Nucleotides. Part LXV. Synthesis of 2'-Deoxyribonucleoside 5'-Phosphoramidites: New Building Blocks for the Inverse (5'-3')-Oligonucleotide Approach

Article abstract of DOI:10.1002/1522-2675(20000809)83:8<2023::AID-HLCA2023>3.0.CO;2-P

The syntheses of the 3'-O-(4,4'-dimethoxytrityl)-protected 5'-phosphoramidites 25-28 and 5'-(hydrogen succinates) 29-32, which can be used as monomeric building blocks for the inverse (5'-3')-oligodeoxyribonucleotide synthesis are described (Scheme). These activated nucleosides and nucleotides were obtained by two slightly different four-step syntheses starting with the base-protected nucleosides 13-20. For the protection of the aglycon residues, the well-established 2-(4-nitrophenyl)ethyl (npe) and [2-(4-nitrophenyl)ethoxy]carbonyl (npeoc) groups were used. The assembly of the oligonucleotides required a slightly increased coupling time of 3 min in application of the common protocol (see Table 1). The use of pyridinium hydrochloride as an activator (instead of 1H-tetrazole) resulted in an extremely shorter activation time of 30 seconds. We established the efficiency of this inverse strategy by the synthesis of the oligonucleotide 3'-conjugates 33 and 34 which carry lipophilic caps derived from cholesterol and vitamin E, respectively, as well as by the formation of (3'-3')- and (5'-5')-internucleotide linkages (see Table 2).

Full text of DOI:10.1002/1522-2675(20000809)83:8<2023::AID-HLCA2023>3.0.CO;2-P

Helvetica Chimica Acta ± Vol. 83 (2000)
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Nucleotides
Part LXV1)
Synthesis of 2'-Deoxyribonucleoside 5'-Phosphoramidites: New Building Blocks
for the Inverse (5'-3')-Oligonucleotide Approach
by Thomas Wagner and Wolfgang Pfleiderer*
Fakultät für Chemie, Universität Konstanz, Postfach 5560, D-78434 Konstanz
With best personal wishes dedicated to Prof. Dr. Albert Eschenmoser on the occassion of his 75th birthday
The syntheses of the 3'-O-(4,4'-dimethoxytrityl)-protected 5'-phosphoramidites 25 ± 28 and 5'-(hydrogen
succinates) 29 ± 32, which can be used as monomeric building blocks for the inverse (5'-3')-oligodeoxyribonu-
cleotide synthesis are described (Scheme). These activated nucleosides and nucleotides were obtained by two
slightly different four-step syntheses starting with the base-protected nucleosides 13 ± 20. For the protection of
the aglycon residues, the well-established 2-(4-nitrophenyl)ethyl (npe) and [2-(4-nitrophenyl)ethoxy]carbonyl
(npeoc) groups were used. The assembly of the oligonucleotides required a slightly increased coupling time of
3 min in application of the common protocol (see Table 1). The use of pyridinium hydrochloride as an activator
(instead of 1H-tetrazole) resulted in an extremely shorter activation time of 30 seconds. We established the
efficiency of this inverse strategy by the synthesis of the oligonucleotide 3'-conjugates 33 and 34 which carry
lipophilic caps derived from cholesterol and vitamin E, respectively, as well as by the formation of (3'-3')- and
(5'-5')-internucleotide linkages (see Table 2).
1. Introduction. ± In nature, oligonucleotides are synthesized in the 5' ! 3' direction,
while the chemical build-up of these biopolymers, due to the easy accessibility of the
corresponding monomeric building blocks, are carried out in DNA synthesizers by the
opposite 3' ! 5' approach. Even though primary phosphoramidites are often used for
the synthesis of (3'-3')- and (5'-5')-internucleotide linkages [2][3], very little is known
in literature [4] about the detailed specification of the synthesis of the appropriate 5'-
phosphoramidites suitable for the built-up of oligonucleotides in the 5' ! 3' direction.
Beside the use of oligonucleotides in medicinal chemistry as potential antisense and
antigene drugs, there is increasing interest in their use in oligonucleotide-chip
technology [5 ± 10], diagnostic tools, biological applications, and nanotechnology
[11]. For these purposes, efficient and diverse oligonucleotide syntheses are essential.
Application of the npe/npeoc strategy (npe ˆ 2-(4-nitrophenyl)ethyl, npeoc ˆ [2-(4-
nitrophenyl)ethoxy]carbonyl) [12] allows also the inverse (5'! 3')-directed built-up of
oligonucleotides of high purity starting with the appropriately protected 5'-phosphor-
amidites. By this strategy, totally unprotected oligonucleotides still attached to the solid
support are available, and final cleavage with ammonia affords the oligonucleotides for
direct use without further purification.
1
)
Part LXIV: [1].

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Helvetica Chimica Acta ± Vol. 83 (2000)
In this paper, we describe the syntheses of new 5'-phosphoramidites and 5'-
(hydrogen succinates) and show their efficiency in inverse (5'-3')-oligodeoxyribonu-
cleotide syntheses [13]. Furthermore, an improved coupling step, brought about by the
use of pyridinium chloride [14] instead of the commonly applied 1H-tetrazole, is
recommended.
2- Syntheses. ± At first, the aglycone functions of the common nucleosides were
protected in the usual manner by the 2-(4-nitrophenyl)ethyl (npe) and the [2-(4-
nitrophenyl)ethoxy]carbonyl (npeoc) group ( ! 1 ± 4) [15], which have already been
shown to be very efficient for the synthesis of oligodeoxynucleotides [16 ± 24]. The
modification of the sugar moiety was then undertaken by the introduction at the 5'-
position of a protecting group compatible with the dimethoxytrityl residue. In our first
route, the primary OH function of 1 ± 4 was blocked by the bulky dimethyl(thexyl)silyl
(tds) group after treatment with [dimethyl(1,1,2-trimethylpropyl)silyl] chloride (tdsCl)
and 1H-imidazole in DMF to furnish the singly protected nucleosides 5 ± 8 in 78 ± 86%
besides 8 ± 17% of the doubly silylated derivatives 5a ± 8a, which could be seperated
easily by silica-gel column chromatography (Scheme). Subsequent dimethoxytrityla-
tion with (MeO)2TrCl in pyridine at 608 led to the fully protected derivatives 13 ± 16 in
almost quantitative yields. Due to the steric hindrance of the secondary alcohol group,
the higher temperature was necessary to obtain complete conversion in 2 ± 4 h.
Desilylation was then achieved by fluoride ion in the presence of small amounts of
AcOH to avoid b-elimination of the aglycone protecting groups. Although an extended
reaction time had to be tolerated, the 3'-O-(dimethoxytrityl)nucleosides 21 ± 24 were
obtained in high yields (87 ± 94%).
In an alternative approach, the primary-alcohol function was protected with the
base-labile (2-dansylethoxy)carbonyl (ˆ(2-{[5-(dimethylamino)naphthalen-1-yl]sulfo-
nyl}ethoxy)carbonyl; dnseoc) [25] or npeoc group to give compounds 9 ± 12.
Subsequent dimethoxytritylation of 9 at 608 and of 10 ± 12 at room temperature due
to the sensitivity of the dnseoc group led to the fully protected nucleosides 17 ± 20. The
precursors 21 ± 24 of the monomeric building blocks were then obtained in high yields
by a mild b-elimination process with 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in
MeCN. The phosphitylations to 25 ± 28 and succinoylations to 29 ± 32 were carried out
following published procedures [26].
The loading of the LCAMA-CPG support ((long-chain-alkyl)methylamine con-
trolled-pore glass [12]) with 29 ± 32 was performed in the presence of 2-{[cyano-
(ethoxycarbonyl)-methylidene]amino}-1,1,3,3-tetramethyluronium tetrafluoroborate
(TOTU) and N-methylmorpholine in MeCN followed by a capping process with
Ac2O and N,N-dimethylpyridin-4-amine (DMAP) in pyridine leading to supports
loaded with 29 ± 35 mmol/g.
Standard solid-phase phosphoramidite chemistry [27 ± 30] was applied for the
assembly of the oligonucleotides in a commercial DNA synthesizer (ABI 392),
changing only the coupling time to 3 min for the phosphoramidites 25 ± 28 to achieve
high coupling efficiencies. Since the stability and handling of the 5'-phosphoramidites
are comparatively similar to that of their 3'-isomers, no special precautions were necessary.
The following slightly modified protocol was used (washing steps not shown): 1)
Detritylation of the terminal dimethoxytrityl group with 3% CCl3COOH in CH2Cl2 for

Helvetica Chimica Acta ± Vol. 83 (2000)
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Scheme

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Helvetica Chimica Acta ± Vol. 83 (2000)
135 s; 2) coupling with 0.1m phosphoramidite 25 ± 28 and 0.5m 1H-tetrazole in MeCN
for 180 s; 3) capping with Ac2O/2,6-dimethylpyridine/1-methyl-1H-imidazole in THF
for 15 s; 4) Oxidation with 0.05m I2 in THF/pyridine/H2O for 32 s. After the synthesis,
the cyanoethyl, npe, and npeoc groups were removed with 1m DBU and washed away,
and then the deprotected oligonucleotide was detached from the solid support by
common ammonia treatment. The oligonucleotides A ± J obtained were checked by
HPLC (Fig. 1) and demonstrated high purity so that they did not require further
purification (Table 1).
In an analogous manner, we also synthesized the oligomers K ±M (see Table 2) in the
presence of the coupling catalyst pyridinium chloride instead of 1H-tetrazole and found
the same high quality of the oligonucleotide with a reduced coupling time of 30 s (Fig. 1).
Fig. 1. HPLCTraces of the crude oligo-2 '-deoxyribonucleotides D, H, and M. AU ˆ arbitrary unit.

Helvetica Chimica Acta ± Vol. 83 (2000)
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Table 1. Oligo-2'-deoxyribonucleotide Syntheses by the Inverse 5' ! 3' Approach
Phosphor-
amidite
Oligonucleotide sequence
Coupling Average
time [s] condensation yield [%] [mmol] OD units quality
Overall Size
Yield in HPLC
yield [%]
25
25
26
26
27
27
5'-TTT-TT-3'
5'-TTT-TTT-T-3'
5'-d(CCC-CCC)-3'
5'-d(CCC-CCC-CCC)-3'
5'-d(AAA-AAA)-3'
5'-d(AAA-AAA-AAA)-3' (F) 180
5'-d(CAC-AGC-GGC-
CGC)-3'
(A) 40
96
88
89
0.24
0.16
0.21
0.17
0.17
0.16
0.18
8.0
5.8
12
6.9
6.2
10.4
11.2
1 ± 2
(B) 120
(C) 120
(D) 180
(E) 180
99.9
95.6
100
95.4
100
100
1
2
1
1
1
1
86.7
100
84
100
86
26, 27, 28
(G) 180
25, 26, 27, 28 5'-d(TGA-GCA-GA)-3'
(H) 180
(I) 180
99
98.6
93
83.7
0.23
0.19
11.1
9.9
1
1
25, 28
5'-d(TTG-TGT-GTG-
TGT-G)-3'
25, 26
5'd(TCC-CCC-CCC-CC)-3' (J) 180
98.5
95.1
0.22
18
1 ± 2
Furthermore, the monomeric building blocks 25 ± 32 could be used for the
introduction of so-called (3'-3')- and/or (5'-5')-end terminal caps (Tab. 2). Loading of
the LCAMA-CPG support in the usual manner with one of the 5'-succinates 29 ± 32 and
subsequent elongation of the oligonucleotides with the common 3'-phosphoramidites
led to (3'-3')-phosphodiester bonds. The opposite (5'-5')-phosphodiester linkages were
achieved by synthesizing the oligonucleotide in the common 3' ! 5' direction with
subsequent condensation of one of the 5'-phosphoramidites 25 ± 28. The combination of
both approaches led to oligonucleotides carrying a (3'-3')- as well as a (5'-5')-terminal
cap structure (see Table 2).
Table 2. Modified Oligo-2'-deoxyribonucleotide Sequences
Sequence
Sequence
5'-d(TCC-CCC-CC)-3' (K)
5'-d(AAA-AA)-3' (L)
T-5'-5'-d(CAC-CGA-CGG-CGC)
dC-5'-5'-d(CAC-CGA-CGG-CGC)
5'-d(TTG-TGT-GT)-3' (M)
dC-5'-5'-d(CAC-CGA-CGG-CGC)
dC-5'-5'-d(CAC-CGA-CGG-CGC)
5'-d(CAC-CGA-CGG-CGC)-3'-3'-T
5'-d(CAC-CGA-CGG-CGC)-3'-3'-dC
5'-d(CAC-CGA-CGG-CGC)-3'-3'-dA
5'-d(CAC-CGA-CGG-CGC)-3'-3'-dG
T-5'-5'-d(CAC-CGA-CGG-CGC)-3'-3'-T
dC-5'-5'-d(CAC-CGA-CGG-CGC)-3'-3'-dC
dA-5'-5'-d(CAC-CGA-CGG-CGC)-3'-3'-dA
dG-5'-5'-d(CAC-CGA-CGG-CGC)-3'-3'-dG
The described inverse strategy allowed also for easy formation of 3'-O-conjugates,
as demonstrated by the reaction of cholesteryl [31] or a-tocopheryl 2-cyanoethyl
diisopropylphosphoramidite [32], in the last step by prolonged condensation time and
the use of pyridinium chloride as activator to form the modified oligonucleotides.
Deprotection and cleavage from the solid support in the usual manner led to the 3'-O-
conjugates 33 and 34 with a lipophilic cap at the 3'-terminal end in 38 and 55% yield,
respectively. The HPLC traces of the crude oligonucleotide 3'-O-conjugates (Fig. 2)
indicated that the attachment of cholesterol and vitamin E is not complete due to steric
effects, but purification of the conjugates could be easily achieved since the retention
times of the unmodified oligonucleotides and their functionalized derivatives are quite
different.

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Fig. 2. HPLCTraces of the crude oligonucleotide 3 '-O-conjugates 33 and 34. AU ˆ arbitrary unit.
Experimental Part
General. Products were dried under high vacuum. Oligonucleotide syntheses: Applied-Biosystems-392
DNA/RNA synthesizer. TLC: precoated silica-gel thin-layer sheets F1500 LS 254 from Schleicher & Schuell.
Flash chromatography (FC): silica gel (Baker, 30 ± 60 mm); 0.2 ± 0.3 bar. M.p.: Gallenkamp melting-point
apparatus; no corrections. UV/VIS: Perkin-Elmer Lambda 15; lmax in nm (log e). 1H-NMR: Bruker AC250 ; d
in ppm rel. to SiMe4 or CDCl3 ((D6)DMSO) as internal standard. 31P-NMR: Joel 400 MHz; d in ppm rel. to
H3PO4.

Helvetica Chimica Acta ± Vol. 83 (2000)
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1. 5'-O-[Dimethyl(1,1,2-trimethylpropyl)silyl]thymidine (5) and 3',5'-Bis-O-[dimethyl(1,1,2-trimethylpro-
pyl)silyl]thymidine (5a). Thymidine (1; 2.42 g, 10 mmol) was co-evaporated with dry pyridine (2 Â 10 ml) and
then taken up in abs. DMF (60 ml). Then, 1H-imidazole (2 g, 30 mmol) and dimethyl(1,1,2-trimethylpropyl)silyl
chloride (ˆdimethyl(thexyl)silyl chloride; 3 ml, 15 mmol) were added and stirred for 2 h at r.t. After dilution
with AcOEt (200 ml), the soln. was extracted with H2O (3 Â 150 ml), the org. layer dried (Na2SO4) and
evaporated, and the residue purified by FC (16 Â 3.5 cm, toluene/AcOEt/MeOH 4 :1:0 (0.5 l) and 10 :10 :1
(0.5 l)): 3.4 g (86%) of 5 and 430 mg (8%) of 5a.
Data of 5: M.p. 178 ± 1808. UV (MeOH): 266 (4.04). 1H-NMR ((D6)DMSO): 11.3 (br. s, NH); 7.4
(s, HÀC(6)); 6.12 (t, HÀC(1')); 5.25 (d, OHÀC(3')); 4.17 (m, HÀC(3')); 3.77 ± 3.69 (m, HÀC(4'), 2 HÀC(5'));
2.07 ± 2.02 (m, 2 HÀC(2')); 1.76 (s, MeÀC(5)); 1.65 ± 1.55 (m, Me2CH); 0.85 ± 0.81 (m, 2 Me2C); 0.094
(s, Me2Si). Anal. calc. for C18H32N2O5Si (384.6): C 56.32, H 8.39, N 7.28; found: C 56.32, H 8.33, N 7.38.
Data of 5a: M.p. 115 ± 1188. UV (MeOH): 266 (4.06). 1H-NMR (CDCl3): 8.42 (br. s, NH); 7.42
(s, HÀC(6)); 6.34 ± 6.28 (t, HÀC(1')); 4.41 ± 4.39 (m, HÀC(3')); 3.94 ± 3.72 (m, HÀC(4'), 2 HÀC(5')); 2.23 ±
2.18 (m, 1 HÀC(2')); 2.05 ± 1.90 (m, 1 HÀC(2'), MeÀC(5)); 1.68 ± 1.54 (m, 2 Me2CH); 0.91 ± 0.82 (m, 4 Me2C);
0.15 ± 0.07 (s, 2 Me2Si). Anal. calc. for C26H50N2O5Si2 (526.8): C 59.27, H 9.57, N 5.32; found: C 58.93, H 9.47,
N 5.29.
2. 2'-Deoxy-5'-O-[dimethyl(1,1,2-trimethylpropyl)silyl]-N4-{[2-(4-nitrophenyl)ethoxy]carbonyl}cytidine (6)
and 2'-Deoxy-3',5'-bis-O-[dimethyl(1,1,2-trimethylpropyl)silyl]-N4-{[2-(4-nitrophenyl)ethoxy]carbonyl}cytidine
(6a). As described in Exper. 1, with 2 (1.26 g, 3 mmol), dry DMF (30 ml), 1H-imidazole (600 mg, 9 mmol),
and dimethyl(thexyl)silyl chloride (1.2 ml, 6 mmol; 2 h, r.t.). Purification by FC (12 Â 3.5 cm, toluene/AcOEt
1:1 (0.5 l) and 1:10 (0.5 l)) gave 1.19 g (71%) of 6 and 320 mg (15%) of 6a.
1
Data of 6: Crystals. M.p. 1488. UV (MeOH): 211 (4.49), 241 (4.27), 280 (4.20). H-NMR ((D6)DMSO):
10.8 (br. s, NH); 8.18 ± 8.13 (m, HÀC(6), 2 H o to NO2); 7.59 (d, 2 H m to NO2); 6.97 (d, HÀC(5)); 6.06
(t, HÀC(1')); 5.29 (d, OHÀC(3')); 4.34 (t, CH2CH2OCO); 4.20 ± 4.12 (m,HÀC(3')); 3.90 ± 3.73 (m, HÀC(4'),
2 HÀC(5')); 3.06 (t, CH2CH2OCO); 2.34 ± 2.24 (m, 1 HÀC(2')); 2.07 ± 1.92 (m, 1 HÀC(2')); 1.58 ± 1.53
(m, Me2CH); 0.83 ± 0.78 (m, 2 Me2C); 0.09 (s, Me2Si). Anal. calc. for C26H38N4O8Si (562.7): C 55.50, H 6.81,
N 9.96; found: C 55.55, H 6.87, N 9.95.
Data of 6a: Oil. UV (MeOH): 211 (4.47), 240 (4.27), 280 (4.18). 1H-NMR ((D6)DMSO): 10.81 (br. s, NH);
8.16 ± 8.10 (m, HÀC(6), 2 H o to NO2); 7.58 (d, 2 H m to NO2); 6.98 (d, HÀC(5)); 6.06 (t, HÀC(1')); 4.37 ± 4.32
(m, CH2CH2OCO, HÀC(3')); 3.86 ± 3.66 (m, HÀC(4'), 2 HÀC(5')); 3.06 (t, CH2CH2OCO); 2.30 ± 2.22
(m, 1 HÀC(2')); 2.14 ± 2.07 (m, 1 HÀC(2')); 1.62 ± 1.51 (m, 2 Me2CH); 0.85 ± 0.77 (m, 4 Me2C); 0.11 ± 0.01
(m, 2 Me2Si). Anal. calc. for C34H56N4O8Si2 (705.0): C 57.92, H 8.00, N 7.95; found: C 57.59, H 8.11, N 7.56.
3. 2'-Deoxy-5'-O-[dimethyl(1,1,2-trimethylpropyl)silyl]-Nt-{[2-(4-nitrophenyl)ethoxy]carbonyl}adenosine
(7) and 2'-Deoxy-3',5'-bis-O-[dimethyl(1,1,2-trimethylpropyl)silyl]-N6-{[2-(4-nitrophenyl)ethoxy]carbonyl}ade-
nosine (7a). As described in Exper. 1, with 3 (1.33 g, 3 mmol), dry DMF (30 ml), 1H-imidazole (0.6 g, 9 mmol),
and dimethyl(thexyl)silyl chloride (1.2 ml, 6 mmol; 2 h, r.t.). Purification by FC (12 Â 3.5 cm, toluene/AcOEt
1:1 (0.5 l) and 1:10 (0.5 l)) gave 1.36 g (77%) of 7 and 380 mg (17%) of 7a.
Data of 7: M.p. 110 ± 1148. UV (MeOH): 206 (4.58), 266 (4.46). 1H-NMR ((D6)DMSO): 10.57 (br. s, NH);
8.60, 8.54 (2s, HÀC(2), HÀC(8)); 8.14 (d, 2 H o to NO2); 7.59 (d, 2 H m to NO2); 6.41 (t, HÀC(1')); 5.38
(d, OHÀC(3')); 4.40 ± 4.35 (m, HÀC(3'), CH2CH2OCO); 3.88 ± 3.65 (m, HÀC(4'), 2 HÀC(5')); 3.09
(t, CH2CH2OCO); 2.82 ± 2.76 (m, 1 HÀC(2')); 2.38 ± 2.32 (m, 1 HÀC(2')); 1.55 ± 1.49 (m, Me2CH); 0.81 ± 0.76
(m, 2 Me2C); 0.02 (s, Me2Si). Anal. calc. for C27H38N6O7Si (586.7): C 55.27, H 6.52, N 14.32; found: C 55.28,
H 6.60, N 14.21.
Data of 7a: M.p. 141 ± 1428. UV (MeOH): 208 (4.56), 266 (4.47). 1H-NMR (CDCl3): 8.72 (s, HÀC(2));
8.26 ± 8.13 (m, HÀC(8), NH, 2 H o to NO2); 7.45 ± 7.41 (m, 2 H m to NO2); 6.45 (t, HÀC(1')); 4.58 ± 4.49
(m, HÀC(3'), CH2CH2OCO); 4.02 ± 3.98 (m, HÀC(4')); 3.86 ± 3.69 (m, 2 HÀC(5')); 3.13 (t, CH2CH2OCO);
2.64 ± 2.58 (m, 1 HÀC(2')); 2.46 ± 2.34 (m, 1 HÀC(2')); 1.64 ± 1.57 (m, 2 Me2CH); 0.89 ± 0.83 (m, 4 Me2C);
0.11 ± 0.09 (m, 2 Me2Si). Anal. calc. for C35H56N6O7Si2 (729.1): C 57.66, H 7.74, N 11.53; found: C 57.28, H 7.67,
N 11.27.
4. 2'-Deoxy-5'-O-[dimethyl(1,1,2-trimethylpropyl)silyl]-N2-{[2-(4-nitrophenyl)ethoxy]carbonyl}-O6-[2-(4-
nitrophenyl)ethyl]guanosine (8) and 2'-Deoxy-3',5'-bis-O-[dimethyl(1,1,2-trimethylpropyl)silyl]-N2-{[2-(4-nitro-
phenyl)ethoxy]carbonyl}-O6-[2-(4-nitrophenyl)ethyl]guanosine (8a). As described in Exper. 1, with 4 (1.22 g,
2 mmol), dry DMF (20 ml), 1H-imidazole (0.4 g, 6 mmol), and dimethyl(thexyl)silyl chloride (0.8 ml, 4 mmol;
2 h, r.t.). Purification by FC (10 Â 3.5 cm, toluene/AcOEt 1:1 (0.5 l) and 1:10 (0.5 l)) gave 1.14 g (75%) of 8 and
200 mg (11%) of 8a.

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Helvetica Chimica Acta ± Vol. 83 (2000)
Data of 8: M.p. 184 ± 1858. UV (MeOH): 214 (4.66), 268 (4.56). 1H-NMR ((D6)DMSO): 10.3 (br. s, NH);
8.30 (1s, HÀC(8)); 8.17 ± 8.14 (m, 4 H o to NO2); 7.64 ± 7.59 (m, 4 H m to NO2); 6.37 (t, HÀC(1')); 5.31
(d, OHÀC(3')); 4.74 (t, CH2CH2OCO); 4.38 ± 4.33 (m, HÀC(3'), CH2CH2O of O6-npe); 3.85 ± 3.63
(m, HÀC(4'), 2 HÀC(5')); 3.31 (t, CH2CH2OCO); 3.09 (t, CH2CH2O of O6-npe); 2.86 ± 2.75 (m, 1 HÀC(2'));
2.27 ± 2.23 (m, 1 HÀC(2')); 1.53 ± 1.45 (m, Me2CH); 0.79 ± 0.74 (m, 2 Me2C); 0.04 (s, Me2Si). Anal. calc. for
C35H45N7O10Si (751.9): C 55.91, H 6.03, N 13.06; found: C 55.90, H 6.12, N 12.86.
Data of 8a: UV (MeOH): 214 (4.68), 268 (4.57). 1H-NMR (CDCl3): 8.18 ± 8.06 (m, HÀC(8), 4 H o to
NO2); 7.49/7.40 (m, 4 H m to NO2); 7.30 (br. s, NH); 6.33 (t, HÀC(1')); 4.79 (t, CH2CH2OCO); 4.58 ± 4.45
(m, HÀC(1'), CH2O of O6-npe); 3.96 ± 3.94 (m, HÀC(4')); 3.82 ± 3.68 (m, 2 HÀC(5')); 3.29 (t, CH2CH2OCO);
3.10 (t, CH2CH2O of O6-npe); 2.64 ± 2.57 (m, 1 HÀC(2')); 2.39 ± 2.30 (m, 1 HÀC(2')); 1.64 ± 1.54 (m, 2 Me2CH);
0.89 ± 0.83 (m, 4 Me2C), 0.11 ± 0.06 (m, 2 Me2Si). Anal. calc. for C48H63N7O10Si2 (894.2): C 57.76, H 7.10, N 10.97;
found: C 57.65, H 7.14, N 10.75.
5. 3'-O-(4,4'-Dimethoxytrityl)-5'-O-[dimethyl(1,1,2-trimethylpropyl)silyl]thymidine (13). In dry pyridine
(10 ml), 5 (812 mg, 2.1 mmol) was co-evaporated, taken up in dry pyridine (20 ml), and treated with
(MeO)2TrCl (1.43 g, 4.2 mmol). After stirring for 2 h at 608, the soln. was diluted with CH2Cl2 (60 ml) and
extracted with sat. NaHCO3 soln. (3 Â 20 ml). The aq. phase was extracted with CH2Cl2 (20 ml), the combined
org. layer dried (Na2SO4), evaporated, and co-evaporated with toluene (2 Â 20 ml), and the residue purified by
FC (15 Â 2.5 cm, toluene/AcOEt 10 :1 (0.4 l and 4 :1 (0.5 l)): 1.35 g (94%) of 13. Slightly yellowish foam. UV
1
(MeOH): 234 (4.38), 275 (4.27). H-NMR (CDCl3): 8.37 (s, NH); 7.50 ± 7.13 (m, HÀC(6), 9 H of (MeO)2Tr);
6.87 (d, 4 H o to MeO); 6.45 ± 6.38 (m,HÀC(1')); 4.34 ± 4.30 (m, HÀC(3')); 4.08 ± 4.06 (m, HÀC(4')); 3.84
(s, 2 MeO); 3.71, 3.69 (dd, 1 HÀC(5')); 3.41, 3.39 (dd, 1 HÀC(5')); 1.91 (s, MeÀC(5)); 1.73 ± 1.53
(m, 2 HÀC(2'), Me2CH); 0.84 ± 0.80 (m, 2 Me2C); 0.04 ± 0.01 (m, Me2Si). Anal. calc. for C39H51N2O7Si
(687.9): C 68.09, H 7.47, N 4.07; found: C 68.32, H 7.43, N 4.33.
6. 2'-Deoxy-3'-O-(4,4'-dimethoxytrityl)-5'-O-[dimethyl(1,1,2-trimethylpropyl)silyl]-N4{[2-(4-nitrophenyl)-
ethoxy]carbonyl}cytidine (14). As described in Exper. 5, with 6 (670 mg, 1.21 mmol), dry pyridine (20 ml),
and (MeO)2TrCl (820 mg, 2.42 mmol; 2 h, 608). Purification by FC (15 Â 2.5 cm, toluene/AcOEt 1:1 (0.7 l) and
1:4 (0.2 l)) gave 950 mg (95%) of 14. Slightly yellowish foam. UV (MeOH): 203 (4.96), 236 (4.59), 275 (4.27),
282 (sh, 4.27). 1H-NMR (CDCl3): 8.17 ± 8.08 (m, 2 H o to NO2, HÀC(6)); 7.72 (br. s, NH); 7.72 ± 7.16
(m, HÀC(5), 9 H of (MeO)2Tr, 2 H m to NO2); 7.05 (d, HÀC(5)); 6.79 (d, 4 H o to MeO); 6.43 ± 6.35
(m, HÀC(1')); 4.39 (t, CH2CH2OCO); 4.23 ± 4.20 (m, HÀC(3')); 3.93 ± 3.90 (m, HÀC(4')); 3.76 (s, 2 MeO);
3.58, 3.54 (dd, 1 HÀC(5')); 3.22, 3.18 (dd, 1 HÀC(5')); 3.08 (t, CH2CH2OCO); 2.29 ± 2.17 (m, 1 HÀC(2'));
1.68 ± 1.42 (m, 1 HÀC(2'), Me2CH); 0.85 ± 0.70 (m, 2 Me2C); 0.02 (d, Me2Si). Anal. calc. for C47H54N4O10Si
(863.1): C 65.40, H 6.31, N 6.49; found: C 65.68, H 6.62, N 6.64.
7. 2'-Deoxy-3'-O-(4,4'-dimethoxytrityl)-5'-O-[dimethyl(1,1,2-trimethylpropyl)silyl]-N6-{[2-(4-nitrophenyl)-
ethoxy]carbonyl}adenosine (15). As described in Exper. 5, with 7 (1.04 g, 1.76 mmol), dry pyridine (20 ml),
and (MeO)2TrCl (1.18 g, 3.52 mmol; 2 h, 608). Purification by FC (silica gel, 15 Â 2.5 cm, toluene/AcOEt 10 :1
(0.5 l) and 1 :4 (0.2 l)) gave 5 g (96%) of 15. Slightly yellowish foam. UV (MeOH): 203 (4.98), 236 (4.46), 267
(4.50). 1H-NMR (CDCl3): 8.72 (s, NH); 8.33 (s, HÀC(2)); 8.16 ± 8.13 (m, 2 H o to NO2, HÀC(8)); 7.47 ± 7.16
(m, 9 H of (MeO)2Tr, 2 H m to NO2); 7.05 (d, HÀC(5)); 6.83, 6.80 (dd, 4 H o to MeO); 6.56 ± 6.50
(m, HÀC(1')); 4.50 (t, CH2CH2OCO); 4.40 ± 4.38 (m, HÀC(3')); 4.09 ± 4.07 (m, HÀC(4')); 3.76 (s, 2 MeO);
3.61, 3.60 (dd, 1 HÀC(5')); 3.39, 3.36 (dd, 1 HÀC(5')); 3.11 (t, CH2CH2OCO); 2.12 ± 1.93 (m, 2HÀC(2')); 1.58 ±
1.42 (m, Me2CH); 0.76 ± 0.72 (m, 2 Me2C); 0.01 (d, Me2Si). Anal. calc. for C48H56N6O9Si (889.1): C 64.84,
H 6.35, N 9.45; found: C 64.73, H 6.33, N 9.25.
8. 2'-Deoxy-3'-O-(4,4'-dimethoxytrityl)-5'-O-[dimethyl(1,1,2-trimethylpropyl)silyl]-N2-{[2-(4-nitrophenyl)-
ethoxy]carbonyl}-O6-[2-(4-nitrophenyl)ethyl]guanosine (16). As described in Exper. 5, with
8 (570 mg,
0.76 mmol), dry pyridine (10 ml), and (MeO)2TrCl (500 mg, 1.44 mmol; 2 h, 608). Purification by FC (11 Â
2.5 cm, toluene/AcOEt 20 :1 (0.5 l), 10 :1 (0.4 l), and 1:4 (0.2 l)) gave 730 mg (95%) of 16. Slightly yellowish
1
foam. UV (MeOH): 203 (5.02), 237 (4.54), 268 (4.60). H-NMR (CDCl3): 8.21 ± 8.12 (m, 4 H o to NO2); 8.00
(s, HÀC(8)); 7.51 ± 7.13 (m, 9 H of (MeO)2Tr, 4 H m to NO2); 6.85 ± 6.82 (m, 4 H o to MeO); 6.44 ± 6.39
(m, HÀC(1')); 4.80 (t, CH2CH2OCO); 4.46 (t, CH2CH2O of O6-npe); 4.43 ± 4.39 (m, HÀC(3')); 4.09 ± 4.07
(m, HÀC(4')); 3.79 (s, 2 MeO); 3.65, 3.61 (dd, 1 HÀC(5')); 3.39 ± 3.32 (m, 1 HÀC(5'), CH2CH2OCO); 3.14
(t, CH2CH2O of O6-npe); 2.07 ± 2.00 (m, 1 HÀC(2')); 1.98 ± 1.83 (m, 1 HÀC(2')); 1.56 ± 1.48 (m, Me2CH); 0.78 ±
0.74 (m, 2 Me2C); 0.09 ± 0.00 (m, Me2Si). Anal. calc. for C56H63N7O12Si (1054.1): C 63.80, H 6.02, N 9.30; found:
C 63.91, H 6.08, N 9.06.
9. 3'-O-(4,4'-Dimethoxytrityl)-5'-O-{[2-(4-nitrophenyl)ethoxy]carbonyl}thymidine (17). As described in
Exper. 5, with 9 (400 mg, 0.9 mmol), dry pyridine/CH2Cl2 1:1 (10 ml), and (MeO)2TrCl (610 mg, 1.8 mmol; 2 h,

Helvetica Chimica Acta ± Vol. 83 (2000)
2031
608). Purification by FC (14 Â 2 cm, toluene/AcOEt/MeOH 10 :1:0 (0.4 l) and 5 :5 :1 (0.2 l)) gave 640 mg
(94%) of 7. Colorless foam. UV (MeOH): 203 (4.85), 236 (4.40), 266 (4.33), 282 (sh, 4.14). 1H-NMR (CDCl3):
8.18 (d, 2 H o to NO2); 7.46 ± 7.17 (m, 2 H m to NO2, 9 H of (MeO)2Tr, HÀC(6)); 6.84 (d, 4 H o to MeO); 6.38 ±
6.37 (m, HÀC(1')); 4.39 ± 4.31 (m, CH2CH2OCO); 4.25 ± 4.21 (m, HÀC(3')); 4.02 ± 3.84 (m, HÀC(4'),
1 HÀC(5')); 3.80 (s, 2 MeO); 3.73 ± 3.68 (m, 1 HÀC(5')); 3.06 (t, CH2CH2OCO); 2.12 ± 1.98 (m, 1 HÀC(2'));
1.82 (s, MeÀC(5)); 1.78 ± 1.64 (m, 1 HÀC(2')). Anal. calc. for C40H39N3O11 (737.8): C 65.12, H 5.33, N 5.69;
found: C 65.35, H 5.42, N 5.64.
10. 2'-Deoxy-3'-O-(4,4'-dimethoxytrityl)-5'-O-{[5-(dimethylamino)naphthalen-1-yl]sulfonyl}-N4-{[2-(4-ni-
trophenyl)ethoxy]carbonyl}cytidine (18). As described in Exper. 5, with 10 (1.01 g, 1.39 mmol), dry pyridine/
CH2Cl2 1:1 (10 ml), and (MeO)2TrCl (940 mg, 2.79 mmol; 32 h, r.t.). Purification by FC (silica gel, 14 Â 2.5 cm,
toluene/AcOEt 1:1 (0.5 l) and 1:4 (0.4 l)) gave 1.33 g (93%) of 18. Yellow foam. UV (MeOH): 204 (5.05), 235
1
(4.57), 278 (sh, 4.56), 346 (sh, 3.58). H-NMR ((D6)DMSO): 10.8 (br. s, NH); 8.45 (d, HÀC(2)(dns)); 8.19 ±
8.12 (m, 2 H o to NO2, HÀC(4)(dns), HÀC(8)(dns)); 7.72 (d, HÀC(6)); 7.62 ± 7.56 (m, 2 H m to NO2,
HÀC(3)(dns), HÀC(7)(dns)); 7.41 ± 7.12 (m, 9 H of (MeO)2Tr, HÀC(6)(dns)); 6.91 ± 6.86 (m, 4 H o to MeO,
HÀC(5)); 6.12 (t, HÀC(1')); 4.33 ± 4.31 (m, 2 CH2CH2OCO); 4.12 ± 4.10 (m, HÀC(3')); 3.85 ± 3.65
(m, HÀC(4'), 2 HÀC(5'), 2 MeO, SO2CH2); 3.06 (t, CH2CH2OCO); 2.77 (s, Me2N); 1.98 ± 1.90
(m, 1 HÀC(2')); 1.64 ± 1.56 (m, 1 HÀC(2')). Anal. calc. for C54H53N5O14S (1028.1): C 63.09, H 5.20, N 6.81;
found: C 63.01, H 5.30, N 6.89.
11. 2'-Deoxy-5'-O-{[5-(dimethylamino)naphthalen-1-yl]sulfonyl}-3'-O-(4,4'-dimethoxytrityl)-N6-{[2-(4-ni-
trophenyl)ethoxy]carbonyl}adenosine (19). As described in Exper. 5, with 11 (680 mg, 0.91 mmol), dry
pyridine/CH2Cl2 1:1 (10 ml); and (MeO)2TrCl (610 mg, 1.82 mmol; 32 h, r.t.). Purification by FC (14 Â 2.5 cm,
toluene/AcOEt 4 :1 (0.5 l), 1:1 (0.4 l), and 1:4 (0.4 l)) gave 900 mg (94%) of 19. Yellow foam. UV (MeOH):
204 (5.08), 238 (4.57), 264 (4.60), 346 (sh, 3.59). 1H-NMR ((D6)DMSO): 10.6 (br. s, NH); 8.57 ± 8.43
(m, HÀC(2), HÀC(8), HÀC(2)(dns)); 8.19 ± 8.11 (m, 2 H o to NO2, HÀC(4)(dns), HÀC(8)(dns)); 7.63 ± 7.10
(m, 2 H m to NO2, HÀC(3)(dns), HÀC(7)(dns), 9 H of (MeO)2Tr, HÀC(6)(dns)); 6.89 (d, 4 H o to MeO);
6.44 (t, HÀC(1')); 4.39 ± 4.22 (m, 2 CH2CH2OCO, HÀC(3')); 3.81 ± 3.71 (m, HÀC(4'), 2 HÀC(5'), 2 MeO,
SO2CH2); 3.09 (t, CH2CH2OCO); 2.74 (s, Me2N); 2.58 ± 2.47 (m, 1 HÀC(2')); 2.05 ± 1.97 (m, 1 HÀC(2')). Anal.
calc. for C55H53N7O13S (1052.1): C 62.77, H 5.08, N 9.32; found: C 62.68, H 5.17, N 9.20.
12. 2'-Deoxy-5'-O-{[5-(dimethylamino)naphthalen-1-yl]sulfonyl}-3'-O-(4,4'-dimethoxytrityl)-N2-{[2-(4-ni-
trophenyl)ethoxy]carbonyl}-O6-[2-(4-nitrophenyl)ethyl]guanosine (20). As described in Exper. 5, with 12
(880 mg, 0.96 mmol), dry pyridine/CH2Cl2 1:1 (10 ml), and (MeO)2TrCl (650 mg, 1.92 mmol; 32 h, r.t.).
Purification by FC (14 Â 2.5 cm, toluene/AcOEt 4 :1 (0.4 l) and 1:1 (0.3 l)) gave 1.03 g (87%) of 20. Yellow
foam. UV (MeOH): 203 (5.12), 238 (4.63), 264 (4.68), 346 (sh, 3.59). 1H-NMR ((D6)DMSO): 10.31 (br. s, NH);
8.43 (d, HÀC(2)(dns)); 8.19 ± 8.10 (m, 4 H o to NO2, HÀC(4)(dns), HÀC(8)(dns), HÀC(8)); 7.63 ± 7.53
(m, 4 H m to NO2, HÀC(3)(dns), HÀC(7)(dns)); 7.44 ± 7.14 (m, 9 H of (MeO)2Tr, HÀC(6)(dns)); 6.87 (d, 4 H
o to MeO); 6.31 (t, HÀC(1')); 4.73 (t, CH2CH2OCO); 4.31 ± 4.17 (m, CH2CH2OCO, CH2CH2O of O6-npe,
HÀC(3')); 3.87 ± 3.70 (m, HÀC(4'), 2 HÀC(5'), 2 MeO, SO2CH2); 3.27 (t, CH2CH2OCO); 3.06 (t, CH2CH2O of
O6-npe); 2.73 (s, 2 Me2N); 2.53 ± 2.48 (m, 1 HÀC(2')); 1.93 ± 1.87 (m, 1 HÀC(2')). Anal. calc. for C63H61N8O16S
(1217.3): C 62.16, H 4.97, N 9.20; found: C 62.30, H 5.09, N 8.68.
13. 3'-O-(4,4'-Dimethoxytrityl)thymidine (21). 13.1. To a soln. of (Bu4N)F ´ H2O in THF (100 ml), 13 (10.2 g,
14.80 mmol) was added. After stirring overnight at r.t., the soln. was diluted with AcOEt (250 ml) and washed
with H2O (3 Â 100 ml) and the org. layer dried (Na2SO4) and evaporated. The crude product was dissolved in
CH2Cl2 (50 ml) and added dropwise to pentane (600 ml). The resulting precipitate was collected and dried at 608
in vacuo: 7.7 g (96%) of 21.
13.2. Compound 17 (1.3 g, 1.8 mmol) was dissolved in 0.5m DBU in MeCN (30 ml) and stirred for 2 h at r.t.
The reaction was stopped by addition of 0.5m aq. AcOH (30 ml), the mixture diluted with H2O (10 ml) and
extracted with CH2Cl2 (3 Â 10 ml), the org. layer washed with sat. NaHCO3 soln. (2 Â 20 ml), the aq. layer
extracted back with CH2Cl2 (10 ml), the combined org. layer dried (Na2SO4) and evaporated, and the residue
purified by FC (10 Â 2.5 cm, toluene/AcOEt 10 :1 (0.3 l) and 1:1 (0.2 l)): 850 mg (90%) of 21. Colorless foam.
UV (MeOH): 236 (4.37), 265 (4.12). 1H-NMR ((D6)DMSO): 11.30 (br. s, NH); 7.62 (s, HÀC(6)); 7.43 ± 7.19
(m, 9 H of (MeO)2Tr); 6.94 ± 6.90 (d, 4 H o to MeO); 6.20 ± 6.19 (m, HÀC(1')); 4.98 (t, OHÀC(5')); 4.25 ± 4.23
(m, HÀC(3')); 3.78 ± 3.74 (m, HÀC(4'), 2 MeO); 3.49 ± 3.15 (m, 2 HÀC(5')); 1.80 ± 1.40 (m, 2 HÀC(2'),
MeÀC(5)). Anal. calc. for C31H33N2O7 (544.6): C 68.24, H 6.10, N 5.13; found: C 68.28, H 6.07, N 4.87.
14. 2'-Deoxy-3'-O-(4,4'-dimethoxytrityl)-N4-{[2-(4-nitrophenyl)ethoxy]carbonyl}cytidine (22). 14.1. As
described in Exper. 13.1, with (Bu4N)F ´ H2O (4.6 g, 14.63 mmol), AcOH (2.8 ml, 48.8 mmol), THF (100 ml),

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and 14 (8.42 g, 9.76 mmol; 12 d, r.t.). The crude product was purified by FC (18 Â 5.5 cm, toluene/AcOEt 1:1
(1.0 l), 1:4 (1.0 l), and 1:8 (1.0 l)): 6.35 g (90%) of 22. Colorless foam.
14.2. As described in Exper. 13.2, with 0.1m DBU (22 ml) in MeCN, 18 (1.13 g, 1.1 mmol; 1 min, r.t.), and
0.1m aq. AcOH (22 ml). Purification by FC (14 Â 2.5 cm, toluene/AcOEt/MeOH 4 :1:0 (0.3 l) and 5 :4 :1 (0.2 l))
gave 760 mg (90%) of 22. Colorless foam. UV (MeOH): 238 (4.63), 275 (4.25), 280 (sh, 4.25) 1H-NMR
((D6)DMSO): 10.74 (br. s, NH); 8.17 ± 8.14 (m, HÀC(6), 2 H o to NO2); 7.57 (d, 2 H m to NO2); 7.42 ± 7.12
(m, 9 H of (MeO)2Tr); 6.95 ± 6.88 (m, HÀC(5), 4 H o to MeO); 6.19 ± 6.14 (m, HÀC(1')); 4.89 (t, OHÀC(5'));
4.33 (t, CH2CH2OCO); 4.23 ± 4.21 (m,HÀC(3')); 3.74 ± 3.72 (m, HÀC(4'), 2 MeO); 3.32 ± 3.30 (m, 1 HÀC(5'));
3.16 ± 3.03 (m, 1 HÀC(5'), CH2CH2OCO); 1.92 ± 1.80 (m, 1HÀC(2')); 1.70 ± 1.58 (m, 1 HÀC(2')). Anal. calc.
for C39H36N4O10 (720.8): C64.99, H 5.03, N 7.77; found: C 64.94, H 5.50, N 7.69.
15. 2'-Deoxy-3'-O-(4,4'-dimethoxytrityl)-N6-{[2-(4-nitrophenyl)ethoxy]carbonyl}adenosine (23). 15.1. As
described in Exper. 13.1, with (Bu4N)F ´ H2O (2.22 g, 7.05 mmol), AcOH (1.34 ml, 23.5 mmol), THF (50 ml),
and 15 (4.18 g, 4.7 mmol; 12 d, r.t.). The crude product was dissolved in toluene/CH2Cl2 3 :1 (50 ml) and the
soln. added dropwise to pentane (500 ml). The resulting residue was collected and dried at 608: 3.37 g (96%) of
23.
15.2. As described in Exper.13.2, with 0.1m DBU (14.5 ml) in MeCN, 19 (770 mg, 0.73 mmol; 1 min, r.t.),
and 0.1m aq. AcOH (14.5 ml). Purification by FC (8 Â 2.5 cm, toluene/AcOEt/MeOH 4 :1:0 (0.3 l) and 5 :4 :1
1
(0.2 l)) gave 520 mg (95%) of 23. Colorless foam. UV (MeOH): 203 (4.96), 236 (4.46), 268 (4.50). H-NMR
((D6)DMSO): 10.60 (br. s, NH); 8.58, 8.54 (2s, HÀC(2), HÀC(8)); 8.13 (d, 2 H o to NO2); 7.59 (d, 2 H m to
NO2); 7.47 ± 7.12 (m, 9 H of (MeO)2Tr); 6.91 (d, 4 H o to MeO); 6.48 ± 6.42 (m, HÀC(1')); 5.00 (t, OHÀC(5'));
4.37 ± 4.34 (m, CH2CH2OCO, HÀC(3')); 3.83 ± 3.72 (m, HÀC(4'), 2 MeO); 3.38 ± 3.35 (m, 1 HÀC(5')); 3.21 ±
3.18 (m, 1 HÀC(5')); 3.08 (t, CH2CH2OCO); 2.45 ± 2.36 (m, 1 HÀC(2')); 1.90 ± 1.83 (m, 1 HÀC(2')). Anal. calc.
for C40H38N6O9 (746.8): C 64.33, H 5.13, N 11.25; found: C 64.12, H 5.26, N 10.65.
16.
2'-Deoxy-3'-O-(4,4'-dimethoxytrityl)-N2-{[2-(4-nitrophenyl)ethoxy]carbonyl}-O6-[2-(4-nitrophenyl)-
ethyl]guanosine (24). 16.1. As described in Exper. 13.1, with (Bu4N)F ´ H2O (2.78 g, 9.84 mmol), AcOH
(1.69 ml, 29.5 mmol), THF (70 ml), and 16 (6.22 g, 5.9 mmol; 12 d, r.t.). The crude product was dissolved in
CH2Cl2 (15 ml) and the soln. added dropwise to Et2O/pentane (500 ml). The resulting precipitate was collected
and co-evaporated with CH2Cl2: 5.10 g (95%) of 24.
16.2. As described in Exper. 13.2, with 0.1m DBU (15.2 ml) in MeCN, 20 (930 mg, 0.76 mmol; 1 min, r.t.),
and 0.1m aq. AcOH (15.2 ml). Purification by FC (9 Â 2.5 cm, toluene/AcOEt/MeOH 10 :1:0 (0.3 l) and 5 :4 :1
1
(0.3 l)) gave 680 mg (95%) of 24. Colorless foam. UV (MeOH): 203 (5.01), 236 (4.53), 268 (4.57). H-NMR
((D6)DMSO): 10.29 (br. s, NH); 8.47 (s, HÀC(8)); 8.14 (d, 4 H o to NO2); 7.63 ± 7.58 (m, 4 H m to NO2); 7.46 ±
7.20 (m, 9 H of (MeO)2Tr); 6.90 (d, 4 H o to MeO); 6.35 ± 6.29 (m, HÀC(1')); 4.81 ± 4.71 (m, CH2CH2OCO,
OHÀC(5')); 4.36 ± 4.32 (m, CH2CH2O of O6-npe, HÀC(3')); 3.83 ± 3.72 (m, HÀC(4'), 2 MeO); 3.37 ± 3.22
(m, 2 HÀC(5'), CH2CH2OCO); 3.08 (t, CH2CH2O of O6-npe); 2.40 ± 2.32 (m, 1 HÀC(2')); 1.79 ± 1.72
(m, 1 HÀC(2')). Anal. calc. for C48H45N7O12 (911.9): C 63.15, H 5.08, N 10.74; found: C 63.41, H 5.21, N 10.06.
17. 3'-O-(4,4'-Dimethoxytrityl)thymidine 5'-(2-Cyanoethyl Diisopropylphosphoramidite) (25). A mixture of
21 (1.63 g, 3 mmol), 2-cyanoethyl tetraisopropylphosphorodiamidite (1.35 g, 4.5 mmol) and 1H-tetrazole
(110 mg, 1.5 mmol) was stirred in abs. MeCN (3 ml) under N2 for 1 h. After dilution with CH2Cl2 (20 ml), the
soln. was washed with phosphate buffer (pH 7; 2 Â 10 ml), the aq. phase extracted with CH2Cl2 (2 Â 10 ml), the
combined org. layer dried (Na2SO4) and evaporated, and the crude foam purified by FC (20 Â 2.5 cm, toluene/
AcOEt (1% Et3N) 4 :1 (0.3 l) and 2 :1 (0.3 l). The product fractions were co-evaporated with CH2Cl2: 1.63 g
1
(73%) of 25. Colorless foam. UV (MeOH): 203 (4.85), 235 (4.35), 268 (4.09). H-NMR ((D6)DMSO): 11.30
(br. s, NH); 7.42 ± 7.12 (m, 9 H of (MeO)2Tr, HÀC(6)); 6.89 (d, 4 H o to MeO); 6.21 ± 6.14 (m, HÀC(1')); 4.21 ±
4.17 (m, HÀC(3')); 3.96 ± 3.89 (m, HÀC(4')); 3.72 ± 3.33 (m, 2 MeO, 2 Me2CH, CH2CH2OP, 2 HÀC(5')); 2.72 ±
2.60 (m, CH2CH2OP); 1.71 ± 1.62 (m, MeÀC(5), 2 HÀC(2')); 1.10 ± 0.93 (m, 2 Me2CH)). 31P-NMR
((D6)DMSO): 148.86, 148.26. Anal. calc. for C40H49N4O8P (744.8): C 64.50, H 6.63, N 7.52; found: C 64.25,
H 6.52, N 7.20.
18.2. 2'-Deoxy-3'-O-(4,4'-dimethoxytrityl)-N4-{[2-(4-nitrophenyl)ethoxy]carbonyl}cytidine 5'-(2-Cyanoethyl
Diisopropylphosphoramidite) (26). As described in Exper. 17, with 22 (720 mg, 1 mmol), 2-cyanoethyl
tetraisopropylphosphorodiamidite (450 mg, 1.5 mmol), 1H-tetrazole (33 mg, 0.5 mmol), and MeCN (3 ml).
Purification by FC (14 Â 2.5 cm, AcOEt (1% Et3N) (0.1 l)) gave 810 mg (88%) of 26. Colorless foam. UV
(MeOH): 202 (4.99), 236 (4.58), 280 (4.25). 1H-NMR ((D6)DMSO): 10.73 (br. s, NH); 8.13 (d, 2 H o to NO2);
7.98 ± 7.94 (m, HÀC(6)); 7.57 (d, 2 H m to NO2); 7.42 ± 7.14 (m, 9 H of (MeO)2Tr); 6.96 ± 6.88 (m, 4 H o to MeO,
HÀC(5)); 6.14 ± 6.09 (m, HÀC(1')); 4.33 (t, CH2CH2OCO); 4.18 ± 4.16 (m, HÀC(3')); 3.97 ± 3.87
(m, HÀC(4')); 3.72 ± 3.39 (m, 2 MeO, 2 Me2CH, CH2CH2OP, 2 HÀC(5')); 3.05 (t, CH2CH2OCO); 2.71 ± 2.60

Helvetica Chimica Acta ± Vol. 83 (2000)
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(m, CH2CH2OP); 2.12 ± 1.91 (m, 1HÀC(2')); 1.64 ± 1.48 (m, 1HÀC(2')); 1.19 ± 0.92 (m, 2 Me2CH). 31P-NMR
((D6)DMSO): 148.74, 148.29. Anal. calc. for C48H55N6O11P (923.0): C 62.46, H 6.00, N 9.11; found: C 61.86,
H 6.05, N 8.81.
19. 2'-Deoxy-3'-O-(4,4'-dimethoxytrityl)-N6-{[2-(4-nitrophenyl)ethoxy]carbonyl}adenosine 5'-(2-Cyanoeth-
yl Diisopropylphosphoramidite) (27). As described in Exper. 17, 23 (700 mg, 0.95 mmol), 2-cyanethyl
tetraisopropylphosphorodiamidite (430 mg, 1.43 mmol), 1H-tetrazole (32 mg, 0.48 mmol), and MeCN (3 ml).
Purification by FC (14 Â 2.5 cm, AcOEt (1% Et3N) (0.1 l)) gave 800 mg (90%) of 27. Colorless foam. UV
(MeOH): 204 (4.96), 237 (4.44), 267 (4.48). 1H-NMR ((D6)DMSO): 10.56 (br. s, NH); 8.57 (d, HÀC(2)); 8.42
(d, HÀC(8)); 8.13 (d, 2 H o to NO2); 7.46 (d, 2 H m to NO2); 7.35 ± 7.16 (m, 9 H of (MeO)2Tr); 6.91 (m, 4 H o to
MeO); 6.50 ± 6.41 (m, HÀC(1')); 4.39 ± 4.35 (m, CH2CH2OCO, HÀC(3')); 4.05 ± 3.96 (m, HÀC(4')); 3.72 ± 3.38
(m, 2 MeO, 2 Me2CH, CH2CH2OP, 2 HÀC(5')); 3.08 (t, CH2CH2OCO); 2.71 ± 2.62 (m, CH2CH2OP); 2.38 ± 2.28
(m, 1 HÀC(2')); 2.11 ± 1.94 (m, 1 HÀC(2')); 1.21 ± 0.84 (m, 2 Me2CH). 31P-NMR ((D6)DMSO): 148.29, 148.12.
Anal. calc. for C48H55N6O11P (947.0): C 62.15, H 5.85, N 11.83; found: C 61.78, H 5.95, N 11.60.
20.
2'-Deoxy-3'-O-(4,4'-dimethoxytrityl)-N2-{[2-(4-nitrophenyl)ethoxy]carbonyl}-O6-[2-(4-nitrophenyl)-
ethyl]guanosine 5'-(2-Cyanoethyl Diisopropylphosphoramidite) (28). As described in Exper. 17, with 24
(810 mg, 0.96 mmol), 2-cyanethyl tetraisopropylphosphorodiamidite (430 mg, 1.43 mmol), 1H-tetrazole
(32 mg, 0.48 mmol), and MeCN (3 ml). Purification by FC (14 Â 2.5 cm, AcOEt (1% Et3N) (0.1 l)) gave
810 mg (83%) of 28. Colorless foam. UV (MeOH): 202 (5.02), 237 (4.51), 269 (4.56). 1H-NMR ((D6)DMSO):
10.29 (br. s, NH); 8.21 ± 8.13 (m, HÀC(8), 4 H o to NO2); 7.63 ± 7.53 (m, 4 H m to NO2); 7.46 ± 7.18 (m, 9 H of
(MeO)2Tr); 6.89 (m, 4 H
o
to MeO); 6.39 ± 6.29 (m, HÀC(1')); 4.73 (t, CH2CH2OCO); 4.34 ± 4.29
(m, CH2CH2O of O6-npe, HÀC(3')); 3.98 ± 3.80 (m, HÀC(4')); 3.71 ± 3.24 (m, 2 MeO, 2 Me2CH, CH2CH2OP,
2 HÀC(5'), CH2CH2OCO); 3.07 (t, CH2CH2 of O6-npe); 2.71 ± 2.60 (m, CH2CH2OP); 2.36 ± 2.28
(m, 1 HÀC(2')); 2.05 ± 1.86 (m, 1 HÀC(2')); 1.21 ± 0.82 (m, 2 Me2CH). 31P-NMR ((D6)DMSO): 148.35. Anal.
calc. for C57H62N9O13P (1112.2): C 61.56, H 5.62, N 11.33; found: C 61.31, H 5.75, N 10.94.
21. 3'-O-(4,4'-Dimethoxytrityl)thymidine 5'-(Hydrogen Butanedioate) (29). In CH2Cl2 (5 ml), 21 (250 mg,
0.46 mmol), succinic anhydride (92 mg, 0.92 mmol), and N,N-dimethylpyridin-4-amine (DMAP; 74 mg,
0.6 mmol) were stirred at r.t. for 2 h. Then the mixture was diluted with CH2Cl2 (20 ml) and washed with
10% citric acid (2 Â 20 ml) and NaCl soln. (2 Â 20 ml). The aq. phase was extracted with CH2Cl2 (2 Â 10 ml), the
combined org. layer dried (Na2SO4) and evaporated, the resulting foam dissolved in CH2Cl2 (5 ml), and the
soln. added dropwise to pentane (70 ml). The resulting residue was collected 290 mg (98%) of 29. Colorless
1
powder. UV (MeOH): 203 (4.83), 234 (4.37), 266 (4.37). H-NMR ((D6)DMSO): 12.23 (br. s, COOH); 11.32
(br. s, NH); 7.43 ± 7.20 (m, 9 H of (MeO)2Tr, HÀC(6)); 6.90 (d, 4 H o to MeO); 6.18 ± 6.12 (m, HÀC(1')); 4.17 ±
4.15 (m, HÀC(3')); 3.95 ± 3.73 (m, HÀC(4'), 2 HÀC(5'), 2 MeO); 2.49 ± 2.41 (m, CH2CH2); 1.78 ± 1.62
(m, 2 HÀC(2'), MeÀC(5)). Anal. calc. for C35H31N2O10 ´ H2O (657.7): C 63.92, H 5.06, N 4.26; found: C 64.08,
H 5.58, N 4.37.
22. 2'-Deoxy-3'-O-(4,4'-dimethoxytrityl)-N4-{[2-(4-nitrophenyl)ethoxy]carbonyl}cytidine 5'-(Hydrogen Bu-
tanedioate) (30). As described in Exper. 21, with 22 (320 mg, 0.44 mmol): 320 mg (89%) of 30. Colorless
powder. UV (MeOH): 202 (4.98), 236 (4.63), 275 (sh, 4.25), 280 (4.25). 1H-NMR ((D6)DMSO): 10.8 (br. s,
NH); 8.15 (d, 2 H o to NO2); 7.89 (d, HÀC(6)); 7.58 (d, 2 H m to NO2); 7.41 ± 7.21 (m, 9 H of (MeO)2Tr); 6.99 ±
6.88 (m, HÀC(5), 4 H o to MeO); 6.13 (t, HÀC(1')); 4.33 (t, CH2CH2OCO); 4.20 ± 4.13 (m, HÀC(3')); 3.94 ±
3.73 (m, HÀC(4'), 2 HÀC(5'), 2 MeO); 3.06 (t, CH2CH2OCO); 2.41 ± 2.39 (m, CH2CH2); 1.99 ± 1.94
(m, 1 HÀC(2')); 1.72 ± 1.67 (m, 1 HÀC(2')). Anal. calc. for C43H40N4O13 (820.8): C 62.92, H 4.91, N 6.83;
found: C 62.54, H 5.15, N 6.81.
23. 2'-Deoxy-3'-O-(4,4'-dimethoxytrityl)-N6-{[2-(4-nitrophenyl)ethoxy]carbonyl}adenosine 5'-(Hydrogen
Butanedioate) (31). As described in Exper. 21, with 23 (260 mg, 0.44 mmol): 320 mg (89%) of 31. Colorless
1
powder. UV (MeOH): 203 (5.02), 236 (4.51), 266 (4.54). H-NMR ((D6)DMSO): 10.50 (br. s, NH); 8.57, 8.49
(2s, HÀC(2), HÀC(8)); 8.14 (d, 2 H o to NO2); 7.59 (d, 2 H m to NO2); 7.48 ± 7.21 (m, 9 H of (MeO)2Tr); 6.90
(d, 4 H o to MeO); 6.44 (m, HÀC(1')); 4.40 ± 4.35 (m, CH2CH2OCO, HÀC(3')); 4.02 ± 3.72 (m, HÀC(4'),
2HÀC(5'), 2 MeO); 3.09 (t, CH2CH2OCO); 2.57 ± 2.36 (m, 1 HÀC(2'), CH2CH2); 1.98 ± 1.91 (m, 1 HÀC(2')).
Anal. calc. for C44H42N6O12 (846.9): C 62.40, H 5.00, N 9.92; found: C 62.20, H 5.28, N 9.46.
24.
2'-Deoxy-3'-O-(4,4'-dimethoxytrityl)-N2-{[2-(4-nitrophenyl)ethoxy]carbonyl}-O6-[2-(4-nitrophenyl)-
ethyl]guanosine 5'-(Hydrogen Butanedioate) (32). As described in Exper. 21, with 24 (320 mg, 0.35 mmol):
330 mg (93%) of 32. Colorless powder. UV (MeOH): 202 (5.00), 236 (4.51), 269 (4.55). 1H-NMR
((D6)DMSO): 12.26 (br. s, COOH); 10.30 (br. s, NH); 8.24 (s, HÀC(8)); 8.15 (d, 4 H o to NO2); 7.63 ± 7.58
(m, 4 H m to NO2); 7.46 ± 7.20 (m, 9 H of (MeO)2Tr); 6.89 (d, 4 H o to MeO); 6.30 (t, HÀC(1')); 4.72
(m, CH2CH2OCO); 4.39 ± 4.28 (m, CH2CH2O of O6-npe, HÀC(3')); 4.02 ± 3.71 (m, 2 HÀC(5'), HÀC(4'),

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Helvetica Chimica Acta ± Vol. 83 (2000)
2 MeO); 3.26 (t, CH2CH2OCO); 3.07 (t, CH2CH2O of O6-npe); 2.51 ± 2.39 (m, 1 HÀC(2'), CH2CH2); 1.86 ± 1.78
(m, 1 HÀC(2')). Anal. calc. for C52H50N7O15 (1013.2): C 61.65, H 4.98, N 9.68; found: C 61.58, H 4.92, N 9.50.
25. Solid-Support Material from 500-Š LCAMA-CPG and 29 ± 32. A mixture of LCAMA-CPG (250 ±
300 mg), 5'-(hydrogen butanedioate) 29 (29 mg, 45 mmol) (or 30 (37 mg, 45 mmol), 31 (38 mg, 45 mmol), or 32
(46 mg, 45 mmol)), TOTU (22 mg, 67 mmol), abs. MeCN (5 ml), and N-methylmorpholine (15 ml, 145 mmol)
was gently shaken for 2 h. The CPG material was collected in a glass funnel and washed with MeOH, DMF,
pyridine, MeOH, acetone, and Et2O. Capping procedure: The nucleoside-functionalized CPG was treated with a
mixture of DMAP (50 mg, 0.41 mmol), abs. pyridine (10 ml), and Ac2O (1 ml, 10.6 mmol) for 45 min at r.t. by
gently shaking. Then the nucleoside-bearing, capped solid support was collected, washed with MeOH, DMF,
MeOH, acetone, and Et2O, and dried in a desiccator. Determination of loading: A defined amount of the
nucleoside-bearing support (ca. 5 mg) was treated in a 10-ml calibrated flask (10 ml) with 0.2m TsOH in MeCN
(10 ml). After 1 min, the absorbance at 498 nm was measured against 0.2m TsOH in MeCN. The loading L
[mmol/g] was calculated by the formula L ˆ A ´ 10 ´ 14.4/m (A ˆ absorbance at 498 nm; m ˆ weight of loaded
CPG material in mg): L ˆ 35 (loaded with 29), 33 (loaded with 30), 34 (loaded with 31) and 29 mmol/g (loaded
with 32).
26. Assembly of Oligonucleotides. The syntheses were carried out in an Applied-Biosystems-392-DNA/RNA
synthesizer. LCAMA-CPG Material loaded with 29, 30, 31, or 32 (see Exper. 25) was packed into a small ABI
column, and cycles of nucleotide addition were carried out by programmed series of reagent and solvent washes
based on recommended procedures with the following main steps: 1) 3'-O-(MeO)2Tr deprotection with 3%
CCl3COOH in CH2Cl2 for 135 s; the eluate from this step was collected and the absorbance at 498 nm measured
to determine the condensation yields. 2) Coupling: 0.1m phosphoramidite and 0.5m 1H-tetrazole in dry MeCN
(or 0.5m pyridinium chloride in dry MeCN), delivered in alternating reagent pushes with a subsequent wait time
of 180 s. 3) Capping: Ac2O/2,6-dimethylpyridine/THF 1:1:8 and 1-methyl-1H-imidazole/THF 16 :84, delivered
in one 10 s push with a subsequent wait time of 5 s. 4) Oxidation: 0.05m I2 in THF/H2O/pyridine 7:2 :1, delivered
in one 10 s push with a subsequent wait time of 15 s.
Then a cleavage program was carried out: 1) Cleavage of the base-protecting groups; 1m DBU in MeCN
delivered in several pushes and following wait steps (total wait time: 11 h). 2) Cleavage from the solid support:
conc. NH3 soln. delivered in one push with a consecutive wait time of 4 Â 900 s (total wait time 2 h). The
reaction soln. containing only the oligonucleotide and NH3 was collected and, after determination of the isolated
amount of oligonucleotide by measurement of the absorbance at 260 nm, lyophilized in a Speedvac concentrator
under high vacuum.
27. Oligonucleotide 3'-Conjugates 33 and 34. After the oligonucleotide synthesis, the solid support was dried
and transferred into a 25-ml flask. Cholesteryl phosphoramidite [31] (52 mg, 88 mmol) or phosphoramidite ester
with vitamin E [32] (52 mg, 82 mmol), resp., was dissolved in dry MeCN/CH2Cl2 (1.6 ml), and pyridinium
chloride (23.8 mg (176 mmol) and 22.7 mg (164 mmol), resp.) in dry MeCN/CH2Cl2 (1 ml) was added. The
heterogenous mixture was gently shaken for 18 h at r.t. After oxidation with 0.05m I2 in THF/H2O/pyridine
7:2 :1 (dropwise addition until the soln. remained brownish), the solid support was washed carefully and then
transferred back into a synthesis column for deprotection and cleavage from the support as described above.
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Received May 4, 2000