Nucleotides: Part LXXV. New types of fluorescence labeling of 2′-deoxycytidine

Article abstract of DOI:10.1002/hlca.200900198

The reactivity of the 2′-deoxy-N⁴-(phenoxycarbonyl) cytidine derivatives 3 and 4 with aromatic amines was studied to form new types of urea derivatives (see 5 - 10). On the same basis, labeling of 3 and 4 with 5-aminofluorescein (14) was achiev

Full text of DOI:10.1002/hlca.200900198

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Helvetica Chimica Acta – Vol. 92 (2009)
Nucleotides
Part LXXV1)
New Types of Fluorescence Labeling of 2’-Deoxycytidine
by Thomas Maier and Wolfgang Pfleiderer*
Fachbereich Chemie, Universitꢀt Konstanz, Postfach 5560, DE-78457 Konstanz
The reactivity of the 2’-deoxy-N4-(phenoxycarbonyl)cytidine derivatives 3 and 4 with aromatic
amines was studied to form new types of urea derivatives (see 5 – 10). On the same basis, labeling of 3
and 4 with 5-aminofluorescein (14) was achieved to give the conjugates 15 and 17, respectively
(Scheme 1). Treatment of 17 with 2-(4-nitrophenyl)ethanol in a Mitsunobu reaction led to double
protection of the fluorescein moiety (!18) and desilylation yielded 19. Dimethoxytritylation (!20) and
subsequent phosphitylations afforded the new building blocks 21 and 22. Synthesis of the fully protected
trimer 28 was achieved by condensation of 21 with 23 to 26 which after detritylation (!27) was coupled
with 25 to give 28 (Scheme 2). Deprotection of all blocking groups was performed with 1,8-
diazabicyclo[5.4.0]undec-7-ene (DBU) in one step to give 29. The synthesis of the decamer 5’-
d(CFluCCG GCC CGC)-3’ (33) started from 30 which was attached to the solid support and then
elongated with 31, 32, and 22 at the 5’-terminal end (CFlu ¼ deprotected phosphate derivative of 22).
Hybridization with the complementary oligomer 5’-d(G GGC CGG GCG)-3’ (34) showed the influence
of the fluorescein label on the stability of the duplex.
1. Introduction. – Radioactive labeling of nucleotides and oligonucleotides [2][3]
has recently been more and more replaced by fluorescent or biochemical markers [4 –
6], especially in research areas where a high degree of automatization is applied. In
these cases, the chromophore is attached at the oligonucleotide chain by a flexible
hydrocarbon linker of several atoms length at the 3’- or 5’-end of an oligonucledotide
[7 – 10], at the phosphate-ribose backbone [11 – 15], or at the nucleobase [16 – 18].
Modified nucleosides are able to bind covalently a fluorophore after completion of the
oligonucleotide synthesis. Examples are N4-(aminoalkyl)-2’-deoxycytidine [19][20], 2’-
deoxy-5-{(1E)-3-oxo-3-{{[6-(trifluoroacetyl)amino]hexyl}amino}prop-1-en-1-yl}uridine
[21][22], 5-(3-aminopropyl)-2’-deoxyuridine [23] and 8-[(aminoalkyl)amino]-2’-de-
oxyadenosine [24 – 26]. The N6-position of 2’-deoxyadenosine was also modified to
incorporate a (6-trifluoroacetylaminohexyl)amino spacer [27], and the ribose moiety
was converted into a 5’-amino- [14][23] and 5’-mercapto derivative [28], respectively.
Our intention was the direct coupling of the highly efficient fluorophor fluorescein onto
the amino functions of all nucleobase moieties by a carbamoyl linker [29], and here we
describe our efforts with 2’-deoxycytidine. The activated phenyl carbamate intermedi-
ates react easily with 5-aminofluorescein (¼ 5-amino-3’,6’-dihydroxyspiro[isobenzofur-
an-1(3H),9’-[9H]xanthen-3-one) to the corresponding dye conjugate. Applications in
1
)
Part LXXIV: [1].
ꢁ 2009 Verlag Helvetica Chimica Acta AG, Zꢂrich

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oligonucleotide syntheses, however, needs further protection of the two acidic
functionalities of the fluorescein moiety which could be achieved by use of the 2-(4-
nitrophenyl)ethyl (npe) and 2-(4-nitrophenylethoxy)carbonyl (npeoc) blocking group
strategy [30].
2. Syntheses. – Analogously to the adenosine strategy [16], 3’,5’-di-O-acetylcytidine
(1) [31] and 2’-deoxy-3’,5’-O-(tetraisopropyldisiloxane-1,3-diyl)-cytidine (2) [32] were
converted into the corresponding N4-(phenoxycarbonyl) derivatives 3 and 4 by
treatment with phenyl 1H-tetrazole-1-carboxylate (Scheme 1). Model reactions of 3
and 4 with aromatic amines such as aniline, 4-nitroaniline, and ethyl 4-aminobenzoate
proceeded well at 708 to give 5 – 10 in good yields. Treatment of 5 – 7 or 8 – 10 with
ammonia or tetrabutylammonium fluoride (Bu4NF), respectively, afforded deprotec-
tion at the sugar moiety yielding 11 – 13. Reaction of 1 and 2 with 5-aminofluorescein
(14) [2] led in excellent yields to the 2’-deoxy-N4-[(fluorescein-5-ylamino)carbonyl]-
cytidines 15 and 17, respectively. Deacetylation of 15 by ammonia and K2CO3 gave 16 in
68% yield. To protect the fluorescein moiety for further reactions, the Mitsunobu
reaction with 2-(4-nitrophenyl)ethanol, triphenylphosphine, and diethyl azodicarbox-
ylate (¼diethyl diazene-1,2-dicarboxylate) was performed and led under twofold
substitution to 18 in excellent yield. Cleavage of the silyl protecting group with Bu4NF
in THF/AcOH afforded 19 which was converted into the oligonucleotide building block
22 first by reaction with dimethoxytrityl chloride to 20 and then by phosphitylation with
2-(4-nitrophenyl)ethyl N,N,N’,N’-tetraisopropylphosphorodiamidite giving 21 and 2-
cyanoethyl N,N,N’,N’-tetraisopropylphosphorodiamidite to yield 22.
For the synthesis of the fully protected 2’-deoxycytidine trimer 28 (Scheme 2)
carrying as a universal pattern the npe and npeoc blocking groups at all functionalities,
2’-deoxy-N4,3’-O-bis{[2-(4-nitrophenyl)ethoxy]carbonyl}cytidine (23) [33] and 2’-de-
oxy-N4,5’-O-bis{[2-(4-nitrophenyl)ethoxy]carbonyl}cytidine 3’-[2-(4-nitrophenyl)ethyl
N,N-diisopropylphosphoramidite] (25) derived from 24 [33] were prepared. In the first
step of the oligonucleotide synthesis, the phosphoramidite 21 was treated with 23 under
tetrazole activation and subsequent oxidation to give the dimer 26. Detritylation of 26
led to 27 which was then coupled with 25 to achieve the fully protected trimer 28 in 83%
yield. The total deprotection of 28 was achieved by 1,8-diazabicyclo[5.4.0]undec-7-ene
(DBU) in pyridine in one step, and workup proceeded by DEAE-Sephadex
chromatography with triethylammonium hydrogen carbonate buffer followed by
conversion of the resulting triethylammonium salt into the ammonium salt 29. Its purity
was established by reversed-phase HPLC (buffer pH 9/MeOH/H2O 1:1:2) yielding
one peak with a retention time of 16.8 min and detection either at 260 nm or 480 nm.
Cleavage of 29 by snake-venom phosphodiesterase (SVP) furnished a mixture which
showed in the HPLC, expectedly, only two peaks of 2’-deoxycytidine and 16.
The labeled 5’-d(CFluCCG GCC CGC)-3’ (33) decamer was prepared by automated
solid-support synthesis on glyceryl-CPG (500 ꢃ) which was activated with phenyl 1H-
tetrazole-1-carboxylate and subsequently loaded with the N1,N6-dimethylhexane-1,6-
diamine spacer [34], followed by reaction with 2’-deoxy-5’-O-(4,4’-dimethoxytrityl)-
N4-{[2-(4-nitrophenyl)ethoxy]carbonyl}cytidine 3’-succinate (30). The next cycles for
the built-up of the oligonucleotide chain were performed with 2’-deoxy-5’-O-(4,4’-
dimethoxytrityl)-N2-{[2-(4-nitrophenyl)ethoxy]carbonyl}-O6-[2-(4-nitrophenyl)ethyl]-

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Scheme 1

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Scheme 2

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Fig. 1. HPLC of the mixture obtained after enzymatic-hydrolysis of 33 by SVP
guanosine 3’-(2-cyanoethyl N,N-diisopropylphosphoramidite) (31) [35], 2’-deoxy-5’-O-
(4,4’-dimethoxytrityl)-N4-{[2-(4-nitrophenyl)ethoxy]carbonyl}cytidine 3’-(2-cyanoethyl
N,N-diisopropylphosphoramidite) (32) [35] and with 22 as the last building block.
Deprotection was achieved first with CHCl2COOH to remove the dimethoxytrityl
group, then with DBU to cleave the ce, npe, and npeoc groups, and finally by washing
with ammonia to detach the oligomer from the solid support. Enzymatic degradation
by snake venom diesterase (SVP) showed in HPLC the three expected peaks of dC,
dG, and 16 (Fig. 1). The complementary 5’-d(G GGC CGG GCG)-3’ (34) and
unmasked 5’-d(C CCG GCC CGC)-3’ (35) decamers were analogously assembled and
obtained after deprotection. The HPLC trace of 33 and 35 showed only one peak, thus
establishing these very efficient and clean syntheses (Fig. 2).
Hybridization experiments in sodium phosphate buffer solution (pH 7.4) between
33 and 34 showed a Tm of 57 – 588, whereas the Tm of the combination 34 · 35 was
slightly higher (60 – 618; Table).
3. Structures. – The newly synthesized 2’-deoxycytidine derivatives were charac-
1
terized and their structures established by elemental analyses, H-NMR and UV
spectra.

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Fig. 2. HPLC of 5’-d(CFluCCG GCC CGC)-3’ (33) and 5’-d(G GGC CGG GCG)-3’ (35)
Table. Oligodeoxynucleotide Sequences and Tm of Hybridizationsa)
Sequence
Hybrid
Tm [8]
5’-d(CFlu CCG GCC CGC)-3’ (33)
33 · 34
34 · 35
56 – 57
60 – 61
5’-d(G GGC CGG GCG)-3’ (34)
5’-d(C CCG GCC CGC)-3’ (35)
a) Salt concentration: 0.12m (Naþ).
Experimental Part
General. Products were dried under high vacuum. All solvents used were of anh. grade. DNA
Synthesizer from Applied Biosystems, model 392. TLC: precoated silica gel thin-layer sheets 60 F 254
from Merck. Flash Chromatography (FC): silica gel (SiO2, 30 – 60 mm; Baker); 0.2 – 0.3 bar. CC ¼ Col-
umn chromatography. HPLC: pump L 6200, autosampler AS 4000, UV detector L 4000, Merck-Hitachi;
column Lichrosorb RP 18, (125 ꢀ 4 mm, 5 mm; Merck); elution: A ¼ 0.1m buffer (pH 9) (2 min); B ¼ 0.1m
buffer (pH 9)/MeOH/H2O 1:1:2 (20 min); C ¼ MeOH (20 min); flow rate 1 ml/min. Ion-exchange CC:
DEAE-Sephadex A25, from Pharmacia Fine Chemicals. M.p.: Gallenkamp melting-point apparatus; no
corrections. UV/VIS Spectra: Perkin-Elmer Lambda 5; lmax in nm (log e). 1H-NMR Spectra: Bruker AC
250; d in ppm rel. to Me4Si or CDCl3 ((D6)DMSO) as internal standard. 31P-NMR Spectra: Jeol JMN-
GX400.
3’,5’-Di-O-acetyl-2’-deoxy-N4-(phenoxycarbonyl)cytidine (¼ Phenyl {1-{4-(Acetyloxy)-5-[(acetyl-
oxy)methyl]tetrahydrofuran-2-yl}-1,2-dihydro-2-oxopyrimidin-4-yl}carbamate; 3). To a soln. of 3’,5’-di-

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O-acetyl-2’-deoxycytidine (1) [31] (6.0 g, 20 mmol) in dioxane (100 ml ) was added phenyl 1H-tetrazole-
1-carboxylate [36] (5.0 g. 26 mmol), and the mixture was stirred at 408 for 1 h. The soln. was
concentrated, the residue suspended in cold abs. CHCl3 (50 ml) and kept at 08 for 2 h. The tetrazole was
filtered off and washed with little cold CHCl3, and then the filtrate again concentrated. The residue was
purified by CC (SiO2 (3 ꢀ 25 cm), toluene (200 ml), toluene/acetone 9 :1 (200 ml), toluene/acetone 7:3
(200 ml), and toluene/acetone 1:1 (200 ml)). The residue of the main fraction was dissolved in CH2Cl2
and the soln. concentrated: 8.0 g (92%) of 3. Solid foam. Rf 0.48 (toluene/acetone 1:1). UV (CH2Cl2):
240 (4.19), 300 (3.87), 310 (sh, 3.76). 1H-NMR ((D6)DMSO): 11.30 (br. s, NH); 8.11 (d, HꢁC(6)); 7.44
(ꢄtꢅ, 2 arom. H); 7.30 – 7.15 (m, 3 arom. H); 6.98 (d, HꢁC(5)); 6.11 (dd, HꢁC(1’)); 5.18 (m, HꢁC(3’));
4.28 – 4.20 (m, HꢁC(4’), CH2(5’)); 2.54 – 2.46 (m, 1 HꢁC(2’)); 2.40 – 2.26 (m, 1 HꢁC(2’)); 2.05 (s,
2 AcO). Anal. calc. for C20H21N3O8 (431.4): C 55.68, H 4.91, N 9.73; found: C 55.11, H 4.96, N 9.54.
2’-Deoxy-3’,5’-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)cytidine (¼ Phenyl N-[1,2-Dihydro-2-oxo-
1-(tetrahydro-2,2,4,4-tetraisopropyl-6H-furo[3,2-f]-1,3,5,2,4-trioxadisilocin-8-yl)pyrimidin-4-yl]carba-
mate; 4). As described for 3, with 2’-deoxy-3’,5’-di-O-1,1,3,3-(tetraisopropyldisiloxane-1,3-diyl)cytidine
(2) [32] (4.7 g, 10 mmol) and phenyl 1H-tetrazole-1-carboxylate (2.5 g, 13 mmol) in dry dioxane
(100 ml). After purification by CC (SiO2 (4.7 ꢀ 13 cm), toluene (400 ml), toluene/acetone 95 :5 (200 ml),
toluene/acetone 9 :1 (400 ml), and toluene/acetone 7:3 (400 ml)), the residue of the main fraction was
dissolved in CH2Cl2 and the soln. concentrated: 5.52 g (93%) of 4. Solid foam. UV (CH2Cl2): 238 (4.18),
301 (3.90), 309 (sh, 3.81). 1H-NMR ((D6)DMSO): 11.29 (br. s, NH); 8.07 (d, HꢁC(6)); 7.46 (ꢄtꢅ, 2 arom.
H); 7.30 – 7.10 (m, 3 arom. H); 6.97 (d, HꢁC(5)); 5.99 (dd, HꢁC(1’)); 4.41 (m, HꢁC(3’)); 4.10 (dd,
1 HꢁC(5’)); 3.01 (dd, 1 HꢁC(5’)); 3.80 (m, HꢁC(4’)); 2.50 – 2.28 (m, CH2(2’)); 1.05 – 0.90 (m,
4 Me2CH). Anal. calc. for C28H43N3O7Si2 (589.8): C 57.02, H 7.34, N 7.12; found: C 56.57, H 7.30, N 7.19.
3’,5’-Di-O-acetyl-2’-deoxy-N4-[(phenylamino)carbonyl]cytidine (¼ N-{1-{4-(Acetyloxy)-5-[(acetyl-
oxy)methyl]tetrahydrofuran-2-yl}-1,2-dihydro-2-oxo-pyrimidin-4-yl}-N’-phenylurea; 5). A soln. of
3
(1.0 g, 2.3 mmol) in abs. pyridine (20 ml) was treated with aniline (0.34 g, 3.6 mmol) at 708 for 3 h.
The mixture was concentrated, 3 ꢀ the residue dissolved in toluene and the soln. concentrated, and the
residue recrystallized from EtOH: 0.7 g (71%) of 5. Colorless crystals. M.p. 176 – 1778. UV (MeOH): 214
1
(sh, 4.19), 282 (4.17), 293 (4.25). H-NMR ((D6)DMSO): 11.20 (br. s, NH); 11.02 (br. s, NH); 7.95 (d,
HꢁC(6)); 7.75 (m, 3 arom. H); 7.37 (m, 2 arom. H); 7.10 (d, HꢁC(5)); 6.30 (dd, HꢁC(1’)); 5.22 (m,
HꢁC(3’)); 4.37 (m, HꢁC(4’), CH2(5’)); 2.79 – 2.70 (m, 1 HꢁC(2’)); 2.20 – 2.11 (m, 1 HꢁC(2’)); 2.09 (s,
2 AcO). Anal. calc. for C20H22N4O7 (430.4): C 55.81, H 5.15, N 13.01; found: C 55.39, H 5.13, N 12.85.
3’,5’-Di-O-acetyl-2’-deoxy-N4-{[(4-nitrophenyl)amino]carbonyl}cytidine (¼ N-{1-{4-(Acetyloxy)-5-
[(acetyloxy)methyl]tetrahydrofuran-2-yl]-1,2-dihydro-2-oxopyrimidin-4-yl}-N’-(4-nitrophenyl)urea; 6).
As described for 5, with 3 (0.6 g, 1.4 mmol) and 4-nitroaniline (0.23 g, 1.67 mmol). The residue was
washed with EtOH, filtered, and recrystallized from DMF (2 ml): 0.2 g (35%) of 6. Yellowish crystals.
M.p. 2498 (dec.). UV (MeOH): 214 (sh, 4.29), 236 (4.07), 315 (4.39). 1H-NMR ((D6)DMSO): 11.80 (br.
s, NH); 11.42 (br. s, NH); 8.24 (d, 2 arom. H); 8.08 (d, HꢁC(6)); 7.71 (d, 2 arom. H); 6.48 (d, HꢁC(5));
6.11 (dd, HꢁC(1’)); 5.19 (m, HꢁC(3’)); 4.25 (m, HꢁC(4’), CH2(5’)); 2.50 – 2.30 (m, CH2(2’)); 2.04 (s,
2 AcO). Anal. calc. for C20H21N5O9 (475.4): C 50.57, H 4.45, N 14.73; found: C 50.16, H 4.51, N 14.73.
3’,5’-Di-O-acetyl-2’-deoxy-N4-{{[4-(ethoxycarbonyl)phenyl]amino}carbonyl}cytidine (¼ Ethyl 4-{3-
{1-{4-(Acetyloxy)-5-[(acetyloxy)methyl]tetrahydrofuran-2-yl}-1,2-dihydro-2-oxopyrimidin-4-yl}ureido}-
benzoate; 7). To a soln. of 3 (1.0 g, 2.3 mmol) in abs. pyridine (20 ml) at 708 was added dropwise a soln. of
ethyl 4-benzoate (0.38 g, 2,3 mmol) in pyridine (15 ml). After heating for 2 h, the mixture was
concentrated, 3 ꢀ the residue dissolved in toluene and the soln. concentrated, and the residue
recrystallized from EtOH: 0.8 g (70%) of 7. Colorless crystals. M.p. 2168. UV (MeOH): 210 (sh, 4.36),
244 (4.13), 297 (4.50). 1H-NMR ((D6)DMSO): 11.31 (br. s, NH); 11.23 (br. s, NH); 8.00 (m, HꢁC(6), 2
arom. H); 7.79 – 7.67 (m, HꢁC(5), 2 arom. H); 6.28 (dd, HꢁC(1’)); 5.22 (m, HꢁC(3’)); 4.42 – 4.30 (m,
HꢁC(4’), CH2(5’), CH2); 2.47 – 2.30 (m, 1 HꢁC(2’)); 2.21 – 2.11 (m, 1 HꢁC(2’)); 2.09 (s, 2 AcO); 1.38 (t,
Me). Anal. calc. for C23H26N4O7 (502.5): C 54.98, H 5.22, N 11.15; found: C 55.07, H 5.25, N 11.20.
2’-Deoxy-N4-[(phenylamino)carbonyl]-3’,5’-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)cytidine
(¼ N-[1,2-Dihydro-2-oxo-1-(tetrahydro-2,2,4,4-tetraisopropyl-6H-furo[3,2-f]-1,3,5,2,4-trioxadisilocin-8-
yl)pyrimidin-4-yl]-N’-phenylurea; 8). As described for 5, with 4 (1.0 g, 2.3 mmol), aniline (0.34 g,
3.6 mmol), and abs. pyridine (20 ml): 0.7 g (71%) of 8. Colorless crystals. M.p. 200 – 2018. UV (MeOH):

Helvetica Chimica Acta – Vol. 92 (2009)
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1
214 (sh, 4.27), 228 (sh, 4,21), 294 (4.26). H-NMR (CDCl3): 11.5 (br. s, NH); 11.3 (br. s, NH); 8.18 (d,
HꢁC(6)); 7.78 (m, 3 arom. H); 7.31 (m, 2 arom. H); 7.08 (d, HꢁC(5)); 6.14 (dd, HꢁC(1’)); 4.44 (m,
HꢁC(3’)); 4.21 (m, 1 HꢁC(5’)); 4.06 (m, 1 HꢁC(5’)); 3.83 (m, HꢁC(4’)); 2.50 – 2.28 (m, CH2(2’)); 0.96
(m, 4 Me2CH). Anal. calc. for C28H44N4O6Si2 · 0.5 H2O (593.3): C 56.67, H 7.55, N 9.43; found: C 56.49, H
7.47, N 9.36.
2’-Deoxy-N4-{[(4-nitrophenyl)amino]carbonyl}-3’,5’-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)cy-
tidine (¼ N-[1,2-Dihydro-2-oxo-1-(tetrahydro-2,2,4,4-tetraisopropyl-6H-furo[3,2-f]-1,3,5,2,4-trioxadisilo-
cin-8-yl)pyrimidin-4-yl]-N’-(4-nitrophenyl)urea; 9). As described for 5, with 4 (0.2 g, 0.34 mmol), 4-
nitroaniline (30 mg, 0.2 mmol), and abs. pyridine (10 ml): 55 mg (88%) of 9. Colorless crystals. M.p. 2358
(dec.). UV (MeOH): 214 (4.23), 236 (sh, 4.06), 314 (4.40). 1H-NMR ((CDCl3): 11.65 (br. s, NH); 10.35
(br. s, NH); 8.15 (d, 2 arom. H); 7.90 (d, HꢁC(6)); 7.56 (d, 2 arom. H); 7.32 (d, HꢁC(5)); 5.93 (dd,
HꢁC(1’)); 4.31 (m, HꢁC(3’)); 3.96 (m, 1 HꢁC(5’)); 3.83 (m, 1 HꢁC(5’)); 3.67 (m, HꢁC(4’)); 2.48 – 2.28
(m, CH2(2’)); 1.00 – 0.83 (m, 4 Me2CH). Anal. calc. for C28H43N5O8Si2 (633.9): C 53.06, H 6.84, N 11.05;
found: C 52.55, H 6.82, N 10.93.
2’-Deoxy-N4-{{[4-(ethoxycarbonyl)phenyl]amino}carbonyl}-3’,5’-O-(1,1,3,3-tetraisopropyldisiloxane-
1,3-diyl)cytidine (¼ Ethyl 4-{3-[1,2-Dihydro-2-oxo-1-(tetrahydro-2,2,4,4-tetraisopropyl-6H-furo[3,2-f]-
1,3,5,2,4-trioxadisilocin-8-yl]pyrimidin-4-yl]ureido}benzoate; 10). As described for 5, with 4 (1.0 g,
2.3 mmol), ethyl 4-aminobenzoate (0.38 g, 2.3 mmol), and pyridine (20 ml): 0.8 g (70%) of 10. Colorless
crystals. M.p. 2438 (dec.). UV (MeOH): 203 (4.37), 211 (4.37), 244 (4.12), 297 (4.51). 1H-NMR (CDCl3):
11.4 (br. s, NH); 11.3 (br. s, NH); 8.23 (d, HꢁC(6)); 8.00 (d, 2 arom. H); 7.78 (d, 2 arom. H); 7.61 (d,
HꢁC(5)); 6.15 (dd, HꢁC(1’)); 4.35 (m, HꢁC(3’), CH2); 4.23 (m, 1 HꢁC(5’)); 4.05 (m, 1 HꢁC(5’)); 3.86
(m, HꢁC(4’)); 2.60 – 2.42 (m, 1 HꢁC(2’)); 2.40 – 2.25 (m, 1 HꢁC(2’)); 1.39 (t, Me); 1.10 – 0.90 (m,
4 Me2CH). Anal. calc. for C31H48N4O8Si2 · 0.5 H2O (669.9): C 55.58, H 7.37, N 8.37; found: C 55.55, H 7.33,
N 8.59.
2’-Deoxy-N4-[(phenylamino)carbonyl]cytidine (¼ N-{1,2-Dihydro-2-oxo-1-[tetrahydro-4-hydroxy-5-
(hydroxymethyl)furan-2-yl]pyrimidin-4-yl}-N’-phenylurea; 11). a) A soln. of 5 (0.55 g, 1.2 mmol) in sat.
methanolic ammonia (15 ml) was stirred overnight. After evaporation, the residue was recrystallized
from EtOH: 0.3 g (73%) of 11. Colorless crystals. M.p. 1758.
b) A soln. of 8 (0.9 g, 1.5 mmol) and Bu4NF · 3 H2O (1.2 g, 3.8 mmol) in THF (20 ml) was stirred at
r.t. for 15 min. The solvent was evaporated, the residue treated with little H2O, and then the precipitate
collected. Recrystallization from EtOH gave 0.34 g (65%) of 11. Colorless crystals. M.p. 1758. UV
(MeOH): 214 (sh, 4.26), 227 (sh, 4.19), 294 (4.24). 1H-NMR ((D6)DMSO): 11.30 (br. s, NH); 10.20 (br. s,
NH); 8.23 (d, HꢁC(6)); 7.46 (m, 2 arom. H); 7.34 (m, 1 arom. H); 7.05 (t, 1 arom. H); 6.41 (d, HꢁC(5));
6.13 (dd, HꢁC(1’)); 5.28 (d, OHꢁC(3’)); 5.05 (t, OHꢁC(5’)); 4.22 (m, HꢁC(3’)); 3.86 (m, HꢁC(4’));
3.69 – 3.51 (m, CH2(5’)); 2.30 – 2.20 (m, 1 HꢁC(2’)); 2.09 – 1.98 (m, 1 HꢁC(2’)). Anal. calc. for
C16H18N4O5 (346.3): C 55.49, H 5.24, N 16.18; found: C 55.62, H 5.34, N 16.12.
2’-Deoxy-N4-{[(4-nitrophenyl)amino]carbonyl}cytidine (¼ N-{1,2-Dihydro-2-oxo-1-[tetrahydro-4-
hydroxy-5-(hydroxymethyl)furan-2-yl]pyrimidin-4-yl}-N’-(4-nitrophenyl)urea; 12). a) A suspension of
6 (0.08 g, 0.17 mmol) in sat. methanolic ammonia (10 ml) was stirred overnight. The solvent was
evaporated and the residue treated with EtOH to give, after drying 0.05 g (73%) of 12. Yellowish solid.
M.p. 205 – 2068 (dec.).
b) As described for 11 (Exper. b), with 9 (0.4 g, 0.6 mmol), Bu4NF · 3 H2O (0.5 g, 1.5 mmol), and
THF (20 ml) for 20 min: 0.13 g (55%) of 12. Yellowish crystals. M.p. 205 – 2068 (dec.). UV (MeOH): 214
(sh, 4.29), 236 (sh, 4.07), 316 (4.39). 1H-NMR ((D6)DMSO): 11.87 (br. s, NH); 10.34 (br. s, NH); 8.27 (m,
2 arom. H, HꢁC(6)); 7.73 (d, 2 arom. H); 6.44 (d, HꢁC(5)); 6.11 (dd, HꢁC(1’)); 5.31 (d, OHꢁC(3’));
5.09 (dd, OHꢁC(5’)); 4.23 (m, HꢁC(3’)); 3.88 (m, HꢁC(4’)); 3.66 – 3.55 (m, CH2(5’)); 2.31 – 2.24 (m,
1 HꢁC(2’)); 2.05 – 1.98 (m, 1 HꢁC(2’)). Anal. calc. for C16H17N5O7 · 0.5 H2O (400.3): C 48.05, H 4.54, N
17.48; found: C 48.19, H 4.53, N 17.20.
2’-Deoxy-N4-{{[4-(ethoxycarbonyl)phenyl]amino}carbonyl}cytidine (¼ Ethyl 4-{3-{1,2-Dihydro-2-
oxo-1-[tetrahydro-4-hydroxy-5-(hydroxymethyl)furan-2-yl]pyrimidin-4-yl}ureido}benzoate; 13). a) As
described for 12 (Exper. a), with 7 (0.7 g, 1.4 mmol) in sat. methanolic ammonia (10 ml): 0.45 g (78%) of
13. Colorless solid. M.p. 1698 (dec.).

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Helvetica Chimica Acta – Vol. 92 (2009)
b) As described for 11 (Exper. b), with 10 (2.0 g, 3 mmol), Bu4NF · 3 H2O (2.37 g, 7.5 mmol), and
THF (20 ml) for 30 min (treatment with little H2O and EtOH): 0.95 g (76%) of 13. Colorless crystals.
1
M.p. 1698 (dec.). UV (MeOH): 203 (4.36), 212 (4.37), 244 (4.11), 297 (4.50). H-NMR ((D6)DMSO):
11.7 (br. s, NH); 10.3 (br. s, NH); 8.28 (d, HꢁC(6)); 7.93 (d, 2 arom. H); 7.59 (d, 2 arom. H); 6.43 (d,
HꢁC(5)); 6.12 (dd, HꢁC(1’)); 5.30 (d, OHꢁC(3’)); 5.06 (dd, OHꢁC(5’)); 4.35 – 4.21 (m, HꢁC(3’),
CH2); 3.88 (m, HꢁC(4’)); 3.31 – 3.18 (m, CH2(5’)); 2.31 – 2.18 (m, 1 HꢁC(2’)); 2.10 – 1.99 (m,
1 HꢁC(2’)); 1.28 (t, Me). Anal. calc. for C19H22N4O7 (418.4): C 54.54, H 5.30, N 13.39; found: C 53.95,
H 5.30, N 13.29.
3’,5’-Di-O-acetyl-2’-deoxy-N4-[(fluorescein-5-ylamino)carbonyl]cytidine (¼ 3’,5’-Di-O-acetyl-2’-de-
oxy-N4-{[(3’,6’-dihydroxy-3-oxospiro[isobenzofuran-1(3H),9’-[9H]xanthen]-5-yl)amino]carbonyl}cyti-
dine; 15). As described for 5, with 5-aminofluorescein (14; 0.33 g, 0.9 mmol), 3 (0.6 g, 1.5 mmol), and
pyridine (50 ml). Workup with toluene (3 ꢀ 50 ml) and purification by CC (SiO2 (5 ꢀ 20 cm), CH2Cl2/
MeOH 9.5 :0.5 (150 ml), CH2Cl2/MeOH 9 :1 (150 ml), and CH2Cl2/MeOH 7:3 (1 l)). The main fraction
was concentrated, and the residue treated with EtOH, washed with Et2O, and dried in a vacuum
desiccator: 0.6 g (93%) of 15. Orange solid. UV (MeOH): 230 (4.64), 291 (4.36), 358 (sh, 3.23), 458 (sh,
3.82), 489 (3.92). 1H-NMR ((D6)DMSO): 11.52 (br. s, NH); 10.15 (br. s, NH, 2 OH); 8.18 (d, HꢁC(4iso));
8.00 (d, HꢁC(6)); 7.70 (dd, HꢁC(6iso)); 7.21 (d, HꢁC(7iso)); 6.65 – 6.45 (m, HꢁC(5), 6 arom. H (xan));
6.18 (dd, HꢁC(1’)); 5.22 (m, HꢁC(3’)); 4.27 (m, HꢁC(4’), CH2(5’)); 2.50 – 2.30 (m, CH2(2’)); 2.10 (s,
2 AcO). Anal. calc. for C34H28N4O12 · H2O (702.6): C 58.12, H 4.30, N 7.97; found: C 58.34, H 4.34, N 7.95.
2’-Deoxy-N4-[(fluorescein-5-ylamino)carbonyl]cytidine (¼2’-Deoxy-N4-{[(3’,6’-dihydroxy-3-oxo-
spiro[isobenzofuran-1(3H),9’-[9H]xanthen]-5-yl)amino]carbonyl}cytidine; 16). A soln. of 15 (0.45 g,
0.64 mmol) in sat. methanolic ammonia was stirred at r.t. for 15 h. Then K2CO3 (0.1 mmol) was added,
and stirring continued for another 24 h. The solvent was evaporated, the residue dissolved in phosphate
buffer (pH 7; 20 ml) followed by AcOH, and the resulting precipitate washed with little H2O, EtOH, and
Et2O, and dried: 0.25 g (68%) of 16. Orange solid. UV (MeOH): 210 (4.70), 238 (4.73), 289 (4.49), 347
(sh, 3.87), 467 (sh, 4.51), 4.92 (4.91). 1H-NMR ((D6)DMSO): 11.73 (br. s, NH); 10.35 (br. s, NH, 2 OH);
8.26 (d, HꢁC(4iso)); 8.18 (d, HꢁC(6)); 7.65 (dd, HꢁC(6iso)); 7.21 (d, HꢁC(7iso)); 6.65 – 6.45 (m, HꢁC(5),
6 arom. H (xan)); 6.13 (dd, HꢁC(1’)); 5.31 (d, OHꢁC(3’)); 5.15 (t, OHꢁC(5’)); 4.22 (m, HꢁC(3’)); 3.85
(m, HꢁC(4’)); 3.59 (m, CH2(5’)); 2.30 (m, 1 HꢁC(2’)); 2.08 (m, 1 HꢁC(2’)). Anal. calc. for
C30H24N4O10 · H2O (618.6): C 58.25, H 4.23, N 9.05; found: C 57.82, H 4.44, N 8.87.
2’-Deoxy-N4-[(fluorescein-5-ylamino)carbonyl]-3’,5’-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)cy-
tidine (¼2’-Deoxy-N4-{[(3’,6’-dihydroxy-3-oxospiro[isobenzofuran-1(3H),9’-[9H]xanthen]-5-yl)ami-
no]carbonyl}-3’,5’-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)cytidine; 17). As described for 5, with 4
(7.0 g, 12 mmol), 14 (2.6 g, 7.5 mmol), and pyridine (100 ml). Workup with toluene (3 ꢀ 80 ml) and
purification of the oil by CC (SiO2 (3 ꢀ 30 cm), toluene/AcOEt 1:1 (300 ml), toluene/AcOEt/MeOH
4.75 :4.75 :1 (600 ml), and toluene/AcOEt/MeOH 4.5 :4.5 :1 (600 ml)). The main fraction was
concentrated to 50 ml and the suspension kept overnight. The precipitate was dried under high vacuum:
5.8 g (92%) of 17. Orange solid. UV (MeOH/CH2Cl2 1:1): 219 (4.42), 231 (4.65), 291 (4.39), 371 (sh,
3.19), 455 (sh, 3.86), 485 (4.93). 1H-NMR ((D6)DMSO): 11.69 (br. s, NH); 10.08 (br. s, NH, 2 OH); 8.21
(d, HꢁC(4iso)); 8.03 (d, HꢁC(6)); 7.72 (dd, HꢁC(6iso))); 7.22 (d, HꢁC(7iso)); 6.65 – 6.40 (m, HꢁC(5), 6
arom. H (xan)); 6.03 (dd, HꢁC(1’)); 4.51 (m, HꢁC(3’)); 4.12 – 3.98 (m, CH2(5’)); 3.83 (m, HꢁC(4’));
2.39 (m, CH2(2’)); 1.10 – 0.95 (m, 4 Me2CH). Anal. calc. for C42H50N4O11Si2 · H2O (861.1): C 58.58, H 6.09,
N 6.50; found: C 58.74, H 6.08, N 6.62.
2’-Deoxy-N4-{{{3-{[2-(4-nitrophenyl)ethoxy]carbonyl}-4-{6-[2-(4-nitrophenyl)ethoxy]-3-oxo-3H-
xanthen-9-yl}-phenyl}amino}carbonyl}-3’,5’-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-diyl)cytidine (18). To
a mixture of 17 (4.7 g, 5.5 mmol), 2-(4-nitrophenyl)ethanol (4.6 g, 28 mmol), and diethyl azodicarbox-
ylate (1.93 g, 11 mmol) in abs. dioxane (100 ml) was added dropwise at 608 PPh3 in abs. dioxane (5 ml),
and the mixture was stirred for 1 h. The mixture was concentrated, the residue dissolved in CH2Cl2, the
soln. washed with 1% KH2PO4 soln. (2 ꢀ 50 ml) and H2O (2 ꢀ 50 ml), dried (Na2SO4), and concentrated,
and the residue purified by CC (SiO2 (6 ꢀ 30 cm), CH2Cl2 (100 ml), CH2Cl2/MeOH 195 :5 (600 ml),
CH2Cl2/MeOH 95 :5 (400 ml), CH2Cl2/MeOH 94 :6 (800 ml), and CH2Cl2/MeOH 93 :7 (400 ml)). The
residue of the product fraction was treated with MeOH (80 ml) and stirred for 1 h, and the precipitate
collected: 5.2 g (84%) of 18. Orange-red solid. UV(MeOH/CH2Cl2 1:1): 225 (4.75), 232 (4.76), 270 (sh,

Helvetica Chimica Acta – Vol. 92 (2009)
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4.68), 276 (4.69), 294 (sh, 4.60), 308 (sh, 4.45), 355 (4.03), 406 (sh, 3.98), 434 (sh, 4.29), 458 (4.44), 487
1
(4.32). H-NMR ((D6)DMSO): 11.63 (br. s, NH); 10.22 (br. s, NH); 8.24 (d, HꢁC(2arom. )); 8.16 (d, 2
arom. H o to NO2); 8.03 (d, 2 arom. H o to NO2, HꢁC(6)); 7.89 (dd, HꢁC(6arom. )); 7.63 (d, 2 arom. H m to
NO2); 7.36 (d, HꢁC(5arom. ), 2 arom. H m to NO2); 7.15 (d, HꢁC(xan); 6.88 – 6.79 (m, 3 HꢁC(xan)); 6.48
(d, HꢁC(5)): 6.34 – 6.14 (m, 2 HꢁC(xan)); 6.03 (m, HꢁC(1’)); 4.59 – 4.40 (m, HꢁC(3’), CH2(a)); 4.28
(t, CH2(a)); 4.05 (m, CH2(5’)); 3.80 (m, HꢁC(4’)); 3.21 (t, CH2(b)); 2.81 (t, CH2(b)); 2.40 (m, CH2(2’));
1.10 – 0.95 (m, 4 Me2CH). Anal. calc. for C58H63N6O15Si2 (1140.3): C 61.09, H 5.57, N 7.37; found: C 61.03,
H 5.74, N 7.55.
2’-Deoxy-N4-{{{3-{[2-(4-nitrophenyl)ethoxy]carbonyl}-4-{6-[2-(4-nitrophenyl)ethoxy]-3-oxo-3H-
xanthen-9-yl}phenyl}amino}carbonyl}cytidine (19). A soln. of 18 (2.5 g, 3.13 mmol) in THF (30 ml) was
stirred for 15 h in AcOH (3.1 ml) in the presence of Bu4NF · 3 H2O (1.68 g, 5.33 mmol). The soln. was
diluted with CH2Cl2 (200 ml) and washed with H2O (100 ml) whereby a part of the product separated out.
The solid was filtered off, and the org. phase dried (Na2SO4) and concentrated. Both solid fractions were
combined and treated with EtOH (30 ml) under stirring for 5 h. The solid was collected and dried: 1.8 g
(92%) of 19. Orange solid. UV(MeOH/CH2Cl2 9 :1): 227 (sh, 4.74), 232 (4.75), 271 (sh, 4.67), 276 (4.68),
1
294 (sh, 4.58), 306 (sh, 4.48), 354 (4.02), 406 (sh, 3.97), 433 (sh, 4.28), 459 (4.44), 487 (4.33). H-NMR
((D6)DMSO): 11.80 (br. s, NH); 10.38 (br. s, NH); 8.30 (d, HꢁC(2arom.)); 8.16 (d, 2 arom. H o to NO2);
8.03 (d, 2 arom. H o to NO2, HꢁC(6)); 7.89 (d, HꢁC(6arom. )); 7.61 (d, 2 arom. H m to NO2); 7.37 (d,
HꢁC(5arom. ), 2 arom. H m to NO2); 7.16 (d, 1 HꢁC(xan); 6.81 (m, 3 HꢁC(xan)); 6.46 (d, HꢁC(5)): 6.33
(d, 1 HꢁC(xan); 6.17 (m, HꢁC(1’), 1 HꢁC(xan)); 5.30 (d, OHꢁC(3’)); 4.45 (t, CH2(a)); 4.23 (t,
HꢁC(3’), CH2(a)); 3.88 (m, HꢁC(4’)); 3.60 (m, CH2(5’)); 3.21 (t, CH2(b)); 2.83 (t, CH2(b)); 2.28 (m,
1 HꢁC(2’)); 2.10 (m, 1 HꢁC(2’)). Anal. calc. for C46H38N6O14 (898.8): C 61.47, H 4.26, N 9.34; found: C
61.24, H 4.49, N 9.20.
2’-Deoxy-5’-O-(4,4’-dimethoxytrityl)-N4-{{{3-{[2-(4-nitrophenyl)ethoxy]carbonyl}-4-{6-[2-(4-nitro-
phenyl)ethoxy]-3-oxo-3H-xanthen-9-yl}phenyl}amino}carbonyl}cytidine (20). A soln. of 19 (6.3 g,
7 mmol) in dry pyridine was concentrated, and 2 ꢀ the residue dissolved in pyridine, and the soln.
concentrated. The residue was dissolved in dry pyridine (200 ml), and then 4,4’-dimethoxytrityl chloride
(2.86 g, 8.4 mmol) was added, and the mixture stirred at r.t. overnight. The soln. was diluted with CH2Cl2
(400 ml) and extracted with sat. NaHCO3 soln. (3 ꢀ 100 ml). The org. phase was dried (Na2SO4) and
concentrated and the residue subject to FC (SiO2, 150 g), CH2Cl2/MeOH 99 :1 (300 ml), CH2Cl2/MeOH
98 :2 (300 ml), CH2Cl2/MeOH 97:3 (300 ml), CH2Cl2/MeOH 95 :5 (600 ml), and CH2Cl2/MeOH 96 :4
(400 ml)). The residue of the product fractions was dried: 7.0 g (83%) of 20. Orange-red solid. UV
(MeOH/CH2Cl2 1:1): 210 (4.68), 227 (sh, 4.87), 232 (4.88), 270 (sh, 4.70), 275 (4.71), 294 (sh, 4.60), 306
(sh, 4.50), 354 (4.04), 406 (sh, 3.98), 434 (sh, 4.29), 459 (4.44), 487 (4.24). 1H-NMR ((D6)DMSO): 11.80
(br. s, NH); 10.45 (br. s, NH); 8.35 (d, HꢁC(2arom.)); 8.13 (d, 2 arom. H o to NO2, HꢁC(6)); 8.06 (d, 2
arom. H o to NO2); 7.89 (d, HꢁC(6arom.)); 7.60 (d, 2 arom. H m to NO2); 7.41 – 7.20 (m, 12 H, HꢁC(5arom. ),
2 arom. H m to NO2, 4 arom. H m to MeO, 5 arom. H); 7.13 (d, HꢁC(xan)); 6.96 – 6.78 (m, 3 HꢁC(xan),
4 arom. H o to MeO); 6.37 (d, HꢁC(5), HꢁC(xan)); 6.15 (m, HꢁC(1’), HꢁC(flu)); 5.31 (d,
OHꢁC(3’)); 4.41 – 4.25 (m, HꢁC(3’), 2 CH2(a)); 3.99 (m, HꢁC(4’)); 3.71 (s, 2 MeO); 3.31 – 3.15 (m,
CH2(5’), CH2(b)); 2.82 (t, CH2(b)); 2.50 – 2.10 (m, CH2(2’)). 31P-NMR ((D6)DMSO): 147.72; 147.63.
Anal. calc. for C67H56N6O16 (1201.2): C 66.99, H 4.70, N 6.99; found: C 66.87, H 4.91, N 6.93.
2’-Deoxy-5’-O-(4,4’-dimethoxytrityl)-N4-{{{3-{[2-(4-nitrophenyl)ethoxy]carbonyl}-4-{6-[2-(4-nitro-
phenyl)ethoxy]-3-oxo-3H-xanthen-9-yl}phenyl}amino}carbonyl}cytidine 3’-[2-(4-Nitrophenyl)ethyl N,N-
Diisopropylphosphoramidite] (21). To a soln. of 20 (0.8 g 0.67 mmol) and 1H-tetrazole (38 mg,
0.54 mmol) in CH2Cl2 (10 ml) under N2 was added MeCN (10 ml) and then 2-(4-nitrophenyl)ethyl
N,N,N’,N’-tetraisopropylphosphorodiamidite [37] (0.8 g, 2 mmol). The mixture was stirred at r.t. for 3 h,
then diluted with CH2Cl2 (100 ml), and washed with sat. NaHCO3 soln. The org. layer was dried
(Na2SO4) and concentrated, and the residue purified by CC (SiO2 (3 ꢀ 10 cm), hexane/acetone 1:1
(200 ml), hexane/acetone 1:3 (200 ml), and hexane/acetone 1:4 (400 ml)). The residue of the product
fractions was dissolved in little CH2Cl2 and slowly added dropwise into abs. Et2O. The resulting
precipitate was collected under N2 and dried under high vacuum: 0.818 g (82%) of 21. Orange-red
powder. UV (MeOH/CH2Cl2 1:1): 216 (4.68), 235 (4.82), 275 (4.77), 293 (sh, 4.65), 355 (sh, 4.04), 415

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Helvetica Chimica Acta – Vol. 92 (2009)
(4.05), 436 (sh, 4.29), 459 (4.44), 487 (4.33). 1H-NMR ((D6)DMSO): 11.79 (br. s, NH); 10.41 (br. s, NH);
8.30 (d, HꢁC(2arom. )); 8.20 – 8.00 (d, 4 arom. H o to NO2, HꢁC(6)); 7.90 (d, HꢁC(6arom.)); 7.62 (d, 2 arom.
H m to NO2); 7.50 – 7.19 (m, HꢁC(5arom. ), 2 arom. H m to NO2, 4 arom. H m to MeO, 5 arom. H); 7.14 (s,
1 HꢁC(xan); 6.90 – 6.78 (m, 3 HꢁC(xan), 4 arom. H o to MeO); 6.33 (2d, HꢁC(5), HꢁC(xan)); 6.15
(m, HꢁC(1’), HꢁC(xan)); 4.41 (t, HꢁC(3’), CH2(a)); 4.27 (t, CH2(a)); 4.03 (m, HꢁC(4’)); 3.72 (m,
CH2OꢁP); 3.69 (s, 2 MeO); 3.41 (m, 2 Me2CH); 3.21 (m, CH2(5’), CH2(b)); 3.00 – 2.79 (m, 2 CH2(b));
2.49 – 2.10 (m, CH2(2’)); 1.10 – 0.90 (m, 2 Me2CH). Anal. calc. for C81H77N8O19P (1497.5): C 64.97, H 5.18,
N 7.48; found: C 63.82, H 5.51, N 7.18.
2’-Deoxy-5’-O-(4,4’-dimethoxytrityl)-N4-{{{3-{[2-(4-nitrophenyl)ethoxy]carbonyl}-4-{6-[2-(4-nitro-
phenyl)ethoxy]-3-oxo-3H-xanthen-9-yl}phenyl}amino}carbonyl}cytidine 3’-[2-Cyanoethyl N,N-Diisopro-
pylphosphoramidite] (22). As described for 21, with 20 (0.8 g 0.67 mmol), 1H-tetrazole (38 mg,
0.54 mmol), CH2Cl2 and MeCN under N2, and 2-cyanoethyl N,N,N’,N’-tetraisopropylphosphorodiami-
dite [38][39] (0.6 g, 2 mmol): 0.75 g (80%) of 22. Orange-red solid. UV (MeOH/CH2Cl2 1:1): 217 (4.65),
234 (4.81), 276 (4.69), 294 (sh, 4.57), 356 (sh, 3.98), 409 (sh, 3.95), 436 (sh, 4.28), 459 (4.44), 487 (4.34).
1H-NMR ((D6)DMSO): 11.75 (br. s, NH); 10.40 (br. s, NH); 8.31 (d, HꢁC(2arom. )); 8.18 (d, 2 arom. H o to
NO2, HꢁC(6)); 8.04 (d, 2 arom. H o to NO2); 7.88 (d, HꢁC(6arom. )); 7.62 (d, 2 arom. H m to NO2); 7.41 –
7.19 (m, HꢁC(5arom.), 2 arom. H m to NO2, 4 arom. H m to MeO, 3 arom. H); 7.16 (s, 1 HꢁC(xan); 6.92 –
6.80 (m, 3 HꢁC(xan), 4 arom. H o to MeO); 6.31 (d, HꢁC(5), 1 HꢁC(xan)); 6.13 (m, HꢁC(1’),
1 HꢁC(xan)); 4.55 (t, HꢁC(3’)); 4.40 (t, CH2(a)); 4.24 (t, CH2(a)); 4.12 (m, HꢁC(4’)); 3.72 (s, 2 MeO);
3.70 – 3.40 (m, CH2OꢁP, 2 M e 2CH, CH2(5’)); 3.22 (m, CH2(b)); 2.84 – 2.60 (m, CH2CN, CH2(b)); 2.37
(m, CH2(2’)); 1.16 – 0.95 (m, 2 Me2CH). 31P-NMR ((D6)DMSO): 148.62; 148.31. Anal. calc. for
C76H73N8O17P (1401.4): C 65.14, H 5.25, N 7.99; found: C 64.90, H 5.71, N 7.34.
2’-Deoxy-N4,3’-O-bis{[2-(4-nitrophenyl)ethoxy]carbonyl}cytidine (23). A soln. of 2’-deoxy-5’-O-
(monomethoxytrityl)-N4,3’-O-bis{[2-(4-nitrophenyl)ethoxy]carbonyl}cytidine (0.885 g, 1 mmol) in 1%
TsOH in CH2Cl2/MeOH 4 :1 (40 ml) was stirred at r.t. for 30 min. The mixture was diluted with CHCl3
(60 ml) and washed with phosphate buffer (pH 7; 3 ꢀ 100 ml), the org. phase dried (Na2SO4) and
concentrated, and the residue purified by CC (SiO2 (2.5 ꢀ 25 cm), CH2Cl2, CHCl3/MeOH 100 :1, and
CHCl3/MeOH 100 :2). The residue of the product fractions was dried under high vacuum: 0.57 g (93%)
of 23. Colorless solid foam. Recrystallization from AcOEt gave crystals with m.p. 143 – 1458. UV
(MeOH): 247 (4.36), 273 (4.39). 1H-NMR ((CDCl3): 8.40 (br. s, NH); 8.20 (d, HꢁC(6)); 8.12 (d, 2 arom.
H o to NO2); 8.10 (d, 2 arom. H o to NO2); 7.38 (d, 2 arom. H m to NO2); 7.34 (d, 2 arom. H m to NO2);
7.15 (d, HꢁC(5)); 6.16 (m, HꢁC(1’)); 5.27 (d, HꢁC(3’)); 4.37 (t, 2 CH2(a)); 4.20 (br. s, HꢁC(4’)); 3.8
(m, CH2(5’)); 3.75 (br. s, OHꢁC(5’)); 3.05 (t, 2 CH2(b)); 2.64 (m, 1 HꢁC(2’)); 2.37 (m, 1 HꢁC(2’)).
Anal. calc. for C27H27N5O12 (613.5): C 52.86, H 4.44, N 11.41; found: C 52.86, H 4.50, N 11.32.
2’-Deoxy-N4,5’-O-bis{[2-(4-nitrophenyl)ethoxy]carbonyl}cytidine (24). A soln. of 2’-deoxy-N4-{[2-
(4-nitrophenyl)ethoxy]carbonyl}cytidine [40] (4.1 g, 9.7 mmol) in abs. pyridine (40 ml) was cooled to
ꢁ 258, and then 2-(4-nitrophenyl)ethyl carbonochloridate (3.36 g, 14.6 mmol) in abs. CH2Cl2 (40 ml) was
added dropwise. After stirring for 3 h at ꢁ 208, the mixture was diluted with CH2Cl2 (300 ml) and washed
with H2O, the org. layer dried (Na2SO4) and concentrated, and the residue purified by CC (SiO2 (4.5 ꢀ
20 cm), CH2Cl2 (300 ml), CH2Cl2/MeOH 98 :2 (600 ml), and CH2Cl2/MeOH 96 :4 (600 ml)). The
residue of the product fractions was dried under high vacuum: 3.6 g (61%) of 24. Colorless solid. UV
(MeOH): 213 (4.55), 246 (sh, 4.39), 273 (4.32). 1H-NMR ((CDCl3): 8.42 (br. s, NH); 8.15 (dd, 4 arom. H
o to NO2); 7.99 (d, HꢁC(6)); 7.37 (d, 4 arom. H m to NO2); 7.11 (d, HꢁC(5)); 6.25 (m, HꢁC(1’)); 4.70
(br. s, OHꢁC(3’)); 4.41 – 4.20 (m, HꢁC(3’), HꢁC(4’), CH2(5’), 2 CH2(a)); 3.09 (t, 2 CH2(b)); 2.70 (m,
1 HꢁC(2’)); 2.08 (m, 1 HꢁC(2’)). Anal. calc. for C27H27N5O12 (613.5): C 52.86, H 4.44, N 11.41; found: C
53.03, H 4.57, N 10.94.
2’-Deoxy-N4,5’-O-bis{[2-(4-nitrophenyl)ethoxy]carbonyl}cytidine 3’-[2-(4-Nitrophenyl)ethyl N,N-
Diisopropylphosphoramidite] (25). As described for 21, with 24 (1.0 g 1.6 mmol), 1H-tetrazole (70 mg,
1 mmol), CH2Cl2 (20 ml), MeCN (10 ml) and 2-(4-nitrophenyl)ethyl N,N,N’,N’-tetraisopropylphosphoro-
diamidite [37] (1.4 g, 3.6 mmol), for 6 h. After CC (SiO2 (3 ꢀ 15 cm), hexane/AcOEt 1:1 (200 ml),
hexane/AcOEt 1:2 (100 ml), and hexane/AcOEt 1:4 (400 ml)), the residue of the product fractions was
dried under high vacuum: 1.2 g (81%) of 25. Colorless powder. UV (MeOH): 203 (4.54), 211 (sh, 4.46),
1
250 (sh, 4.26), 272 (4.34). H-NMR (CDCl3): 8.20 – 8.15 (m, 6 arom. H o to NO2); 7.43 (d, HꢁC(6));

Helvetica Chimica Acta – Vol. 92 (2009)
2733
7.44 – 7.36 (m, 6 arom. H m to NO2); 7.13 (br. s, NH); 7.08 (d, HꢁC(5)); 6.19 (m, HꢁC(1’)); 4.49 – 4.19 (m,
HꢁC(3’), HꢁC(4’), CH2(5’), 2 CH2(a)); 3.90 – 3.71 (m, CH2OꢁP); 3.60 – 3.48 (m, 2 Me2CH); 3.16 – 2.99
(m, 3 CH2(b)); 2.65 (m, 1 HꢁC(2’)); 2.03 (m, 1 HꢁC(2’)); 1.13 – 1.04 (m, 2 Me2CH). 31P-NMR: 148.79;
148.66. Anal. calc. for C41H48N7O15P (909.8): C 54.12, H 5.32, N 10.77; found: C 53.60, H 5.33, N 10.45.
2’-Deoxy-5’-O-(4,4’-dimethoxytrityl)-N4-{{{3-{[2-(4-nitrophenyl)ethoxy]carbonyl}-4-{6-[2-(4-nitro-
phenyl)ethoxy]-3-oxo-3H-xanthen-9-yl}phenyl}amino}carbonyl}-OP-[2-(4-nitrophenyl)ethyl]cytidylyl-
3’ ! 5’)-2’-deoxy-N4,3’-O-bis{[2-(4-nitrophenyl)ethoxy]carbonyl}cytidine (26). A mixture of 23 (0.35 g,
0.57 mmol) and 1H-tetrazole (0.1 g, 1.5 mmol) was dissolved in CH2Cl2 (20 ml) and MeCN (10 ml) under
N2. Then 21 (0.57 g, 0.38 mmol) was added, and the mixture was stirred at r.t. for 3 h, followed by
treatment with I2 (0.5 g) in CH2Cl2/pyridine/H2O 1:3 :1 (4 ml) for 30 min. The mixture was diluted with
CH2Cl2 (150 ml) and the soln. extracted with a sat. soln. of Na2S2O3/NaCl (3 ꢀ 100 ml). The aq. phases
were reextracted with CH2Cl2 (50 ml). The combined org. extract was dried (Na2SO4) and concentrated,
3 ꢀ the residue dissolved in toluene (100 ml), and the soln. concentrated, and the residue purified by CC
(SiO2 (1 ꢀ 10 cm), CH2Cl2 (100 ml), CH2Cl2/MeOH 98 :2 (100 ml), CH2Cl2/MeOH 96 :4 (200 ml), and
CH2Cl2/MeOH 95 :5 (100 ml)). The residue of the product fractions was dissolved in little CH2Cl2/
MeOH 1:1, and this soln. slowly added dropwise to cold (08) MeOH (25 ml). The precipitate was washed
with MeOH and Et2O and dried under high vacuum: 0.585 g (76%) of 26. Orange-red solid. UV
(MeOH): 224 (4.97), 275 (4.91), 348 (sh, 3.99), 409 (sh, 3.96), 439 (sh, 4.28), 460 (4.44), 488 (4.33).
1H-NMR ((D6)DMSO): 11.71 (s, NH); 10.80 (s, NH); 10.40 (s, NH); 8.29 (s, HꢁC(2arom. )); 8.19 – 7.95 (m,
2 HꢁC(6), 10 arom. H o to NO2); 7.89 (d, HꢁC(6arom. )); 7.65 – 7.18 (m, 21 H, 10 arom. H m to NO2, 9
arom. H, HꢁC(5arom. ), HꢁC(5’xan)); 6.96 (d, HꢁC(5)); 6.90 – 6.80 (m, HꢁC(1’xan), HꢁC(7’xan),
HꢁC(8’xan)), 4 arom. H o to MeO); 6.18 – 6.01 (m, 2 HꢁC(1’), HꢁC(3’xan), HꢁC(4’xan)); 5.83 (m,
HꢁC(5), HꢁC(5’xan)); 5.03 (m, HꢁC(3’)); 4.91 (m, HꢁC(3’)); 4.50 – 4.10 (m, 14 H, 5 CH2(a),
2 HꢁC(4’), CH2(5’)); 3.70 (s, 2 MeO); 3.30 – 2.83 (m, 5 CH2(b), CH2(5’)); 2.50 – 2.20 (m, 2 CH2(2’)). 31P-
NMR ((D6)DMSO): ꢁ 1.51. Anal. calc. for C102H89N12O32P (2025.9): C 60.47, H 4.43, N 8.30; found: C
60.26, H 4.58, N 8.23.
2’-Deoxy-N4-{{{3-{[2-(4-nitrophenyl)ethoxy]carbonyl}-4-{6-[2-(4-nitrophenyl)ethoxy]-3-oxo-3H-
xanthen-9-yl}phenyl}amino}carbonyl}-OP-[2-(4-nitrophenyl)ethyl]cytidylyl-(3’ ! 5’)-2’-deoxy-N4,3’-O-
bis{[2-(4-nitrophenyl)ethoxy]carbonyl}cytidine (27). To 1% TsOH in CH2Cl2/MeOH 4 :1 (20 ml) was
added 26 (0.48 g, 0.24 mmol) and stirred at r.t. for 1 h. The mixture was diluted with CH2Cl2 (150 ml), and
the soln. extracted with sat. NaHCO3 soln. (3 ꢀ 80 ml). The aq. extracts were reextracted with CH2Cl2
(50 ml). Then the combined org. phase was dried (Na2SO4) and concentrated and the resulting solid
purified by CC (SiO2 (1.5 ꢀ 8 cm), CH2Cl2 (50 ml), CH2Cl2/MeOH 98.5 :1:5 (50 ml), CH2Cl2/MeOH
96 :4 (50 ml), and CH2Cl2/MeOH 94 :6 (100 ml)). The residue of the product fractions was dissolved in
little CH2Cl2/MeOH 1:1, and the soln. slowly and dropwise added to cold (08) MeOH (25 ml). The
precipitate was washed with Et2O and dried under high vacuum: 0.35 g (85%) of 27. Orange-red powder.
UV (MeOH): 224 (4.85), 275 (4.88), 352 (4.01), 407 (sh, 3.94), 438 (sh, 4.28), 459 (4.44), 487 (4.33).
1H-NMR ((D6)DMSO): 11.75 (s, NH); 10.80 (s, NH); 10.49 (s, NH); 8.28 (s, HꢁC(2arom. )); 8.23 – 7.99 (m,
2 HꢁC(6), 10 arom. H o to NO2); 7.88 (d, HꢁC(6arom. )); 7.65 – 7.48 (m, 8 arom. H m to NO2); 7.40 – 7.30
(m, 2 arom. H m to NO2, HꢁC(5arom. )); 7.17 (s, HꢁC(5’xan)); 6.98 (d, HꢁC(5)); 6.83 (m, HꢁC(1’xan),
HꢁC(7’xan), HꢁC(8’xan)); 6.41 (d, HꢁC(5)); 6.36 (d, HꢁC(2’xan)); 6.19 – 6.00 (m, 2 HꢁC(1’),
HꢁC(4’xan)); 5.19 (t, OHꢁC(5’)); 5.10 (m, HꢁC(3’)); 4.88 (m, HꢁC(3’)); 4.50 – 4.10 (m, 5 CH2(a),
2 HꢁC(4’), CH2(5’)); 3.58 (m, CH2(5’)); 3.30 – 2.80 (m, 5 CH2(b)); 2.43 – 2.21 (m, 2 CH2(2’)). 31P-NMR
((D6)DMSO): ꢁ 1.53; ꢁ 1.42. Anal. calc. for C81H71N12O30P (1723.5): C 56.45, H 4.15, N 9.75; found: C
56.02, H 4.31, N 9.69.
2’-Deoxy-N4,5’-O-bis{[2-(4-nitrophenyl)ethoxy]carbonyl}-OP-[2-(4-nitrophenyl)ethyl]cytidylyl-
(3’ ! 5’)-2’-deoxy-N4-{{{3-{[2-(4-nitrophenyl)ethoxy]carbonyl}-4-{6-[2-(4-nitrophenyl)ethoxy]-3-oxo-
3H-xanthen-9-yl}phenyl}amino}carbonyl}-OP-[2-(4-nitrophenyl)ethyl]cytidylyl-(3’ ! 5’)-2’-deoxy-N4,3’-
O-bis{[2-(4-nitrophenyl)ethoxy]carbonyl}cytidine (28). A mixture of 27 (0.22 g, 0.13 mmol) and 1H-
tetrazole (95 mg, 1.35 mmol) in abs. CH2Cl2 (10 ml) was concentrated, and 2 ꢀ the residue dissolved in
abs. toluene (10 ml), and the soln. concentrated. The residue was dissolved in CH2Cl2 (15 ml) and MeCN
(8 ml), and under N2, 25 (0.45 g, 0.5 mmol) was added and the mixture stirred overnight. After treatment
with I2 (0.5 g) in CH2Cl2/pyridine/H2O 1:3 :1 (4 ml), the mixture was stirred for 30 min, diluted with

2734
Helvetica Chimica Acta – Vol. 92 (2009)
CH2Cl2 (150 ml), and extracted with a sat. soln. of Na2S2O3/NaCl (3 ꢀ 100 ml). The aq. phases were
reextracted with CH2Cl2 (50 ml). The combined org. extract was dried (Na2SO4) and concentrated, and
the residue 3 ꢀ dissolved in toluene (50 ml), and the soln. concentrated. The residue was dissolved in
CH2Cl2 (8 ml) and MeOH (2 drops) and purified by CC (SiO2 (1 ꢀ 20 cm), CH2Cl2 (100 ml), CH2Cl2/
MeOH 98 :2 (100 ml), CH2Cl2/MeOH 96 :4 (200 ml), CH2Cl2/MeOH 95 :5 (300 ml), and CH2Cl2/MeOH
93 :7)). The residue of the product fractions was dissolved in little CH2Cl2/MeOH and then dropwise
added to MeOH (25 ml) with stirring. The precipitate was washed with little Et2O and dried under high
vacuum: 0.27 g (83%) of 28. Orange-red solid. UV (MeOH): 216 (4.86), 274 (5.02), 350 (sh, 4.01), 408
1
(sh, 3.93), 436 (sh, 4.26), 459 (4.43), 484 (4.32). H-NMR ((D6)DMSO): 11.70 (s, NH); 10.80 (s, NH);
10.38 (s, NH); 8.27 (s, HꢁC(2arom. )); 8.22 – 7.90 (m, 3 HꢁC(6), 16 arom. H o to NO2); 7.86 (d,
HꢁC(6arom. )); 7.70 – 7.30 (m, 16 arom. H m to NO2, HꢁC(5arom.)); 7.19 (s, HꢁC(5’xan)); 6.98 (m,
2 HꢁC(5)); 6.83 (m, HꢁC(1’xan), HꢁC(7’xan), HꢁC(8’xan)); 6.40 (m, HꢁC(5), HꢁC(2’xan)); 6.12 (m,
3 HꢁC(1’), HꢁC(4’)); 5.10 (m, HꢁC(3’)); 4.92 (m, 2 HꢁC(3’)); 4.45 – 4.20 (m, 8 CH2(a), 3 HꢁC(4’),
3 CH2(5’)); 3.26 (t, CH2(b)); 3.12 (m, 6 CH2(b)); 2.81 (t, CH2(b)); 2.45 – 2.20 (m, 3 CH2(2’)). 31P-
NMR((D6)DMSO): ꢁ 1.48; ꢁ 1.45; ꢁ 1.36. Anal. calc. for C113H104N18O46P2 (2512.0): C 54.03, H 4.17, N
10.04; found: C 53.81, H 4.19, N 9.83.
2’-Deoxycytidylyl-(3’ ! 5’)-2’-deoxy-N4-[(fluorescein-5-ylamino)carbonyl]cytidylyl-(3’ ! 5’)-2’-de-
oxycytidine Ammonium Salt (1:2) (29). A soln. of 28 (0.1 g, 0.039 mmol) in abs. pyridine (20 ml) was
concentrated, the residue again dissolved in abs. pyridine (20 ml), DBU (1.4 ml, 9.4 mmol) added, and
then the mixture stirred at r.t. for 3 d. The mixture was neutralized with AcOH and concentrated. The
residue was dissolved in H2O (10 ml), the soln. extracted with CH2Cl2 (2 ꢀ 5 ml), and the aq. layer
subjected to ion-exchange CC (DEAE-Sephadex A-25 (HCOꢁ3 form, 1.5 ꢀ 60 cm), H2O (200 ml), then
linear gradient of 0 – 0.3m triethylammonium hydrogencarbonate buffer (pH 7.5, 5 l)). The product
fractions eluted with 0.24 – 0.26m buffer, and their residue was several times dissolved in MeOH and the
soln. evaporated. The conversion into the ammonium salt was achieved by dissolving the residue in little
ammonia and purifying by paper chromatography (six large paper sheets (30 ꢀ 52 cm; Schleicher &
Schuell), iPrOH/25% ammonia/H2O 1:3 :1). The product bands were cut out and extracted with dilute
ammonia. The combined extract was concentrated: 2200 OD490 (65%, 0.025 mmol) of 29. Orange-red
solid. HPLC (RP-18, system A followed by B and C): tR 16.8 min (detection at 260 and 480 nm).
1H-NMR (D2O): 8.04 (d, HꢁC(6)); 7.90 (s, HꢁC(2arom. )); 7.73 (d, HꢁC(6)); 7.61 (d, HꢁC(6)); 7.43 (d,
HꢁC(6arom. )); 7.21 (d, HꢁC(5arom. )); 7.09 (d, HꢁC(5)); 7.00 (d, HꢁC(5)); 6.53 (m, HꢁC(2’),
HꢁC(7’xan)); 6.42 (s, HꢁC(4’xan), HꢁC(6’xan)); 6.22 (d, HꢁC(5)); 6.15 (dd, HꢁC(1’xan)); 5.98 (dd,
HꢁC(1’)); 5.89 (m, HꢁC(1’xan), HꢁC(5’xan)); 5.71 (d, HꢁC(5)); 4.39 (m, HꢁC(3’)); 4.21 (m,
2 HꢁC(3’)); 4.05 – 3.90 (m, 3 CH2(5’)); 3.63 (m, 3 HꢁC(4’)); 2.60 – 2.00 (m, 3 CH2(2’)).
2’-Deoxy-5’-O-(4,4’-dimethoxytrityl)-N4-{[2-(4-nitrophenyl)ethoxy]carbonyl}cytidine 3’-(Hydrogen
Butanedioate) (¼1-{Tetrahydro-5-{4-{{[2-(4-nitrophenyl)ethoxy]carbonyl}amino}-2-oxopyrimidin-
1(2H)-yl}-2-{[bis(4-methoxyphenyl)phenylmethoxy]methyl}furan-3-yl} Hydrogen Butanoedioate; 30).
A soln. of 2’-deoxy-5’-O-(4,4’-dimethoxytrityl)-N4-{[2-(4-nitrophenyl)ethoxy]carbonyl}cytidine [41]
(0.723 g, 1 mmol), succinic anhydride (0.2 g, 2 mmol), and N,N-dimethylpyridin-4-amine (0.16 g,
1.3 mmol) in abs. CH2Cl2 (5 ml) was stirred until the starting material had disappeared. The mixture
was diluted with CH2Cl2 (50 ml), the soln. extracted subsequently with cold 10% aq. citric acid (30 ml),
followed by sat. NaHCO3 soln. (30 ml) and H2O (30 ml). The aq. phases were reextracted with CH2Cl2.
The combined org. phase was dried (Na2SO4) and concentrated, and the residue dried under high
vacuum: 0.658 g (80%) of 30. UV (MeOH): 214 (4.48), 236 (4.45), 275 (4.17), 282 (sh, 4.17). 1H-NMR
(CDCl3): 8.25 – 8.15 (m, 2 arom. H o to NO2, NH, HꢁC(6)); 7.45 – 7.12 (m, 11 arom. H), 6.95 (d,
HꢁC(5)); 6.85 (m, 4 arom. H o to MeO); 6.24 (dd, HꢁC(1’)); 4.52 (m, HꢁC(3’)); 4.40 (t, OCH2CH2);
4.26 (m, HꢁC(4’)); 3.78 (s, MeO); 3.45 – 3.35 (m, CH2(5’)); 3.09 (t, OCH2CH2); 2.85 (m, 1 HꢁC(2’));
2.70 – 2.50 (m, OCCH2CH2CO); 2.35 – 2.20 (m, 1 HꢁC(2’)). Anal. calc. for C43H42N4O13 (822.8): C 62.77,
H 5.15, N 6.81; found: C 62.62, H 4.95, N 6.60.
5’-d(CFluCCG GCC CGC)-3’ DNA (33). The automated solid-phase syntheses of the various
oligodeoxyribonucleotides were performed in a DNA synthesizer. The solid phase was glyceryl-CPG
(500 ꢃ) from Fluka which was activated by phenyl 1H-tetrazole-1-carboxylate and coupled with N1,N6-
dimethylhexane-1,6-diamine [42]. Then 30 was attached and led to a loading of 14 mmol/g. This solid

Helvetica Chimica Acta – Vol. 92 (2009)
2735
support (43 mg, 0.6 mmol) was applied in the synthesizer for chain elongation with 31 [35], 32 [35], and 22
as 0.095m soln. in CH2Cl2 in the usual manner. The overall yield was 89.3% which corresponds to a
stepwise yield of 98.4%. The isolation of the oligomer was achieved by treatment of the solid-support
material first with DBU to deprotect the ce, npe, and npeoc groups, followed by washing and finally
treatment with ammonia to cleave 33 from the support. HPLC (A ¼ 0.1m Et4N(OAc), B ¼ Et4N(OAc)/
MeCN 1:1; elution: 0 – 2 min 95% A þ 5% B; 2 – 32 min 60% A þ 40% B; 32 – 52 min 100% B): tR
28.2 min.
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Received May 25, 2009