Nucleotides. Part LXXVIII: Double labeling of nucleosides and nucleotides

Article abstract of DOI:10.1002/hlca.201000253

Several N(-hydroxyalkyl)-2,4-dinitroanilines were transformed into their phosphoramidites (see 5 and 6 in Scheme 1) in view of their use as fluorescence quenchers, and modified 2-aminobenzamides (see 9, 10, 18, and 19 in Scheme 1) were applied in model re

Full text of DOI:10.1002/hlca.201000253

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Nucleotides
Part LXXVIII1)
Double Labeling of Nucleosides and Nucleotides
by Thomas Maier and Wolfgang Pfleiderer*
Fachbereich Chemie, Universitꢀt Konstanz, Postfach 5560, D-78457 Konstanz
Dedicated to Prof. Albert Eschenmoser on the occasion of his 85th birthday and in admiration of his
outstanding scientific achievements
Several N(-hydroxyalkyl)-2,4-dinitroanilines were transformed into their phosphoramidites (see 5
and 6 in Scheme 1) in view of their use as fluorescence quenchers, and modified 2-aminobenzamides (see
9, 10, 18, and 19 in Scheme 1) were applied in model reactions as fluorophors to determine the relative
fluorescence quantum yields of the 3’-Aba and 5’-Dnp-3’-Aba conjugates (Aba ¼ aminobenzamide,
Dnp ¼ dinitroaniline). Thymidine was alkylated with N-(2-chloroethyl)-2,4-dinitroaniline (24) to give 25
which was further modified to the building blocks 27 and 28 (Scheme 3). The 2-amino group in 29 was
transformed by diazotation into the 2-fluoroinosine derivative 30 used as starting material for several
reactions at the pyrimidine nucleus (! 31, 33, and 35; Scheme 4). The 3’,5’-di-O-acetyl-2’-deoxy-N2-
[(dimethylamino)methylene]guanosine (37) was alkylated with methyl and ethyl iodide preferentially at
N(1) to 43 and 44, and similarly reacted N-(2-chloroethyl)-2,4-dinitroaniline (24) to 38 and the N-(2-
iodoethyl)-N-methyl analog 50 to 53 (Scheme 5). The 2’-deoxyguanosine derivative 53 was transformed
into 3’,5’-di-O-acetyl-2-fluoro-1-{2-[(2,4-dinitrophenyl)methylamino]ethyl}inosine (54; Scheme 5) which
reacted with 2,2’-[ethane-1,2-diylbis(oxy)]bis[ethanamine] to modify the 2-position with an amino spacer
resulting in 56 (Scheme 6). Attachment of the fluorescein moiety 55 at 56 via a urea linkage led to the
doubly labeled 2’-deoxyguanosine derivative 57 (Scheme 6). Dimethoxytritylation to 58 and further
reaction to the 3’-succinate 59 and 3’-phosphoramidite 60 afforded the common building blocks for the
oligonucleotide synthesis (Scheme 6). Similarly, 30 reacted with N-(2-aminoethyl)-2,4-dinitroaniline (61)
thus attaching the quencher at the 2-position to yield 62 (Scheme 7). The amino spacer was again
attached at the same site via a urea bridge to form 64. The labeling of 64 with the fluorescein derivative
55 was straigthforward giving 65. and dimethoxytritylation to 66 and further phosphitylation to 67
followed known procedures (Scheme 7). Several oligo-2’-deoxynucleotides containing the doubly
labeled 2’-deoxyguanosines at various positions of the chain were formed in a DNA synthesizer, and their
fluorescence properties and the Tms in comparison to their parent duplexes were measured (Tables 1 – 5).
Introduction. – Since 1992 [2], it is known from peptide chemistry that the
fluorescence of 2-aminobenzamides can intramolecularly be quenched by 2,4-
dinitroaniline residues. This fact was applied to a nucleotide dimer [3] carrying at
the 5’-end a dinitroaniline and at the 3’-end a 2-aminobenzamide residue. Fluorescence
could only be observed after splitting of the nucleotide linkage. To evaluate the
capacity of 2-aminobenzamides as fluorescence markers for oligonucleotides and to
study the interaction with 2,4-dinitroaniline derivatives, a series of labeled oligo-2’-
deoxynucleotides were prepared by solid-phase synthesis.
1
)
Part LXXVII: [1].
ꢁ 2010 Verlag Helvetica Chimica Acta AG, Zꢂrich

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Synthesis. – N-Substituted 2,4-dinitroanilines 2 – 4 were easily available from 1-
chloro-2,4-dinitrobenzene (1) and the corresponding amines (Scheme 1). The hydroxy
functions of 3 and 4 were coupled with 2-(4-nitrophenyl)ethyl tetraisopropylphos-
phorodiamidite to the phosphoramidites 5 and 6 which were obtained as yellow oils.
The succinic acid derivative 9 for solid-support attachment was synthesized from isatoic
anhydride (¼2H-3,1-benzoxazine-2,4(1H)-dione; 7) with propylamine via 2-amino-N-
propylbenzamide (8) and subsequent reaction with succinic anhydride. Coupling of 9
with 2-(4-nitrophenyl)ethylamine and TOTU (¼ N-{{[(1-cyano-2-ethoxy-2-oxoethyl-
idene)amino]oxy}(dimethylamino)methylene}-N-methylmethanaminium tetrafluoro-
borate(1ꢀ)) yielded 10 which was used as model substance for cleavage studies from
solid-support material. Unfortunately, the amide bond turned out to be too stable for
further use. Also the oxalic acid derived linker 11 showed negative results.
Scheme 1
Next, we converted 7 with 3-aminopropan-1-ol into 12 which reacted with
trifluoroacetic anhydride to the bistrifluoroacetyl derivative 13 which led, under
selective cleavage of the ester bond, to N-(3-hydroxypropyl)-2-(2,2,2-trifluoroacet-
amido)benzamide (14) (Scheme 1). Silylation of 14 with dimethylthexylsilyl chloride

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2367
(¼chlorodimethyl(1,1,2-trimethylpropyl)silane ¼ tds-Cl) gave 15 which was subse-
quently hydrolyzed with K2CO3 in a one-pot reaction to 2-amino-N-{3-[(dimethyl-
thexylsilyl)oxy]propyl}benzamide (16). Treatment of 16 with oxirane led to 17 which
was acylated with succinic anhydride and then desilylated with fluoride ion yielding 18.
Dimethoxytritylation gave the linker in form of its triethylammonium salt 19 for
further attachment to the solid-support material.
The modified LCAMA-CPG (¼(long-chain-alkyl)methylamine controlled-pore
ˆ
glass), lCAA-CPg 500A [4], was used and loaded with 19 in the usual manner up to
15 mm/g (!20; Scheme 2). Stability tests showed that this material 20 was well suited
for oligonucleotide syntheses since it was stable against the applied reagents and gave,
after detritylation and subsequent cleavage with ammonia, chromatographically pure
2-[(2-hydroxyethyl)amino]-N-(3-hydroxypropyl)benzamide (21).
Scheme 2
After deprotection, short oligo-T-nucleotides with an aminobenzamide (Aba) cap
at the 3’-end (see 22) and an additional dinitroaniline (Dnp) cap at the 5’-end (see 23)
were obtained as products of high purity (Scheme 2 and Fig. 1).
A series of aminobenzamide conjugates with nucleic acid building blocks were
synthesized to determine their influence on the fluorescence quantum yield of 21 as the
reference (Table 1). The purine nucleosides dG and dA showed a small quenching
effect by the Fçrster or short-range mechanism [5][6], whereas the pyrimidine
nucleosides dC and T quenched by ca. 80%. This effect was less severe if the chain
started with dG and did not alter on elongation.
Additional attachment of a 2,4-dinitroaniline spacer to the 5’-end, besides the
aminobenzamide cap at the 3’-end, caused a strong quenching effect specially in short
oligonucleotide chains (Table 2). Since 2,4-dinitroaniline shows a transition at 348 nm
and a shoulder at 406 nm, there is an overlap with the fluorescence maximum of the
aminobenzamide explaining the quench effect by a short-range and/or long-range
mechanism.
Since the 2,4-dinitroaniline-derived phosphoramidites 5 and 6 can only be attached
to the 5’-end of an oligodeoxynucleotide, we also modified thymidine at the N(3)
position by alkylation with N-(2-chloroethyl)-2,4-dinitroaniline (24) to give 25
(Scheme 3). The latter was dimethoxytritylated to 26 followed by phosphitylation to

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Fig. 1. HPLC (RP 18) of 22 and 23
Table 1. Relative Fluorescence Quantum Yields of 3’-Aba Conjugates. Aba ¼ Aminobenzamide, i.e., 2-
[(2-hydroxyethyl)amino]-N-(3-hydroxypropyl)benzamide (21).
Conjugate
Aba
Relative quantum yield
1.0
dG-Aba
dA-Aba
0.97
0.84
dC-Aba
T-Aba
TT-Aba
TTT-Aba
TTTT-Aba
0.18
0.14
0.14
0.14
0.14
d(GG)-Aba
d(AG)-Aba
d(CG)-Aba
d(TG)-Aba
0.62
0.68
0.68
0.67
d(AAA GGG AAC AAA AGC TGG GTA G)-Aba
0.60
the phosphoramidite 27. The reaction of 26 with succinic anhydride gave the building
block 28.
A few oligodeoxynucleotides carrying the base-modified thymidine moiety dTDnp
(see 25) at the 3’- and the 5’-end as well as in the middle of the chain were synthesized,

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Table 2. Relative Fluorescence Quantum Yields of 5’-Dnp-3’-Aba Conjugates. For Aba, see Table 1.
Dnp ¼ N-Substituted 2,4-dinitroaniline.
Conjugate
Relative quantum yield
Rel. to 21
TT-Aba
Dnp-TT-Aba
1.00
0.05
(0.14)
d(TG)-Aba
d(Dnp-TG)-Aba
1.00
0.03
(0.69)
(0.14)
(0.61)
TTTT-Aba
Dnp-TTTT-Aba
1.00
0.26
d(AAA GGG AAC AAA AGC TGG GTA G)-Aba
d(Dnp-AAA GGG AAC AAA AGC TGG GTA G)-Aba
1.00
0.76
Scheme 3
and the melting points of the duplexes with the complementary sequence of the 21-
mers were determined (Table 3). Attachments at the modified thymidine base at the
ends of the oligodeoxynucleotides harmed the duplex formation very little, but
attachment in the middle produced a strong hyperchromicity effect of 20%.
Furthermore, we found that the thymidine quencher dTDnp (see 25) reduced the
fluorescence quantum yields of fluorescein-labeled nucleosides in dimers tremen-
dously, but separation of the two modifiers by increasing chain lengths reduced the
interaction as expected (Table 4). If adjoined modified dimers were part of an
oligodeoxynucleotide chain, the quenching effect could partly be reversed by duplex
formation (Table 4).
If such modified oligodeoxynucleotide systems are used in DNA diagnostics,
complications may arise due to the fact that the neighborhood of the modified units will
play an important role, and special sequences have to be chosen to get reliable results.

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Table 3. Melting Points of Duplexes with Modified Oligodeoxynucleotides. See 25 for dTDnp. Phosphate
buffer pH 7.0: c(Naþ) ¼ 0.12m.
Duplex
Tm [8]
d(TCA CCA GCT TTT GTT CCC TTT)
d(ATG GGT CGA AAA CAA GGG AAA)
61.2
d(TCA CCA GCT TTT GTT CCC TTTDpn
d(ATG GGT CGA AAA CAA GGG AAA)
)
60.6
61.1
56.4
d(TDnpCA CCA GCT TTT GTT CCC TTT)
d(A TG GGT CGA AAA CAA GGG AAA)
d(TCA CCA GCTDnp TTT GTT CCC TTT)
d(ATG GGT CGA AAA CAA GGG AAA)
Table 4. Relative Fluorescence Quantum Yields in Comparison to Fluorescein. Flu ¼ Fluorescein; Dnp ¼
N-substituted 2,4-dinitroaniline. See 25 for dTDnp
.
Oligonucleotide Fluorescence Oligonucleotide conjugates
Fluorescence
conjugates
quantum yields
quantum yields
Fluorescein
dCFlu
1.00
0.99
1.01
1.03
0.05
0.06
0.05
d(AAA GGG AAC AAA AGCFluTDnp GGG TA) 0.16
dGFlu
d(AAA GGG AAC AAA AGCFluTDnp GGG TA) 0.27
dAFlu
d(TTT CCC TTG TTT TCG
A
CCC AT)
Dnp-dT-dCFlu
Dnp-dT-dGFlu
Dnp-dT-dAFlu
d(ACT GCT GTDnpCFlu GAT TTC CCA C)
d(ACT GCT GTDnpCFlu GAT TTC CCA C)
0.19
0.43
Dnp-(dT)2-dCFlu 0.11
Dnp-(dT)4-dCFlu 0.25
Dnp-(dT)4-dGFlu 0.23
Dnp-(dT)4-dAFlu 0.30
Dnp-(dT)6-dCFlu 0.38
Dnp-(dT)10-dCFlu 0.77
Dnp-(dT)15-dCFlu 0.85
Dnp-(dT)20-dCFlu 0.85
Dnp-(dT)30-dCFlu 0.83
d(TGA CGA CA
G
CTA AAG GGT G)
d(ACT GCT GTDnpCFlu GAT TTC CCA C)
d(ACT GCT GTDnpCFlu GAT TTC CCA C)
0.31
0.53
d(TGA CGA CA
G
CTA AAG GGT G)
To overcome these potential difficulties, we started to develop new modified
monomeric building blocks carrying both functions – the fluorophor and the quencher
– at the same molecule.
Various strategies involving modifications at N(1) and NH2ꢀC(2) of 2’-deoxy-
guanosine were followed. Starting with 3’,5’-di-O-acetyl-2’-deoxy-O6-[2-(4-nitrophen-
yl)ethyl]guanosine (29) [7], the amino group was converted by diazotation with tert-
butyl nitrite in HF [8] into 3’,5’-di-O-acetyl-2’-deoxy-2-fluoro-O6-[2-(4-nitrophenyl)-
ethyl]inosine (30) (Scheme 4). Treatment with DBU (¼1,8-diazabicyclo[5.4.0]undec-
7-ene) eliminated the 2-(4-nitrophenyl)ethyl (npe) group to give 31 which was
alkylated with N-(2-iodoethyl)-2,4-dinitroaniline/K2CO3 in DMF forming a mixture of
3’,5’-di-O-acetyl-2’-deoxy-O6-{2-[(2,4-dinitrophenyl)amino]ethyl}-2-fluoroinosine (33)
and 3’,5’-di-O-acetyl-2’-deoxy-N2-(2,4-dinitrophenyl)-1,N2-ethanoguanosine (34), and

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ammonia converted 33 into 2’-deoxy-O6-{2-[(2,4-dinitrophenyl)amino]ethyl}guanosine
(35). A Mitsunobu reaction of 3’,5’-di-O-acetyl-2’-deoxyguanosine with 2-[(2,4-
dinitrophenyl)amino]ethanol (32; see Scheme 1) [9] led directly in high yield to 3’,5’-
di-O-acetyl-2’-deoxy-O6-{2-[(2,4-dinitrophenyl)amino]ethyl}guanosine (36) which
could be deacetylated by ammonia to give 35 again.
Scheme 4
Based upon former results in the pterin series [10], we applied the Mitsunobu
reaction also to 3’,5’-di-O-acetyl-2’-deoxy-N2-[(dimethylamino)methylene]guanosine
[11] (37) and obtained with 2-[(2,4-dinitrophenyl)amino]ethanol (32; see Scheme 1) a
mixture of the 1- and O6-{2-[(2,4-dinitrophenyl)amino]ethyl}-substituted derivatives 38
and 39) in 60 and 17% yield, respectively (Scheme 5). Furthermore, 2’-deoxy-N2-
[(dimethylamino)methylene]guanosine (40) reacted with N-(2-chloroethyl)-2,4-dini-
troaniline in DMF, in the presence of K2CO3 under N(1) substitution which gave, after
base treatment, 2’-deoxy-1-{2-[(2,4-dinitrophenyl)amino]ethyl}guanosine (41). Mild
acetylation of 41 led to the protection of the sugar moiety yielding 42 in 84% yield.
Compound 37 was also subjected to alkylations with methyl and ethyl iodide,
respectively, leading in high yields to N(1) substitution (see 43 and 44), and after
cleavage of the blocking groups to 1-methyl- (45) and 1-ethyl-2’-deoxyguanosine (46)
[12]. Structural assignments of N(1)- and O6-alkylated 2’-deoxyguanosines can be
performed by 1H-NMR and UV spectra. Thus, the chemical shift of the a-protons of the
alkyl substituents is characteristically at d 4.21 for 41 and at d 4.65 for 35. The UV
spectrum of 35 shows a maximum at 248 and a shoulder at 264 nm, whereas 41 exhibits
an absorption maximum at 257 nm.

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Scheme 5
Diazotation of 42 in 60% HF solution with 1 equiv. of tert-butyl nitrite led in low
yield to 3’,5’-di-O-acetyl-2’-deoxy-1-{2-[(2,4-dinitrophenyl)nitrosoamino]ethyl}guano-
sine (47), and with 5 equiv. of tert-butyl nitrite to the 2-fluoroinosine analog 48
(Scheme 5). Both compounds were not isolated in pure form but the proposed
structures were derived from NMR studies. These experiments showed that the amino
function of the side-chain has to be protected to achieve good yields on diazotation. For
this purpose, N-methyl-2,4-dinitro-N-(2-iodoethyl)aniline (50) was synthesized from 1-
chloro-2,4-dinitrobenzene with 2-(methylamino)ethanol and via the corresponding
chloro derivative 49. Alkylation of 37 with 50 in DMF/K2CO3 led in 76% yield to 3’,5’-
di-O-acetyl-2’-deoxy-N2-[(dimethylamino)methylene]-1-{2-[(2,4-dinitrophenyl)me-
thylamino]ethyl}guanosine (51) which was first treated with ammonia to form 52, then
acetylated mildly with Ac2O to give N2-deprotected 53, and finally converted with tert-
butyl nitrite in 60% HF solution into 3’,5’-di-O-acetyl-2’-deoxy-2-fluoro-1-{2-[(2,4-
dinitrophenyl)methylamino]ethyl)inosine (54) in 78% yield.

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Compound 54 was then treated with 2,2’-[ethane-1,2-diylbis(oxy)]bis[ethanamine]
to introduce the spacer into position 2, followed by deprotection of the sugar moiety to
give N2-{{2-[2-(2-aminoethoxy)ethoxy]ethyl}amino}-2’-deoxy-1-{2-[(2,4-dinitrophenyl)-
methylamino]ethyl}guanosine (56) in 77% yield (Scheme 6). In two subsequent steps,
56 was first treated with 2-(4-nitrophenyl)ethyl 5-[(phenoxycarbonyl)amino]-2-{6-[2-
(4-nitrophenyl)ethoxy]-3-oxo-3H-xanthen-9-yl}benzoate (55) in presence of N,N-
dimethylpyridin-4-amine (DMAP) to introduce the fluorescent label yielding 57, then
dimethoxytritylation proceeded in the usual manner to give 58 in high (88%) yield. In
TLC (thin-layer chromatography) experiments, we established that 58 reacted with the
Scheme 6

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capping mixture (Ac2O/1-methyl-1H-imidazole/lutidine/THF; used subsequently in the
oligonucleotide synthesis) only at the sugar moiety, whereas the NHꢀC(2) function
was fortunately not protected. Acylation at NHꢀC(2) during capping would have
created problems in oligonucleotide synthesis since N2-deacetylation with ammonia
would have required too long reaction times and higher temperatures. Compound 58
was, therefore, a perfect starting material for the syntheses of the two building blocks,
the 3’-succinate 59 and the 3’-phosphoramidite 60.
A second strategy of double labeling of 2’-deoxyguanosine consisted in the
introduction of both the quencher and fluorophor at the 2-amino group. A
corresponding synthesis started from 30 (see Scheme 4) which was treated with N-(2-
aminoethyl)-2,4-dinitroaniline (¼ N-(2,4-dinitrophenyl)ethane-1,2-diamine; 61) yield-
ing 62 (Scheme 7). Activation of the secondary amino group by phenyl carbono-
chloridate gave 3’,5’-di-O-acetyl-2’-deoxy-N2-{2-[(2,4-dinitrophenyl)amino]ethyl}-O6-
[2-(4-nitrophenyl)ethyl]-N2-(phenoxycarbonyl)guanosine (63) which reacted with the
2,2’-[ethane-1,2-diylbis(oxy)]bis[ethanamine] spacer to the urethane 64. Deacetylation
by ammonia in MeOH/dioxane and subsequent treatment with 55 under DMAP
catalysis led in 78% yield to 65. Finally, the building block 67 was obtained in two more
steps by dimethoxytritylation to 66 and its interconversion into the corresponding 3’-(2-
cyanoethyl N,N-diisopropylphosphoramidite) 67.
Structures. – The structures of the newly synthesized compounds were confirmed by
elemental analyses as well as UV and NMR spectra reported in the Exper. Part.
Oligonucleotide Syntheses. – The syntheses of the doubly labeled 2’-deoxyguano-
sines were based on the idea that in a single-strand oligo-2’-deoxynucleotide a ꢃrandom-
coilꢄ conformation exists allowing the quencher and fluorophor to adopt a close
location to quench the fluorescence. In the corresponding duplex, both functionalities
should be so far apart that the quench effect is limited. To corroborate this hypothesis, a
series of marked oligo-2’-deoxynucleotides, 68 – 72, were assembled in a DNA
synthesizer (Table 5). These oligomers were paired with the unlabeled complementary
sequence and their melting points determined and compared with those of the
unmodified duplexes which showed as expected, a Tm higher by 8 – 108 (Table 5).
Comparing the relative fluorescence quantum yields of the modified single strands
with their duplexes, we see in the case of 68, 70, and 72 an increase of the quantum yield
by a factor of 1.6 – 1.8, and in the case of 69 and 71 a remarkably higher increase by a
factor of 2.9 and 4.4, respectively (Table 6). This effect was temperature-dependent,
and close to the Tm, the increased fluorescence in the duplexes was reduced to the level
of the single strain (Fig. 2).
Experimental Part
General. Products were dried under high vacuum. All solvents used were of anh. grade. DNA
Syntheses: Eppendorf-Biotronik-Ecosyn-D-300 and Applied-Biosystems-392 synthesizer. TLC: pre-
coated silica gel thin-layer sheets 60 F 254 (Merck). Flash chromatography (FC): silica gel (30 – 60 mm;
Baker); 0.2 – 0.3 bar. Column chromatography ¼ CC. M.p.: Bꢀchi-510 melting-point apparatus; no
corrections. UV/VIS: Perkin-Elmer Lambda 15; lmax in nm (log e). Temp.-dependent UV/VIS: Perkin-

Helvetica Chimica Acta – Vol. 93 (2010)
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Scheme 7
1
Elmer Lambda 2 with PTP-6 (Peltier temp. programmer). H-NMR: Bruker AC 250; d in ppm rel. to
Me4Si, or CDCl3, or (D6)DMSO) as internal standard. 31P-NMR: Joel JNM-GX400.
1. N-Propyl-2,4-dinitrobenzenamine (2). A soln. of 1-chloro-2,4-dinitrobenzene (1.01 g, 5 mmol) in
dry CH2Cl2 (10 ml) was treated with propan-1-amine (0.325 g, 5.5 mmol) at r.t. for 1 h. The mixture was
concentrated, then AcOH (1.5 ml) added, and the precipitate collected and recrystallization from EtOH/

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Table 5. Melting Points of Modified and Unmodified Oligo-2’-deoxynucleotide Duplexes. Dnp ¼ N-
Substituted 2,4-dinitroaniline; Flu ¼ fluorescein. Phosphate buffer pH 8, c(Naþ) ¼ 0.12m.
Tm [8]
modified
52.5
unmodified
60.5
2
68
69
70
71
72
5’-d(AAA GGG AAC AAA AG1-Dnp,N -FluC TGC GTA)-3’
3’-d(TTT CCC TTG TTT TC
G ACC CAT)-5’
2
2
5’-d(AAA GGG AAC AAA AGN -Dnp,N -FluC TGG GTA)-3’
51.1
53.2
51.9
62.6
60.5
62.7
62.7
70.0
3’-d(TTT CCC TTG TTT TC
G ACC CAT)-5’
2
5’-d(ACT GCT GTC G1-Dnp,N -FluAT TTC CCA C)-3’
3’-d(TGA CGA CAG C
TA AAG GGT G)-5’
5’-d(ACT GCT GTC GN -Dnp,N -FluAT TTC CCA C)-3’
3’-d(TGA CGA CAG C TA AAG GGT G)-5’
5’-d(CGA CCC CCA TG1-Dnp,N -FluG AGC CCC GC)-3’
3’-d(GCT GGG GGC AC C TCG GGG CG)-5’
2
2
2
Table 6. Relative Fluorescence Quantum Yields of Modified Oligo-2’-deoxynucleotides. Dnp ¼ N-
Substituted 2,4-dinitroaniline; Flu¼ fluorescein. Phosphate buffer pH 8, c(Naþ) ¼ 0.12m. Temperature 58.
Relative quantum yield
Factor
2
68
68
5’-d(AAA GGG AAC AAA AG1-Dnp,N -FluC TGC GTA)-3’
0.30
0.48
2
5’-d(AAA GGG AAC AAA AG1-Dnp,N -FluC TGC GTA)-3’
1.6
3’-d(TTT CCC TTG TTT TC
G ACC CAT)-5’
2
2
69
69
5’-d(AAA GGG AAC AAA AGN -Dnp,N -FluC TGG GTA)-3’
0.14
0.41
2
2
5’-d(AAA GGG AAC AAA AGN -Dnp,N -FluC TGG GTA)-3’
2.9
1.4
4.4
1.6
3’-d(TTT CCC TTG TTT TC
G ACC CAT)-5’
2
70
70
5’-d(ACT GCT GTC G1-Dnp,N -FluAT TTC CCA C)-3’
0.31
0.42
2
5’-d(ACT GCT GTC G1-Dnp,N -FluAT TTC CCA C)-3’
3’-d(TGA CGA CAG C
TA AAG GGT G)-5’
2
2
71
71
5’-d(ACT GCT GTC GN -Dnp,N -FluAT TTC CCA C)-3’
0.11
0.48
2
2
5’-d(ACT GCT GTC GN -Dnp,N -FluAT TTC CCA C)-3’
3’-d(TGA CGA CAG C TA AAG GGT G)-5’
2
72
72
5’-d(CGA CCC CCA TG1-Dnp,N -FluG AGC CCC GC)-3’
0.33
0.52
2
5’-d(CGA CCC CCA TG1-Dnp,N -FluG AGC CCC GC)-3’
3’-d(GCT GGG GGC AC
C TCG GGG CG)-5’
H2O with charcoal: 1.1 g (98%) of 2. Yellow crystals. M.p. 97 – 998. UV (MeOH): 212 (4.08), 259 (3.93),
348 (4.22), 406 (sh, 3.79). 1H-NMR ((D6)DMSO): 8.81 (t, NH); 8.76 (d, HꢀC(3)); 8.16 (dd, HꢀC(5));
7.15 (d, HꢀC(6)); 3.41 (q, MeCH2CH2); 1.63 (sext., MeCH2CH2); 0.93 (t, MeCH2CH2). Anal. calc. for
C9H11N3O4 (225.2): C 48.00, H 4.92, N 18.65; found: C 47.94, H 4.92, N 18.52.
2. 3-[(2,4-Dinitrophenyl)amino]propan-1-ol (3). To a mixture of 3-aminopropan-1-ol (2.25 g,
30 mmol) and Et3N (13 ml) in dry CH2Cl2 (100 ml) was added dropwise a soln. of 1-chloro-2,4-
dinitrobenzene (2.02 g, 10 mmol) in CH2Cl2 (10 ml) and stirred at r.t. for 2 h. After evaporation, the

Helvetica Chimica Acta – Vol. 93 (2010)
2377
Fig. 2. Temperature-dependent fluorescence quantum yields of 69 and 71 in their single strain (ꢁ) and
&
duplex ( )
residue was dissolved again in CH2Cl2 (50 ml) and the soln. washed with NaHCO3 soln. (3ꢁ), dried
(Na2SO4), and concentrated. The red syrup was dissolved in little EtOH and kept in an icebox to give
2.0 g (83%) of 3. Orange-red crystals. M.p. 738. UV (MeOH): 212 (4.11), 260 (3.94), 349 (4.23), 400 (sh,
1
3.82). H-NMR ((D6)DMSO): 9.01 (t, NH); 8.80 (d, HꢀC(3)); 8.22 (dd, HꢀC(5)); 7.12 (d, HꢀC(6));

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Helvetica Chimica Acta – Vol. 93 (2010)
4.80 (br. t, OH); 3.55 (m, CH2CH2CH2OH); 1.80 (m, CH2CH2CH2OH). Anal. calc. for C9H11N3O5
(241.2): C 44.82, H 4.60, N 17.41; found: C 44.91, H 4.60, N 17.33.
3. 4-[(2,4-Dinitrophenyl)amino]butan-1-ol (4). As described for 3, with 4-aminobutan-1-ol (2.7 g,
30 mmol). The oily residue was crystallized from EtOH: 2.0 g (78%) of 4. Orange crystals. M.p. 94 – 968.
1
UV (MeOH): 212 (4.13), 259 (3.94), 348 (4.23), 399 (sh, 3.82). H-NMR ((D6)DMSO): 8.88 (t, NH);
8.81 (d, HꢀC(3)); 8.21 (dd, HꢀC(5)); 7.21 (d, HꢀC(6)); 4.53 (br. t, OH); 3.53 – 3.42 (m,
CH2CH2CH2CH2OH); 1.71 – 1.42 (m, CH2CH2CH2CH2OH). Anal. calc. for C10H13N3O5 (255.2): C
47.06, H 5.13, N 16.46; found: C 47.43, H 5.21, N 16.70.
4. 3-[(2,4-Dinitrophenyl)amino]propyl 2-(4-Nitrophenyl)ethyl N,N-Diisopropylphosphoramidite
(5). A mixture of 3 (0.12 g, 0.5 mmol) and sublimed 1H-tetrazole (17.5 mg, 0.25 mmol) in MeCN/
CH2Cl2 1:1 (4 ml) was treated with N2 for 5 min. Then, 2-(4-nitrophenyl)ethyl N,N,N’,N’-tetraisopro-
pylphosphorodiamidite (0.338 g, 0.85 mmol) was added, and after stirring at r.t. for 2 h, the mixture was
diluted with CH2Cl2. The soln. was washed with sat. NaHCO3/NaCl soln., dried (Na2SO4), and
concentrated. The residue was dissolved in little hexane/acetone and subjected to FC (silica gel (3 ꢁ
20 cm), hexane/acetone 4 :1 (200 ml) and hexane/acetone 1:1 (400 ml) containing 1.5% Et3N). The
residue of the product fractions was co-concentrated with CH2Cl2: 0.18 g (67%) of 5. Red oil. UV
(MeOH): 210 (4.40), 264 (4.26), 348 (4.22), 398 (sh, 3.82). 1H-NMR (CDCl3): 9.12 (d, HꢀC(3)); 8.69 (t,
NH); 8.70 (dd, HꢀC(5)); 8.12 (d, 2 H o to NO2); 7.43 (d, 2 H m to NO2); 6.45 (d, HꢀC(6)); 3.96 – 3.60
(m, 2 Me2CH, CH2CH2O); 3.59 – 3.51 (m, CH2CH2CH2O); 3.02 (t, CH2CH2O); 2.03 (m, CH2CH2CH2O);
1.11 (m, 2 Me2CH). 31P-NMR (CDCl3): 147.2.
5. 4-[(2,4-Dinitrophenyl)amino]butyl 2-(4-Nitrophenyl)ethyl N,N-Diisopropylphosphoramidite (6).
As described for 5, with 4 (0.5 g, 2 mmol), 1H-tetrazole (35 mg, 0.5 mmol), CH2Cl2/MeCN 1:1 (6 ml),
and 2-(4-nitrophenyl)ethyl N,N,N’,N’-tetraisopropylphosphorodiamidite (1.2 g, 3 mmol) (stirring over-
night). FC (hexane/acetone 9 :1 (200 ml), hexane/acetone 4 :1 (100 ml), and hexane/acetone 7:3
(400 ml) containing 1.5% Et3N), and co-evaporation gave 0.75 g (68%) of 6. Red oil. UV (MeOH): 210
(4.41), 263 (4.28), 347 (4.23), 399 (sh, 3.83). 1H-NMR (CDCl3): 9.11 (d, HꢀC(3)); 8.71 (t, NH); 8.70 (dd,
HꢀC(5)); 8.10 (d, 2 H o to NO2); 7.45 (d, 2 H m to NO2); 6.47 (d, HꢀC(6)); 3.98 – 3.60 (m, 2 Me2CH,
CH2CH2O); 3.61 – 3.50 (m, CH2CH2CH2CH2O); 3.00 (t, CH2CH2O); 2.05 (m, CH2CH2CH2CH2O); 1.09
(m, 2 Me2CH). 31P-NMR (CDCl3): 148.1. Anal. calc. for C24H34N5O8P (551.5): C 52.27, H 6.21, N 12.70;
found: C 52.78. H 6.22, N 13.00.
6. 2-Amino-N-propylbenzamide (8). To a soln. of 3H-1,3-benzoxazine-2,4(1H)-dione (7; 4.09 g,
25 mmol) in dry DMF (40 ml) was added dropwise propan-1-amine (2.07 g, 25 mmol) in DMF (5 ml)
within 20 min. The mixture was stirred for 3 h and then concentrated. The residue was recrystallized from
H2O (50 ml): 4.0 g (89%) of 8. Colorless crystals. M.p. 1048. UV (MeOH): 213 (4.33), 247 (3.92), 322
1
(3.57). H-NMR (CDCl3): 7.33 – 7.20 (2d, HꢀC(3), HꢀC(5)); 6.75 – 6.62 (m, HꢀC(4), HꢀC(6)); 6.08
(br. s, NH); 3.37 (q, MeCH2CH2); 1.65 (sext., MeCH2CH2); 1.00 (t, MeCH2CH2). Anal. calc. for
C10H14N2O (179.1): C 67.39, H 7.92, N 15.71; found: C 67.02, H 7.81, N 15.60.
7. 4-Oxo-4-{{2-[(propylamino)carbonyl]phenyl}amino}butanoic Acid (9). A suspension of 8 (1.07 g,
6 mmol) in dry toluene (25 ml) was treated with succinic anhydride (0.8 g, 8 mmol) at 408 for 3 h. The
precipitate was recrystallized from little H2O: 1.4 g (85%) of 9. Colorless crystals. M.p. 1678. UV
(MeOH): 213 (4.37), 251 (4.16), 296 (3.43). 1H-NMR ((D6)DMSO): 12.19 (br. s, COOH); 11.34 (br. s,
NH); 8.69 (t, PrNH); 8.36 (d, HꢀC(3)); 7.72 (dd, HꢀC(6)); 7.46 (dd, HꢀC(5)); 7.12 (t, HꢀC(4)); 3.21 (q,
C(O)CH2CH2COOH); 2.55 (m, C(O)CH2CH2COOH, MeCH2CH2NH); 1.58 (sext., MeCH2CH2NH);
0.91 (t, MeCH2CH2NH). Anal. calc. for C14H18N2O4 (278.3): C 60.42, H 6.52, N 10.06; found: C 60.23, H
6.46, N 10.04.
8. N1-[2-(4-Nitrophenyl)ethyl]-N4-{2-[(propylamino)carbonyl]phenyl}butanediamide (10). A mix-
ture of 9 (0.278 g, 1 mmol) and 2-(4-nitrophenyl)ethanamine hydrochloride (0.202 g, 1 mmol) in dry
CH2Cl2/DMF 1:1 (10 ml) was treated with TOTU (0.328 g, 1 mmol), followed by N-methylmorpholine
(0.22 ml, 2 mmol). After stirring at r.t. for 2 h, the mixture was concentrated and the residue dissolved in
CH2Cl2 (30 ml). The soln. was washed with sat. NaHCO3 soln. (3ꢁ), dried (Na2SO4), and concentrated
and the residue subjected to FC (silica gel (2.5 ꢁ 20 cm), toluene/AcOEt 1:1 (200 ml), toluene/AcOEt/
MeOH 49 :49 :2 (200 ml), toluene/AcOEt/MeOH 48.5 :48.5 :3 (200 ml), toluene/AcOEt/MeOH
48 :48 :4 (200 ml), and toluene/AcOEt/MeOH 47.5 :47.5 :5 (200 ml)). The residue of the product

Helvetica Chimica Acta – Vol. 93 (2010)
2379
fractions was recrystallized from toluene (20 ml): 0.388 g (91%) of 10. Colorless crystals. M.p. 159 – 1608.
UV (MeOH): 213 (4.47), 254 (4.30), 285 (sh, 4.05). 1H-NMR (CDCl3): 11.22 (br. s, 2 NH); 8.49 (d,
HꢀC(6)); 8.05 (d, 2 H o to NO2); 7.45 (m, HꢀC(3), HꢀC(5)); 7.33 (d, 2 H m to NO2); 7.09 (t, HꢀC(4));
6.44 (br. t, NHCO); 6.28 (s, ArNHCO); 3.56 (q, CH2NH); 3.39 (q, CH2NH); 2.94 (t, ArCH2); 2.74 (t,
CH2(Suc)); 2.52 (t, CH2(Suc)); 1.70 – 1.60 (sext., MeCH2CH2); 1.01 (t, MeCH2CH2). Anal. calc. for
C22H26N4O5 (426.5): C 61.96, H 6.14, N 13.14; found: C 61.89, H 6.15, N 13.37.
9. 2-Oxo-2-{{2-[(propylamino)carbonyl]phenyl}amino}acetic Acid (11). A mixture of 8 (1.06 g,
6 mmol) and oxalic acid dihydrate (7.56 g, 60 mmol) in toluene (100 ml) was heated under reflux under a
water separator for 3 h. The toluene was evaporated, the residue dissolved in little DMFand then diluted
with H2O (100 ml). The resulting precipitate was recrystallized from EtOH/H2O 1:1 (100 ml): 1.1 g
(73%) of 11. Colorless crystals. M.p. 1868 (dec.). UV (MeOH): 209 (4.16), 225 (4.10), 263 (4.03), 296
1
(3.82). H-NMR ((D6)DMSO): 14.40 (br. s, COOH); 12.67 (br. s, ArNHCO); 8.83 (br. s, PrNHCO);
8.51 (d, HꢀC(3)); 7.77 (d, HꢀC(6)); 7.55 (t, HꢀC(5)); 7.23 (t, HꢀC(4)); 3.23 (q, MeCH2CH2); 1.55
(sext., MeCH2CH2); 0.92 (t, MeCH2CH2). Anal. calc. for C12H14N2O4 (250.2): C 57.60, H 5.64, N 11.19;
found: C 57.25, H 5.64, N 11.09.
10. N-(3-Hydroxypropyl)-2-[(2,2,2-trifluoroacetyl)amino]benzamide (14). To a soln. of 7 (8.18 g,
50 mmol) in dry DMF (30 ml) was added dropwise 3-aminopropan-1-ol (4.5 g, 60 mmol) in DMF (5 ml).
After stirring for 3 h, the mixture was concentrated and the residue of 12 dissolved in CH2Cl2 (30 ml).
The soln. was cooled to 08, Et3N (10 ml) and then dropwise 2,2,2-trifluoroacetic anhydride (15 ml,
110 mmol) were added. The soln. was slowly warmed to r.t. and then concentrated to give crude 13. The
solid was dissolved in pyridine/ H2O 1:1 (100 ml), then Et3N (10 ml) was added and the mixture kept
overnight to cleave the ester bond. The mixture was concentrated and co-concentrated four times with
toluene. The residue was dissolved in CH2Cl2 (150 ml), the soln. washed with sat. NaHCO3 soln. (3ꢁ),
dried (Na2SO4), and then again concentrated. The solid was dissolved in toluene/CH2Cl2 and subjected to
FC (silica gel (4 ꢁ 40 cm), toluene/AcOEt 1:1 (200 ml), toluene/AcOEt 1:1 þ 2% MeOH (200 ml),
toluene/AcOEt 1:1 þ 4% MeOH (200 ml), toluene/AcOEt 1:1 þ 6% MeOH (200 ml), toluene/AcOEt
1:1 þ 8% MeOH (400 ml), and toluene/AcOEt 1:1 þ 10% MeOH (400 ml)). The residue of the product
fractions was recrystallized from MeOH/H2O: 9.3 g (65%) of 14. Colorless fine needles. M.p. 88 – 898.
UV (MeOH): 215 (4.31), 251 (4.19), 293 (3.66). 1H-NMR ((D6)DMSO): 13.32 (br. s, CF3CONH); 8.97
(br. s, PrNHCO); 8.34 (d, HꢀC(3)); 7.87 (d, HꢀC(6)); 7.60 (t, HꢀC(5)); 7.23 (t, HꢀC(4)); 4.51 (t, OH);
3.46 (t, CH2CH2CH2OH); 3.32 (t, CH2CH2CH2OH); 1.69 (sext., CH2CH2CH2OH). Anal. calc. for
C12H13F3N2O3 (291.3): C 49.66, H 4.51, N 9.65; found: C 49.77, H 4.56, N 9.66.
11. 2-Amino-N-{3-{[dimethyl(1,1,2-trimethylpropyl)silyl]oxy}propyl}benzamide (16). Compound 14
(2.9 g, 10 mmol) was co-concentrated twice with dry pyridine and then dissolved in this solvent (30 ml).
Chlorodimethyl(1,1,2-trimethylpropyl)silane (2.55 ml, 13 mmol) was added and the mixture stirred at r.t.
for 3 h. After evaporation and co-evaporation with toluene (3ꢁ), the residue was dissolved in CH2Cl2
(100 ml), the soln. washed with sat. NaHCO3 soln. (2ꢁ) and H2O, dried (Na2SO4), and again
concentrated. The residue of 15 was dissolved in MeOH/H2O 9 :1 (100 ml), then K2CO3 (5.0 g, 36 mmol)
was added and the mixture heated under reflux for 3 h. After cooling, the mixture was neutralized with
AcOH and concentrated, the residue suspended in CH2Cl2 (100 ml), the mixture washed with sat.
NaHCO3 soln. (2ꢁ), the org. phase dried (Na2SO4) and concentrated, and the residue dissolved in little
toluene and subjected to FC (silica gel (4 ꢁ 30 cm), toluene (200 ml), toluene/AcOEt 95 :5 (200 ml),
toluene/AcOEt 9 :1 (200 ml), toluene/AcOEt 4 :1 (400 ml), toluene/AcOEt 3 :2 (400 ml), and toluene/
AcOEt 5 :5 (200 ml)): 3.0 g (89%) of 16. Colorless oil. UV (MeOH): 212 (4.32), 247 (sh, 3.94), 323
(3.58). 1H-NMR ((D6)DMSO): 8.12 (br. t, NHCO); 7.45 (d, HꢀC(6)); 7.08 (t, HꢀC(4)); 6.66 (d,
HꢀC(3)); 6.49 (t, HꢀC(5)); 6.34 (br. s, NH2); 3.61 (t, CH2CH2CH2O); 3.26 (q, CH2CH2CH2O); 1.71 (m,
CH2CH2CH2O); 1.58 (sept., Me2CH); 0.86 (m, MeCHC(Me)2Si); 0.06 (2s, Me2Si). Anal. calc. for
C18H32N2O2Si (336.5): C 64.24, H 9.58, N 8.52; found: C 64.16, H 9.48, N 8.31.
12. 2-[(2-Hydroxyethyl)amino]-N-{3-{[dimethyl(1,1,2-trimethylpropyl)silyl]oxy}propyl}benzamide
(17). To a cold soln. (08) of 16 (2.35 g, 7 mmol) in THF/H2O 2 :1 (30 ml) was added AcOH (1.8 g,
30 mmol), then oxirane (3.3 ml, 70 mmol, prepared at ꢀ 708) was added at 08, and the mixture was
stirred overnight at r.t. The mixture was concentrated, the residue dissolved in CH2Cl2 (100 ml), the soln.
washed with sat. NaHCO3 soln. (2ꢁ), dried (Na2SO4), and again concentrated, and the residue purified

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Helvetica Chimica Acta – Vol. 93 (2010)
by FC (silica gel (2.5 ꢁ 35 cm), toluene/AcOEt 1:1 (150 ml), toluene/AcOEt 1:1 þ 2% MeOH (150 ml),
toluene/AcOEt 1:1 þ 4% MeOH (150 ml), toluene/AcOEt 1:1 þ 7% MeOH (300 ml), and toluene/
AcOEt 1:1 þ 10% MeOH (300 ml): 1.7 g (53) of 17. Colorless oil. UV (MeOH): 215 (4.37), 256 (4.07),
338 (3.68). 1H-NMR ((D6)DMSO): 8.20 (br. t, NHCO); 7.81 (t, ArNH); 7.47 (d, HꢀC(6)); 7.20 (t,
HꢀC(4)); 6.63 (d, HꢀC(3)); 6.51 (t, HꢀC(5)); 4.77 (t, OH); 3.70 – 3.60 (m, CH2CH2CH2O,
CH2CH2OH); 3.28 (q, CH2NH); 3.12 (q, CH2NH); 1.74 (m, CH2CH2CH2O); 1.56 (sept., Me2CH);
0.88 (m, Me2CHC(Me)2Si); 0.10 (2s, Me2Si). Anal. calc. for C20H36N2O3Si (380.6): C 63.11, H 9.53, N 7.36;
found: C 63.04, H 9.54, N 8.03.
13. 4-{2-{{2-{[(3-Hydroxypropyl)amino]carbonyl}phenyl}amino}ethoxy}-4-oxobutanoic Acid (18).
A mixture of 17 (1.14 g, 3 mmol) and DMAP (0.55 g, 4.5 mmol) was co-concentrated twice with toluene.
Then, the residue was dissolved in CH2Cl2 (10 ml), succinic anhydride (0.42 g, 4.2 mmol) added, and the
mixture stirred for 24 h at r.t. The soln. was diluted with CH2Cl2 (50 ml) and washed with 10% aq. citric
acid (2ꢁ) and H2O (2ꢁ). The aq. layers were re-extracted with CH2Cl2 (50 ml). Then, the combined org.
phase was dried (Na2SO4) and concentrated, the residue dissolved in MeOH (15 ml), NH4F (0.222 g,
6 mmol) added, and the mixture heated under reflux for 5 h. AcOH (1 ml) was added, the mixture
concentrated, and the residue subjected to FC (silica gel (2.5 ꢁ 30 cm), CH2Cl2 þ 1% AcOH (100 ml),
CH2Cl2/MeOH 98 :2 þ 1% AcOH (100 ml), CH2Cl2/MeOH 96 :4 þ 1% AcOH (200 ml), and CH2Cl2/
MeOH 94 :6 þ 1% AcOH (200 ml): 0.75 g (75%) of 18. Colorless oil. UV (MeOH): 214 (4.35), 256
(4.06), 332 (3.66). 1H-NMR (CDCl3): 7.32 (m, HꢀC(6), HꢀC(4)); 7.20 – 6.50 (m, 2 OH, 2 NH); 6.63 (d,
HꢀC(3)); 6.52 (t, HꢀC(5)); 4.31 (t, CH2CH2OCO); 3.71 (t, CH2CH2CH2OH); 3.57 (q, CH2CH2OCO);
3.43 (t, CH2CH2CH2OH); 2.61 (m, 4 H, Suc); 1.71 (m, CH2CH2CH2OH). Anal. calc. for C16H22N2O6
(338.4): C 56.80, H 6.55, N 8.28; found: C 56.76, H 6.54, N 8.30.
14. Triethylammonium 4-{2-{{2-{{{3-[(4,4’-Dimethoxytrityl)oxy]propyl}amino}carbonyl}phenyl}-
amino}ethoxy}-4-oxobutanoate (19). Compound 18 (0.6 g, 1.78 mmol) in dry pyridine was concentrated
twice. Then, the residue was dissolved in dry pyridine (50 ml), 4,4’-dimethoxytrityl chloride (0.663 g,
1.96 mmol) added, and the mixture stirred overnight. After evaporation, Et3N (5 ml) was added and the
mixture concentrated twice with toluene. The solid was dissolved in little CH2Cl2 and subjected to FC
(silica gel (2 ꢁ 25 cm), CH2Cl2/MeOH 99 :1 (100 ml), CH2Cl2/MeOH 98 :2 (100 ml), CH2Cl2/MeOH
96 :4 (200 ml), CH2Cl2/MeOH 95 :5 (100 ml), and CH2Cl2/MeOH 93 :7 (200 ml)). The residue of the
product fractions was dissolved in little CH2Cl2 and at 08 added dropwise to Et2O/Et3N 39 :1. All
triethylammonium salts different from 19 precipitated. After filtration, the soln. was evaporared: 1.2 g
(91%) of 19. Colorless solid foam. UV (MeOH): 205 (4.76), 253 (sh, 4.18), 334 (3.68). 1H-NMR
((D6)DMSO): 8.20 (br. t, Et3NH); 7.48 (m, HꢀC(6)); 7.45 – 7.25 (m, Ph, 4 H m to MeO, HꢀC(4)); 6.88
(2d, 4 H o to MeO); 6.79 (d, HꢀC(3)); 6.58 (t, HꢀC(5)); 4.22 (t, CH2CH2OCO); 3.66 (2s, 2 MeO); 3.41
(t, CH2CH2OCO); 3.38 (q, CH2CH2CH2OTr(OMe)2); 3.05 (t, CH2CH2CH2OTr(MeO)2); 2.63 (q,
(MeCH2)3N); 2.54 – 2.42 (m, 4 H, Suc); 1.81 (m, CH2CH2CH2OTr(OMe)2); 1.01 (t, (MeCH2)3N). Anal.
calc. for C43H5N3O8 · 0.3 H2O (747.9): C 68.82, H 7.52, N 5.60; found: C 69.05, H 7.50, N 5.61.
15. N-(2-Chloroethyl)-2,4-dinitrobenzenamine (24). The 2-chloroethanamine hydrochloride (21.0 g,
0.18 mol) was co-concentrated with dry DMF and then dissolved in DMF (300 ml). Then, 1-chloro-2,4
dinitrobenzene (36.5 g, 0.18 mol) was added. Under vigorous stirring, Et3N (75 ml, 0.54 mol) was added
slowly and dropwise within 1 h, and after another 3 h stirring at r.t., the mixture was concentrated. The
red oily residue was treated with CH2Cl2 (250 ml), the soln. washed with sat. aq. NaHCO3 soln. (2 ꢁ
400 ml) and H2O, dried (Na2SO4), and again concentrated. The residue was recrystallized from EtOH/
acetone 95 :5 (100 ml): 33.1 g (75%) of 24. Yellow crystalline powder. M.p. 918. UV (MeOH): 211 (4.11),
259 (3.94), 347 (4.22), 392 (sh, 3.82). 1H-NMR (((D6)DMSO): 8.91 (br. t, NH); 8.82 (d, HꢀC(3)); 8.22
(dd, HꢀC(5)); 7.30 (d, HꢀC(6)); 3.86 (s, CH2CH2). Anal. calc. for C8H8ClN3O4 (245.6): C 39.12, H 3.28,
N 17.10; found: C 39.26, H 3.32, N 16.68.
16. 3-{2-[(2,4-Dinitrophenyl)amino]ethyl}thymidine (25). A mixture of thymidine (2.42 g, 10 mmol)
and 24 (3.7 g, 15 mmol) was co-concentrated with dry DMF under high vacuum. The residue was
dissolved in dry DMFat 1008, dry K2CO3 (4.15 g, 30 mmol) added, and the mixture stirred at 1008 for 3 h.
After cooling, AcOH (3.6 ml, 60 mmol) was added, and the mixture concentrated under high vacuum.
The residue was recrystallized from H2O/EtOH 1:4: 4.1 g (89%) of 25. Yellow crystals. M.p. 200 – 2018.
UV (MeOH): 210 (4.31), 263 (4.21), 347 (4.23), 390 (sh, 3.84). 1H-NMR ((D6)DMSO): 8.95 (br. t, NH);

Helvetica Chimica Acta – Vol. 93 (2010)
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8.82 (s, HꢀC(3) (Ar)); 8.25 (d, HꢀC(5) (Ar)); 7.78 (s, HꢀC(6)); 7.30 (d, HꢀC(6) (Ar)); 6.15 (ꢃtꢄ,
HꢀC(1’)); 5.23 (s, OHꢀC(3’)); 5.04 (dd, OHꢀC(5’)); 4.23 (m, HꢀC(3’)); 4.09 (m, CH2(5’)); 3.77 (m,
HꢀC(4’)); 3.74 (q, CH2CH2NH); 3.52 (m, CH2CH2NH); 2.12 – 2.01 (m, CH2(2’)); 1.80 (s, MeꢀC(5)).
Anal. calc. for C18H21N5O9 (451.4): C 47.90, H 4.68, N 15.15; found: C 47.47, H 4.63, N 15.17.
17. 5’-O-(4,4’-Dimethoxytrityl)-3-{2-[(2,4-dinitrophenyl)amino]ethyl}thymidine (26). Compound 25
(4.0 g, 8.9 mmol) was twice co-concentrated with dry pyridine and then the residue dissolved in dry
pyridine (30 ml). Dimethoxytrityl chloride (3.3 g, 9.8 mmol) was added and the mixture stirred at 48 for
3 days. After evaporation, the mixture was twice co-concentrated with toluene, the residue dissolved in
CH2Cl2 (100 ml) and the soln. washed with sat. aq. NaHCO3 soln. (2 ꢁ 300 ml). The aq. layer was
reextracted with CH2Cl2 (50 ml) and the combined CH2Cl2 phase dried (Na2SO4) and concentrated. The
residue was dissolved in little CH2Cl2 and subjected to FC (silica gel (2 ꢁ 40 cm), toluene/AcOEt 1:1
(80 ml), toluene/AcOEt 1:1 þ 1% MeOH (80 ml), toluene/AcOEt 1:1 þ 2% MeOH (80 ml), toluene/
AcOEt 1:1 þ 3% MeOH (80 ml), and toluene/AcOEt 1:1 þ 4% MeOH (80 ml)). The residue of the
product fractions was dissolved in CH2Cl2 and the soln. filtered through cellulose and again concentrated:
5.9 g (89%) of 26. Yellow foam. UV (MeOH): 204 (sh, 4.87), 230 (sh, 4.51), 264 (4.25), 347 (4.23), 393
(sh, 3.83). 1H-NMR (CDCl3): 9.11 (d, HꢀC(3) (Ar)); 8.86 (br. t, NH); 8.29 (dd, HꢀC(5) (Ar)); 7.65 (s,
HꢀC(6)); 7.42 – 7.18 (m, 5 arom. H, 4 H o to MeO); 7.12 (d, HꢀC(6) (Ar)); 6.84 (2d, 4 H m to MeO);
6.20 (dd, HꢀC(1’)); 4.61 (m, HꢀC(3’)); 4.32 (m, CH2CH2NH); 4.03 (m, HꢀC(4’)); 3.87 (d, 1 HꢀC(5’));
3.80 (d, 1 HꢀC(5’)); 3.78 (2s, 2 MeO); 3.73 (q, CH2CH2NH); 2.41 – 2.35 (m, CH2(2’)); 1.53 (s,
MeꢀC(5)). Anal. calc. for C39H39N5O11 · 0.3 H2O (759.7): C 61.66, H 5.19, N 9.22; found: C 61.42, H 5.19,
N 9.14.
18. 5’-O-(4,4’-Dimethoxytrityl)-3-{2-[(2,4-dinitrophenyl)amino]ethyl}thymidine 3’-(2-Cyanoethyl
N,N-Diisopropylphosphoramidite) (27). Dry 26 (1.28 g. 1.7 mmol) was dissolved in CH2Cl2/MeCN 1:3
(10 ml) and then, under N2, 2-cyanoethyl N,N,N’,N’-tetraisopropylphosphorodiamidite (1.0 g, 3.4 mmol)
and 1H-tetrazole (0.256 g, 0.85 mmol) were added. After stirring for 12 h, the mixture was diluted with
CH2Cl2 (50 ml) and the soln. washed with sat. aq. NaHCO3 soln. (2 ꢁ 150 ml), dried (Na2SO4), and
concentrated. The residue was dissolved in little toluene and subjected to FC (silica gel (2 ꢁ 20 cm),
toluene/AcOEt 3 :1 (300 ml) and toluene/AcOEt 1:1 (300 ml)): 1.24 g (76%) of 27. Yellow solid foam.
UV (MeOH): 204 (4.88), 231 (sh, 4.50), 265 (4.37), 347 (4.21), 390 (sh, 3.83). 1H-NMR (CDCl3): 9.13 (d,
HꢀC(3) (Ar)); 8.82 (br. t, NH); 8.31 (dd, HꢀC(5) (Ar)); 7.70 (s, HꢀC(6)); 7.41 – 7.15 (m, 5 arom. H, 4 H
o to MeO); 7.12 (d, HꢀC(6) (Ar)); 6.84 (2d, 4 H m to MeO); 6.41 (dd, HꢀC(1’)); 4.68 (m, HꢀC(3’));
4.32 (m, CH2CH2NH); 4.19 (m, HꢀC(4’)); 3.73 (2s, 2 MeO); 3.88 – 3.29 (m, CH2CH2NH, CH2(5’),
OCH2CH2CN, 2 Me2CH); 2.65 – 2.30 (m, CH2(2’), OCH2CH2CN); 1.48 (s, MeꢀC(5)); 1.24 – 1.00 (m,
2 Me2CH). 31P-NMR (CDCl3): 149.5; 149.26. Anal. calc. for C48H56N7O12P (953.9): C 60.44, H 5.92, N
10.27; found: C 60.51, H 5.92, N 9.91.
19. 5’-O-(4,4’-Dimethoxytrityl)-3-{2-[(2,4-dinitrophenyl)amino]ethyl}thymidine 3’-(Hydrogen Buta-
noate) (28). A mixture of dry 26 (0.6 g, 0.8 mmol) and DMAP (0.176 g, 1.44 mmol) in dry CH2Cl2 (10 ml)
was treated with succinic anhydride (0.136 g, 1.36 mmol) for 3 days at r.t. The mixture was diluted with
CH2Cl2 (30 ml) and washed with 10% citric acid soln. (3 ꢁ 50 ml). The aq. phases were reextracted with
CH2Cl2 (20 ml) and the combined CH2Cl2 phase was washed with H2O (2ꢁ), dried (Na2SO4), and
concentrated: 0.65 g (95%) of 28. Yellow foam. UV (MeOH): 205 (sh, 4.82), 231 (sh, 4.42), 263 (4.28),
347 (4.23), 401 (sh, 3.81). 1H-NMR (CDCl3): 9.16 (d, HꢀC(3) (Ar)); 8.88 (br. t, NH); 8.29 (dd, HꢀC(5)
(Ar)); 7.67 (s, HꢀC(6)); 7.41 – 7.19 (m, 5 arom. H, 4 H o to MeO); 7.12 (d, HꢀC(6) (Ar)); 6.83 (2d, 4 H m
to MeO); 6.42 (dd, HꢀC(1’)); 5.49 (m, HꢀC(3’)); 4.39 (m, CH2CH2NH); 4.17 (m, HꢀC(4’)); 3.78 (2s,
2 MeO); 3.70 (q, CH2CH2NH); 3.49 (m, CH2(5’)); 2.73 (m, 4 H, Suc); 2.53 – 2.40 (m, CH2(2’)); 1.43 (s,
MeꢀC(5)). Anal. calc. for C43H43N5O14 · 0.5 H2O (862.8): C 59.85, H 5.06, N 8.11; found: C 59.58, H 5.11,
N 8.15.
20. 3’,5’-O-Diacetyl-2’-deoxy-2-fluoro-O6-[2-(4-nitrophenyl)ethyl]inosine (30). In a Teflon flask, 70%
HF in pyridine (10 ml) was cooled to ꢀ 508. Then, dry pyridine (1.9 ml) was dropwise added under
stirring for 30 min, followed by addition of 3’,5’-di-O-acetyl-2’-deoxy-O6-[2-(4-nitrophenyl)ethyl]gua-
nosine [7] (29; 1.5 g, 3 mmol). After 3 min, 95% tert-butyl nitrite in tBuOH (0.53 ml, 0.433 g, 4.2 mmol)
was added and stirred for 30 min at ꢀ 508. The reaction was stopped by ice/H2O 1:1 (200 ml). Then, the
mixture was extracted with CH2Cl2 (2 ꢁ 80 ml) and the org. phase again extracted with ice/phosphate

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Helvetica Chimica Acta – Vol. 93 (2010)
buffer (pH 7) 1:10 (3 ꢁ 200 ml), dried (Na2SO4), and concentrated. The residue was dissolved in iPrOH
and the soln. concentrated and kept at ꢀ 108 for crystallization. The precipitate was washed with cold
EtOH and Et2O: 1.4 g (93%) of 30. Colorless crystals from EtOH. M.p. 1318. UV (MeOH): 203 (4.46),
257 (4.29), 276 (sh, 4.05). 1H-NMR ((D6)DMSO): 8.58 (s, HꢀC(8)); 8.16 (d, 2 H o to NO2); 7.61 (d, 2 H
m to NO2); 6.35 (dd, HꢀC(1’)); 5.39 (m, HꢀC(3’)); 4.82 (t, CH2CH2O); 4.30 – 4.15 (m, HꢀC(4’),
CH2(5’)); 3.32 (t, CH2CH2O); 3.12 – 3.00 (m, HaꢀC(2’)); 2.60 – 2.50 (m, HbꢀC(2’)); 2.10 (s, Ac); 2.00 (s,
Ac). Anal. calc. for C22H22FN5O8 (503.4): C 52.49, H 4.41, N 13.91; found: C 52.52, H 4.44, N 13.62.
21. 3’,5’-Di-O-acetyl-2’-deoxy-2-fluoroinosine (31). Compound 30 (1.0 g, 2 mmol) was dried by co-
concentration with dry pyridine (3 ꢁ 4 ml) and then dissolved in dry pyridine (4 ml), DBU (0.6 ml,
4 mmol) was added and stirred at r.t. for 4 h. After neutralization with AcOH, the mixture was
concentrated and twice co-concentrated with toluene and the residue dissolved in little CH2Cl2 and
subjected to FC (silica gel (1 ꢁ 10 cm) with toluene/AcOEt 1:1 þ 10% MeOH (100 ml), toluene/AcOEt
1:1 þ 15% MeOH (160 ml), toluene/AcOEt 1:1 þ 20% MeOH (80 ml), and toluene/AcOEt 1:1 þ 25%
MeOH (80 ml)). The oily residue of the product fractions was dissolved in CH2Cl2 and the soln.
concentrated: 0.39 g (55%) of 31. Colorless foam. UV (MeOH): 204 (4.15), 262 (3.87), 304 (sh, 4.15).
1H-NMR ((D6)DMSO): 8.31 (s, HꢀC(8)); 6.21 (dd, HꢀC(1’)); 5.32 (m, HꢀC(3’)); 4.30 – 4.12 (m,
HꢀC(4’), CH2(5’)); 3.05 – 2.92 (m, HaꢀC(2’)); 2.60 – 2.50 (m, HbꢀC(2’)); 2.08 (s, Ac); 2.02 (s, Ac). Anal.
calc. for C14H15FN4O6 · 0.5 H2O (363.3): C 46.29, H 4.44, N 15.41; found: C 46.36, H 4.45, N 15.73.
22. 2-[(2,4-Dinitrophenyl)amino]ethanol (32) [8]. A soln. of 1-chloro-2,4-dinitrobenzene (36.5 g,
0.18 mol) in DMF (250 ml) was treated with 2-aminoethanol (14 ml, 0.23 mol), followed by Et3N (50 nl,
0.36 mol). Reaction took place under warming to 508, and after stirring for 2 h at r.t., the mixture was
evaporated under high vacuum. The oily residue was dissolved in CH2Cl2 (200 ml), the soln. washed with
H2O (4 ꢁ 150 ml), dried (Na2SO4), and concentrated, and the residue recrystallized from EtOH (80 ml):
29 g (72%) of 32. Yellow crystals. M.p. 89 – 908. UV (MeOH): 212 (4.11), 259 (3.93), 347 (4.21), 399 (sh,
3.78). 1H-NMR ((D6)DMSO): 8.88 (br. t, NH); 8.79 (d, HꢀC(3)); 8.22 (dd, HꢀC(5)); 7.22 (d, HꢀC(6));
5.08 (t, OH); 3.69 (d, CH2); 3.56 (q, CH2). Anal. calc. for C8H9N3O5 (227.2): C 42.30, H 4.00, N 18.49;
found: C 42.28, H 4.02, N 18.39.
23. 3’,5’-Di-O-acetyl-2’-deoxy-2-fluoro-O6-{2-[(2,4-dinitrophenyl)amino]ethyl}inosine (33) and 3-
(3’,5’-Di-O-acetyl-2’-deoxy-b-d-ribofuranosyl)-5-(2,4-dinitrophenyl)-3,5,6,7-tetrahydro-9H-imidazo[1,2-
a]purin-9-one (34). A mixture of 31 (0.85 g, 2.4 mmol), N-(2-iodoethyl)-2,4-dinitroaniline (0.97 mg,
2.9 mmol) and K2CO3 (0.8 g, 5.8 mmol) in dry DMF (10 ml) was heated under stirring to 1008 for 1 h.
After cooling, the solid was filtered off, the filtrate mixed with little flash silica gel and then concentrated
under high vacuum, and the residue put onto a FC column (SiO2 (3 ꢁ 25 cm), toluene/AcOEt 1:1
(100 ml), toluene/AcOEt 1:1 þ 2% MeOH (100 ml), toluene/AcOEt 1:1 þ 5% MeOH (100 ml),
toluene/AcOEt 1:1 þ 8% MeOH (100 ml), toluene/AcOEt 1:1 þ 12% MeOH (100 ml), toluene/AcOEt
1:1 þ 15% MeOH (100 ml), toluene/AcOEt 1:1 þ 20% MeOH (100 ml), and toluene/AcOEt 1:1 þ
30% MeOH (100 ml)). The first product fractions (Rf 0.52; toluene/AcOEt/MeOH 4.5 :4.5 :1) were
concentrated, and the residue was treated with little MeOH: 0.85 g (61%) of 33. The second product
fractions (Rf 0.13) were also concentrated, and the residue was recrystallized from MeOH (20 ml) þ
acetone (2 ml): 0.19 g (15%) of 34.
Data of 33: Amorphous yellow solid. M.p 1628. UV (MeOH): 203 (4.38), 254 (4.28), 346 (4.22), 402
1
(sh, 3.76). H-NMR ((D6)DMSO): 9.00 (br. t, NH); 8.82 (d, HꢀC(3) (Ar)); 8.22 (dd, HꢀC(5) (Ar));
8.08 (s, HꢀC(8)); 7.43 (d, HꢀC(6) (Ar)); 6.32 (dd, HꢀC(1’)); 5.40 (m, HꢀC(3’)); 4.82 (t, CH2CH2O);
4.30 – 4.12 (m, HꢀC(4’), CH2(5’)); 4.02 (q, CH2CH2O); 3.12 – 3.00 (m, HaꢀC(2’)); 2.61 – 2.50 (m,
HbꢀC(2’)); 2.09 (s, Ac); 2.00 (s, Ac). Anal. calc. for C22H22FN7O10 (563.4): C 46.90, H 3.94, N 17.39;
found: C 47.09, H 4.01, N 16.92.
Data of 34: Yellow crystals. M.p. > 1118 (dec.). UV (MeOH): 204 (4.42), 253 (4.37), 336 (3.76), 367
(sh, 3.71). 1H-NMR ((D6)DMSO): 8.80 (d, HꢀC(3) (Ar)); 8.61 (dd, HꢀC(5) (Ar)); 8.06 (s, HꢀC(2));
7.92 (d, HꢀC(6) (Ar)); 6.01 (dd, HꢀC(1’)); 5.21 (m, HꢀC(3’)); 4.45 (t, NCH2CH2N); 4.29 (t,
NCH2CH2N); 4.12 (m, HꢀC(4’)); 3.96 (m, CH2(5’)); 2.85 – 2.72 (m, HaꢀC(2’)); 2.50 – 2.41 (m,
HbꢀC(2’)); 2.10 (s, Ac); 1.92 (s, Ac). Anal. calc. for C22H21N7O10 · 0.5 H2O (552.4): C 47.83, H 4.01, N
17.74; found: C 47.93, H 3.94, N 17.66.

Helvetica Chimica Acta – Vol. 93 (2010)
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24. 2’-Deoxy-O6-{2-[(2,4-dinitrophenyl)amino]ethyl}guanosine (35). A soln. of 36 (0.2 g, 0.36 mmol)
in dioxane/MeOH/conc. NH3 soln. 1:1:1 (50 ml) was stirred at r.t. for 3 h and then concentrated. The
residue was dissolved in dioxane/MeOH 1:1 and subjected to FC (silica gel (1 ꢁ 10 cm), CH2Cl2/MeOH
85 :15 (100 ml), CH2Cl2/MeOH 8 :2 (100 ml), CH2Cl2/MeOH 75 :25 (50 ml), and CH2Cl2/MeOH 7:3
(50 ml)). The product fractions were concentrated till a precipitate separated. After cooling in the icebox
overnight, the solid was collected and recrystallized from a mixture of acetone (30 ml), CHCl3 (20 ml),
and little MeOH: 0.13 g (76%) of 35. Yellow crystals. M.p. 171 – 1738 (dec.). UV (MeOH): 211 (4.54),
1
248 (4.24), 264 (sh, 4.10), 346 (4.21), 397 (sh, 3.79). H-NMR ((D6)DMSO): 9.00 (br. t, NH); 8.82 (d,
HꢀC(3) (Ar)); 8.31 (dd, HꢀC(5) (Ar)); 8.09 (s, HꢀC(8)); 7.41 (d, HꢀC(6) (Ar)); 6.48 (br. s, NH2); 6.21
(dd, HꢀC(1’)); 5.27 (d, OHꢀC(3’)); 4.99 (dd, OHꢀC(5’)); 4.65 (t, OCH2CH2NH); 4.31 (m, HꢀC(3’));
3.96 (q, OCH2CH2NH); 3.81 (m, HꢀC(4’)); 3.61 – 3.48 (m, CH2(5’)); 2.62 – 2.50 (m, HaꢀC(2’)); 2.28 –
2.18 (m, HbꢀC(2’)). Anal. calc. for C18H20N8O8 · 0.5 H2O (485.4): C 44.54, H 4.36, N 23.07; found: C
44.40, H 4.41, N 22.85.
25. 3’,5’-Di-O-acetyl-2’-deoxy-O6-{2-[(2,4-dinitrophenyl)amino]ethyl}guanosine (36). A mixture of
3’,5’-di-O-acetyl-2’-deoxyguanosine (0.705 g, 2 mmol) and 32 (0.909 g, 4 mmol) was treated in dry
dioxane (10 ml) by ultrasound. The suspension was concentrated, co-concentrated twice with dry
dioxane, and then suspended in dry dioxane (10 ml) by heating to 608. Triphenylphosphine (0.813 g,
3 mmol) was added, followed by dropwise addition of diisopropyl azodicarboxylate (0.6 ml, 3 mmol).
After stirring at 608 for 1 h, the mixture was concentrated and the residue washed with CH2Cl2 (10 ml)
and Et2O: 0.98 g (88%) of crude 36. Recrystallization from acetone/EtOH 1:4 gave 0.78 g (70%) of 36.
Yellow crystals. M.p. 2158. UV (MeOH): 210 (4.53), 248 (4.26), 266 (sh, 4.12), 346 (4.23), 400 (sh, 3.79).
1H-NMR ((D6)DMSO): 9.02 (br. t, NH); 8.84 (d, HꢀC(3) (Ar)); 8.31 (dd, HꢀC(5) (Ar)); 8.11 (s,
HꢀC(8)); 7.41 (d, HꢀC(6) (Ar)); 6.55 (br. s, NH2); 6.21 (dd, HꢀC(1’)); 5.32 (m, HꢀC(3’)); 4.64 (t,
OCH2CH2NH); 4.28 – 4.10 (m, HꢀC(4’), CH2(5’)); 3.92 (q, OCH2CH2NH); 3.10 – 2.90 (m, HaꢀC(2’));
2.51 – 2.40 (m, HbꢀC(2’)); 2.08 (s, Ac); 2.01 (s, Ac). Anal. calc. for C22H24N8O10 (560.4): C 47.15, H 4.32,
N 19.98; found: C 46.79, H 4.31, N 19.49.
26. 3’,5’-Di-O-acetyl-2’-deoxy-N2-[(dimethylamino)methylene]guanosine (37) and 2’-Deoxy-N2-[(di-
methylamino)methylene]guanosine (40). The 2’-deoxyguanosine dihydrate (0.2 g, 0.36 mmol) was co-
concentrated with dry pyridine, the residue suspended in MeOH (8.5 ml), N,N-dimethylformamide
dimethyl acetal (1.67 g, 14 mmol) added, and the mixture treated by ultrasound for 30 min. After stirring
for 30 h, the precipitate of 40 was washed with little MeOH and Et2O. The solid was co-concentrated with
dry pyridine again and then suspended in DMF/pyridine 1:1 (10 ml). Ac2O (2 ml) was added and the
mixture stirred at r.t. for 5 h. After evaporation, the residue was dissolved in CH2Cl2 (30 ml), the soln.
washed with H2O, dried (Na2SO4), and concentrated, and the residue dissolved in MeOH (5 ml). Et2O
(30 ml) was added and the mixture cooled in an icebox overnight. The crystals were collected: 1.1 g
(78%) of 37. Colorless needles. M.p. 1138. pKa 11.0. UV (pH 8): 206 (4.15), 232 (4.11), 276 (4.18), 298
(4.31). 1H-NMR (CDCl3): 9.40 (br. s, NH); 8.68 (s, Me2NCH¼N)); 7.72 (s, HꢀC(8)); 6.24 (dd,
HꢀC(1’)); 5.59 (m, HꢀC(3’)); 4.46 – 4.20 (m, HꢀC(4’), CH2(5’)); 3.21 (s, MeN); 3.11 (s, MeN); 3.11 –
3.00 (m, HaꢀC(2’)); 2.60 – 2.50 (m, HbꢀC(2’)); 2.16 (s, Ac); 2.11 (s, Ac). Anal. calc. for C17H22N6O6
(406.4): C 50.24, H 5.46, N 20.68; found: C 49.80, H 5.55, N 20.64.
27. 3’,5’-Di-O-acetyl-2’-deoxy-N2-[(dimethylamino)methylene]-1-{2-[(2,4-dinitrophenyl)amino]-
ethyl}guanosine (38) and 3’,5’-Di-O-acetyl-2’-deoxy-N2-[(dimethylamino)methylene]-O6-{2-[(2,4-dinitro-
phenyl)amino]ethyl}guanosine (39). A mixture of 37 (0.16 g, 0.34 mmol), 32 (0.136 g, 0.6 mmol), and
triphenylphosphine (0.157 g, 0.6 mmol) was co-concentrated with dry dioxane (3 ꢁ 10 ml) and then the
residue dissolved in dry dioxane (10 ml). To the soln., diisopropyl azodicarboxylate (0.12 g, 0.6 mmol)
was added and stirred at 508 for 4 h. The suspension was concentrated and the residue dissolved in DMF
(20 ml). Flash silica gel was added, the mixture concentrated under high vacuum, and the solid put onto a
FC column (SiO2 (3 ꢁ 12 cm), toluene/AcOEt 1:1 þ 5% MeOH (200 ml), toluene/AcOEt 1:1 þ 7%
MeOH (200 ml), toluene/AcOEt 1 :1 þ 9% MeOH (200 ml), toluene/AcOEt 1:1 þ 11% MeOH
(200 ml), toluene/AcOEt 1 :1 þ 15% MeOH (200 ml), and toluene/AcOEt 1 :1 þ 18% MeOH
(200 ml)). The residue of the fractions of the main product (Rf 0.37) was recrystallized from MeOH
(10 ml): 0.125 g (60%) of 38. The product fractions with Rf 0.26 gave, after evaporation and
recrystallization from MeOH (30 ml) þ H2O (1 ml), 36 mg (17%) of 39.

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Helvetica Chimica Acta – Vol. 93 (2010)
Data of 38: Yellow crystals. M.p. 118 – 1218. UV (MeOH): 237 (4.35), 267 (4.26), 315 (4.38), 342 (sh,
4.28), 394 (sh, 3.77). 1H-NMR ((D6)DMSO): 9.02 (br. t, NH); 8.82 (d, HꢀC(3) (Ar)); 8.49 (s,
Me2NCH¼N)); 8.17 (dd, HꢀC(5) (Ar)); 8.05 (s, HꢀC(8)); 7.30 (d, HꢀC(6) (Ar)); 6.26 (dd, HꢀC(1’));
5.49 (m, HꢀC(3’)); 4.55 (t, NCH2CH2NH); 4.30 – 4.10 (m, HꢀC(4’), CH2(5’)); 3.81 (m, NCH2CH2NH);
3.18 (s, MeN); 2.94 (s, MeN); 3.12 – 3.02 (m, HaꢀC(2’)); 2.50 – 2.42 (m, HbꢀC(2’)); 2.10 (s, Ac); 1.98 (s,
Ac). Anal. calc. for C25H29N9O10 (615.6): C 48.78, H 4.75, N 20.47; found: C 48.65, H 4.76, N 20.19.
Data of 39: Yellow crystals. M.p. > 2158 (dec.). UV (MeOH): 211 (4.43), 273 (4.53), 290 (4.53), 345
(sh, 4.22), 396 (sh, 3.79). 1H-NMR ((D6)DMSO): 8.97 (br. t, NH); 8.81 (d, HꢀC(3) (Ar)); 8.55 (s,
Me2NCH¼N)); 8.28 (s, HꢀC(8)); 8.21 (dd, HꢀC(5) (Ar)); 7.57 (d, HꢀC(6) (Ar)); 6.32 (dd, HꢀC(1’));
5.43 (m, HꢀC(3’)); 4.78 (t, OCH2CH2N); 4.32 – 4.12 (m, HꢀC(4’), CH2(5’)); 3.92 (m, OCH2CH2N); 3.12
(s, MeN); 3.14 – 3.05 (m, HaꢀC(2’)); 3.03 (s, MeN); 2.58 – 2.48 (m, HbꢀC(2’)); 2.10 (s, Ac); 2.01 (s, Ac).
Anal. calc. for C25H29N9O10 (615.7): C 48.78, H 4.75, N 20.47; found: C 49.13, H 4.79, N 20.19.
28. 2’-Deoxy-1-{2-[(2,4-dinitrophenyl)amino]ethyl}guanosine (41). To a mixture of 40 (6.65 g,
21.7 mmol) and 24 (16.0 g, 65.1 mmol) in dry DMF (100 ml), K2CO3 (18.0 g) was added and the
suspension treated with ultrasound and then heated under stirring to 1208 for 2 h. After cooling, aq. conc.
ammonia (100 ml) was added and the mixture stirred at 558 overnight. After concentration to half of the
volume, the mixture was neutralized with AcOH and then concentrated. The residue was suspended in
little DMF, flash silica gel added, and the mixture concentrated and put on a FC column (SiO2 (3 ꢁ
20 cm), CH2Cl2/MeOH 85 :15 (200 ml), CH2Cl2/MeOH 82 :18 (200 ml), CH2Cl2/MeOH 8 :2 (200 ml),
CH2Cl2/MeOH 75 :25 (200 ml), and CH2Cl2/MeOH 7:3 (200 ml)). The product fractions were
concentrated to 1/5 and then kept in an icebox overnight: 7.0 g (75%) of 41. Yellow crystals. M.p.
174 – 1758. UV (MeOH): 205 (4.46), 257 (4.34), 346 (4.23), 401 (sh, 3.79). 1H-NMR ((D6)DMSO): 8.98
(br. t, NH); 8.83 (d, HꢀC(3) (Ar)); 8.26 (dd, HꢀC(5) (Ar)); 7.93 (s, HꢀC(8)); 7.28 (d, HꢀC(6) (Ar));
7.23 (br. s, NH2); 6.10 (dd, HꢀC(1’)); 5.29 (d, OHꢀC(3’)); 4.91 (dd, OHꢀC(5’)); 4.33 (m, HꢀC(3’));
4.21 (t, NCH2CH2NH); 3.81 (m, HꢀC(4’)); 3.71 (m, NCH2CH2NH); 3.58 – 3.49 (m, CH2(5’)); 2.60 – 2.48
(m, HaꢀC(2’)); 2.20 – 2.10 (m, HbꢀC(2’)). Anal. calc. for C18H20N8O8 · H2O (485.4): C 43.73, H 4.49, N
22.66; found: C 43.68, H 4.27, N 22.38.
29. 3’,5’-Di-O-acetyl-2’-deoxy-1-{2-[(2,4-dinitrophenyl)amino]ethyl}guanosine (42). A soln. of 41
(6.5 g, 13.6 mmol) was co-concentrated with dry pyridine (2 ꢁ 100 ml) and then dissolved in dry pyridine
(200 ml). Under stirring at r.t., Ac2O (45 ml) was dropwise added, and after 3 h, the mixture was
concentrated. The red residue was dissolved in CH2Cl2/acetone 95 :5 (400 ml), the soln. washed with sat.
aq. NaHCO3 soln. (3 ꢁ 200 ml) and H2O (200 ml), the aq. phase reextracted with CH2Cl2/acetone 9 :1
(100 ml), and the combined org. phase dried (Na2SO4) and concentrated. The residue was treated with
hot EtOH/H2O 3 :1 (400 ml) and then the mixture kept at ꢀ 108 for two days: 6.39 g (84%) of 42. Yellow
solid from EtOH. M.p. 124 – 1268 (dec.). UV (MeOH): 205 (4.46), 257 (4.36), 346 (4.23), 399 (sh, 3.81).
1H-NMR ((D6)DMSO): 8.98 (br. t, NH); 8.82 (d, HꢀC(3) (Ar)); 8.28 (dd, HꢀC(5) (Ar)); 7.90 (s,
HꢀC(8)); 7.31 (d, HꢀC(6) (Ar)); 7.28 (br. s, NH2); 6.10 (dd, HꢀC(1’)); 5.30 (m, HꢀC(3’)); 4.28 – 4.11
(m, HꢀC(4’), CH2 (5’)); 4.18 (t, NCH2CH2NH); 3.71 (m, NCH2CH2NH); 3.00 – 2.89 (m, HaꢀC(2’));
2.50 – 2.21 (m, HbꢀC(2’)); 2.07 (s, Ac); 2.01 (s, Ac). Anal. calc. for C22H24N8O10 · 2 H2O (596.4): C 44.30,
H 4.73, N 18.78; found: C 44.68, H 4.66, N 18.17.
30. 3’,5’-Di-O-acetyl-2’-deoxy-N2-[(dimethylamino)methylene]-1-methylguanosine (43). Compound
37 (1.5 g, 4 mmol) was co-concentrated with dry DMF and then dissolved in dry DMF (20 ml). K2CO3
(1.2 g, 8.8 mmol) was added under stirring, followed by dropwise addition of MeI (0.28 ml, 4.4 mmol).
Stirring was continued for 20 h at 358. The solid was filtered off and washed with DMF, and the filtrates
were concentrated under high vacuum. The residue was dissolved in CH2Cl2 (50 ml) and the soln. washed
with sat. aq. NaHCO3 soln. (2 ꢁ 50 ml), dried (Na2SO4), and concentrated to a small volume which was
put on a FC column (silica gel (3 ꢁ 20 cm), toluene/AcOEt 1:1 þ 12% MeOH (200 ml), toluene/AcOEt
1:1 þ 14% MeOH (200 ml), toluene/AcOEt 1:1 þ 16% MeOH (200 ml), and toluene/AcOEt 1:1 þ
18% MeOH (200 ml)). The residue of the product fractions was recrystallized from EtOH (30 ml)/
Et2O (70 ml): 1.4 g (82%) of 43. Colorless crystals. M.p. 1718. UV (MeOH): 202 (4.32), 237 (4.24), 281
(sh, 4.08), 306 (4.30). 1H-NMR ((D6)DMSO): 8.58 (s, Me2NCH¼N)); 7.98 (s, HꢀC(8)); 6.25 (dd,
HꢀC(1’)); 5.47 (m, HꢀC(3’)); 4.38 – 4.10 (m, HꢀC(4’), CH2(5’)); 3.51 (s, MeꢀN(1)); 3.20 (s, MeN); 3.09

Helvetica Chimica Acta – Vol. 93 (2010)
2385
(s, MeN); 3.10 – 3.00 (m, HaꢀC(2’)); 2.59 – 2.40 (m, HbꢀC(2’)); 2.13 (s, Ac); 1.98 (s, Ac). Anal. calc. for
C18H24N6O6 (420.4): C 51.43, H 5.75, N 19.98; found: C 51.29, H 5.75, N 20.01.
31. 3’,5’-Di-O-acetyl-2’-deoxy-N2-[(dimethylamino)methylene]-1-ethylguanosine (44). As described
for 43, with 37 (1.5 g, 4 mmol) in dry DMF (20 ml), K2CO3 (1.2 g, 8.8 mmol), and iodoethane (0.36 ml,
4.4 mmol): 1.5 g (85%) of 44. Colorless crystals. M.p. 1628. UV (MeOH): 203 (4.30), 238 (4.24), 280 (sh,
4.08), 307 (4.31). 1H-NMR ((D6)DMSO): 8.62 (s, Me2NCH¼N)); 8.00 (s, HꢀC(8)); 6.23 (dd, HꢀC(1’));
5.50 (m, HꢀC(3’)); 4.35 – 4.10 (m, HꢀC(4’), CH2(5’), MeCH2N); 3.21 (s, MeN); 3.08 (s, MeN); 3.10 –
3.00 (m, HaꢀC(2’)); 2.55 – 2.43 (m, HbꢀC(2’)); 2.11 (s, Ac); 1.96 (s, Ac); 1.13 (t, MeCH2N). Anal.
calc. for C19H26N6O6 (434.4): C 52.53, H 6.03, N 19.34; found: C 52.53, H 6.05, N 19.45.
32. 2’-Deoxy-1-methylguanosine (45) [12]. Compound 43 (0.84 g, 2 mmol) was stirred in a closed
round-bottom flask with a mixture of sat. methanolic ammonia (40 ml) and conc. aq. ammonia (80 ml) at
r.t. for 4 days. After evaporation, the residue was dissolved in MeOH (50 ml), and then Et2O was added
until a precipitate started to separate. The mixture was kept in an icebox for 2 days: 0.45 g (80%) of 45.
Colorless crystals. M.p. > 2308 (dec.). UV (MeOH): 204 (4.31), 256 (4.14), 267 (sh, 4.06). 1H-NMR
((D6)DMSO): 7.92 (s, HꢀC(8)); 7.00 (s, NH2); 6.11 (ꢃtꢄ, HꢀC(1’)); 5.21 (d, OHꢀC(3’)); 4.93 (t,
OHꢀC(5’)); 4.37 (m, HꢀC(3’)); 3.77 (m, HꢀC(4’)); 3.60 – 3.45 (m, CH2(5’)); 3.28 (s, MeꢀN(1)); 2.60 –
2.45 (m, HaꢀC(2’)); 2.22 – 2.10 (m, HbꢀC(2’)). Anal. calc. for C11H15N5O4 (281.2): C 46.98, H 5.38, N
24.89; found: C 46.81, H 5.41, N 25.26.
33. 2’-Deoxy-1-ethylguanosine (46) [12]. As described for 45, with 44 (0.59 g, 2 mmol): 0.44 g (75%)
1
of 46. Colorless crystals. M.p. 153 – 1548. UV (MeOH): 204 (4.36), 257 (4.10), 268 (sh, 4.00). H-NMR
((D6)DMSO): 7.88 (s, HꢀC(8)); 7.06 (s, NH2); 6.12 (ꢃtꢄ, HꢀC(1’)); 5.20 (d, OHꢀC(3’)); 4.93 (t,
OHꢀC(5’)); 4.32 (m, HꢀC(3’)); 3.96 (q, MeCH2); 3.79 (m, HꢀC(4’)); 3.56 – 3.41 (m, CH2(5’)); 2.45 (m,
HaꢀC(2’)); 2.25 – 2.13 (m, HbꢀC(2’)); 1.12 (t, MeCH2). Anal. calc. for C12H17N5O4 · 0.3 H2O (301.3): C
47.84, H 5.91, N 23.23; found: C 47.84, H 6.11, N 23.01.
34. N-(2-Chloroethyl)-N-methyl-2,4-dinitrobenzenamine (49). A mixture of 2-(methylamino)ethan-
ol (15.3 g, 0.2 mol) and 1-chloro-2,4-dinitrobenzene (20.2 g, 0.1 mol) was co-concentrated with dry
pyridine (2 ꢁ 80 ml) and the residue dissolved in dry pyridine (80 ml) and stirred at 708 for 3 h. After
evaporation and co-evaporation with toluene (3 ꢁ 100 ml), the residue was taken up in CH2Cl2 (80 ml)
and extracted with sat. aq. NaHCO3 soln. (2 ꢁ 100 ml). The org. phase was dried (Na2SO4) and
concentrated and the red sirup treated with thionyl chloride (20 ml) under reflux for 2 h. The reaction
soln. was added dropwise unter vigorous stirring into H2O (300 ml). The resulting suspension was
extracted with CH2Cl2 (2 ꢁ 100 ml), the extract washed with sat. NaHCO3 soln. (2 ꢁ 100 ml), dried
(Na2SO4), and again concentrated: 18.0 g (70%) of crude 49 which was pure enough for the next reaction
to 50. A sample of crude 49 (1.0 g) in little CH2Cl2 was subjected to FC (silica gel (25 g), toluene
(400 ml)). The residue of the product fraction gave, on drying under high vacuum, a yellow oil which
solidified: 0.85 g of a yellow solid. M.p. 61 – 638. UV (MeOH): 206 (4.11), 362 (4.20). 1H-NMR
((D6)DMSO): 8.57 (d, HꢀC(2)); 8.19 (dd, HꢀC(5)); 7.39 (d, HꢀC(6)); 3.91 (t, CH2CH2Cl); 3.72 (t,
CH2CH2Cl); 2.94 (s, MeN). Anal. calc. for C9H10ClN3O4 (259.6): C 41.64, H 3.88, N 16.18; found: C 42.01,
H 3.87, N 16.07.
35. N-(2-Iodoethyl)-N-methyl-2,4-dinitrobenzenamine (50). A mixture of crude 49 (13.0 g, 0.05 mol),
NaI (37.0 g, 0.25 mol), and NaHCO3 (21.0 g, 0.25 mol) was refluxed in dry butan-2-one (350 ml) for 10 h.
After cooling, the mixture was filtered and the residue washed with acetone till the solvent was colorless.
The filtrate and extract were concentrated, and the residue was suspended in CH2Cl2 (80 ml), the
suspension washed with H2O (3 ꢁ 200), the org. phase dried (Na2SO4) and concentrated, and the
resulting solid recrystallized from EtOH (200 ml) with acetone (30 ml): 16.5 g (94%) of 50. Yellow
crystals. M.p. 978. UV (MeOH): 204 (4.07), 364 (4.18). 1H-NMR ((D6)DMSO): 8.56 (d, HꢀC(2)); 8.21
(dd, HꢀC(5)); 7.36 (d, HꢀC(6)); 3.76 (t, CH2CH2I); 3.43 (t, CH2CH2I); 2.92 (s, MeN). Anal. calc. for
C9H10IN3O4 (351.1): C 30.79, H 3.10, N 11.94; found: C 30.81, H 2.85, N 11.98.
36. 3’,5’-Di-O-acetyl-2’-deoxy-N2-[(dimethylamino)methylene]-1-{2-[(2,4-dinitrophenyl)methylami-
no]ethyl}guanosine (51). A mixture of 37 (0.406 g, 1 mmol), 50 (0.421 g, 1.2 mol), and K2CO3
(0.331 g, 2.4 mol) was co-concentrated with dry DMF (3 ꢁ 20 ml) and then the residue dissolved in
dry DMF (20 ml) and heated to 1008 for 6 h. After cooling to r.t., the solid was filtered off and washed
with DMF, and the filtrates were evaporated. The residue was dissolved in CH2Cl2 (30 ml), the soln.

2386
Helvetica Chimica Acta – Vol. 93 (2010)
washed with sat. aq. NaHCO3 soln. (3 ꢁ 60 ml), dried (Na2SO4), and then concentrated again. The
residue, dissolved in little CH2Cl2, was subjected to FC (silica gel (2 ꢁ 12 cm), toluene/AcOEt/MeOH
47:47:6 (200 ml), toluene/AcOEt/MeOH 46 :46 :8 (200 ml), and toluene/AcOEt/MeOH 45 :45 :10
(200 ml)). The residue of the product fractions was co-concentrated several times with CH2Cl2: 0.47 g
1
(76%) of 51. Yellow solid foam. UV (MeOH): 203 (4.42), 234 (4.33), 311 (4.28), 371 (4.13). H-NMR
((D6)DMSO): 8.58 (s, Me2NCH¼N)); 8.49 (d, HꢀC(3) (Ar)); 8.12 (dd, HꢀC(5) (Ar)); 7.98 (s,
HꢀC(8)); 7.37 (d, HꢀC(6) (Ar)); 6.22 (dd, HꢀC(1’)); 5.47 (m, HꢀC(3’)); 4.52 (t, NCH2CH2NMe);
4.37 – 4.12 (m, HꢀC(4’), CH2(5’)); 3.73 (m, NCH2CH2NMe); 3.19 (s, MeN); 3.09 – 2.90 (m, HaꢀC(2’));
2.99 – 2.95 (2s, Me2N); 2.50 – 2.36 (m, HbꢀC(2’)); 2.09 (s, Ac); 1.98 (s, Ac). Anal. calc. for C26H31N9O10
(629.6): C 49.60, H 4.96, N 20.01; found: C 49.70, H 4.96, N 19.75.
37. 2’-Deoxy-N2-[(dimethylamino)methylene]-1-{2-[(2,4-dinitrophenyl)methylamino]ethyl}guano-
sine (52). A soln. of 51 (0.651 g, 1 mmol) in dioxane (10 ml), MeOH (10 ml), and conc. aq. NH3 soln.
was kept in an icebox overnight and then concentrated. The residue was treated with MeOH (10 ml):
0.53 g (95%) of 52. Yellow fine crystals. M.p. 2198. UV (MeOH): 205 (4.43), 233 (4.35), 311 (4.30), 370
(4.14). 1H-NMR ((D6)DMSO): 8.53 (s, Me2NCH¼N)); 8.51 (d, HꢀC(3) (Ar)); 8.09 (dd, HꢀC(5) (Ar));
8.00 (s, HꢀC(8)); 7.38 (d, HꢀC(6) (Ar)); 6.21 (dd, HꢀC(1’)); 5.33 (d, OHꢀC(3’)): 4.92 (t, OHꢀC(5’));
4.53 (t, NCH2CH2NMe); 4.41 (m, HꢀC(3’)); 3.82 (m, HꢀC(4’)); 3.77 (m, NCH2CH2NMe); 3.54 (m,
CH2(5’)); 3.21 (s, MeN); 3.03 – 3.00 (2s, Me2N); 2.62 – 2.50 (m, HaꢀC(2’)); 2.29 – 2.18 (m, HbꢀC(2’)).
Anal. calc. for C22H27N9O8 (545.5): C 48.44, H 4.99, N 23.10; found: C 48.56, H 4.96, N 22.86.
38. 3’,5’-Di-O-acetyl-2’-deoxy-1-{2-[(2,4-dinitrophenyl)methylamino]ethyl}guanosine (53). To a soln.
of 52 (2.65 g, 5 mmol) in DMF (300 ml), conc. aq. NH3 soln. (300 ml) was added and heated to 558 for
6 h. After concentration to half of the volume, more conc. aq. NH3 soln. (300 ml) was added and heating
to 558 continued for 12 h. The mixture was concentrated, the residue co-concentrated with dry pyridine
(3 ꢁ 150 ml) and then dissolved in dry pyridine (150 ml), and Ac2O (5.1 g, 50 mmol) added. After stirring
at r.t. for 3 h, the mixture was concentrated, the residue dissolved in CH2Cl2 (100 ml), the soln. washed
with sat. aq. NaHCO3 soln. (2 ꢁ 150 ml), dried, (Na2SO4), and concentrated, and the residue purified by
FC (silica gel (2.5 ꢁ 30 cm), CH2Cl2 (150 ml), CH2Cl2/MeOH 97:3 (150 ml), CH2Cl2/MeOH 95 :5
(150 ml), CH2Cl2/MeOH 93 :7 (150 ml), and CH2Cl2/MeOH 9 :1 (150 ml)). The residue of the product
fractions was treated with little MeOH to give 1.8 g (63%) of 53. Fine yellow crystals. M.p. 1158 (dec.).
1
UV (MeOH): 204 (4.40), 251 (4.24), 369 (4.13). H-NMR ((D6)DMSO): 8.48 (d, HꢀC(3) (Ar)); 8.04
(dd, HꢀC(5) (Ar)); 7.83 (s, HꢀC(8)); 7.41 (d, HꢀC(6) (Ar)); 7.20 (br. s, NH2); 6.04 (dd, HꢀC(1’)); 5.29
(d, HꢀC(3’)); 4.34 – 4.13 (m, HꢀC(4’), CH2(5’), NCH2CH2NMe); 3.67 (m, NCH2CH2NMe); 3.09 (s,
MeN); 3.00 – 2.86 (m, HaꢀC(2’)); 2.46 – 2.32 (m, HbꢀC(2’)); 2.08 (s, Ac); 2.03 (s, Ac). Anal. calc. for
C23H26N8O10 · 0.5 H2O (583.5): C 47.35, H 4.66, N 19.19; found: C 47.42, H 4.72, N 18.74.
39. 3’,5’-Di-O-acetyl-2’-deoxy-2-fluoro-1-{2-[(2,4-dinitrophenyl)methylamino]ethyl}inosine (54). In
a Teflon flask, 70% HF in pyridine (8 ml) was cooled to ꢀ 508. Then, dry pyridine (1.5 ml) was added
dropwise under stirring within 30 min, followed by the addition of 53 (0.862 g, 1.5 mmol). After 3 min,
t
95% tert-butyl nitrite in BuOH (1.87 ml, 15 mmol) was added and stirred for 30 min at ꢀ 508 with
occasional shaking. Then, cold dry Et3N (17 ml) was added dropwise within 15 min. The reaction was
stopped by ice (50 g). Then, the mixture was extracted twice with CH2Cl2 (2 ꢁ 50 ml), the org. phase
washed with cold phosphate buffer (pH 7) (250 ml) and sat. aq. NaCl soln. followed by reextraction of
the aq. phases with CH2Cl2. The combined org. phase was dried (Na2SO4), and concentrated and the
residue dissolved in iPrOH (50 ml). The soln. was concentrated until the beginning of crystallization and
then kept in an icebox overnight. The yellow crystals were collected and recrystallized from EtOH
(100 ml) by addition of acetone (40 ml): 0.675 g (78%) of 53 which was pure enough for the subsequent
reactions. A sample of 53 (0.3 g) was purified by FC (silica gel (1 ꢁ 10 cm), toluene/AcOEt 1:1 (50 ml),
then toluene/AcOEt/MeOH 49 :49 :2 ! 47:47:6). The residue of the product fractions was recrystal-
lized from EtOH/acetone: 0.25 g of 54. Fine yellow crystals. M.p. 174 – 1758 (dec.). UV (MeOH): 203
1
(4.40), 238 (4.22), 364 (4.11). H-NMR ((D6)DMSO): 8.52 (d, HꢀC(3) (Ar)); 8.31 (s, HꢀC(8)); 8.20
(dd, HꢀC(5) (Ar)); 7.43 (d, HꢀC(6) (Ar)); 6.19 (dd, HꢀC(1’)); 5.32 (d, HꢀC(3’)): 4.31 – 4.14 (m,
HꢀC(4’), CH2(5’), NCH2CH2NMe); 3.67 (m, NCH2CH2NMe); 2.95 (s, MeN); 3.00 – 2.88 (m, HaꢀC(2’));
2.61 – 2.50 (m, HbꢀC(2’)); 2.08 (s, Ac); 2.01 (s, Ac). Anal. calc. for C23H24FN7O10 (577.4): C 47.48, H 4.19,
N 16.96; found: C 47.85, H 4.19, N 16.81.

Helvetica Chimica Acta – Vol. 93 (2010)
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40. 2-(4-Nitrophenyl)ethyl 2-{6-[2-(4-Nitrophenyl)ethoxy]-3-oxo-3H-xanthen-9-yl}-5-[(phenoxycar-
bonyl)amino]benzoate (55). To a mixture of 2-(4-nitrophenyl)ethyl 5-amino-2-{6-[2-(4-nitrophenyl)-
ethoxy]-3-oxo-3H-xanthen-9-yl}benzoate [1] (1.0 g, 1.55 mmol) and DMAP (0.244 g, 2 mmol) in dry
pyridine (50 ml) at 08, phenyl carbonochloridate (0.19 ml, 1.55 mmol) was added dropwise and the
mixture and stirred at r.t. for 3 h. After evaporation and co-evaporation with toluene (3 ꢁ 20 ml), the
residue was suspended in CH2Cl2/MeOH 1:1 (40 ml) by ultrasound. The suspension was cooled to 08 and
the red solid collected and washed with EtOH: 1.1 g (93%) of 55. Red powder. UV (CH2Cl2/MeOH 1:1):
227 (4.80), 250 (4.73), 299 (sh, 4.31), 357 (3.96), 438 (sh, 4.26), 460 (4.42), 487 (sh, 4.31). 1H-NMR
((D6)DMSO): 10.80 (br. s, NH); 8.43 (d, HꢀC(6)); 8.18 (d, 2 H o to NO2); 8.00 (d, 2 H o to NO2); 7.87
(dd, HꢀC(4)); 7.63 (d, 2 H m to NO2); 7.46 (d, 2 H m to NO2); 7.38 (d, HꢀC(3)); 7.37 – 7.27 (m, Ph); 7.18
(d, HꢀC(5) (Xan)); 6.85 – 6.80 (m, HꢀC(8), HꢀC(7), HꢀC(1) (all Xan)); 6.36 (dd, HꢀC(2) (Xan));
6.18 (d, HꢀC(4) (Xan)); 4.46 (t, CH2CH2O); 4.25 (t, CH2CH2O); 3.27 (t, CH2CH2O); 2.81 (t,
CH2CH2O). Anal. calc. for C43H31N3O11 (765.7): C 67.48, H 4.08, N 5.48; found: C 67.01, H 4.18, N 5.48.
41. N2-{{2-[2-(2-Aminoethoxy)ethoxy]ethyl}amino}-2’-deoxy-1-{2-[(2,4-dinitrophenyl)methylami-
no]ethyl}guanosine (56). To a soln. of 54 (2.31 g, 4 mmol) in dry CH2Cl2 (20 ml) at 08, 2,2’-[ethane-
1,2-diylbis(oxy)]bis[ethanamine] (2.37 g, 16 mmol) was added and stirred vigorously for 10 min. The
yellow soln. was then treated with sat. NH3/MeOH (80 ml) and, in a closed system, stirred at 408 for 24 h.
The mixture was concentrated and the residue, dissolved in little CH2Cl2, subjected to FC (silica gel
(2.5 ꢁ 20 cm), CH2Cl2/MeOH 85 :15 þ 2% Et3N (200 ml), CH2Cl2/MeOH 8 :2 þ 2% Et3N (200 ml), and
CH2Cl2/MeOH 75 :25 þ 2% Et3N (400 ml)). The residue of the product fractions was dissolved in CH2Cl2
and the soln. filtered through cellulose powder and concentrated: 1.9 g (77%) of 56. Yellowish solid
foam. UV (MeOH): 204 (4.43), 251 (4.23), 284 (sh., 4.00), 3.67 (4.14). 1H-NMR ((D6)DMSO): 8.41 (d,
HꢀC(3) (Ar)); 7.96 (dd, HꢀC(5) (Ar)); 7.72 (s, HꢀC(8)); 7.31 (d, HꢀC(6) (Ar)); 7.25 (t, NHꢀC(2)
(Pur)); 6.02 (dd, HꢀC(1’)); 4.30 (d, HꢀC(3’), NCH2CH2NMe); 3.71 (m, HꢀC(4’)); 3.70 – 3.20 (m,
CH2(5’), NCH2CH2NMe, CH2CH2O, OCH2CH2O); 3.32 (t, OCH2CH2NH2); 3.01 (s, MeN); 2.61 (t,
OCH2CH2NH2); 2.60 – 2.50 (m, HaꢀC(2’)); 2.18 – 2.11 (m, HbꢀC(2’)).
42. 2-(4-Nitrophenyl)ethyl 5-{{{{2-{2-{2-{{9-(2’-Deoxy-b-d-ribofuranosyl)-1-{2-[(2,4-dinitrophenyl)-
methylamino]ethyl}-6,9-dihydro-6-oxo-1H-purin-2-yl}amino}ethoxy}ethoxy}ethyl}amino}carbonyl}ami-
no}-2-{6-[2-(4-nitrophenyl)ethoxy]-3-oxo-3H-xanthen-9-yl}benzoate (57). A mixture of 55 (1.53 g,
2 mmol) and DMAP (0.488 g, 4 mmol) in dry DMF (50 ml) was concentrated and then the residue
dissolved in dry DMF (50 ml) and dry pyridine (20 ml). After addition of 56 (1.0 g, 1.6 mmol), the
mixture was heated to 708 for 45 min. After evaporation under high vacuum to remove DMF completely,
the residue was dissolved in CH2Cl2 (8 ml) and the soln. subjected to FC (silica gel (3 ꢁ 30 cm), CH2Cl2/
MeOH 95 :5 (100 ml), CH2Cl2/MeOH 93 :7 (100 ml), CH2Cl2/MeOH 9 :1 (100 ml), CH2Cl2/MeOH
87:13 (200 ml), and CH2Cl2/MeOH 85 :15 (200 ml)). The product fractions which contained also some
DMAP, were concentrated, and the red sirup was dissolved in little CH2Cl2/MeOH 95 :5, the soln. filtered
through cellulose powder, and the filtrate added dropwise under stirring to cold MeOH (08). The
resulting suspension was kept in an icebox overnight and the precipitate collected and dried in a vacuum
desiccator: 1.7 g (84%) of 57. Orange-red powder. UV (CH2Cl2/MeOH 1:1): 230 (4.88), 260 (4.81), 304
(sh, 4.23), 364 (4.37), 437 (sh, 4.33), 459 (4.45), 487 (sh, 4.34). 1H-NMR ((D6)DMSO): 9.08 (br. s,
NHCONH); 8.38 (d, HꢀC(3) (Ar)); 8.31 (s, HꢀC(2) (Ar)); 8.18 (d, 2 H o to NO2); 8.03 (d, 2 H o to
NO2); 7.96 (d, HꢀC(5) (Ar)); 7.82 (s, HꢀC(8) (Pur)); 7.68 (d, HꢀC(6) (Ar)); 7.63 (d, 2 H m to NO2);
7.35 – 7.21 (m, HꢀC(5) (Ar), HꢀC(6) (Ar), 2 H m to NO2, NHꢀC(2) (Pur)); 7.38 (d, HꢀC(3)); 7.16 (d,
HꢀC(5) (Xan)); 6.90 – 6.77 (m, HꢀC(8), HꢀC(7), HꢀC(1) (all Xan)); 6.36 (dd, HꢀC(2) (Xan); t,
NHCONH); 6.13 (d, HꢀC(4)); 6.06 (ꢃtꢄ, HꢀC(1’)); 5.31 (br. d, OHꢀC(3’)); 4.82 (br. t, OHꢀC(5’));
4.50 – 4.22 (m, OCH2CH2, NCH2CH2, HꢀC(3’)); 4.18 (t, OCH2CH2); 3.81 (m, HꢀC(4’)); 3.70 – 3.45 (m,
NCH2CH2NMe, CH2(5’), NHCH2CH2OCH2CH2OCH2CH2NH); 3.25 (m, CH2CH2O, OCH2CH2NH);
3.04 (s, MeN); 2.82 (t, CH2CH2O); 2.20 – 2.10 (m, CH2(2’)). Anal. calc. for C62H60N12O20 (1293.2): C 57.59,
H 4.68, N 12.99; found: C 57.50, H 4.62, N 12.75.
43. 2-(4-Nitrophenyl)ethyl 5-{{{{2-{2-{2-{{9-[2’-Deoxy-5’-O-(4,4’-dimethoxytrityl)-b-d-ribofura-
nosyl]-1-{2-[(2,4-dinitrophenyl)methylamino]ethyl}-6,9-dihydro-6-oxo-1H-purin-2-yl}amino}ethoxy}eth-
oxy}ethyl}amino}carbonyl}amino}-2-{6-[2-(4-nitrophenyl)ethoxy]-3-oxo-3H-xanthen-9-yl}benzoate (58).
Compound 57 (1.5 g, 1.2 mmol) was co-concentrated with dry pyridine (3 ꢁ 30 ml) and then dissolved in

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Helvetica Chimica Acta – Vol. 93 (2010)
the same solvent (30 ml). After addition of 4,4’-dimethoxytrityl chloride (0.474 g, 1.4 mmol), the
mixture was stirred overnight, then concentrated, and co-concentrated with toluene (50 ml). The residue
was dissolved in CH2Cl2 (100 ml), the soln. washed with sat. aq. NaHCO3 soln. (3 ꢁ 200 ml), dried
(Na2SO4), and concentrated, and the residue dissolved in little CH2Cl2/MeOH and subjected to FC (silica
gel (2.5 ꢁ 30 cm), CH2Cl2 (100 ml), CH2Cl2/MeOH 98 :2 (100 ml), CH2Cl2/MeOH 96 :4 (100 ml),
CH2Cl2/MeOH 94 :6 (100 ml), CH2Cl2/MeOH 92 :8 (100 ml), and CH2Cl2/MeOH 99 :1 (100 ml)). The
red residue of the product fractions was dissolved in CH2Cl2/MeOH 95 :5, the soln. filtered through
cellulose powder, and the filtrate dropwise added to cold MeOH (50 ml) at 08. The resulting suspension
was kept in an icebox overnight, the solid collected, washed with Et2O, and dried: 1.64 g (88%) of 58.
Orange-red powder. UV (CH2Cl2/MeOH 1:1): 231 (4.96), 260 (4.83), 304 (sh, 4.22), 367 (4.38), 436 (sh,
4.33), 458 (4.46), 488 (sh, 4.34). 1H-NMR ((D6)DMSO): 9.08 (br. s, NHCONH); 8.39 (d, HꢀC(3) (Ar));
8.33 (s, HꢀC(2) (Ar)); 8.19 (d, 2 H o to NO2); 8.06 (d, 2 H o to NO2); 7.96 (d, HꢀC(5) (Ar)); 7.80 (s,
HꢀC(8) (Pur)); 7.68 (d, HꢀC(6)); 7.65 (d, 2 H m to NO2); 7.41 – 7.14 (m, HꢀC(5) (Ar), HꢀC(6) (Ar),
HꢀC(5) (Xan), HꢀC(3) (Ar), 2 H m to NO2, 4 H o to MeO, C6H5 ((MeO)2Tr) , NHꢀC(2) (Pur));
6.90 – 6.72 (m, HꢀC(8), HꢀC(7), HꢀC(1) (all Xan), 4 H m to MeO); 6.35 (dd, HꢀC(2) (Xan); t,
NHCONH); 6.15 (d, HꢀC(4) (Xan)); 6.12 (ꢃtꢄ, HꢀC(1’)); 5.35 (br. d, OHꢀC(3’)); 4.48 – 4.25 (m,
CH2CH2O, NCH2CH2NMe, HꢀC(3’)); 4.21 (t, CH2CH2O); 3.92 (m, HꢀC(4’)); 3.70 – 3.00 (m,
NCH2CH2NMe, CH2(5’), NHCH2CH2OCH2CH2OCH2CH2NH, 2 MeO, OCH2CH2NH, CH2CH2O);
3.03 (s, MeN); 2.81 (t, CH2CH2O); 2.75 – 2.65 (m, HaꢀC(2’)); 2.30 – 2.15 (m, HbꢀC(2’)). Anal. calc. for
C83H78N12O22 (1595.5): C 62.48, H 4.93, N 10.52; found: C 62.43, H 4.93, N 10.34.
44. 2-(4-Nitrophenyl)ethyl 5-{{{{2-{2-{2-{{9-[3’-O-(3-Carboxy-1-oxopropyl)-2’-deoxy-5’-O-(4,4’-di-
methoxytrityl)-b-d-ribofuranosyl]-1-{2-[(2,4-dinitrophenyl)methylamino]ethyl}-6,9-dihydro-6-oxo-1H-
purin-2-yl}amino}ethoxy}ethoxy}ethyl}amino}carbonyl}amino}-2-{6-[2-(4-nitrophenyl)ethoxy]-3-oxo-
3H-xanthen-9-yl}benzoate (59). A mixture of 58 (0.109 g, 0.07 mmol) and DMAP (34 mg, 0.28 mmol)
was dissolved in dry CH2Cl2 (5 ml), succinic anhydride (20 mg, 0.2 mmol) added, and the mixture stirred
at r.t. for 24 h. The mixture was diluted with CH2Cl2 (10 ml), the soln. washed with 10% citric acid soln.
(3 ꢁ 30 ml) and H2O (2 ꢁ 20 ml), dried (Na2SO4), and concentrated. The red residue was dissolved in
little CH2Cl2/MeOH 95 :5 and dropwise added to MeOH (10 ml) at 08. The precipitate was collected,
washed with Et2O, and dried under vacuum: 0.114 g (98%) of 59. Orange-red powder. UV (CH2Cl2/
MeOH 1:1): 230 (4.93), 260 (5.23), 304 (sh, 4.25), 365 (4.34), 433 (sh, 4.32), 458 (4.44), 486 (sh, 4.32).
1H-NMR ((D6)DMSO): 12.21 (s, COOH); 9.09 (br. s, NHCONH); 8.37 (d, HꢀC(3) (Ar)); 8.31 (s,
HꢀC(2) (Ar)); 8.15 (d, 2 H o to NO2); 8.03 (d, 2 H o to NO2); 7.98 (d, HꢀC(5) (Ar)); 7.78 (s, HꢀC(8)
(Pur)); 7.68 (d, HꢀC(6)); 7.62 (d, 2 H m to NO2); 7.37 – 7.12 (m, HꢀC(5), HꢀC(6) (Ar), HꢀC(5)
(Xan), 2 H m to NO2, 4 H o to MeO, C6H5 ((MeO)2Tr) , NHꢀC(2) (Pur)); 6.88 – 6.77 (m, HꢀC(8),
HꢀC(7), HꢀC(1) (all Xan), 4 H m to MeO); 6.38 (dd, HꢀC(2) (Xan); t, NHCONH); 6.18 – 6.10 (m,
HꢀC(1’), HꢀC(4) (Xan)); 5.31 (m, HꢀC(3’)); 4.50 – 4.21 (t, CH2CH2O, NCH2CH2NMe); 4.20 (t,
CH2CH2O); 4.08 (m, HꢀC(4’)); 3.70 – 3.00 (m, NCH2CH2NMe, CH2(5’), NHCH2CH2OCH2-
CH2OCH2CH2NH, 2 MeO, OCH2CH2NH, CH2CH2O); 3.02 (s, MeN); 2.82 (t, CH2CH2O); 2.60 – 2.50
(m, HaꢀC(2’), C(O)CH2CH2COOH); 2.45 – 2.35 (m, HbꢀC(2’)). Anal. calc. for C87H82N12O25 (1695.6):
C 61.63, H 4.87, N 9.90; found: C 61.79, H 4.86, N 9.84.
45. 2-(4-Nitrophenyl)ethyl 5-{{{{2-{2-{2-{{9-[3’-O-[(2-Cyanoethoxy)(diisopropylamino)phosphino]-
2’-deoxy-5’-O-(4,4’-dimethoxytrityl)-b-d-ribofuranosyl}-1-{2-[(2,4-dinitrophenyl)methylamino]ethyl}-
6,9-dihydro-6-oxo-1H-purin-2-yl}amino}ethoxy}ethoxy}ethyl}amino}carbonyl}amino}-2-{6-[2-(4-nitro-
phenyl)ethoxy]-3-oxo-3H-xanthen-9-yl}benzoate (60). A mixture of 59 (0.45 g, 0.3 mmol), sublimed 1H-
tetrazole (10.5 mg, 0.15 mmol), and 2-cyanoethyl N,N,N’,N’-tetraisopropylphosphorodiamidite (0.165 g,
0.55 mmol) in dry MeCN (5 ml) was stirred under N2 at r.t. for 3 h. After dilution with CH2Cl2 (50 ml),
the soln. was washed with NaCl-sat. NaHCO3 soln. (2 ꢁ 50 ml), dried (Na2SO4), and concentrated and
the residue dissolved in little CH2Cl2 and subjected to FC (flash silica gel (3 ꢁ 8 cm), hexane/acetone 1:1
(200 ml), hexane/acetone 9 :1 (200 ml), and acetone (100 ml)). The oily sirup of the product fractions
was dissolved again in little CH2Cl2 and dropwise added to dry Et2O at 08. The precipitate was collected
under N2 and dried under vacuum: 0.454 g (77%) of 60. UV (CH2Cl2/MeOH 1:1): 231 (4.97), 261 (4.85),
1
302 (sh, 4.30), 367 (4.40), 435 (sh, 4.36), 458 (4.46), 486 (sh, 4.35). H-NMR ((D6)DMSO): 9.09 (br. s,
NHCONH); 8.36 (d, HꢀC(3) (Ar)); 8.31 (s, HꢀC(2) (Ar)); 8.17 (d, 2 H o to NO2); 8.02 (d, 2 H o to

Helvetica Chimica Acta – Vol. 93 (2010)
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NO2); 8.00 (s, HꢀC(8) (Pur)); 7.96 (d, HꢀC(5) (Ar)); 7.69 (d, 2 H m to NO2); 7.65 (d, HꢀC(6)); 7.37 –
7.17 (m, HꢀC(5), HꢀC(6) (Ar), HꢀC(5) (Xan), 2 H m to NO2, 4 H o to MeO, C6H5 ((MeO)2Tr) ,
NHꢀC(2) (Pur)); 6.86 – 6.75 (m, HꢀC(8), HꢀC(7), HꢀC(1) (all Xan), 4 H m to MeO); 6.31 (dd,
HꢀC(2) (Xan); t, NHCONH); 6.15 (m, HꢀC(1’), HꢀC(4) (Xan)); 4.65 (m, HꢀC(3’)); 4.48 – 4.25 (t,
CH2CH2O, NCH2CH2NMe); 4.33 (m, OCH2CH2CN); 4.21 (t, CH2CH2O); 4.08 (m, HꢀC(4’)); 3.70 – 3.00
(m, NCH2CH2NMe, CH2(5’), NHCH2CH2OCH2CH2OCH2CH2NH, 2 MeO, Me2CH, OCH2CH2NH,
2 CH2CH2O); 3.03 (s, MeN); 2.81 (t, CH2CH2O); 2.82 (m, HaꢀC(2’)); 2.50 (m, HbꢀC(2’)); 1.26 – 1.00 (m,
2 Me2CH). Anal. calc. for C92H95N14O23P (1795.7): C 61.54, H 5.33, N 10.91; found: C 60.55, H 5.37, N
10.66.
46. N1-(2,4-Dinitrophenyl)ethane-1,2-diamine (61). To a mixture of ethane-1,2-diamine dihydro-
chloride (33.3 g, 0.25 mol) and Et3N (124.5 ml, 0.9 mol) in DMF/H2O 1:1 (400 ml) was added dropwise
at 08 under vigorous stirring a soln. of 1-chloro-2,4-dinitrobenzene (10.1 g, 0.05 mol) in DMF (50 ml).
After stirring at r.t. for 2 h, the mixture was concentrated under high vacuum. The residue was heated
with H2O (500 ml) and conc. HCl soln. (50 ml) to boiling, and then the hot soln. was filtered. The
precipitate contained the bis-substituted less soluble by-product and was, therefore, treated with hot
MeOH/acetone, the mixture filtered, and the filtrate co-concentrated with the firstly obtained filtrate.
After concentration to 80 ml, the soln. was cooled, the resulting solid (HCl salt) collected and treated
with H2O (90 ml) and conc. NH3 soln. (10 ml). The soln. was kept overnight in an icebox, the precipitate
filtered off by suction, washed with little H2O, and dried in a vacuum desiccator: 7.8 g (69%) of 61. Yellow
powder. Mp. 928. 1H-NMR ((D6)DMSO): 8.80 (d, HꢀC(3)); 8.19 (d, HꢀC(5)); 7.20 (d, HꢀC(6)); 3.49
(t, CH2); 2.82 (t, CH2).
Data of 61 · HCl: UV (MeOH): 209 (4.07), 259 (3.93), 349 (4.20), 393 (sh, 3.77). 1H-NMR
((D6)DMSO): 8.87 (br. t, NH); 8.83 (d, HꢀC(3)); 8.22 (br. s, CH2NHþ3 ); 8.20 (d, HꢀC(5)); 7.41 (d,
HꢀC(6)); 3.84 (q, NHCH2CH2NH3þ ); 2.93 (t, NHCH2CH2NHþ3 ). Anal. calc. for C8H10N4O4 · HCl (262.6):
C 36.59, H 4.22, N 21.37; found: C 36.67, H 4.33, N 21.34.
47. 3’,5’-Di-O-acetyl-2’-deoxy-N2-{2-[(2,4-dinitrophenyl)amino]ethyl}-O6-[2-(4-nitrophenyl)ethyl]-
guanosine (62). A mixture of 61 (1.1 g, 4.8 mmol) and 30 (1.0 g, 2 mmol) was co-concentrated with
dry pyridine (30 ml). The residue was dissolved in dry pyridine (10 ml) and then heated to 808 for 8 h.
After cooling to r.t., the mixture was concentrated and co-concentrated with toluene (3 ꢁ 20 ml), the
residue dissolved in CH2Cl2 (50 ml), and the soln. washed with sat. aq. NaHCO3 soln. (3 ꢁ 80 ml). The
aq. phase was re-extracted with CH2Cl2 (2 ꢁ 20 ml), the combined org. phase dried (Na2SO4) and
concentrated, and the residue purified by FC (silica gel (2 ꢁ 25 cm), CH2Cl2/MeOH 99 :1). The residue of
the product fractions was recrystallized from MeOH (80 ml)/acetone (2 ml): 1.05 g (74%) of 62. Red
powder. UV (MeOH): 211 (4.63), 254 (4.48), 350 (4.23), 407 (sh, 3.82). 1H-NMR ((D6)DMSO;
measured at 608): 8.89 (br. t, NH); 8.82 (d, HꢀC(3) (Ar)); 8.20 (d, HꢀC(5) (Ar)); 8.13 (d, 2 H o to
NO2); 8.04 (s, HꢀC(8)); 7.60 (d, 2 H m to NO2); 7.31 (d, HꢀC(6) (Ar)); 7.17 (br. t, NH); 6.26 (dd,
HꢀC(1’)); 5.43 (m, HꢀC(3’)); 4.70 (t, CH2CH2O); 4.30 – 4.13 (m, HꢀC(4’), CH2(5’)); 3.71 (t, CH2); 3.65
(t, CH2); 3.22 (t, CH2CH2O); 3.11 – 3.02 (m, HaꢀC(2’)); 2.55 – 2.45 (m, HbꢀC(2’)); 2.08 (s, Ac); 2.00 (s,
Ac). Anal. calc. for C30H31N9O12 (709.6): C 50.78, H 4.40, N 17.76; found: C 50.67, H 4.48, N 17.37.
48. Phenyl N-{9-(3’,5’-Di-O-acetyl-2’-deoxy-b-d-ribofuranosyl)-6-[2-(4-Nitrophenyl)ethoxy]-9H-pu-
rin-2-yl}-N-{2-[(2,4-dinitrophenyl)amino]ethyl}carbamate (63). Compound 62 (0.4 g, 0.56 mmol) was co-
concentrated with dry pyridine (3 ꢁ 10 ml) and the residue dissolved in dry pyridine (2 ml) and CH2Cl2
(10 ml). A small amount of molecular sieve (4ꢅ) and phenyl carbonochloridate (0.22 ml, 1.68 mmol) was
added at 08 and the mixture stored for 5 days in an icebox (08). After evaporation and co-evaporation
with toluene (3 ꢁ 10 ml), the residue was purified by FC (silica gel (1.5 ꢁ 20 cm), CH2Cl2/MeOH 97:3):
0.17 g (37%) of 63. Yellow foam. UV (MeOH): 202 (4.68), 218 (sh, 4.58), 262 (4.50), 348 (4.13), 403 (sh,
3.65). 1H-NMR ((D6)DMSO): 8.92 (br. t, NH); 8.67 (d, HꢀC(3) (Ar)); 8.50 (s, HꢀC(8)); 8.20 (d,
HꢀC(5) (Ar)); 8.12 (d, 2 H o to NO2); 7.50 (d, 2 H m to NO2); 7.57 – 7.10 (m, C6H5, HꢀC(6) (Ar)); 6.28
(dd, HꢀC(1’)); 5.32 (m, HꢀC(3’)); 4.68 (t, CH2CH2O); 4.35 (m, NHCH2CH2NꢀC(2)); 4.30 – 4.18 (m,
HꢀC(4’), CH2(5’)); 3.91 (q, NHCH2CH2NꢀC(2)); 3.20 (t, CH2CH2O); 3.14 – 3.02 (m, HaꢀC(2’)); 2.55 –
2.49 (m, HbꢀC(2’)); 2.08 (s, Ac); 1.99 (s, Ac). Anal. calc. for C37H35N9O14 (829.7): C 53.56, H 4.25, N
15.19; found: C 53.61, H 4.25, N 14.97.

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Helvetica Chimica Acta – Vol. 93 (2010)
49. N’-{2-[2-(2-Aminoethoxy)ethoxy]ethyl}-N-{9-(3’,5’-di-O-acetyl-2’-deoxy-b-d-ribofuranosyl}-6-[2-
(4-nitrophenyl)ethoxy]-9H-purin-2-yl}-N-{2-[(2,4-dinitrophenyl)amino]ethyl}urea (64). Compound 63
(0.25 g, 0.3 mmol) was co-concentrated with dry pyridine (3 ꢁ 10 ml) and then dissolved in dry pyridine
(10 ml). Then, 2,2’-[ethane-1,2-diylbis(oxy)]bis[ethanamine] (0.18 ml, 1.2 mmol) was added and stirred
at 508 for 30 min. After evaporation and co-evaporation with toluene (2 ꢁ 10 ml), the residue was
purified by FC (SiO2 (1.5 ꢁ 30 cm), CH2Cl2/MeOH 97:3 þ 1% Et3N (100 ml), CH2Cl2/ MeOH 92 :8 þ
1% Et3N (100 ml), CH2Cl2/MeOH 85 :15 þ 1% Et3N (200 ml), and CH2Cl2/MeOH 8 :2 þ 1% Et3N
(200 ml)). The residue of the product fractions was treated with little MeOH and the solid collected and
dried to give 0.145 g (56%) of 64. UV (MeOH): 205 (4.63), 254 (4.51), 349 (4.23), 402 (sh, 3.84).
1H-NMR ((D6)DMSO): 9.62 (br. t, NH); 9.00 (br. t, NH); 8.78 (d, HꢀC(3) (Ar)); 8.41 (s, HꢀC(8)); 8.20
(d, HꢀC(5) (Ar)); 8.13 (d, 2 H o to NO2); 7.61 (d, 2 H m to NO2); 7.29 (m, HꢀC(6) (Ar)); 6.36 (dd,
HꢀC(1’)); 5.36 (m, HꢀC(3’)); 4.80 (t, CH2CH2O); 4.51 (m, NHCH2CH2NꢀC(2)); 4.40 – 4.10 (m,
HꢀC(4’), CH2(5’)); 3.83 (q, NHCH2CH2NꢀC(2)); 3.60 – 3.20 (m, CH2CH2O, H2NCH2CH2OCH2-
CH2OCH2CH2); 3.09 – 2.95 (m, HaꢀC(2’)); 2.60 – 2.49 (m, HbꢀC(2’)); 2.08 (s, Ac); 1.98 (s, Ac). Anal.
calc. for C37H45N11O15 (883.8): C 50.28, H 5.13, N 17.43; found: C 50.23, H 5.14, N 17.08.
50. 2-(4-Nitrophenyl)ethyl 5-{{{{2-{2-{2-{{{{9-(2’-Deoxy-b-d-ribofuranosyl)-6-[2-(4-nitrophenyl)-
ethoxy]-9H-purin-2-yl}{2-(2,4-dinitrophenyl)amino]ethyl}amino}carbonyl}amino}ethoxy}ethoxy}ethyl}-
amino}carbonyl}amino}-2-{6-[2-(4-nitrophenyl)ethoxy]-3-oxo-3H-xanthen-9-yl}benzoate (65). A soln. of
64 (0.15 g, 0.177 mmol) in conc. NH3 soln./H2O/dioxane 1:1:1 (20 ml) was kept overnight in an icebox.
After evaporation, the residue and DMAP (65 mg, 0.27 mmol) was co-concentrated with dry DMF and
dissolved in dry DMF (4 ml) and dry pyridine (1 ml). Then, 55 (0.203 g, 0.27 mmol) was added and
heated to 608 for 4 h. The mixture was concentrated, and the oily sirup dissolved in little CH2Cl2/MeOH
95 :5 and subjected to FC (silica gel (1 ꢁ 10 cm), CH2Cl2/MeOH 95 :5 (80 ml), CH2Cl2/MeOH 93 :7
(50 ml), CH2Cl2/MeOH 9 :1 (100 ml), and CH2Cl2/MeOH 85 :15 (100 ml)). The residue of the product
fractions was treated with cold MeOH and kept in an icebox to give 0.2 g (78%) of 65. Orange-red solid.
UV (CH2Cl2/MeOH 1:1): 205 (4.60), 211 (4.65), 217 (4.68), 225 (4.93), 262 (4.90), 302 (sh, 4.36), 354
1
(4.45), 436 (sh, 4.38), 459 (4.46), 487 (sh, 4.37). H-NMR ((D6)DMSO): 9.69 (br. t, NH); 9.07 (br. t,
NH); 9.02 (br. t, NH); 8.75 (d, HꢀC(3) (Ar)); 8.44 (s, HꢀC(8) (Pur)); 8.28 (d, HꢀC(2)); 8.21 (d,
HꢀC(5) (Ar)); 8.17 (d, 6 H o to NO2); 7.69 (d, 2 H m to NO2); 7.66 – 7.58 (2d, 2 H m to NO2); 7.31 (m, 2 H
m to NO2, HꢀC(5) (Ar)); 7.30 (m, HꢀC(6) (Ar)); 7.12 (d, HꢀC(5) (Xan)); 6.88 – 6.78 (m, HꢀC(8),
HꢀC(7), HꢀC(1) (all Xan)); 6.36 – 6.25 (m, HꢀC(1’), HꢀC(2) (Xan), NH); 6.13 (d, HꢀC(4) (Xan));
5.32 (d, OHꢀC(3’)); 4.94 (br. t, OHꢀC(5’)); 4.79 (t, CH2CH2O); 4.48 (m, NHCH2CH2NꢀC(2) (Pur));
4.45 (m, CH2CH2O, HꢀC(3’)); 4.21 (t, CH2CH2O); 3.89 (m, HꢀC(4’), NHCH2CH2NꢀC(2) (Pur));
3.60 – 3.20 (m, 2 CH2CH2O, CH2(5’), H2NCH2CH2OCH2CH2OCH2CH2); 2.82 (t, CH2CH2O); 2.70 – 2.59
(m, HaꢀC(2’)); 2.39 – 2.28 (m, HbꢀC(2’)). Anal. calc. for C70H66N14O23 (1471.3): C 57.14, H 4.52, N 13.32;
found: C 56.77, H 4.63, N 13.20.
51. 2-(4-Nitrophenyl)ethyl 5-{{{{2-{2-{2-{{{{9-[2’-Deoxy-5’-O-(4,4’-dimethoxytrityl)-b-d-ribofurano-
syl]-6-[2-(4-nitrophenyl)ethoxy]-9H-purin-2-yl}{2-[(2,4-dinitrophenyl)amino]ethyl}amino}carbonyl}-
amino}ethoxy}ethoxy}ethyl}amino}carbonyl}amino}-2-{6-[2-(4-nitrophenyl)ethoxy]-3-oxo-3H-xanthen-
9-yl}benzoate (66). As described for 58, with 65 (1.5 g, 1 mmol) in dry pyridine (30 ml) and 4,4’-
dimethoxytrityl chloride 0.45 g, 1,35 mmol). Purification by FC (silica gel (2.5 ꢁ 30 cm), CH2Cl2/MeOH
97:3 (300 ml) and CH2Cl2/MeOH 96 :4 (400 ml)) gave 1.45 g (80%) of 66. Orange red crystals. UV
(CH2Cl2/MeOH 1:1): 207 (4.64), 226 (5.03), 262 (4.90), 306 (sh, 4.30), 353 (4.43), 436 (sh, 4.36), 459
(4.45), 487 (sh, 4.34). 1H-NMR ((D6)DMSO): 9.71 (br. s, NHCONH); 8.96 (br. t, NH); 8.77 (d, HꢀC(3)
(Ar)); 8.34 (s, HꢀC(8) (Pur)); 8.29 (s, HꢀC(2) (Ar)); 8.18 (dd, HꢀC(5) (Ar)); 8.15 (d, 4 H o to NO2);
8.02 (d, 2 H o to NO2); 7.69 (d, HꢀC(6)); 7.66 – 7.58 (2d, 4 H m to NO2); 7.30 – 7.08 (m, HꢀC(5) (Ar),
HꢀC(6) (Ar), HꢀC(5) (Xan), HꢀC(3) (Ar), 2 H m to NO2, 4 H o to MeO, C6H5 ((MeO)2Tr)); 6.8 –
6.67 (m, HꢀC(8) (Xan), HꢀC(7), HꢀC(1) (Xan), 4 H m to MeO); 6.38 (dd, HꢀC(1’)); 6.33 (dd,
HꢀC(2) (Xan)); 6.21 (t, NHCONH); 6.13 (d, HꢀC(4) (Xan)); 5.32 (br. d, OHꢀC(3’)); 4.79 (m,
CH2CH2O); 4.42 (m, NHCH2CH2NꢀC(2) (Pur), CH2CH2O, HꢀC(3’)); 4.21 (t, CH2CH2O); 3.99 (m,
HꢀC(4’)); 3.78 (m, NHCH2CH2NꢀC(2) (Pur)); 3.68 (2s, 2 MeO); 3.60 – 3.18 (m, NHCH2CH2OCH2-
CH2OCH2CH2NH, OCH2CH2NH, CH2CH2O); 3.16 (m, CH2(5’)); 2.81 (t, CH2CH2O); 2.85 – 2.73 (m,

Helvetica Chimica Acta – Vol. 93 (2010)
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HaꢀC(2’)); 2.50 – 2.38 (m, HbꢀC(2’)). Anal. calc. for C91H84N14O25 · H2O (1782.7): C 61.31, H 4.86, N
10.99; found: C 61.02, H 4.85, N 10.84.
52. 2-(4-Nitrophenyl)ethyl 5-{{{{2-{2-{2-{{{{9-{3’-O-[(2-Cyanoethoxy)(diisopropylamino)phosphi-
no]-2’-deoxy-5’-O-(4,4’-dimethoxytrityl)-b-d-ribofuranosyl}-6-[2-(4-nitrophenyl)ethoxy]-9H-purin-2-
yl}{2-[(2,4-dinitrophenyl)amino]ethyl}amino}carbonyl}amino}ethoxy}ethoxy}ethyl}amino}carbonyl}-
amino}-2-{6-[2-(4-nitrophenyl)ethoxy]-3-oxo-3H-xanthen-9-yl}benzoate (67). As described for 60, with
66 (0.25 g, 0.14 mmol), 1H-tetrazole (2.4 mg, 0.035 mmol), and 2-cyanoethyl N,N,N’,N’-tetraisopropyl-
phosphorodiamidite (77 mg, 0.25 mmol): 0.2 g (72% of 67. Red solid foam. UV (CH2Cl2/MeOH 1:1):
228 (4.99), 263 (4.91), 299 (sh, 4.40), 354 (4.45), 435 (sh, 4.37), 458 (4.46), 486 (sh, 4.35). 1H-NMR
((D6)DMSO): 9.80 (br. s, NHCONH); 9.08 (br. s, NH); 9.00 (br. t, NH); 8.72 (d, HꢀC(3) (Ar)); 8.32 (s,
HꢀC(8) (Pur)); 8.28 (s, HꢀC(2) (Ar)); 8.18 (m, HꢀC(5) (Ar), 4 H o to NO2); 8.01 (d, 2 H o to NO2);
7.96 (d, HꢀC(5) (Ar)); 7.64 (d, HꢀC(6)); 7.62 – 7.54 (2d, 4 H m to NO2); 7.30 – 7.08 (m, HꢀC(5) (Ar),
HꢀC(6) (Ar), HꢀC(5) (Xan), 2 H m to NO2, 4 H o to MeO, C6H5 ((MeO)2Tr)); 6.85 – 6.70 (m,
HꢀC(8), HꢀC(7), HꢀC(1) (all Xan), 4 H m to MeO); 6.41 (m, HꢀC(1’)); 6.32 (dd, HꢀC(2) (Xan));
6.30 (br. t, NHCONH); 6.12 (d, HꢀC(4) (Xan)); 4.81 (t, CH2CH2O); 4.70 – 4.60 (m, HꢀC(3’)); 4.42 (m,
CH2CH2O, NHCH2CH2NꢀC(2) (Pur)); 4.25 (t, OCH2CH2CN); 4.20 (t, CH2CH2O); 4.11 (m, HꢀC(4’));
3.79 (m, NHCH2CH2NꢀC(2) (Pur)). 3.66 (2s, MeO); 3.69 – 3.15 (m, CH2(5’), NHCH2CH2OCH2-
CH2OCH2CH2NH, OCH2CH2CN, 2 CH2CH2O); 3.00 – 2.90 (m, HaꢀC(2’)); 2.89 – 2.50 (m, HbꢀC(2’),
CH2CH2O, OCH2CH2CN, 2 Me2CH); 1.18 – 1.00 (m, 2 Me2CH). 31P-NMR ((D6)DMSO): 148.4; 149.0.
Anal. calc. for C100H101N16O26P (1973.9): C 60.85, H 5.16, N 11.35; found: C 60.15, H 5.26, N 11.04.
53. Oligonucleotide Synthesis. Solid-support material 500 ꢅ LCAMA-CPG [4] (200 mg) was loaded
with the appropriate nucleoside 3’-succinate (30 mmol) by TOTU (10 mg, 30 mmol) in dry MeCN (3 ml)
and N-methylmorpholine (8 ml, 80 mml) and gentle shaking for 1.5 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 1 h at r.t. by gently shaking. The material was collected,
washed with MeOH, DMF, MeOH, acetone, and Et2O and dried in a vacuum desiccator. Assembly of
oligodeoxynucleotides: The syntheses were carried out in an Eppendorf-Biotronik-Ecosyn-D-300 or
Applied-Biosystems-392-DNA synthesizer applying the functionalized CPG material packed into a small
ABI column and followed by cycles of nucleotide addition according to a programmed series of reagents
and solvent washes based on recommended procedures with the following main steps: 1) 5’-O-(MeO)2Tr
deprotection in 135 s. 2) Coupling: 0.1m phosphoramidite and 0.5m 1H-tetrazole in dry MeCN, delivered
in alternating reagent pushes with a subsequent wait time of 60 s. 3) Capping: Ac2O/2,6-dimethylpyr-
idine/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 npe groups by 1m
DBU in MeCN delivered in several pushes and following wait steps (total wait time 20 min). 2) Cleavage
of the base-labile groups and cleavage from the support: conc. NH3 soln. delivered in one push with a
consecutive wait time of 1 h. The reaction soln. was collected and lyophilized in a Speed-vac concentrator
under high vacuum.
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Received July 5, 2010