coevaporation with anhydrous toluene (3 × 20 mL)
under reduced pressure. Dry 5a was then dissolved in
anhydrous THF (50 mL), and 100 mL of 2.0 M borane-methyl
sulfide complex in THF (200 mmol) was added to the solution
by use of a syringe under an inert atmosphere. The reaction
Modeling studies were performed in the CHARMm force
field. The test sequence 5′-d(GCGTTN*TTGCG) where N* is
(26) Shaw, G.; Warrener, R. N. J . Chem. Soc. 1958, 153-156.

8624 J . Org. Chem., Vol. 61, No. 24, 1996
Boal et al.
mixture was heated to reflux for at least 40 min. Dimethyl
sulfide that escaped condensation was trapped with a 5%
aqueous solution of sodium hypochlorite. The solution was
then cooled to 0 °C, and methanol was carefully added until
the evolution of hydrogen had subsided. Volatiles were
removed by evaporation under reduced pressure, and the
residue was dissolved in dichloromethane (400 mL), which was
extracted with 5% aqueous sodium bicarbonate saturated with
sodium chloride (200 mL). The aqueous phase was back-
extracted twice with ethyl ether (200 mL) to ensure complete
extraction of the reaction product. The combined organic
extracts were dried over anhydrous sodium sulfate and
evaporated to dryness in vacuo. Crude 6a (9.02 g, 22.4 mmol,
94%) was used in the next step without further purification.
The â-epimer 6b was prepared under similar conditions and
yield. The aminoethylated glycoside derivatives 6a and 6b
were characterized after conversion to their corresponding
diacetates 7a and 7b by treatment with acetic anhydride and
pyridine (1:1, v/v). 7a : 1H- and 13C-NMR data have been
reported (ref 4a). FAB-HRMS: calcd for C30H34NO5 (MH+)
488.2437, found 488.2438. 7b: 1H-NMR (300 MHz, CDCl3), δ
1.71 (m, 2H), 1.89 (s, 3H), 1.92 (m, 1H), 2.05 (s, 3H), 2.10 (m,
1H), 3.18 (dd, J ) 9.9, 4.8 Hz, 1H), 3.26 (dd, J ) 9.9, 3.8 Hz,
1H), 3.30 (ddd, J ) 10.5, 3.8, 3.4 Hz, 1H), 3.59 (m, 1H), 4.06
(ddd, J ) 4.2, 4.0, 2.0 Hz, 1H), 4.18 (m, 1H), 5.17 (m, 1H),
6.30 (t, J ) 5.2 Hz, 1H), 7.24 (t, J ) 6.8 Hz, 3H), 7.30 (t, J )
7.7 Hz, 6H), 7.45 (d, J ) 7.0 Hz, 6H). 13C-NMR (75 MHz,
CDCl3): δ 21.1, 23.4, 34.0, 38.0, 38.9, 64.5, 76.4, 78.7, 84.1,
86.7, 127.1, 127.8, 128.7, 143.8, 170.0, 170.5. FAB-HRMS:
calcd for C30H34NO5 (MH+) 488.2437, found 488.2440.
N-[(2-Deoxy-r- a n d â-D-er yth r o-p en tofu r a n osyl)eth yl]-
N′-(3-m eth oxy-2-m eth yla cr yloyl)u r ea (9a a n d 9b). The
preparation of 9a from 6a and its characterization by 1H- and
13C-NMR spectroscopies have been reported (ref 4a). FAB-
HRMS: calcd for C13H23N2O6 (MH+) 303.1556, found 303.1552.
The â-epimer 9b was prepared (35%) under conditions similar
to those used for the preparation of 9a . 1H-NMR (300 MHz,
CDCl3): δ 1.67 (m, 1H), 1.75 (s, 3H), 1.80 (m, 1H), 1.93 (m,
1H), 1.98 (m, 1H), 3.30 (m, 1H), 3.63 (m, 2H), 3.76 (m, 1H),
3.85 (s, 3H), 3.95 (m, 1H), 4.15 (m, 1H), 4.26 (m, 1H), 7.35 (s,
1H), 8.3 (bs, 1H), 9.21 (dd, J ) 6.5, 3.2 Hz, 1H). 13C-NMR (75
MHz, CDCl3): δ 8.7, 34.2, 38.0, 41.6, 61.5, 63.7, 72.4, 78.5,
87.9, 106.9, 153.8, 158.9, 169.5. FAB-HRMS: calcd for
C13H23N2O6 (MH+) 303.1556, found 303.1559.
hood (Caution! Hydrogen cyanide is generated). After removal
of the solvents under reduced pressure, the residue was
dissolved in dichloromethane (200 mL), and the solution
filtered through silica gel (100 mL) covered with a layer of
anhydrous sodium sulfate. The filtering pad was washed with
dichloromethane (ca. 1 L). The filtrates were evaporated to
dryness in vacuo to give an anomeric mixture of 15a and 15b
(9.58 g). The mixture was loaded onto a column of silica gel
and eluted with a gradient of ethyl acetate (3-14%) in hexane.
The â-anomer 15b (4.06 g, 10.7 mmol) emerged first and was
recrystallized from ethanol. Mp: 109-110 °C (lit.18b mp 110-
111 °C). FAB-HRMS: calcd for C22H22NO5 (MH+) 380.1498,
found 380.1504. Further elution of the column with an
increasing gradient of ethyl acetate (14-50%) afforded the
R-anomer 15a (4.66 g, 12.3 mmol) that was also recrystallized
from ethanol. Mp: 143-144 °C (lit.18b mp 145-146 °C). FAB-
HRMS: calcd for C22H22NO5 (MH+) 380.1498, found 380.1497.
1H- and 13C-NMR data for 15a and 15b are in agreement with
reported values.18a
(2-Deoxy-3,5-d i-O-p-tolu oyl-r- a n d -â-D-er yth r o-p en to-
fu r a n osyl)a m in om eth a n e (16a a n d 16b). The preparation
of the hydrochloride salt of the aminomethylated R-glycoside
16a from 15a , and its characterization by 1H- and 13C-NMR
spectroscopies have been described in detail (reference 4b). The
aminomethylated â-glycoside 16b was obtained from 15b and
isolated in a manner similar to that described for 16a . 1H-
NMR (300 MHz, CDCl3) (free amine): δ 1.73 (b, 2H), 2.13 (m,
2H), 2.40 (s, 3H), 2.41 (s, 3H), 2.80 (m, 1H), 3.01 (m, 1H), 4.30
(m, 1H), 4.39 (m, 1H), 4.51 (m, 2H), 5.49 (m, 1H), 7.22 (d, J )
8.0 Hz, 2H), 7.23 (d, J ) 8.0 Hz, 2H), 7.86 (d, J ) 8.0 Hz, 2H),
7.87 (d, J ) 8.0 Hz, 2H). 13C-NMR (CDCl3): δ 21.6, 35.3, 45.4,
64.6, 76.6, 80.4, 82.5, 127.0, 127.1, 129.1, 129.4, 129.6, 130.0,
143.7, 143.9, 166.0, 166.2. Both 16a and 16b were sufficiently
pure to be used as is.
N-[(2-Deoxy-3,5-di-O-p-tolu oyl-r- an d -â-D-er yth r o-pen to-
fu r a n osyl)m e t h yl]-N ′-(3-m e t h oxy-2-m e t h yla cr yloyl)-
u r ea (17a a n d 17b). Under an inert atmosphere, dry silver
cyanate (7.19 g, 48 mmol) was added to a solution of 3-meth-
oxy-2-methylacryloyl chloride (2.96 g, 22 mmol) in dry benzene
(31 mL). The suspension was stirred under reflux for 30 min
and then allowed to cool to room temperature. The superna-
tant was cannulated into an addition funnel and added
dropwise over a period of 30 min to a cold solution (0 °C) of
16b (3.5 g, 8.4 mmol) and triethylamine (1.96 g, 2.7 mL, 19.4
mmol) in anhydrous benzene (50 mL). Upon completion of the
addition, the stirred reaction mixture was allowed to warm to
ambient temperature and further stirred overnight under an
argon atmosphere. Volatile components were then removed
by use of a rotary evaporator. The residue was dissolved in
chloroform (200 mL) and extracted, successively, with aqueous
5% sodium bicarbonate (200 mL) and water (200 mL). The
organic phase was dried over anhydrous sodium sulfate and
evaporated to dryness under reduced pressure. The crude
product was purified by silica gel chromatography using a
gradient of methanol (0.5-1.5%) in chloroform as an eluent.
Pure 17b was isolated as a white foam in 80% yield (3.5 g, 6.7
mmol). 1H-NMR (300 MHz, CDCl3): δ 1.70 (s, 3H), 2.14 (m,
J ) 13.7, 10.5, 5.8 Hz, 1H), 2.25 (m, J ) 13.6, 5.3 Hz, 1H),
2.40 (s, 3H), 2.42 (s, 3H), 3.61 (m, 2H), 3.83 (s, 3H), 4.45 (m,
1H), 4.52 (d, J ) 4.0 Hz, 2H), 5.54 (m, 1H), 7.22 (d, J ) 8.0
Hz, 2H), 7.26 (d, J ) 8.0 Hz, 2H), 7.27 (m, 1H), 7.94 (d, J )
8.0 Hz, 4H), 8.28 (b, 1H), 9.07 (bt, 1H). 13C-NMR (75 MHz,
CDCl3): δ 8.6, 21.6, 35.6, 42.4, 61.4, 64.8, 76.9, 77.8, 82.6,
106.8, 126.9, 127.0, 129.0, 129.1, 129.6, 143.5, 144.0, 154.6,
158.5, 165.9, 166.0, 169.0. FAB-HRMS: calcd for C28H33N2O8
(MH+) 525.2237, found 525.2241. The R-anomer 17a was
prepared and isolated as described for 17b. 1H-NMR (300
MHz, CDCl3): δ 1.75 (s, 3H), 2.05 (m, J ) 14.1, 5.1, 3.3 Hz,
1H), 2.41 (s, 6H), 2.65 (m, J ) 14.1, 7.4 Hz, 1H), 3.59 (m, 2H),
3.83 (s, 3H), 4.48 (m, 2H), 4.56 (m, 1H), 5.51 (m, 1H), 7.22 (d,
J ) 7.9 Hz, 4H), 7.91 (d, J ) 7.9 Hz, 2H), 7.94 (d, J ) 7.9 Hz,
2H), 8.28 (b, 1H), 9.04 (bt, 1H). 13C-NMR (75 MHz, CDCl3):
δ 8.7, 21.7, 35.2, 43.7, 61.4, 64.6, 76.4, 77.9, 81.9, 107.0, 126.8,
127.0, 129.1, 129.6, 143.7, 143.9, 154.5, 158.5, 166.1, 166.2,
169.1. FAB-HRMS: calcd for C28H33N2O8 (MH+) 525.2237,
found 525.2246.
1-(2-Deoxy-r- a n d â-D-er yth r o-p en tofu r a n osyl)-2-(th y-
m in -1-yl)eth a n e (1a a n d 1b). The acryloylurea derivative
9a (1.1 g, 3.6 mmol) was dissolved in concentrated ammonium
hydroxide (80 mL) and heated in a glass-lined stainless steel
pressure vessel at 50 °C for 12 h. The solution was then
evaporated to a gum under reduced pressure. The crude
product was dissolved in a minimum amount of ethanol (5 mL),
to which ethyl ether (150 mL) was added. The resulting
suspension was cooled to 0 °C until complete precipitation of
the nucleoside was obtained. The precipitate was filtered and
dried under vacuum. The nucleoside analogue 1a (0.9 g, 3.3
mmol) was isolated in 92% yield. UV (H2O, pH 7): λmax 272
nm (ꢀ 8900), λmin 238 nm (ꢀ 1600). [R]25: +50.2 (c 1.0, CH3-
OH). FAB-HRMS: calcd for C12H19N2O5 (MH+) 271.1294,
found 271.1293. The â-nucleoside 1b was similarly prepared
and isolated with a similar yield. UV (H2O, pH 7): λmax 272
nm (ꢀ 8700), λmin 238 nm (ꢀ 2400). [R]25: -14.3 (c 1.8, CH3-
OH). FAB-HRMS: calcd for C12H19N2O5 (MH+) 271.1294,
found 271.1294.
1H- and 13C-NMR peak assignments for 1a and 1b are listed
in Tables 1 and 2.
2-Deoxy-3,5-d i-O-p-tolu oyl-r- a n d -â-D-er yth r o-p en to-
fu r a n osyl cya n id e (15a a n d 15b). The cyanoglycosides 15a
and 15b were prepared by slight modification of a procedure
published by Iyer et al.18a To a cold solution (0 °C) of 2-deoxy-
3,5-di-o-toluoyl-D-erythro-pentofuranosyl chloride (7, 10.39 g,
26.2 mmol) in dry THF (35 mL) was added, dropwise, 40 mL
of 1.0 M diethylaluminum cyanide in toluene (40.0 mmol) over
a period of 30 min under an inert atmosphere. The solution
was then allowed to stir at ambient temperature for 7 h.
Excess diethylaluminum cyanide was destroyed upon slow
addition of methanol (15 mL) at 0 °C in a well-ventilated fume

Formation of DNA-DNA and DNA-RNA Hybrids
J . Org. Chem., Vol. 61, No. 24, 1996 8625
(2-Deoxy-3,5-d i-O-p-tolu oyl-r- a n d â-D-er yth r o-p en to-
fu r a n osyl)(th ym in -1-yl) m eth a n e (18a a n d 18b). The
preparation of 18a from 17a and its characterization by NMR
spectroscopy and high-resolution mass spectrometry have been
reported (ref 4b). The preparation of 18b from 17b was
achieved under conditions similar to those reported for 18a ,
and pure 18b was isolated in a similar yield. 1H-NMR (300
MHz, CDCl3): δ 1.75 (s, 3H), 1.98 (ddd, J ) 13.9, 11.0, 6.0
Hz, 1H), 2.33 (dd, J ) 13.9, 4.8 Hz, 1H), 2.42 (s, 6H), 3.55 (dd,
J ) 14.2, 7.0 Hz, 1H), 4.20 (dd, J ) 14.2, 1.5 Hz, 1H), 4.40 (m,
1H), 4.50 (dd, J ) 11.7, 4.5 Hz, 1H), 4.55 (m, 1H), 4.62 (dd, J
) 11.7, 3.1 Hz, 1H), 5.52 (m, 1H), 7.10 (s, 1H), 7.25 (d, J ) 7.9
Hz, 4H), 7.90 (d, J ) 7.9 Hz, 4H), 8.85 (s, 1H). 13C-NMR (75
MHz, CDCl3): δ 12.1, 21.7, 35.6, 50.6, 64.3, 76.4, 77.6, 83.3,
110.2, 126.6, 126.8, 129.2, 129.3, 129.5, 129.7, 141.6, 144.3,
151.1, 163.9, 166.1, 166.2. FAB-HRMS: calcd for C27H29N2O7
(MH+) 493.1975, found 493.1979.
37.6, 51.0, 55.2, 64.0, 73.8, 76.7, 86.0, 111.2, 113.1, 126.9, 127.9,
128.0, 129.9, 135.7, 141.6, 144.5, 150.8, 158.5, 164.0. FAB-
HRMS: calcd for C32H34N2O7 (M•+) 558.2366, found 558.2369.
1-[2-Deoxy-5-O-(ter t-b u t yld im et h ylsilyl)-r-D-er yth r o-
p en tofu r a n osyl]-2-(th ym in -1-yl)eth a n e (21a ). A general
procedure for the preparation of [2-deoxy-5-O-(tert-butyldi-
methylsilyl)-R- and -â-D-erythro-pentofuranosyl](thymin-1-yl)-
alkanes (21a ,b and 22a ,b) is available as Supporting Infor-
mation. 1H-NMR (300 MHz, CDCl3): δ 0.26 (s, 6H), 0.88 (s,
9H), 1.63 (ddd, J ) 12.7, 6.2, 4.8 Hz, 1H), 1.84 (s, 3H), 1.92
(m, 2H), 2.34 (ddd, J ) 12.7, 7.0, 6.4 Hz, 1H), 3.52 (dd, J )
10.6, 5.7 Hz, 1H), 3.67 (dd, J ) 10.6, 3.8 Hz, 1H), 3.70 (m,
1H), 3.82 (m, 1H), 3.86 (m, 1H), 4.02 (m, 1H), 4.29 (m, 1H),
7.05 (s, 1H). 13C-NMR (75 MHz, CDCl3): δ -5.5, 12.2, 18.2,
25.8, 35.2, 40.3, 46.2, 64.3, 74.2, 75.5, 85.2, 110.3, 141.2, 151.0,
164.5. FAB-HRMS: calcd for C18H33N2O5Si (MH+) 385.2159,
found 385.2152. 1-[2-Deoxy-5-O-(ter t-bu tyld im eth ylsilyl)-
â-D-er yth r o-p en tofu r a n osyl]-2-(th ym in -1-yl)eth a n e (21b).
1H-NMR (300 MHz, CDCl3): δ 0.26 (s, 6H), 0.88 (s, 9H), 1.70
(m, 2H), 1.85 (s, 3H), 1.95 (m, 2H), 3.47 (dd, J ) 10.7, 5.6 Hz,
1H), 3.65 (dd, J ) 10.7, 4.0 Hz, 1H), 3.70 (m, 1H), 3.76 (ddd,
J ) 5.6, 4.0, 3.0 Hz, 1H), 3.85 (m, J ) 13.8, 6.5 Hz, 1H), 4.07
(m, 1H), 4.26 (m, 1H), 7.0 (s, 1H). 13C-NMR (75 MHz, CDCl3):
δ -5.5, 12.3, 18.3, 25.9, 34.7, 40.9, 46.1, 64.2, 74.2, 75.2, 87.0,
110.5, 140.9, 151.4, 164.7. FAB-HRMS: calcd for C18H33N2O5-
Si (MH+) 385.2159, found 385.2152. [2-Deoxy-5-O-(ter t-
bu tyld im eth ylsilyl)-r-D-er yth r o-p en tofu r a n osyl](th ym in -
1-yl)m eth a n e (22a ). 1H-NMR (500 MHz, CDCl3): δ 0.26 (s,
6H), 0.88 (s, 9H), 1.75 (m, J ) 13.5, 4.5 Hz, 1H), 1.91 (s, 3H),
2.39 (m, J ) 13.5, 6.9 Hz, 1H), 3.54 (dd, J ) 10.5, 5.8 Hz, 1H),
3.70 (dd, J ) 10.5, 3.9 Hz, 1H), 3.73 (dd, J ) 14.3, 7.9 Hz,
1H), 3.93 (m, 1H), 4.01 (dd, J ) 14.3, 2.7 Hz, 1H), 4.38 (m,
2H), 7.16 (m, 1H), 8.55 (bs, 1H). 13C-NMR (125 MHz, CDCl3):
δ -5.5, 12.3, 25.8, 37.4, 52.0, 64.1, 73.9, 77.1, 86.1, 110.0, 141.8,
151.1, 164.1. FAB-HRMS: calcd for C17H31N2O5Si (MH+)
371.2002, found 371.2004. [2-Deoxy-5-O-(ter t-bu tyld im eth -
ylsilyl)-â-D-er yth r o-p en t ofu r a n osyl](t h ym in -1-yl)m et h -
a n e (22b). 1H-NMR (500 MHz, CDCl3): δ 0.10 (s, 6H), 0.90
(s, 9H), 1.80 (m, 1H), 1.90 (s, 3H), 2.00 (m, 1H), 3.52 (dd, J )
14.3, 7.3 Hz, 1H), 3.54 (dd, J ) 10.3, 5.4 Hz, 1H), 3.70 (dd, J
) 10.5, 3.6 Hz, 1H), 3.88 (m, 1H), 4.13 (d, 14.3 Hz, 1H), 4.32
(m, 1H), 4.41 (m, 1H), 7.17 (m, 1H), 9.03 (bs, 1H). 13C-NMR
(125 MHz, CDCl3): δ -5.4, 12.3, 25.9, 37.9, 51.3, 63.8, 73.5,
76.7, 87.2, 109.9, 141.8, 151.1, 164.2. FAB-HRMS: calcd for
C17H31N2O5Si (MH+) 371.2002, found 371.2006.
1-[2-Deoxy-3-O-(4,4′-dim eth oxytr ityl)-r-D-er yth r o-pen to-
fu r a n osyl]-2-(th ym in -1-yl)eth a n e (25a ). A general proce-
dure for the preparation of [2-deoxy-3-O-(4,4′-dimethoxytrityl)-
R- and -â-D-erythro-pentofuranosyl](thymin-1-yl)alkanes (25a ,b
and 26a ,b) is provided as Supporting Information. 1H-NMR
(300 MHz, CDCl3): δ 1.26 (ddd, J ) 13.5, 5.7, 4.3 Hz, 1H),
1.68 (ddd, J ) 13.5, 6.8, 6.5 Hz, 1H), 1.83 (m, 1H), 1.87 (s,
3H), 2.05 (m, 1H), 3.14 (dd, J ) 11.7, 6.4 Hz, 1H), 3.30 (dd, J
) 11.7, 2.5 Hz, 1H), 3.73 (m, 1H), 3.77 (s, 6H), 3.88 (m, 1H),
3.98 (m, 1H), 4.05 (m, 1H), 6.83 (d, J ) 8.8 Hz, 4H), 7.05 (s,
1H), 7.22 (t, J ) 7.6 Hz, 1H), 7.27 (t, J ) 7.6 Hz, 2H), 7.32 (d,
J ) 8.8 Hz, 4H), 7.43 (d, J ) 7.6 Hz, 2H). 13C-NMR (75 MHz,
CDCl3): δ 12.2, 34.4, 39.8, 46.1, 55.2, 62.4, 75.4, 75.5, 85.4,
87.0, 110.5, 113.2, 126.9, 127.9, 128.3, 130.2, 136.5, 136.6,
140.9, 145.4, 151.1, 158.6, 164.3. FAB-HRMS: calcd for
C33H36N2O7 (M•+) 572.2523, found 572.2525. 1-[2-Deoxy-3-
O-(4,4′-d im et h oxyt r it yl)-â-D-er yth r o-p en t ofu r a n osyl]-2-
(th ym in -1-yl)eth a n e (25b). 1H-NMR (300 MHz, CDCl3): δ
1.17 (ddd, J ) 13.1, 10.7, 6.1 Hz, 1H), 1.33 (ddd, J ) 13.1, 4.9,
1.0 Hz, 1H), 1.65 (m, 2H), 1.90 (s, 3H), 3.15 (dd, J ) 11.7, 4.7
Hz, 1H), 3.47 (dd, J ) 11.8, 2.5 Hz, 1H), 3.72 (ddd, J ) 14.5,
7.6, 7.2 Hz, 1H), 3.77 (s, 6H), 3.83 (ddd, J ) 14.5, 8.4, 5.3 Hz,
1H), 3.90 (ddd, J ) 4.5, 2.9, 2.2 Hz, 1H), 4.11 (m, 2H), 6.82 (d,
J ) 8.6 Hz, 4H), 7.03 (s, 1H), 7.22 (t, J ) 7.4 Hz, 1H), 7.27 (t,
J ) 7.4 Hz, 2H), 7.32 (d, J ) 8.6 Hz, 4H), 7.43 (d, J ) 7.4 Hz,
2H). 13C-NMR (75 MHz, CDCl3): δ 12.3, 34.0, 40.7, 46.2, 55.2,
63.0, 75.6, 75.9, 86.6, 86.8, 110.5, 113.2, 126.9, 127.8, 128.3,
130.2, 136.5, 136.6, 140.8, 145.3, 150.8, 158.6, 164.1. FAB-
HRMS: calcd for C33H36N2O7 (M•+) 572.2523, found 572.2528.
[2-Deoxy-3-O-(4,4′-d im et h oxyt r it yl)-r-D-er yth r o-p en t o-
fu r an osyl](th ym in -1-yl)m eth an e (26a). 1H-NMR (500 MHz,
(2-Deoxy-r- a n d -â-D-er yth r o-p en tofu r a n osyl)th ym in -
1-yl)m eth a n e (2a a n d 2b). The saponification of 18a and
18b by potassium hydroxide in aqueous ethanol has been
described in ref 4b. The characterization of 2a and 2b was
also reported. 1H- and 13C-NMR peak assignments for 2a and
2b are listed in Tables 1 and 2.
1-[2-Deoxy-5-O-(4,4′-dim eth oxytr ityl)-r-D-er yth r o-pen to-
fu r a n osyl]-2-(th ym in -1-yl)eth a n e (19a ). A general proce-
dure for the preparation of [2-deoxy-5-O-(4,4′-dimethoxytrityl)-
R- and -â-D-erythro-pentofuranosyl](thymin-1-yl)alkanes (19a ,b
and 20a ,b) is provided as Supporting Information. 1H-NMR
(300 MHz, CDCl3): δ 1.66 (ddd, J ) 12.7, 7.4, 5.8 Hz, 1H),
1.83 (s, 3H), 1.99 (m, 2H), 2.37 (ddd, J ) 12.7, 6.8, 6.4 Hz,
1H), 3.09 (dd, J ) 9.5, 6.0 Hz, 1H), 3.24 (dd, J ) 9.5, 4.7 Hz,
1H), 3.76 (m, 1H), 3.79 (s, 6H), 3.89 (ddd, J ) 13.3, 8.0, 5.0
Hz, 1H), 3.99 (m, 1H), 4.08 (m, 1H), 4.32 (m, 1H), 6.83 (d, J )
8.9 Hz, 4H), 7.06 (s, 1H), 7.22 (t, J ) 7.1 Hz, 1H), 7.25 (t, J )
7.1 Hz, 2H), 7.30 (d, J ) 8.9 Hz, 4H), 7.42 (d, J ) 7.1 Hz, 2H).
13C-NMR (75 MHz, CDCl3): δ 12.2, 35.2, 40.2, 46.1, 55.2, 64.4,
74.5, 75.3, 84.1, 86.3, 110.3, 113.1, 126.8, 127.9, 128.1, 130.0,
135.9, 141.1, 144.7, 150.8, 158.5, 164.2. FAB-HRMS: calcd
for C33H36N2O7 (M•+) 572.2523, found 572.2521. 1-[2-Deoxy-
5-O-(4,4′-d im eth oxytr ityl)-â-D-er yth r o-p en tofu r a n osyl]-2-
(th ym in -1-yl)eth a n e (19b). 1H-NMR (300 MHz, CDCl3): δ
1.77 (m, 2H), 1.87 (s, 3H), 2.02 (m, 2H), 3.10 (dd, J ) 9.7, 5.5
Hz, 1H), 3.22 (dd, J ) 9.7, 4.5 Hz, 1H), 3.72 (ddd, J ) 14.4,
7.7, 7.2 Hz, 1H), 3.79 (s, 6H), 3.91 (m, 1H), 3.97 (m, 1H), 4.13
(dddd, J ) 8.9, 8.7, 4.8, 4.5 Hz, 1H), 4.31 (m, 1H), 6.83 (d, J )
8.9 Hz, 4H), 7.03 (s, 1H), 7.22 (t, J ) 7.8 Hz, 1H), 7.26 (t, J )
7.8 Hz, 2H), 7.32 (d, J ) 8.9 Hz, 4H), 7.43 (d, J ) 7.8 Hz, 2H).
13C-NMR (75 MHz, CDCl3): δ 12.2, 34.7, 40.8, 46.2, 55.2, 64.6,
74.4, 75.0, 85.8, 86.2, 110.5, 113.1, 126.9, 127.8, 128.1, 130.1,
136.0, 141.0, 144.8, 150.9, 158.5, 164.2. FAB-HRMS: calcd
for C33H36N2O7 (M•+) 572.2523, found 572.2529. [2-Deoxy-5-
O-(4,4′-d im e t h oxyt r it yl)-r-D-er yt h r o-p e n t ofu r a n osyl]-
(th ym in -1-yl)m eth a n e (20a ). 1H-NMR (500 MHz, CDCl3):
δ 1.74 (ddd, J ) 13.5, 5.4, 4.3 Hz, 1H), 1.92 (d, J ) 1.1 Hz,
3H), 2.38 (ddd, J ) 13.5, 7.7, 6.5 Hz, 1H), 3.09 (dd, J ) 9.8,
6.0 Hz, 1H), 3.20 (dd, J ) 9.8, 4.5 Hz, 1H), 3.73 (dd, J ) 14.1,
7.9 Hz, 1H), 3.79 (s, 6H), 4.02 (m, J ) 3.3 Hz, 1H), 4.04 (dd, J
) 14.1, 3.1 Hz, 1H), 4.35 (m, J ) 6.5, 4.1, 3.3 Hz, 1H), 4.40
(dddd, J ) 8.3, 7.9, 5.6, 3.2 Hz, 1H), 6.82 (d, J ) 8.9 Hz, 4H),
7.17 (q, J ) 1.1 Hz, 1H), 7.22 (t, J ) 7.3 Hz, 1H), 7.28 (t, J )
7.3 Hz, 2H), 7.29 (d, J ) 8.9 Hz, 4H), 7.39 (d, J ) 7.3 Hz, 2H),
8.18 (bs, 1H). 13C-NMR (125 MHz, CDCl3): δ 12.3, 37.4, 52.1,
55.2, 64.3, 74.1, 76.9, 85.0, 110.0, 113.1, 126.8, 127.8, 128.0,
129.9, 135.7, 141.8, 144.6, 150.9, 158.5, 163.8. FAB-HRMS:
calcd for C32H34N2O7 (M•+) 558.2366, found 558.2380. [2-
D e o x y -5-O -(4,4′-d im e t h o x y t r it y l)-â-D -er yt h r o-p e n t o -
fu r an osyl](th ym in -1-yl)m eth an e (20b). 1H-NMR (500 MHz,
CDCl3): δ 1.75 (d, J ) 1.1 Hz, 3H), 1.82 (ddd, J ) 13.3, 9.7,
6.1 Hz, 1H), 2.00 (ddd, J ) 13.3, 5.9, 2.5 Hz, 1H), 3.06 (dd, J
) 9.9, 5.6 Hz, 1H), 3.25 (dd, J ) 9.9, 4.5 Hz, 1H), 3.58 (dd, J
) 14.3, 6.8 Hz, 1H), 3.80 (s, 6H), 3.96 (m, J ) 5.6, 4.5, 2.9 Hz,
1H), 4.08 (dd, J ) 14.3, 2.5 Hz, 1H), 4.32 (m, J ) 5.5, 2.5 Hz,
1H), 4.41 (dddd, J ) 8.9, 6.8, 6.5, 2.5 Hz, 1H), 6.83 (d, J ) 8.9
Hz, 4H), 7.07 (q, J ) 1.2 Hz, 1H), 7.23 (t, J ) 7.3 Hz, 1H),
7.29 (t, J ) 7.3 Hz, 2H), 7.32 (d, J ) 8.9 Hz, 4H), 7.41 (d, J )
7.3 Hz, 2H), 8.07 (bs, 1H). 13C-NMR (125 MHz, CDCl3): δ 12.1,

8626 J . Org. Chem., Vol. 61, No. 24, 1996
Boal et al.
CDCl3): δ 1.23 (ddd, J ) 13.9, 3.6, 2.4 Hz, 1H), 1.78 (ddd, J )
13.9, 7.6, 6.6 Hz, 1H), 1.91 (d, J ) 1.0 Hz, 3H), 3.18 (dd, J )
11.8, 5.6 Hz, 1H), 3.39 (dd, J ) 11.8, 1.5 Hz, 1H), 3.79 (dd, J
) 14.0, 9.0 Hz, 1H), 3.80 (s, 6H), 3.94 (dd, J ) 14.0, 3.1 Hz,
1H), 4.04 (m, J ) 5.6, 2.9, 1.5 Hz, 1H), 4.14 (m, J ) 6.6, 2.9,
2.4 Hz, 1H), 4.25 (dddd, J ) 9.0, 7.6, 3.6, 3.1 Hz, 1H), 6.85 (d,
J ) 8.9 Hz, 4H), 7.10 (q, J ) 1.0 Hz, 1H), 7.23 (t, J ) 7.2 Hz,
1H), 7.31 (t, J ) 7.2 Hz, 2H), 7.34 (d, J ) 8.9 Hz, 4H), 7.44 (d,
J ) 7.2 Hz, 2H), 8.17 (bs, 1H). 13C-NMR (125 MHz, CDCl3):
δ 12.4, 37.0, 52.2, 55.3, 62.8, 75.4, 77.2, 86.0, 109.9, 113.3,
127.1, 128.0, 128.1, 130.1, 136.2, 136.3, 141.7, 145.1, 150.9,
158.7, 164.1. FAB-HRMS: calcd for C32H34N2O7 (M•+) 558.2366,
found 558.2368. [2-Deoxy-3-O-(4,4′-d im eth oxytr ityl)-â-D-
er yth r o-p en tofu r a n osyl](th ym in -1-yl)m eth a n e (26b). 1H-
NMR (500 MHz, CDCl3): δ 1.31 (ddd, J ) 13.2, 10.0, 6.1 Hz,
1H), 1.55 (ddd, J ) 13.2, 5.5, 1.4 Hz, 1H), 1.90 (d, J ) 1.2 Hz,
3H), 3.14 (dd, J ) 11.9, 4.4 Hz, 1H), 3.42 (dd, J ) 11.9, 3.0
Hz, 1H), 3.63 (dd, J ) 14.3, 8.3 Hz, 1H), 3.78 (dd, J ) 14.3,
2.7 Hz, 1H), 3.79 (s, 6H), 3.82 (m, 1H), 4.17 (m, 1H), 4.42 (dddd,
J ) 10.0, 8.3, 5.5, 2.7 Hz, 1H), 6.83 (d, J ) 8.9 Hz, 4H), 7.09
(q, J ) 1.2 Hz, 1H), 7.22 (t, J ) 7.2 Hz, 1H), 7.29 (t, J ) 7.2
Hz, 2H), 7.32 (d, J ) 8.9 Hz, 4H), 7.43 (d, J ) 7.2 Hz, 2H),
8.10 (bs, 1H). 13C-NMR (125 MHz, CDCl3): δ 12.3, 37.3, 52.0,
55.2, 64.3, 74.1, 76.9, 85.1, 110.0, 113.1, 126.9, 127.8, 128.0,
130.0, 135.7, 141.8, 144.6, 150.9, 158.5, 163.9. FAB-HRMS:
calcd for C32H35N2O7 (MH+) 559.2444, found 559.2446.
Gen er a l P r oced u r e for th e P r ep a r a tion of th e r- a n d
â-Deoxyr ibon u cleosid e P h osp h or a m id ites 27a ,b-30a ,b.
The preparation of 1-[[2-deoxy-3-O-[(N,N-diisopropylamino)-
(2-cyanoethoxy)phosphinyl]-5-O-(4,4′-dimethoxytrityl)-â-D-eryth-
ro-pentofuranosyl]-2-(thymin-1-yl)ethane (27b) is representa-
tive. Dry 19b (750 mg, 1.31 mmol) and 140 mg of the N,N-
diisopropylammonium salt of 1H-tetrazole (0.82 mmol) were
dissolved in anhydrous dichloromethane (14 mL) under an
inert atmosphere. To the cloudy solution was added 800 mL
of 2-cyanoethyl N,N,N′,N′-tetraisopropylphosphordiamidite
(759 mg, 2.52 mmol) by use of a syringe. The reaction mixture
was stirred at ambient temperature for 4 h and then quenched
by the addition of a solution of triethylamine (2% v/v) in
dichloromethane (80 mL) and brine (80 mL). The two layers
were separated, and the organic phase was washed once with
brine (50 mL). The organic layer was dried (MgSO4) and
evaporated to dryness in vacuo. The crude product was
purified by silica gel chromatography; the â-deoxyribonucleo-
side phosphoramidite 27b was eluted from the column with
benzene:triethylamine (90:10) and was isolated as a white
foam. Purified 27b was triturated with hexane, and the
resulting suspension was cooled to -20 °C for 2 h. The solid
was filtered, washed with cold hexane, and dried to give 840
mg of 27b as a white powder (1.09 mmol, 83%). FAB-HRMS:
calcd for C42H53N4O8P (M•+) 772.3601, found 772.3600. 31P-
NMR (202 MHz, CDCl3): δ 147.2 and 146.9 ppm.27 The
phosphoramidites 27a and 28a ,b-30a ,b were similarly pre-
pared and isolated in 80-90% yield. A mixture of dichlo-
romethane:ethyl acetate:triethylamine (65:25:10) was, how-
ever, used as eluent for the purification of 28a ,b and 30a ,b.
1-[[2-Deoxy-3-O-[(N,N-diisopr opylam in o)(2-cyan oeth oxy)-
ph osph in yl]-5-O-(4,4′-dim eth oxytr ityl)-r-D-er yth r o-pen to-
fu r an osyl]-2-(th ym in -1-yl)eth an e (27a). FAB-HRMS: calcd
for C42H53N4O8P (M•+) 772.3601, found 772.3601. 31P-NMR
(202 MHz, CDCl3): δ 147.4 and 147.1 ppm. [2-Deoxy-3-O-
[(N,N-d iisop r op yla m in o)(2-cya n oeth oxy)p h osp h in yl]-5-
O-(4,4′-d im e t h oxyt r it yl)-r-D-er yt h r o-p e n t ofu r a n osyl]-
an e (29a). FAB-HRMS: calcd for C42H53N4O8P (M•+) 772.3601,
found 772.3600. 31P-NMR (202 MHz, CDCl3): δ 147.9 and
147.6 ppm. 1-[[2-Deoxy-3-O-(4,4′-d im et h oxyt r it yl)-5-O-
[(N,N-d iisop r op yla m in o)(2-cya n oeth oxy)p h osp h in yl]-â-
D-er yth r o-p en t ofu r a n osyl]-2-(t h ym in -1-yl)et h a n e (29b ).
FAB-HRMS: calcd for C42H53N4O8P (M•+) 772.3601, found
772.3601. 31P-NMR (202 MHz, CDCl3): δ 147.6 and 147.4
ppm. [2-Deoxy-3-O-(4,4′-d im et h oxyt r it yl)-5-O-[(N,N-d i-
isopr opylam in o)(2-cyan oeth oxy)ph osph in yl]-r-D-er yth r o-
p en tofu r a n osyl](th ym in -1-yl)m eth a n e (30a ). FAB-HRMS:
calcd for C41H52N4O8P (MH+) 759.3523, found 759.3524. 31P-
NMR (202 MHz, CDCl3): δ 147.2 and 147.1 ppm. [2-Deoxy-
3-O-(4,4′-d im eth oxytr ityl)-5-O-[(N,N-d iisop r op yla m in o)-
(2-cya n oe t h oxy)p h osp h in yl]-â-D-er yt h r o-p e n t ofu r a n -
osyl](th ym in -1-yl)m eth a n e (30b). FAB-HRMS: calcd for
C
41H52N4O8P (MH+) 759.3523, found 759.3527. 31P-NMR (202
MHz, CDCl3): δ 146.9 and 146.6 ppm.
P r ep a r a tion of Oligon u cleotid es. Standard â-cyanoeth-
yl deoxyribonucleoside phosphoramidites and all the reagents
required for solid-phase oligodeoxyribonucleotide syntheses
were purchased from Applied Biosystems Inc. and used as
recommended by the manufacturer. The incorporation of the
modified phosphoramidites 27a ,b-30a ,b into oligonucleotide
31 according to Table 3 was performed under conditions
identical to those used for standard phosphoramidites.
P u r ifica tion of Oligon u cleotid es. Purification of the
oligoribonucleotide 5′-r(UCCAUUUUCAGAAUUGGGUGUC-
GA) (Cruachem), along with the modified and unmodified
oligodeoxyribonucleotides listed in Table 3, was accomplished
by polyacrylamide gel electrophoresis according to literature
protocols.24a,28
Th er m a l Den a tu r a tion P r ofiles. Each DNA oligonucle-
otide was mixed with either a complementary unmodified DNA
or RNA oligomer in PBS buffer (pH 7.2); the total strand
concentration was kept at ca. 5 µM. Thermal denaturation
profiles (absorbance vs temperature) of the resulting DNA-
DNA and DNA-RNA hybrids were measured at 260 nm with
a diode array UV/vis spectrophotometer equipped with a
Peltier temperature controller that was interfaced to a per-
sonal computer. For the temperature range 20-80 °C, 121
measurement points were taken every 0.5 °C, with an equili-
bration time of 30 s for each measurement point. A heating
and a cooling profile were recorded for each complex. The
melting temperature (Tm) was determined from the first
derivative of each of the heating and cooling curves and
reported in Table 3 as the average of these two temperatures.
The reproducibility of these measurements was within (1 °C.
Ack n ow led gm en t. This research was supported in
part by an appointment to the Postgraduate Research
Participation Program at the Center for Biologics Evalu-
ation and Research administered by the Oak Ridge
Institute for Science and Education through an inter-
agency agreement between the U.S. Department of
Energy and the U.S. Food and Drug Administration.
Su p p or tin g In for m a tion Ava ila ble: General procedures
for the preparation of 19a ,b-22a ,b, 25a ,b, and 26a ,b and
NMR spectra of new compounds (84 pages). This material is
contained in libraries on microfiche, immediately follows this
article in the microfilm version of the journal, and can be
ordered from the ACS; see any current masthead page for
ordering information.
(th ym in -1-yl)m eth a n e (28a ). FAB-HRMS: calcd for C41H52
-
N4O8P (MH+) 759.3523, found 759.3530. 31P-NMR (202 MHz,
CDCl3): δ 146.9 and 146.7 ppm. [2-Deoxy-3-O-[(N,N-d iiso-
p r op yla m in o)(2-cya n oet h oxy)p h osp h in yl]-5-O-(4,4′-d i-
m et h oxyt r it yl)-â-D-er yth r o-p en t ofu r a n osyl](t h ym in -1-
yl)m eth an e (28b). FAB-HRMS: calcd for C41H52N4O8P (MH+)
J O961548K
759.3523, found 759.3528. 31P-NMR (202 MHz, CDCl3):
δ
146.7 and 146.4 ppm. 1-[[2-Deoxy-3-O-(4,4′-d im eth oxytr i-
t yl)-5-O-[(N,N-d iisop r op yla m in o)(2-cya n oet h oxy)p h os-
p h in yl]-r-D-er yth r o-p en tofu r a n osyl]-2-(th ym in -1-yl)eth -
(28) Damha, M. J .; Ogilvie, K. K. In Methods in Molecular Biology,
Vol 20: Protocols for Oligonucleotides and Analogs; Agrawal, S., Ed.;
Humana Press: Totowa, 1993; pp 81-114.
(29) (a) Ohrui, H.; J ones, G. H.; Moffatt, J . G.; Maddox, M. L.;
Christensen, A. T.; Byram, S. K. J . Am. Chem. Soc. 1975, 97, 4602-
4613. (b) De Bernardo, S.; Weigele, M. J . Org. Chem. 1977, 42, 109-
112.
(27) 31P-NMR signals reported for 27a ,b-30a ,b are downfield
relative to that of the external reference (85% phosphoric acid in D2O).