Chimera of dimethylene sulfone-, methyl sulfide-, and methyl sulfoxide-linked ribonucleotides and DNA

Full text of DOI:10.1021/jo960669h

7620
J . Org. Chem. 1996, 61, 7620-7626
amidite protocol. This required preparation of 2′-
Ch im er a of Dim eth ylen e Su lfon e-, Meth yl
protected dimer 14 (Scheme 1). Synthesis of this dimeric
building block made use of known olefin 1,4a which after
ozonolysis and in situ reduction of the resulting aldehyde
gave alcohol 2 in 85% yield. Cleavage of the acetal with
iodine/methanol,7 regioselective silylation, and benzoyl-
ation converted 2 to glycosyl donor 5. Silylated uracil
was reacted with 5 under Vorbru¨ggen conditions8 to
afford 3′-homonucleoside 6 in 90% yield. Conventional
ester hydrolysis gave 7, and a Mitsunobu-type hydroxyl
to thioester conversion at the 3′′ position yielded 8, which
was coupled with iodohomothymidine 99 using cesium
carbonate in THF/DMF. Under these conditions, the
liberation of the attacking thiolate occurs via migration
of the protecting acetyl group to the 2′-hydroxyl group.
As expected,4c 30% of the thioether precursor of 11 was
formed as the 2′-unprotected dimer due to intermolecular
transesterification. After oxidation of the crude coupling
product with buffered Oxone, alcohol 10 was isolated by
chromatography, and acetylated with Ac2O/pyridine, and
the combined fractions of 11 were desilylated with HF/
pyridine. The yield of isolated diol 12 was 50% over four
steps. Dimethoxytritylation and phosphitylation with
bis(diisopropylamino) (â-cyanoethoxy)phosphine10 af-
forded phosphoramidite 14 as the expected mixture of
stereoisomers, which was characterized by FAB-MS and
1H and 31P NMR spectroscopy.
Sulfone dimer 14 was incorporated into modified DNA
decamers 17 and 21 (Scheme 2) using a standard phos-
phoramidite protocol (see Experimental Section). Inter-
estingly, the all-pyrimidine oligomer 21 coeluted from
RP18 HPLC columns with shorter “failure sequences” and
had to be purified on an ion-exchange resin. Structure
confirmation for both oligomers was obtained from
MALDI-TOF MS under established conditions.11
Duplexes between the chimeras 17 and 21 and their
complementary DNA strands 22 and 25 had lower
melting points than the double helices of the unmodified
controls (16/22 and 20/25) (Table 1). The lowering of the
melting temperature (∆Tm) was considerably more pro-
nounced for the internally modified 17 than for 21, which
bears a terminal modification. The interpretation of this
phenomenon is difficult in this case, as the oligomers
have different sequences. Examples from the literature
are known, however, where oligomers of a given sequence
have shown a similar melting point depression upon
positioning a terminal nonionic12 or charged13 backbone
modification in the middle of the sequence. Assuming
that regularity in a duplex structure has energetic
significance, a perturbation in backbone structure may
be more destabilizing when in the middle of a strand than
when at an end. It may be argued that the ribonucleoside
units in chimera 17 and 21 cause A-type structural
perturbations to otherwise B-type DNA duplexes, as
Su lfid e-, a n d Meth yl Su lfoxid e-Lin k ed
Ribon u cleotid es a n d DNA
Daniel K. Baeschlin, Birgitte Hyrup,†
Steven A. Benner, and Clemens Richert*,‡
Department of Chemistry, Swiss Federal Institute of
Technology, 8092 Zu¨rich, Switzerland
Received April 10, 1996
In tr od u ction
Modified nucleic acids are important tools for studying
structural and functional aspects of DNA, RNA, and
protein-nucleic acid interactions. They can also help
rationalize why Nature has selected RNA and DNA as
the sole carriers of genetic information.1 Analogs of
nucleosides can also be powerful drugs,2 and analogs of
oligonucleotides have the potential for being used as
sequence-specific inhibitors of gene expression.3
Our studies of modified nucleic acids have focused on
backbone modified oligonucleotides where the phospho-
rus found in DNA and RNA has been replaced by sulfur
and the bridging phosphodiester oxygens by methylene
units.4 Crystal structures have shown that dimethylene
sulfone-bridged ribonucleotides can form A-type, Wat-
son-Crick-paired duplexes5 and, at elevated tempera-
tures, extended single-stranded structures with only
three torsion angles significantly different from A-type
duplex RNA.6 Their preferred conformation appears thus
to be similar to that of natural RNA. It was therefore of
great interest to see how incorporation of this nonionic
modification influences the properties of natural oligo-
mers. We present here the synthesis of chimera of
dimethylene sulfone-, methyl sulfoxide-, and methyl
sulfide-linked RNA and natural DNA. The extent to
which these modifications induce perturbations in double-
stranded DNA and RNA was determined in hybridization
experiments with unmodified oligonucleotide counter-
strands.
Resu lts a n d Discu ssion
Incorporation of a dimethylene sulfone-bridged ribo-
linkage into DNA was achieved using the phosphor-
† Present address: Department of Chemistry, University of Copen-
hagen, 2100 Copenhagen Ø, Denmark.
‡ Present address: Department of Chemistry, Tufts University,
Medford, MA 02155.
* To whom correspondence should be addressed at the Depart-
ment of Chemistry, Tufts University, 62 Talbot Ave., Medford, MA
02155. Tel.: (617) 627-3475. Fax: (617) 627-3443. E-mail: crichert@
emerald.tufts.edu.
(1) See, e.g.: (a) Eschenmoser, A. Pure Appl. Chem. 1993, 65, 1179-
1188. (b) Hunziker, J .; Roth, H.-J .; Bo¨hringer, M.; Giger, A.; Dieder-
ichsen, U.; Go¨bel, M.; Krishnan, R.; J aun, B.; Leumann, C.; Eschen-
moser, A. Helv. Chim. Acta 1993, 76, 259-351. (c) Schneider, K. C.;
Benner, S. A. J . Am. Chem. Soc. 1990, 112, 453-455.
(2) (a) Huryn, D. M.; Okabe, M. Chem. Rev. 1992, 92, 1745-1768.
(b) Perigaud, C.; Gosselin, G.; Imbach, J .-L. Nuclesides Nucleotides
1992, 11, 903-945.
(3) (a) Uhlmann, E.; Peyman, A. Chem. Rev. 1990, 90, 544-584. (b)
Milligan, J . F.; Matteucci, M. D.; Martin, J . C. J . Med. Chem. 1993,
36, 1923-1937.
(4) (a) Huang, Z.; Schneider, K. C.; Benner, S. A. J . Org. Chem. 1991,
56, 3869-3882. (b) Schneider, K. C.; Benner, S. A. Tetrahedron Lett.
1990, 31, 335-338. (c) Richert, C.; Roughton, A. L.; Benner, S. A. J .
Am. Chem. Soc. 1996, 118, 4518-4531.
(5) Roughton, A. L.; Portmann, S.; Benner, S. A.; Egli, M. J . Am.
Chem. Soc. 1995, 117, 7249-7250.
(6) Hyrup, B.; Richert, C.; Schulte-Herbru¨ggen, T.; Benner, S. A.;
Egli, M. Nucleic Acids Res. 1995, 23, 2427-2433.
(7) Szarek, W. A.; Zamojski, A.; Tiwari, K. N.; Ison, E. R. Tetrahe-
dron Lett. 1986, 27, 3827-3830.
(8) (a) Vorbru¨ggen, H.; Bennua, B. Chem. Ber. 1981, 114, 1279-
1286. (b) Vorbru¨ggen, H.; Krolikiewicz, K.; Bennua, B. Chem. Ber.
1981, 114, 1234-1255.
(9) Baeschlin, D. K.; Daube, M;. Bla¨ttler, M. O.; Benner, S. A.;
Richert, C. Tetrahedron Lett. 1996, 37, 1591-1592.
(10) Kierzek, R.; Kopp, D. W.; Edmonds, M.; Caruthers, M. H.
Nucleic Acids Res. 1986, 14, 4751-4764.
(11) Pieles, U.; Zu¨rcher, W.; Scha¨r, M.; Moser, H. E. Nucleic Acids
Res. 1993, 21, 3191-3196.
(12) Habus, I.; Temsamani, J .; Agrawal, S. Bioorg. Med. Chem. Lett.
1994, 4, 1065-1070.
(13) Bjerga˚rde, K.; Dahl, O. Nucleic Acids Res. 1991, 19, 5843-5850.
S0022-3263(96)00669-X CCC: $12.00 © 1996 American Chemical Society

Notes
J . Org. Chem., Vol. 61, No. 21, 1996 7621
Sch em e 1
Sch em e 2
dimethylene sulfone-linked oligoribonucleotides have
been observed to be conformationally similar to natural
RNA,4c,5,6 which is found almost exclusively in an A or
A′ conformation.14 This effect could be expected to lead
to improved binding of the ribo-modified oligomers to
RNA counter strands, because DNA/RNA duplexes are
known to adopt A-type conformations.14 The melting
point of 17 with RNA-target 24 was only slightly ele-
vated, however (∆Tm -14 °C versus -16 °C for 17/22), a
selectivity for RNA considerably less pronounced than
that reported for an ethylenesulfide-linked RNA-DNA
chimera.15 The lowering of the melting points of duplexes
containing 17 and 21 may be caused by increased
stability of self-structure in the modified oligonucleotides
and not by decreased stability of the duplexes containing
them.16 A high-temperature UV transition has been
observed for a non-self-complementary dimethylene sul-
fone-linked RNA octamer, corroborating this assumption.4c
Hybridization experiments with 17 and DNA target
strand 23, which contains a T mismatch opposite the 3′-
nucleoside of UCH
T, led to disappearance of an
2SO2CH2
observable melting point (Table 1). When the experiment
was run at 1 M NaCl, it could be shown that the sequence
specificity of modified oligomer 17 was greater than that
of the control oligomer 16. The lowering of the melting
point induced by the mismatch was greater than 24 °C
for sulfone-linked 17 versus 16 °C for 16. This suggests
involvement of the nucleobases of the UT dimer in
Watson-Crick base-pairing and virtually excludes bulg-
ing. The nonionic linkage in 17 does not reduce the
dependence of the melting point on ionic strength sig-
nificantly, since ∆Tm values remained almost unchanged
when increasing the NaCl content from 0.1 to 1 M (Table
1, compare, e.g., rows 1, 2 and rows 5, 6). As modification
(14) Saenger, W. Principles of Nucleic Acid Structure; Springer-
Verlag: New York, 1984.
(15) Meng, B.; Kawai, S. H.; Wang, D.; J ust, G.; Giannaris, P. A.;
Damha, M. J . Angew. Chem. 1993, 105, 733-735; Angew. Chem., Int.
Ed. Engl. 1993, 32, 729-731.
(16) Vesnaver, G.; Breslauer, K. J . Proc. Natl. Acad. Sci. U.S.A.
1991, 88, 3569-3573.

7622 J . Org. Chem., Vol. 61, No. 21, 1996
Ta ble 1. Meltin g Tem p er a tu r es of Du p lexes Con ta in in g Na tu r a l a n d Mod ified Oligon u cleotid esa
Notes
oligomer
target
[NaCl] (M)
Tm (°C)
∆Tm (°C)
5′-GCGTTTTGCT-3′ (16)
3′-CGCAAAACGA-5′ (22)
3′-CGCAAAACGA-5′ (22)
3′-CGCAAAACGA-5′ (22)
3′-CGCAAAACGA-5′ (22)
3′-CGCAAAACGA-5′ (22)
3′-CGCAAAACGA-5′ (22)
0.1
0.1
0.1
0.1
1
46
30
30
23
53
39
5′-GCGUCH
TTTGCT-3′ (17)
-16
-16
-23
2SO2CH2
5′-GCGUCH STTTGCT-3′ (18)
2
5′-GCGUCH SOTTTGCT-3′ (19)
5′-GCGTTT2TGCT-3′ (16)
5′-GCGUCH
TTTGCT-3′ (17)
1
-14
2SO2CH2
5′-GCGTTTTGCT-3′ (16)
5′-GCGUCH TTTGCT-3′ (17)
3′-CGCATAACGA-5′ (23)
3′-CGCATAACGA-5′ (23)
3′-CGCATAACGA-5′ (23)
3′-CGCATAACGA-5′ (23)
3′-CGCATAACGA-5′ (23)
3′-CGCATAACGA-5′ (23)
0.1
0.1
0.1
0.1
1
24
<15
<15
<15
37
<-9
<-9
<-9
2SO2CH2
5′-GCGUCH STTTGCT-3′ (18)
2
5′-GCGUCH SOTTTGCT-3′ (19)
5′-GCGTTT2TGCT-3′ (16)
5′-GCGUCH
TTTGCT-3′ (17)
1
<15
<-22
2SO2CH2
5′-GCGTTTTGCT-3′ (16)
5′-GCGUCH TTTGCT-3′ (17)
3′-r (CGCAAAACGA)-5′ (24)
3′-r (CGCAAAACGA)-5′ (24)
3′-r (CGCAAAACGA)-5′ (24)
3′-r (CGCAAAACGA)-5′ (24)
0.1
0.1
0.1
0.1
45
31
27
22
-14
-18
-23
2SO2CH2
5′-GCGUCH STTTGCT-3′ (18)
2
5′-GCGUCH SOTTTGCT-3′ (19)
2
5′-TTTTTTTTTT-3′ (20)
3′-AAAAAAAAAA-5′ (25)
3′-AAAAAAAAAA-5′ (25)
3′-AAAAAAAAAA-5′ (25)
3′-AAAAAAAAAA-5′ (25)
0.1
0.1
1
22
18
35
32
5′-UCH
TTTTTTTTT-3′ (21)
-4
-3
2SO2CH2
5′-TTTTTTTTTT-3′ (20)
5′-UCH TTTTTTTTT-3 (21)
1
2SO2CH2
a
Melting temperatures were determined as previously described.24 Solutions were 1:1 mixtures of single strands (ca. 4 µM each), 10
mM NaH2PO4, adjusted to pH 7.0, and 0.1 mM EDTA. An estimate of experimental errors was obtained from experiments run in duplicate,
which gave melting points identical within (1.5 °C.
Sch em e 3
of the DNA backbone with other nonionic linkages in
most cases also lowers the melting temperature,17 aboli-
tion of the phosphate-phosphate repulsion appears to
be a stability factor that is easily overcome by other,
destabilizing effects.
Ac2O/pyridine. Desilylation, dimethoxytritylation, and
phosphitylation were performed as described for 11,
yielding 15, which was used for the solid phase assembly
of oligomer 18 (Scheme 2).
Mass spectrometric analysis of 18 showed that during
DNA synthesis approximately 60% of the thioether had
been oxidized to the sulfoxide 19. This was unexpected,
as thioether-linked nucleoside dimers have been reported
to be resistant to oxidation with I2/pyridine/THF/water
even after prolonged reaction times.15,19 Even more
unexpected was the fact that thioether 18 could readily
be separated from the mixture of sulfoxide diastereomers
19 by reversed-phase HPLC. This leads us to assume
that a major conformational change occurs when the
linking sulfur atom is oxidized. This was confirmed in
the hybridization experiments (Table 1). Thioether-
containing duplex 18/22 gave ∆Tm of -16 °C relative to
the unmodified duplex, whereas the sulfoxide-containing
duplex 19/22 gave a ∆Tm of -23 °C. The presence of two
diastereomers in 19 did not lead to a biphasic melting
curve. The modified oligomers 18 and 19 both displayed
sequence specificity when hybridized to the mismatch-
The influence of backbone modifications on the thermal
stability of oligonucleotide duplexes was subjected to
further scrutiny. Sulfide-linked dinucleoside 15 was
synthesized, which contains the ribonucleoside portion
of 14 but lacks one CH2 group in the ethylene linker of
the 3′-nucleoside (Scheme 3). Synthesis of 15 started
with bromination of thymidine using a modification of a
known procedure.18 When bromide 26 was reacted with
thioester 8, the coupling yield never exceeded 40%.
Silylation of the free 3′-position in 26 gave nucleoside 27,
which reacted smoothly with 8 to afford the desired
thioether-linked dimer in almost 80% combined yield of
2′-acetylated 29 and 2′-deprotected 28. The latter com-
pound was converted to the former by acetylation with
(17) Hunziker, J .; Leumann, C. Nucleic Acid Analogues: Synthesis
and Properties. In Modern Synthetic Methods 1995; Ernst, B., Leu-
mann, C., Eds.; Verlag Helvetica Chimica Acta: Basel, 1995; pp 333-
417.
(18) Verheyden, J . P. H.; Moffatt, J . G. J . Org. Chem. 1972, 37,
2289-2299.
(19) Kawai, S. H.; Wang, D.; Giannaris, P. A.; Damha, M. J .; J ust,
G. Nucleic Acids Res. 1993, 21, 1473-1479.

Notes
J . Org. Chem., Vol. 61, No. 21, 1996 7623
(Macherey-Nagel) with 20 mM KHPO4/CH3CN (4:1) and a
gradient of KCl (0-1 M). Assignments (see the supporting
information) are based on homonuclear decoupling, COSY, and
DEPT experiments performed on selected compounds. Multi-
plets reported without spectral region cover e 0.3 ppm. FAB
spectra were obtained using a 3-nitrobenzyl alcohol (NOBA)
containing counterstrand 23. While solvation effects
have not been excluded, molecular modeling and force
field calculations indicated that the Tm differences be-
tween the two duplexes may be due to different CH2-
S-CH2 angles. The wider angle typically found in
thioethers appears to distort the conformation of the UT
dimer embedded in an A-type duplex less than the
smaller angle in the sulfoxide. Attempts to oxidize the
diastereomeric sulfoxides 19 to the corresponding sulfone
led to decomposition of the oligomer, possibly via a
mechanism proposed for ethylidene sulfone-linked ana-
logs.20
matrix; MALDI-TOF spectra used
a 2,4,6-trihydroxyaceto-
phenone/diammonium hydrogen citrate matrix.11 Experimental
errors of the MALDI-TOF mass peaks are estimated to be
(0.1%. Combustion analyses were performed by the Micro-
analytical Laboratory ETH Zurich. HPLC-purified oligonucleo-
tides 22-25 were obtained from Cruachem, Glasgow, Scotland,
and were used without modification.
3-Deoxy-3-[(b en zoyloxy)m et h yl]-1,2-isop r op ylid en e-r-
r ibofu r a n ose (2). Ozone/O2 gas was introduced into a stirred
solution of alkene 14a (66.3 mg, 0.21 mmol) in dry MeOH (5 mL)
at -73 °C. A white precipitate formed during ozone treatment.
After 30 min the solution turned blue, and the ozone introduction
was terminated. N2 was bubbled through the solution to remove
the excess of ozone. A solution of NaBH4 (21.7 mg, 0.57 mmol)
in EtOH (2 mL) was added. The reaction mixture was allowed
to warm to -15 °C and was stirred for another 60 min. Acetic
acid (0.5 mL) was added dropwise to the solution, and the
mixture was stirred for an additional 20 min at rt. The reaction
mixture was diluted with ethyl acetate (20 mL) and was washed
with aqueous saturated NaHCO3 (20 mL) and brine (5 mL). The
aqueous phase was reextracted twice with ethyl acetate. The
combined organic phases were dried over MgSO4, filtered, and
concentrated in vacuo, and the residue was chromatographed
on silica (10 g) with petroleum ether/ethyl acetate (1:1). Alcohol
2 was obtained as a colorless oil (55.1 mg, 0.178 mmol). Yield:
85%. 1H NMR (300 MHz, CDCl3) δ: 1.33, 1.52 (2s); 2.3 (s, 1H);
2.57 (m, 1H); 3.68 (m, 1H); 3.95 (m, 1H); 4.11 (m, 1H); 4.41; 4.61
(dq, 2H); 4.79 (t, 1H); 5.86 (d, 1H); 7.42 (AA′BB′C-system, BB′-
part, 2H); 7.55 (AA′BB′C-system, C-part, 1H); 8.02 (AA′BB′C-
system, AA′-part, 2H). 13C NMR (75 MHz, CDCl3) δ: 26.3, 26.7,
43.2, 61.4, 62.2, 80.7, 81.2, 105, 112.3, 128.4, 129.5, 129.8, 133.2,
166.4. MS m/z 307 (M + H+, <1); 293 (29); 278 (0.32), 277 (1.9),
In conclusion, we have shown that ribo dimethylene
sulfone, methyl sulfide, and methyl sulfoxide linkages can
be incorporated into DNA oligomers. These linkages do
not prevent the formation of DNA/DNA and DNA/RNA
duplexes, and sequence-specific binding is observed.
Therefore, incorporation of the nonionic dinucleosides
presented here as well as longer sulfone-linked oligomers4c
may become a tool for studying nucleic acids and nucleic
acid protein interactions.21 Their incorporation in syn-
thetic RNA, in particular, merits exploration. In addi-
tion, we have shown that oxidation of a single thioether
linker in a modified oligonucleotide can shift the melting
point of its duplex with complementary DNA by 7 °C.
Methyl sulfide-linked oligomers may thus have applica-
tions as chemotunable hybridization probes. Work in this
area is in progress.22
Exp er im en ta l Section
Gen er a l P r oced u r es. Reactions involving air- or moisture-
sensitive compounds were carried out in glassware dried either
in an oven or under high vacuum (HV) and then placed under a
positive pressure of argon. Molecular sieves (3 or 4 Å; grain
size: 2-3 mm) were activated under high vacuum at 360 °C for
36 h. THF was distilled from sodium. DMF, pyridine and
CH3CN were puriss, absolute, over molecular sieves from Fluka,
Buchs, Switzerland. Dry acetonitrile and 1,2-dichloroethane
were from Rathburn Chemicals Ltd. All reagents were the best
available quality from Fluka. Deoxyribonucleoside phosphor-
amidites, 5′-O-(dimethoxytrityl)thymidine LCAA-CPG, and DNA
desalting cartridges were obtained from Glen Research Corpora-
tion. Diisopropylammonium tetrazolide23 was kindly provided
by B. Eschgfa¨ller (University of Freiburg). Solvents for extrac-
tion and chromatography were distilled from anhydrous CaSO4
(Sikkon, Fluka). Acetate buffer was made up from acetic acid
(3 M), NaOAc (1 M), and deionized water. Analytical thin-layer
chromatography was performed on E. Merck 60 F254 precoated
silica gel plates. Flash chromatography was performed under
0.2-0.3 bar pressure with Merck silica gel 60 (0.040-0.063 mm
mesh). Eluent mixtures for chromatography are expressed as
ratios of volumes. Reversed-phase HPLC purification of oligo-
nucleotides was performed on a Supelcosil LC 18-S (RP 18, 250
× 10 mm) reversed-phase column with HPLC-grade CH3CN
(Merck) and 0.1 M triethylamine/acetate buffer (TEA/A buffer)
pH 7.0. Anion-exchange chromatography was performed on a
Nucleogel SAX 1000-8/46 (50 × 4.6 mm) anion-exchange column
105 (100). Anal. Calcd for
Found: C, 62.05; H, 6.26.
C16H20O6: C, 62.33; H, 6.54.
3-[(Ben zoyloxy)m et h yl]-3-d eoxy-1-O-m et h yl-r- a n d â-
r ibofu r a n ose (3). To a solution of alcohol 2 (5.3 g, 17.2 mmol)
in MeOH (170 mL) was added iodine (0.82 g, 3.2 mmol) under
stirring at rt. The solution was refluxed for 4.5 h. The reaction
mixture was allowed to cool to rt. Pyridine (3.3 mL) and
NaS2O3‚5H2O (1.6 g) were added, and the mixture was stirred
for an additional 15 min before the solvents were removed by
rotary evaporation. The residue was taken up in ethyl acetate
(100 mL) and washed with brine (80 mL). The aqueous phase
was reextracted twice with ethyl acetate. The combined organic
phases were dried over MgSO4. After filtration and concentra-
tion in vacuo, the residue was chromatographed on silica (60 g)
with ethyl acetate/petroleum ether (1:1-3:1). A mixture of the
R- and â-anomers of 3 was obtained as a light yellow oil (3.02 g,
10.7 mmol). Yield: 62%. 1H NMR (â-anomer, 300 MHz, CDCl3)
δ: 2.31 (q, 1H); 2.73 (m, 1H); 3.32 (d, 1H); 3.40 (s, 3H); 3.62,
3.86 (m, 2H); 4.18 (m, 2H); 4.24 (q, 1H); 4.81 (dd, 1H); 4.89 (s,
1H); 7.44 (t, 2H); 7.58 (m, 1H); 8.02 (d, 2H). 1H NMR (R-anomer,
300 MHz, CDCl3) δ: 2.11 (q, 1H); 2.60 (m, 1H); 2.78 (d, 1H);
3.49 (s, 3H); 3.62, 3.86 (m, 2H); 4.18 (m, 2H); 4.39 (m, 1H); 4.62
(dd, 1H); 5.00 (s, 1H); 7.44 (t, 2H); 7.58 (m, 1H); 8.02 (d, 2H).
13C NMR (â-anomer, 75 MHz, CDCl3) δ: 40.9, 55.5, 61.3, 64.1,
75.4, 81.5, 109.2, 128.4, 129.6, 129.7, 133.4, 167.4. 1H NMR (R-
anomer, 75 MHz, CDCl3) δ: 42.4, 55.2, 6.1, 63.9, 72.5, 81.2,
103.1, 128.3, 129.5, 129.9, 133.1, 166.5. MS m/z: 281 (M+, <
1); 263; 251 (4.6); 233 (0.9); 176 (2.8); 105 (100). Anal. Calcd
for C14H17O6: C, 59.57; H, 6.43. Found: C, 59.17; H, 6.48.
3-[(Ben zoyloxy)m et h yl]-5-(ter t-b u t yld ip h en ylsilyl)-3-
d eoxy-1-O-m eth yl-r and â-r ibofu r a n ose (4). To a solution
of diol 3 (2.97 g, 10.5 mmol) and imidazole (1.72 g, 25.2 mmol)
in anhydrous THF (60 mL) was added tert-butyldiphenylsilyl-
chloride (TBDPSCl) (3.33 g, 11.6 mmol) under stirring at 0 °C.
A white precipitate formed during the addition. The reaction
mixture was allowed to warm to rt and was stirred for another
60 min. Dry MeOH (4 mL) was added and the solvents were
removed by rotary evaporation. The residue was taken up in
CH2Cl2 (140 mL), washed quickly with 1 N HCl, saturated
aqueous NaHCO3, and brine (100 mL each), and dried over
(20) J ust, G.; Kawai, S. H. In Carbohydrate Modifications in
Antisense Research; Saghvi, Y. S., Cook, P. D., Eds.; ACS Symposium
Series: Washington, DC, 1994; pp 52-65.
(21) See, e.g.: (a) Gait, M. J .; Grasby, J . A.; Karn, J .; Mersmann,
K.; Pritchard, C. E. Nucleosides Nucleotides 1995, 14, 1133-1144. (b)
Perrin, K. A.; Huang, J .; McElroy, E. B.; Iams, K. P.; Widlanski, T. S.
J . Am. Chem. Soc. 1994, 116, 7427-7428. (c) Strauss, J . K.; Maher, L.
J . Science 1994, 266, 1829-1834. (d) Duggan, L. J .; Hill, T. M.; Wu,
S.; Garrison, K.; Zhang, X.; Gottlieb, P. A. J . Biol. Chem. 1995, 270,
28049-28054.
(22) Baeschlin, D. K.; Hyrup, B.; Benner, S. A.; Richert, C. Unpub-
lished results.
(23) Barone, A. D.; Tang, J .-Y.; Caruthers, M. H. Nucleic Acids Res.
1984, 12, 4051-4061.
(24) Egholm, M.; Nielsen, P.; Buchardt, O.; Berg, R. H. J . Am. Chem.
Soc. 1992, 114, 9677-9678.

7624 J . Org. Chem., Vol. 61, No. 21, 1996
Notes
MgSO4. Filtration and concentration in vacuo yielded crude 4
(5.9 g) in quantitative yield containing approximately 10% silyl
methyl ether. The reaction product was submitted directly to
benzoylation. An analytical sample was prepared by chroma-
tography on silica with petroleum ether/ethyl acetate (2:1). 1H
NMR (â-anomer, 300 MHz, CDCl3) δ: 1.11 (s, 9H); 2.68 (m, 1H);
3.28 (s, 3H); 3.82 (m, 2H); 4.01-4.08 (m, 2H); 4.29 (dd, 1H, J )
11.5, 4.5 Hz), 4.81 (dd, 1H, J ) 11.5, 10.5 Hz); 4.83 (s, 1H); 7.32-
7.49 (m, 8H); 7.58 (m, 1H); 7.71 (m, 4H); 8.02 (d, 2H). 13C NMR
(â-anomer, 75 MHz, CDCl3) δ: 19.2, 26.8, 45.4, 54.5, 61.3, 66.9,
75.3, 80.1, 108.7, 127.7, 128.3, 128.5, 129.8, 133.2, 133.3, 135.6,
129.6, 133.4, 167.5. MS m/z: 519 (M+, <1); 489 (5.6); 463 (6.9);
431 (9.5); 105 (100); 77 (30). Anal. Calcd for C30H36SiO6: C,
69.20; H, 6.97. Found: C, 69.14; H, 6.95.
2-Ben zoyl-3-[(ben zoyloxy)m eth yl]-5-(ter t-bu tyld ip h en yl-
silyl)-3-d eoxy-1-O-m eth yl-r a n d â-r ibofu r a n ose (5). Crude
4 (5.6 g, containing 10.2 mmol of pure compound) was dissolved
in pyridine (105 mL). (Dimethylamino)pyridine (DMAP) (5 mg,
41 µmol) was added, and the mixture was cooled to 0 °C.
Benzoyl chloride (2.37 g, 20.4 mmol) was slowly added, and the
reaction mixture was allowed to warm to rt. After 2.5 h,
saturated aqueous NaHCO3 (40 mL) was added at 0 °C, and the
mixture was stirred for an additional 10 min. The solvent was
partly evaporated before the mixture was diluted with ethyl
acetate (150 mL). The mixture was washed with water, 1 N
HCl, saturated aqueous NaHCO3, and brine (100 mL each) and
dried over MgSO4. After filtration and concentration in vacuo,
the residue was chromatographed on silica (320 g) with petro-
leum ether/ethyl acetate (7:1-2:1). Dibenzoate 5 was obtained
as a colorless oil (5.17 g, 8.27 mmol). Yield: 78% over silylation
and benzoylation. 1H NMR (â-anomer, 300 MHz, CDCl3) δ: 1.08
(s, 9H); 3.04 (m, 1H); 3.35 (s, 3H); 3.88 (m, 2H); 4.34 (quint, 1H);
4.55 (m, 2H), 5.01 (s, 1H,); 5.55 (d, 1H, J ) 4.2 Hz); 7.32-7.47
(m, 10H); 7.55 (m, 2H); 7.71 (m, 4H); 8.02 (m, 4H). 13C NMR
(â-anomer, 75 MHz, CDCl3) δ: 19.2, 26.8, 42.0, 54.8, 61.2, 66.6,
77.8, 82.0, 106.8, 127.7, 128.3, 128.37 128.43, 129.4, 129.6, 129.7,
133.0, 133.2, 133.3, 135.6, 129.4, 133.4, 166.2, 165.6. MS m/z
593 (M+ - OCH3, 28); 567 (20); 547 (2); 105 (100); 77 (30). Anal.
Calcd for C37H40SiO7, C, 71.13; H, 6.45. Found: C, 71.20; H,
6.52.
mg, 0.41 mmol) was dissolved in a mixture of MeOH (5.7 mL)
and THF (5 mL) and treated with aqueous 2 N NaOH (2 mL, 4
mmol). The reaction mixture was stirred at rt for 1 h and diluted
with acetate buffer (1.2 mL). The mixture was concentrated to
one-third of its original volume by rotary evaporation, diluted
with CH2Cl2 (40 mL), and washed with brine (30 mL) and
aqueous 50 mM Na2CO3 (20 mL). The organic layer was
separated, and the aqueous phase was reextracted four times
with CH2Cl2. The combined organic layers were dried in vacuo,
and the residue was chromatographed on silica (30 g) with
CH2Cl2/MeOH (92:8) to yield 7 (143 mg, 0.29 mmol) as a colorless
foam. Yield: 70%. 1H NMR (300 MHz, CDCl3) δ: 1.10 (s, 9H);
2.49 (m, 1H); 3.29 (s, 1H); 3.63-3.89 (m, 3H); 4.22 (d, 1H, J )
15 Hz); 4.41 (d, 1H, J ) 9 Hz); 4.53 (d, 1H, J ) 5 Hz); 5.39 (d,
1H, J ) 8 Hz); 5.51 (s, 1H); 5.80 (s, 1H); 7.36-7.51 (m, 6H);
7.77 (m, 4H); 8.19 (d, 1H); 10.71 (s, 1H). 13C NMR (75 MHz,
CDCl3) δ: 19.3, 27.1, 41.7, 58.1, 62.5, 78.4, 82.1, 92.4, 102.0,
128.0, 128.1, 130.1, 130.3, 132.2, 132.7, 135.4, 135.6, 140.2, 151.3,
164.0. FABMS m/z: 519 (M + Na+, 24); 497 (23); 439 (22); 111
(15); 77 (48); 57 (57). UV (CH2Cl2): λmax 265 (ꢀ 7200).
1-[3′-[(Acetylth io)m eth yl]-5′-(ter t-bu tyld ip h en ylsilyl)-3′-
d eoxy-â-r ibofu r a n osyl]u r a cil (8). Homouridine diol 7 (300
mg, 604 µmol) was coevaporated from toluene, dried under high
vacuum, and dissolved in dry CH3CN (3 mL) and dry THF (2
mL). PPh3 (328 mg, 1.2 mmol) was dissolved in dry CH3CN (2
mL), cooled to 0 °C, and treated with diisopropyl azodicarbox-
ylate (DIAD) (220 µL, 1.15 mmol). After 15 min, the solution of
the diol and thioacetic acid (96 mg, 90 µL, 1.22 mmol) were added
at the same time, and the reaction was allowed to warm to rt.
After 2 h, the solvents were removed by rotary evaporation. The
residue was chromatographed on silica (50 g) with petroleum
ether/THF (3:2 and 9:5) to yield thioester 8 as a colorless foam
(220 mg, 396 µmol). Yield: 65%. 1H NMR (300 MHz, CDCl3)
δ: 1.16 (s, 9H); 2.49 (m, 1H); 2.97 (m, 2H); 3.81 (dd, 1H, J ) 2,
12.5 Hz); 4.13-4.26 (m, 2H); 4.32 (d, 1H, J ) 4.5 Hz); 5.16 (s,
1H); 5.32 (d, 1H, J ) 8.1 Hz); 5.81 (s, 1H); 7.38-7.50 (m, 6H);
7.72 (m, 4H); 8.19 (d, 1H, J ) 8.1 Hz); 10.45 (s, 1H). 13C NMR
(75 MHz, CDCl3) δ: 19.3, 23.7, 27.0, 30.5, 40.5, 62.0, 77.1, 84.7,
92.4, 102.0, 128.0, 128.1, 130.1, 130.2, 132.2, 132.8, 135.4, 135.7,
140.0, 150.8, 164.0, 195.6. FABMS m/z: 577 (M + Na+, 10);
555 (25); 497 (16); 111 (8); 77 (22); 57 (27). UV (CH2Cl2): λmax
264 (ꢀ 10 200).
Com p ou n d 11. Homothymidine iodide 99 (169 mg, 279
µmol), homouridine thioester 8 (170 mg, 307 µmol), and CsCO3
(293 mg, 0.9 mmol) were dried under high vacuum at 50 °C and
then pulverized in the flask with a stirring bar. THF (5 mL)
was added, and the fine white slurry was stirred for 3 h at rt.
DMF (2 mL) was added, and the mixture was stirred for an
additional 2.5 h. Acetate buffer (5 mL) and CH2Cl2 were added,
and the mixture was washed with brine. The aqueous phase
was reextracted three times with CH2Cl2. The combined organic
phases were concentrated in vacuo. The crude product, contain-
ing 2′-OH thioether and 2′-OAc thioether, was submitted directly
to oxidation. The crude mixture of thioethers (approximately
270 µmol) was dissolved in MeOH (40 mL) and THF (6 mL),
and a freshly prepared solution of Oxone (2KHSO5‚K2SO4‚
KHSO4) (737 mg, 1.12 mmol) and NaOAc (428 mg, 5 mmol) in
water (9 mL) was added under stirring. The resulting white
slurry was stirred for 2 h at rt. Then, approximately 2/3 of the
organic solvent was evaporated, and aqueous saturated NaS2O3
and CH2Cl2 were added. The organic phases were separated,
and the aqueous phase was extracted four times with CH2Cl2.
The combined organic phases were concentrated in vacuo, and
the residue was chromatographed on silica (38 g) with CH2Cl2/
ethyl acetate/water (80:20:0.25) and a stepwise MeOH gradient
of 3-8%. Fractions containing 2′-OAc dimer 11 (108 mg, 102
µmol) were concentrated in vacuo. Fractions containing the 2′-
OH dimer 10 (84 mg, 75 µmol) were concentrated in vacuo and
submitted to acetylation. Combined yield over coupling and
oxidation: 63%. The sample of 2′OH dimer 10 (130 mg, 127
µmol) was coevaporated from pyridine, dried under high vacuum
at rt, and dissolved in dry pyridine (2 mL). Acetic anhydride
(500 µL) was added, and the reaction mixture was stirred at rt
overnight. Then MeOH (4 mL) was added, and the solvents were
removed by rotary evaporation. The residue was taken up in
CH2Cl2 and was washed twice with aqueous saturated NaHSO4.
The aqueous phases were reextracted with CH2Cl2, and the
combined organic phases were concentrated in vacuo yielding
1-[2′-Ben zoyl-3′-[(b en zoyloxy)m et h yl]-5′-(ter t-b u t yld i-
ph en ylsilyl)-3′-deoxy-â-r ibofu r an osyl)u r acil (6). Uracil (538
mg, 4.8 mmol) was dried under high vacuum at 60 °C and then
suspended in dry CH3CN (17.5 mL). N-Methyl-N-(trimethyl-
silyl)trifluoroacetamide (MSTFA) (3.5 mL, 19.2 mmol) was added
under stirring, resulting in
a clear solution after 10 min.
Glycosyl donor 5 (1.5 g, 2.4 mmol), which had been coevaporated
with toluene and dried under high vacuum at 50 °C, was
dissolved in dry CH3CN (17.5 mL) and added to the silylated
base. The resulting solution was cooled to 0 °C, and trimeth-
ylsilyl triflate (TMSTf) (0.65 mL, 3.6 mmol) was added. The
solution was warmed to 40 °C over 30 min and stirred at this
temperature. After 3 h and after an additional 4 h, TMSTf (0.4
mL/2.2 mmol, and 0.3 mL/1.7 mmol) was added. After 5 h, the
reaction was cooled to 0 °C and the solution was poured into a
mixture of saturated NaHCO3 solution (160 mL), ice (120 g),
and CH2Cl2 (250 mL). The organic layer was separated, and
the aqueous layer was reextracted twice with CH2Cl2. The
combined organic phases were washed with brine, which was
again reextracted twice with CH2Cl2. The organic layer was
dried over MgSO4, filtered, and concentrated in vacuo and the
residue chromatographed on silica (35 g) with petroleum ether/
ethyl acetate (2:1) and a stepwise MeOH gradient (1-6%).
Removal of residual N-methyltrifluoroacetamide under high
vacuum yielded homouridine 6 (1.6 g, 2.2 mmol) as a colorless
glass: yield 90%. 1H NMR (300 MHz, CDCl3) δ: 1.12 (s, 9H);
3.23 (quint, 1H); 3.86 (dd, 1H, J ) 12.1, 2.5 Hz); 4.22 (dd, 1H, J
) 11.9, 2.2 Hz); 4.34 (m, 1H); 4.42 (m, 2H), 5.42 (dd, 1H, J )
7.1, 2.3 Hz); 5.82 (dd, 1H, J ) 6.8, 3.2 Hz); 6.22 (d, 1H, J ) 3.2);
7.32-7.47 (m, 10H); 7.55 (m, 2H); 7.68 (m, 4H); 7.85 (d, 1H);
7.99 (m, 4H); 8.77 (s, 1H). 13C NMR (50 MHz, CDCl3) δ: 19.8,
27.0, 40.0, 60.6, 63.7, 77.2, 82.7, 89.0, 102.6, 127.2, 127.9, 128.0,
128.2, 128.4, 128.5, 128.7, 129.3, 129.6, 129.9, 130.1, 130.2, 132.0,
132.7, 133.2, 133.7, 135.4, 135.5, 135.6, 135.7, 139.8, 149.9, 162.8,
165.6, 166.2. FABMS m/z: 647 (M+- t-Bu, 10); 627 (4); 593 (22);
105 (100); 77 (29). UV (CH2Cl2): λmax 260 (ꢀ 11 300).
1-[5′-(t er t -Bu t yld ip h e n ylsilyl)-3′-d e oxy-3′-(h yd r oxy-
m et h yl)-â-r ib ofu r a n osyl]u r a cil (7). Homouridine 6 (288.8

Notes
J . Org. Chem., Vol. 61, No. 21, 1996 7625
quantitatively crude 2′-OAc dimer 11. This product was submit-
ted directly to desilylation. 1H NMR (300 MHz, CDCl3) δ: 1.08
(s, 9H); 1.09 (s, 9H); 1. 79 (m, 2H); 1.87 (d, 3H, J ) 1.2 Hz);
2.12 (s, 3H); 2.21 (m, 2H); 2.74-2.94 (m, 3H); 3.18-3.32 (m, 2H);
3.82-3.93 (m, 1H); 4.00-4.10 (m, 3H), 4.12-4.18 (m, 1H); 5.43
(d, 1H, J ) 8.2 Hz); 5.54 (m, 1H); 5.75 (t, 1H, J ) 0.6 Hz); 6.08
(t, 1H, J ) 7 Hz); 6.90 (d, 1H, J ) 1.1 Hz); 7.33-7.49 (m, 12H);
7.51 (d, 1H, J ) 8.2 Hz); 7.61-7.67 (m, 8H); 8.92 (s, 1H); 9.04
(s, 1H). 13C NMR (75 MHz, CDCl3) δ: 12.4, 19.1, 19.3, 20.7,
25.2, 26.88, 26.9, 35.4, 38.5, 49.0, 50.2, 63.4, 75.9, 76.5, 83.7,
84.6, 87.7, 91.0, 103.0, 111.4, 128.0, 128.1, 130.1, 130.2, 130.3,
130.35, 132.2, 132.7, 132.8, 132.9, 135.4, 135.7, 135.9, 136.8,
140.5, 150.0, 150.2, 163.6, 163.7, 169.4. FABMS m/z: 1085 (M
+ Na+, 20%); 1063 (M+, 6); 1005 (12); 937 (13); 623 (18); 603
(46); 569 (23); 511 (21); 491 (21); 461 (11); 401 (17): UV
(CH2Cl2): λmax 261 (ꢀ 17 400).
5.62 (m, 1H); 5.76, 5.80 (dd, 1H); 6.03 (dt, 1H); 6.82 (d, 4H); 7.04
(m, 1H); 7.22 (m, 1H); 7.30 (m, 6H); 7.41 (dd, 2H); 7.66 (dd, 1H).
31P-NMR (121 MHz, CDCl3) δ: 148.8 (s); 149.5 (s). FABMS
m/z: 1661 (0.49); 1190 ((M + (iPr)2N)+, 0.13); 1013 (M + Na+,
0.25); 1090 (M + H+, 0.3); 303 (100).
5′-Br om o-5′-d eoxyth ym id in e (26). (Procedure modified af-
ter ref 18). A mixture of thymidine (441.5 mg, 1.823 mmol) and
triphenylphosphine (956 mg, 3.65 mmol) was coevaporated from
pyridine (20 mL) and dried, in vacuo, at 55 °C for 2 h. The solid
was subsequently dissolved in pyridine (35 mL), and a solution
of tetrabromomethane (1.088 g; 3.28 mmol) in pyridine (5 mL)
was added. The yellow solution was stirred for 1 h (TLC: ethyl
acetate/CH2Cl2/methanol, 60:40:10, Rf thymidine 0.28, Rf product
0.53). The mixture was then evaporated to dryness, in vacuo.
The resulting yellow-brown oil was chromatographed on silica
gel (50 g), with CH2Cl2/ethyl acetate (2:3) and a stepwise MeOH
gradient (4-6%). The first product fractions contained some
triphenyl phosphinoxide that was removed by washing the solid
obtained after evaporation with CH2Cl2 (3 × 5 mL). The
combined product fractions gave 26 (368 mg; 1.21 mmol) as a
colorless foam. Yield: 66%. Mp: 157-158 °C (lit.16 mp 157-
158 °C). 1H-NMR (200 MHz, DMSO-d6) δ: 1.79 (s, 3H); 2.09,
2.28 (2 × m, 2H); 3.65 (dd, 1H, J ) 6.3, 10.2 Hz); 3.75 (dd, 1H,
J ) 5.7, 10.7 Hz); 3.93 (m, 1H); 4.23 (m, 1H,); 5.49 (br d, 1H);
6.23 (t, 1H, J ) 6.4 Hz), 7.51 (s, 1H); 11.33 (br s, 1H). 13C-NMR
(50 MHz, DMSO-d6) δ: 11.78, 33.38, 37.59, 71.57, 83.49, 84.73,
109.47, 135.62, 150.06, 163.25. UV (MeOH): λmax (nm) 266 (ꢀ
18 000); 226 (ꢀ 5 000).
5′-Br om o-5′-d e oxy-3′-(t er t -b u t yld ip h e n ylsilyl)t h ym i-
d in e (27). Bromide 26 (528.3 mg, 1.73 mmol) was dissolved in
warm MeOH and coevaporated from toluene. Imidazole (353.6
mg, 5.19 mmol) was added, and the mixture was dried, in vacuo,
at 50 °C for 1 h. Subsequently, the solid was dissolved in
tetrahydrofuran (10.5 mL), and tert-butyldiphenylsilyl chloride
(665 µL, 2.60 mmol) was slowly added, causing precipitation of
imidazolium chloride. The suspension was heated to 40 °C for
14 h (TLC: CH2Cl2/ethyl acetate/methanol, 75:25:5, Rf 26 0.22,
Rf product 0.79). MeOH (4 mL) was added, and the solution
was stirred for 10 min and then evaporated to dryness, in vacuo.
The resulting solid was taken up in CH2Cl2 (20 mL) and water
(1 mL), and the organic phase was extracted with ice-cold 0.1
M hydrochloric acid (2 × 10 mL) and saturated aqueous sodium
bicarbonate (10 mL). The aqueous phases were back-extracted
with CH2Cl2, and the combined organic phases were evaporated
to dryness. The resulting foam was purified by column chro-
matography on silica (95 g) with CH2Cl2 and a stepwise MeOH
gradient (1.5-7%). Silyl ether 27 was isolated as a white foam
(882 mg, 1.62 mmol, 94% yield). 1H-NMR (200 MHz, CDCl3) δ:
1.10 (s, 9H); 1.89 (s, 3H); 1.99 (m, 1H); 2.37 (ddd, 1H, J ) 2.4,
5.9, 13.1 Hz); 2.84 (dd, 1H, J ) 3.4, 11.3 Hz); 3.34 (dd, 1H, J )
3.5, 11.3 Hz); 4.09 (m, 1H); 4.36 (m, 1H); 6.41 (dd, 1H, J ) 5.8,
8.2 Hz); 7.33 (s, 1H); 7.43 (m, 6H); 7.66 (m, 4H); 8.84 (br s, 1H).
13C-NMR (75 MHz, CDCl3) δ: 12.80, 19.19, 27.10, 33.60, 40.86,
74.86, 84.72, 85.25, 111.67, 128.37, 128.41, 130.58, 130.68,
133.14, 133.38, 135.80, 136.14, 150.76, 164.19; FAB-MS m/z:
545, 543 (M + H+). UV (CH2Cl2) λmax 265 (ꢀ 10 700); 228 (ꢀ
7 100).
Com p ou n d 29. Cesium carbonate (29.4 mg, 0.0901 mmol,
flame dried, in vacuo, for 2 min), bromide 27 (11.3 mg, 0.0207
mmol), and thioester 8 (10.0 mg, 0.0180 mmol) were mixed and
dried, in vacuo, at 40 °C for 1 h. Subsequently, tetrahydrofuran
(0.7 mL) was added, and the suspension was stirred at rt. The
reaction was followed by TLC (methanol/hexane/CH2Cl2, 5:20:
75, Rf bromide 0.51, Rf thioester 0.22, Rf 2′-OAc-product 0.18).
After 10 h acetate buffer (45 µL; 3 M acetic acid, 1 M sodium
acetate) was added, and the reaction mixture was evaporated
to dryness, in vacuo. The resulting solid was taken up in half-
saturated brine (3 mL) and CH2Cl2 (3 mL). The aqueous phase
was extracted with CH2Cl2 (4 × 3 mL). The combined organic
phases were washed with brine (10 mL) and evaporated to
dryness, in vacuo. The residue was purified by column chro-
matography on silica (3 g) with methanol/ethanol/water/CH2Cl2
(1:1:0.2:98, stepwise gradient to 3:3:0.2: 94). Compound 29 (9.6
mg, 0.0094 mmol; 52%) was isolated as a white foam. Fractions
containing compound 28, which eluted after 29, were combined,
dried in vacuo, and acetylated. To this end, a 0.07 M solution
of 28 in pyridine was treated with 2 equiv of acetic anhydride.
The solution was stirred overnight, quenched with MeOH, and
Com p ou n d 12. 2′-OAc dimer 11 (181 mg, 170 µmol) was
dissolved in dry pyridine (0.5 mL) in a 10 mL polypropylene tube
under Ar. An HF/pyridine solution (0.7 mL, 3.29 mmol, 4.7 M
in pyridine) was added, and the resulting solution was stirred
for 3 h at rt before methoxytrimethylsilane (4 mL, 28 mmol) was
added. A white precipitate formed. Stirring continued for 30
min before the mixture was concentrated in vacuo. The residue
was chromatographed on silica (22 g) with CH2Cl2/EtOH/MeOH/
water (86:7:7:0.25 and 83:9:8:0.25) to yield diol 12 as a white
glass (79.5 mg, 135 µmol). Yield: 79%. 1H-NMR (400 MHz,
DMSO) δ: 1.87 (d, 3H, J ) 1.1 Hz); 1.98 (m, 1H); 2.04-2.25 (m,
2H); 2.08 (s, 3H); 2.24 (m, 1H); 3.04 (m, 1H); 3.28 (m, 1H); 3.42
(m, 2H); 3.61 (m, 1H); 3.73 (m, 2H); 3.97 (m, 1H); 4.13 (m, 1H);
4.33 (s, 1H); 5.15 (s, 1H); 5.36 (dd, 1H, J ) 2.6, 6.8 Hz); 5.66 (d,
1H, J ) 8.1 Hz); 5.75 (d, 1H, J ) 2.7 Hz); 6.15 (t, 1H, J ) 7 Hz);
7.42 (d, 1H, J ) 1 Hz); 7.84 (d, 1H, J ) 8 Hz); 11.31 (s, 2H).
13C-NMR (100 MHz, DMSO) δ: 11.9, 20.4, 24.8, 34.5, 38.0, 47.9,
49.9, 60.3, 72.7, 75.6, 83.2, 83.4, 83.7, 89.7, 101.9, 109.9, 136.1,
141.4, 150.0, 150.4, 163.0, 163.6, 169.1. FABMS m/z: 609 (M
+ Na+, 7); 587 (3.5); 391 (3); 369 (3); 329 (3); 307 (11); 289 (7);
176 (21); 154 (64). UV (CH2Cl2/MeOH 1:1) λmax 262 (ꢀ 16 000).
Com p ou n d 13. Dimer 12 (75.8 mg, 129 µmol) was coevapo-
rated with dry pyridine, dried under high vacuum at rt, and
dissolved in dry pyridine (2 mL). TEA (45 mL, 320 µmol), DMAP
(ca. 5 mg), and molecular sieves (6 beads, 4 A) were added, and
the solution was stirred for 15 min at rt before dimethoxytri-
phenyl chloride (DMT-Cl) (90 mg, 258 µmol) was added. The
yellow solution was stirred for 2.5 h at rt. Then, MeOH (2 mL)
was added, and the solution was stirred for an additional 30
min before the mixture was concentrated in vacuo. The residue
was chromatographed on silica (24 g) with CH2Cl2/iPrOH/MeOH/
TEA (94:3:3:0.5 and 92:4:4:0.5) to yield 5′-DMT dimer 13 as a
colorless solid (112.5 mg, 126 µmol). Yield: 97%. 1H-NMR (400
MHz, CDCl3) δ: 1.79 (d, 3H, J ) 0.9 Hz); 1.88-2.05 (m, 1H);
2.04 (s, 3H); 2.15-2.26 (m, 3H); 2.91 (m, 1H); 3.08-3.36 (m, 5H);
3.49 (m, 1H); 3.80 (m, 1H); 3.97 (m, 1H); 3.67 (2Ys, 6H); 4.13
(m, 1H); 5.42 (d, 1H, J ) 8.2 Hz); 5.52 (d, 1H, J ) 1.5 Hz); 5.62
(d, 1H, J ) 6.7 Hz); 6.02 (t, 1H, J ) 6.7 Hz); 6.74 (d, 4H); 6.99
(s, 1H); 7.11 (m, 1H); 7.20 (m, 6H); 7.31 (d, 2H); 7.49 (d, 1H, J
) 8.2 Hz); 9.63 (s, 1H); 9.80 (s, 1H). 13C-NMR (100 MHz, CDCl3)
δSPCLN 12.4, 20.7, 25.1, 36.4, 39.0, 48.9, 49.5, 55.3, 62.5, 73.9,
76.4, 82.3, 83.9, 85.9, 92.6, 102.8, 111.5, 113.4, 127.2, 128.05,
128.12, 130.12, 130.15, 135.21, 135.24, 136.3, 141.7, 144.2, 150.5,
150.6, 158.7, 163.5, 164.0, 169.8. FABMS m/z: 990 (M + H+,
4.7); 888 (1.4); 492 (11); 391 (33); 303 (100). UV (CH2Cl2): λmax
259 (ꢀ 16 900); 238 (ꢀ 18 450).
Com p ou n d 14. Dimer 13 (251 mg, 28 µmol) and diisopro-
pylammonium tetrazolide (DIPAT) (3 mg) were dried under high
vacuum at 40 °C and then dissolved in dry CH3CN (150 µL).
Bis(diisopropylamino)-(â-cyanoethoxy)phosphine (15 µL, 47 µmol)
was added at rt, and the solution was stirred for 3 h. Aqueous
saturated NaHCO3 (5 mL) and CH2Cl2 (5 mL) were added, and
the phases were separated. The organic phase was washed with
brine. The aqueous phase was reextracted twice with CH2Cl2.
The combined organic phases were dried over MgSO4, filtrated,
and concentrated in vacuo to a volume of 1 mL. The product
was precipitated twice with hexane (5 mL) from the concentrated
solution to yield phosphoramidite 14 (two diastereomers) as a
colorless solid (18.5 mg, 17 µmol). Yield: 60%. 1H-NMR
(selected signals of diastereomeric mixture, 300 MHz, CDCl3)
δ: 1.12-1.34 (m, 2H); 1.91 (m, 3H, J ) 0.9 Hz); 2.14 (2Ys, 3H);
2.38, 2.63 (dt, 4H); 3.58 (dt, 4H); 3.69 (2Ys, 6H); 5.45 (d, 1H);

7626 J . Org. Chem., Vol. 61, No. 21, 1996
Notes
evaporated to dryness, in vacuo. The compound was redissolved
in CH2Cl2 and extracted twice with saturated sodium bicarbon-
ate, and evaporated to dryness, in vacuo, affording a second crop
of 29 (90% pure according to TLC), which was combined with
the first crop obtained from the reaction. 1H-NMR (300 MHz,
CDCl3) δ: 1.08 (s, 18H); 1.82 (s, 3H); 1.93 (m, 1H); 2.09 (s, 3H);
2.32 (m, 3H), 2.48 (m, 2H); 2.69 (quintet, 1H, J ) 7 Hz); 3.73
(dd, 1H, J ) 2.2, 11.8 Hz); 4.02 (m, 2H); 4.09 (dd, 1H, J ) 2,
11.8 Hz); 4.26 (m, 1H); 5.35 (d, 1H, J ) 8.1 Hz); 5.42 (dd, 1H, J
) 2.8, 6.3 Hz); 5.91 (d, 1H, J ) 2.9 Hz); 6.28 (t, 1H, J ) 6.8 Hz);
7.11 (s, 1H); 7.41 (m, 12H); 7.63 (m, 8H); 7.77 (d, 1H, J ) 8.1
Hz); 8.99 (br s, 1H); 9.08 (br s, 1H). 13C-NMR (50 MHz, CDCl3)
δ: 12.76, 19.24, 20.87, 27.01, 29.65, 35.11, 40.29, 44.3, 63.86,
74.76, approximately 77 (under CDCl3), 84.42, 85.0, 85.95, 88.92,
102.92, 111.54, 128.36, 130.56, 132.51-133.43, 135.73-136.0,
136.08, 140.1, 150.57, 150.70, 163.65, 164.18, 169.64; FAB-MS
m/z 1017 (M + H+); 959; 905; 891; UV (CH2Cl2, qualitative) λmax
(nm) 264 (100); 227 (84).
Com p ou n d 30. Protected dimer 29 (186.4 mg; 0.183 mmol)
was dissolved in CH2Cl2, transferred to a 15 mL plastic tube,
and evaporated to dryness. The residue was dissolved in
pyridine (1.3 mL), and a 4.7 M solution of hydrogen fluoride in
pyridine (2.9 mL) was added. The solution was stirred overnight
at rt. (TLC: ethyl acetate/CH2Cl2/methanol, 60:40:10, Rf start-
ing material 0.78, Rf product 0.14). Methoxytrimethylsilane (4.8
mL, 34.77 mmol) was added to quench excess HF, and the
solution was stirred for 30 min and evaporated to dryness, in
vacuo. The crude product was used directly for the following
tritylation. An analytical sample of 30 was purified by column
chromatography on silica with CH2Cl2/ethanol/water (85:15:0.5).
1H-NMR (300 MHz, CD3OD) δ: 1.88 (s, 3H); 2.10 (s, 3H); 2.26
(m, 2H); 2.71, 2.81 (2 × m, 3H); 2.89 (dd, 2H, J ) 4.4, 6.2 Hz);
3.75 (dd, 1H, J ) 3.4, 12.5 Hz); 3.92-4.05 (m, 3H); 4.32 (m, 1H);
5.47 (dd, 1H, J ) 2, 5.7 Hz); 5.68 (d, 1H, J ) 8.1 Hz); 5.78 (d,
1H, J ) 2.2 Hz); 6.23 (t, 1H, J ) 6.9 Hz); 7.52 (s, 1H); 7.98 (d,
1H, J ) 8.1 Hz). FAB-MS m/z: 541 (M + H+).
Com p ou n d 31. Dimer 30 (39 mg crude product; approxi-
mately 0.05 mmol) was dissolved in pyridine (1 mL) and
evaporated to dryness, in vacuo. A catalytic amount of 4-(N,N-
dimethylamino)pyridine (DMAP) (ca. 0.5 mg) was added, and
the mixture was dried, in vacuo, at 40 °C for 1 h. Pyridine (1.3
mL) and molecular sieves (4 Å) were added, and the solution
was stirred for 15 min followed by addition of triethylamine (17.4
µL, 0.125 mmol) and 4,4′-dimethoxytriphenylmethyl chloride
(33.9 mg, 0.1 mmol). After full conversion of the educt (2.5 h,
TLC (MeOH/triethylamine/CH2Cl2, 10:0.5:89.5, Rf educt 0.25, Rf
product 0.55), MeOH (2 mL) was added. The solution was
evaporated to dryness, and the residue was chromatographed
on silica (10 g) with MeOH/2-propanol/triethylamine/CH2Cl2 (4:
4:0.5:91.5). Dimer 31 was obtained as a slightly yellow foam
(39.2 mg; 0.0465 mmol; approximately 93%). 1H-NMR (300
MHz, CDCl3) δ: 1.89 (s, 3H); 2.14 (s, 3H); 2.20 (m, 1H); 2.41,
2.62, 3.01 (3 × m, 4H); 2.78 (d, 2H, J ) 6.1 Hz); 3.36 (dd, 1H, J
) 3.2, 10.9 Hz); 3.66 (dd, 1H, J ) 1.9, 10.9 Hz); 3.79 (s, 6H);
4.02 (m, 2H); 4.38 (m, 1H); 5.46 (d, 1H, J ) 8.1 Hz); 5.64 (dd,
1H, J ) 1.6, 5.8 Hz); 5.75 (d, 1H, J ) 1.7 Hz); 6.16 (t, 1H, J )
6.8 Hz); 6.85 (dd, 4H, J ) 1.0, 9.0 Hz); 7.29, 7.40 (2 × m, 10H);
7.82 (d, 1H, J ) 8.2 Hz). 13C-NMR (75 MHz, CDCl3) δ: 12.58,
20.84, 28.32, 34.98, 39.63, 41.32, 55.29, 62.35, 72.81, 77.63, 83.14,
84.88, 85.21, 86.95, 90.89, 102.49, 111.19, 113.31, 127.17, 128.03,
128.14, 130.16, 135.18, 144.26, 158.69, 135.82, 141.08), 150.32,
150.45, 163.54, 163.93, 169.87. FAB-MS m/z: 843 (M + H+).
UV (CH2Cl2): λmax 261 (ꢀ 22 100); 239 (ꢀ 25 600).
aqueous phase was further extracted with CH2Cl2 (2 × 5 mL),
and the combined organic phases were washed with brine (10
mL) and dried over Na2SO4 for 30 min. After filtration, the
solution was concentrated to approximately 1 mL and cooled to
0 °C, and the product was precipitated with hexane (5 mL). After
20 min at -20 °C, the suspension was centrifuged, the solvent
was decanted off, and the residue was dried, in vacuo, overnight.
Phosphoramidite 15 was obtained as a colorless foam (43.2 mg,
0.0414 mmol). Yield: 90%. FAB-MS indicated that some
residual bis(diisopropylamino)(â-cyanoethoxy)phosphine was
present. FAB-MS m/z: 303 (bis(diisopropylamino)(â-cyano-
ethoxy)phosphine); 1043 (M + H+).
Mod ified DNA Oligom er s. Oligonucleotides were prepared
on a 1.0 µmol scale by solid-phase synthesis on a DNA synthe-
sizer using the protocol recommended by the manufacturer
(Pharmacia, Gene Assembler Plus, 1992). Phosphoramidites 14
and 15 were dried for 12 h under vacuum over P2O5 and
dissolved in CH3CN (0.1 M solutions), and molecular sieves (3Å,
ca. 10% v/v of beads) were added to the solutions. Coupling
times were 1 min for natural nucleosides, 2 min for 14 and 10
min for 15. Oligomers were synthesized “trityl-on”. At the end
of the synthesis, oligomers were deprotected and released from
the support with 25% aqueous ammonium hydroxide at 55 °C
overnight. The crude deprotection solutions were decanted from
the CPG beads, evaporated to dryness, in vacuo, rehydrated, and
purified by reversed-phase HPLC. Product-containing fractions
were combined, lyophilized, redissolved in water (100 µL), and
detritylated with acetic acid (400 µL). After 20 min, deprotection
was terminated by lyophilization, followed by redissolving in
water (200-600 µL) and washing with diethyl ether (3 × 500
µL) to remove the DMT alcohol. Following this procedure,
compound 17 was obtained >95% pure by RP-HPLC (4-18%
CH3CN in 30 min, peak at 21 min) and MS analysis. UV
(H2O): λmax 261 nm. MALDITOFMS: calcd average mass
3023.1, found m/z 3020.2 ((M - H+)-, 1508.5 (M - 2H+)2-).
Oligopyrimidine 21 was further purified by anion-exchange
chromatography and desalted. RP-HPLC, 0-25% CH3CN in 30
min, peak at 23 min. UV (H2O): λmax 260 nm. MALDI-
TOFMS: calcd average mass 2977.6, found m/z 2977.0 ((M -
H+)-, 1487.2 (M - 2H+)2-). The mixture of 18 and 19 obtained
following the above-given procedure was further purified on a
Hamilton 79425, 150 × 4.1 mm, 10 µm column, at 50 °C with a
gradient of 100% A for 5 min, 100% A to 100% B in 40 min (A,
5% CH3CN in 0.1 M NH4HCO3, pH 9.0; B, 80% CH3CN in 0.1
M NH4HCO3, pH 9.0); retention times, 19, 3.8 min; 18, 5.1 min.
Compound 18. UV (H2O): λmax 261 nm. MALDITOFMS: calcd
average mass 2978.1, found 2978.4 ((M - H+)-, 1490.2 (M -
2H+)2-). Compound 19. UV (H2O): λmax 260 nm. MALDITOF
MS: calcd average mass 2994.1, found 2994.5 ((M - H+)-, 1497.4
(M - 2H+)2-).
Ack n ow led gm en t. The authors are indebted to H.-
U. Hediger and W. Amrein for the acquisition of
MALDI-TOF mass spectra, M. Daube for contributions
to the synthesis, C. Hammer for help with the DNA
synthesizer, and U. Pieles and M. Bla¨ttler for helpful
discussions. B.H. was supported by The Danish Re-
search Council. C.R. was supported by a Kekule´ Fel-
lowship from the Stipendien Fonds des Verbandes der
Chemischen Industrie, Frankfurt, FRG.
Com p ou n d 15. A mixture of dimer 31 (39.1 mg, 0.0464
mmol) and diisopropylammonium tetrazolide23 (DIPAT) (4 mg,
0.0232 mmol) was dried, in vacuo, at 40 °C for 1 h (a small
amount of DIPAT was probably lost due to sublimation). Most
of the solid was dissolved in acetonitrile (200 µL), and bis-
(diisopropylamino)(â-cyanoethoxy)phosphine (20 µL, 0.063 mmol)
was added. The mixture was stirred at rt for 5 h (TLC: acetone/
hexane/triethylamine, 45:45:10, Rf starting material 0.08, Rf
product 0.34) and then transferred to a separatory funnel with
CH2Cl2 (5 mL) and saturated sodium bicarbonate (5 mL). The
Su p p or tin g In for m a tion Ava ila ble: Assigned NMR and
mass spectral data, selected IR spectral data, copies of NMR
spectra of 6-8, 11, 13, 26, 27, and 30, and MALDI-TOF
spectra of 17-19 and 21 (18 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.
J O960669H