Nucleotides: Part LXXI. A new type of labelling of nucleosides and nucleotides

Article abstract of DOI:10.1002/hlca.200390186

A new labelling technique attaching fluorescein via a carbamoyl linker directly to the amino groups of the nucleobases was developed. The amino groups were first converted to the phenoxycarbonyl derivatives (→ 10, 15, 19, 58), which reacted under mild conditions with 5-aminofluorescein to give the corresponding N-[(fluorescein-5-ylamino)carbonyl] derivatives (→ 11-14, 16, 17, 20, 59, 60). The introduction of the 5-aminofluorescein residue into properly protected adenylyl-adenosine dimers (→ 39, 40) and trimer (→ 50) worked well, and final deprotection of these uniformly blocked precursors led on treatment with DBU (1,8-diazabicyclo[5.4.0]undec-7-ene), in one step to dimer 41 and trimer 51. Synthesis of an appropriately protected monomeric phosphoramidite building block (→ 75) was more difficult, since introduction of the 2-(4-nitrophenyl) ethyl residue into the fluorescein moiety in 59 led mainly to trisubstitution to give 61 including the urea function. Formation of the adenylyl dimer 66 and trimer 67 proceeded in the usual manner by phosphoramidite chemistry; however, deprotection of 67 with DBU was incomplete since the O-alkyl group at the urea moiety was found to be very stable. Finally, the appropriate phosphoramidite building block 75 could be synthesized by the sequence 59 → 72 → 73 → 74 → 75. The phosphoramidite 75 was used for the synthesis of dimer 77 and trimer 79 by solution chemistry, as well as for that of various oligonucleotides by the machine-aided approach on solid support carrying the fluorophore at different positions of the chain (→ 84-87). The attachment of the fluorescein fluorophor via a short carbamoyl linker onto the 6-amino group of 2′-deoxyadenosine enables such molecules to function very well in fluorescence-polarization experiments.

Full text of DOI:10.1002/hlca.200390186

Helvetica Chimica Acta Vol. 86 (2003)
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Nucleotides
Part LXXI1)
A New Type of Labelling of Nucleosides and Nucleotides
by Harald Sigmund and Wolfgang Pfleiderer*)
Fachbereich Chemie, Universit‰t Konstanz, Postfach 5560, D-78457 Konstanz
A new labelling technique attaching fluorescein via a carbamoyl linker directly to the amino groups of the
nucleobases was developed. The amino groups were first converted to the phenoxycarbonyl derivatives ( ! 10,
15, 19, 58), which reacted under mild conditions with 5-aminofluorescein to give the corresponding N-
[(fluorescein-5-ylamino)carbonyl] derivatives ( ! 11 14, 16, 17, 20, 59, 60). The introduction of the 5-
aminofluorescein residue into properly protected adenylyl-adenosine dimers ( ! 39, 40) and trimer ( ! 50)
worked well, and final deprotection of these uniformly blocked precursors led on treatment with DBU (1,8-
diazabicyclo[5.4.0]undec-7-ene), in one step to dimer 41 and trimer 51. Synthesis of an appropriately protected
monomeric phosphoramidite building block ( ! 75) was more difficult, since introduction of the 2-(4-
nitrophenyl)ethyl residue into the fluorescein moiety in 59 led mainly to trisubstitution to give 61 including the
urea function. Formation of the adenylyl dimer 66 and trimer 67 proceeded in the usual manner by
phosphoramidite chemistry; however, deprotection of 67 with DBU was incomplete since the O-alkyl group at
the urea moiety was found to be very stable. Finally, the appropriate phosphoramidite building block 75 could be
synthesized by the sequence 59 ! 72 ! 73 ! 74 ! 75. The phosphoramidite 75 was used for the synthesis of
dimer 77 and trimer 79 by solution chemistry, as well as for that of various oligonucleotides by the machine-
aided approach on solid support carrying the fluorophore at different positions of the chain ( ! 84 87). The
attachment of the fluorescein fluorophor via a short carbamoyl linker onto the 6-amino group of 2'-
deoxyadenosine enables such molecules to function very well in fluorescence-polarization experiments.
1. Introduction. Labelling of nucleosides, nucleotides, and oligonucleotides is a
crucial procedure for identification of nucleic acid components in general. DNA
sequencing and DNA probing play an important role in nucleic acid chemistry and
molecular biology and can be achieved only when the molecules under investigation
can be detected. The starting techniques [2] used radioactive nuclides, which reveal
several disadvantages and have, therefore, been substitued by a broad variety of
fluorophores [3]. The DNA-probing technique for gene analysis that was introduced by
Southern in 1975 [2] applies the principle of hybridization of complementary
oligonucleotide sequences. We have to differentiate between hybridizations in solution
and those on solid-support material, depending on the purpose of the investigation. The
use of nonradioactive markers for oligoncleotides was originally based upon biotin and
its interaction with the proteins avidin and streptavidin [4]; however, this indirect
method affords painful preparations of the probe, whereas the developed digoxigenin
UTP conjugate [5] allows more easily the introduction of a marker into oligonucleo-
tides by chemical and enzymatic means. An ideal label for DNA probing should fulfill
1
)
Part LXX: [1]

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Helvetica Chimica Acta Vol. 86 (2003)
at least four criteria, such as easy introduction into DNA sequences, easy detection in
low concentrations, unequivocal signal appearance showing hybridization, and
reasonable stability towards increased hybridization temperatures. The crucial step is
the former approach, whereby in principle a spacer between the nucleotide unit and the
label is applied by attachment to the 3'- or 5'-end [7] of the oligonucleotide, its
phosphate bridge [8], the nucleobase [9], and the sugar moiety [10]. We will describe a
new type of labelling of nucleosides by omitting the spacer and attaching the
fluorophore directly to the amino group of adenosine, cytidine, or guanosine via an
urea function. These investigations are based upon our former findings [11] that N6-
(phenoxycarbonyl)adenosine and N4-(phenoxycarbonyl)cytidine react under mild
conditions with aromatic amines to the corresponding urea derivatives.
2. Synthesis. The fluorophore of choice for the labelling experiments was 5-
aminofluorescein (7), which was synthesized from 4-nitrophthalic acid (1) and
resorcinol (ˆ benzene-1,3-diol; 2) in a fusion reaction [12] leading to a mixture of 4-
and 5-nitrofluorescein (3 and 4, resp.) (Scheme 1). Acetylation to the diacetyl
derivatives 5 and 6 allowed separation of the isomers by fractional crystallization.
Reduction of 6 is tricky [13] and worked best only with the system Na2S/NaSH to
give 5-aminofluorescein (7) in 77% yield. Esterification of 6 with MeOH/H2SO4 gave
the open-form methylester 8 of 5-nitrofluorescein, which could, however, not been
reduced to the corresponding 5-amino derivative 9. Compound 9 was, therefore,
prepared from 7 by an esterification analogous to that of 6. The informative 1H-NMR
spectra of these compounds show clearly that 5-nitro-3',6'-diacetylfluorescein (6) and 5-
aminofluorescein (7) exist in their lactone forms, whereas 9 is methyl 5-amino-2-(6-
hydroxy-3-oxo-3H-xanthen-9-yl)benzoate. The same structural conclusion can be
drawn from the UV spectra. The UV absorption [14] and emission spectra [15] of
fluorescein itself have been widely investigated in relationship to the pH. Lindquist
[14c] determined the pK values describing the equilibria monocation to neutral form,
neutral form to monoanion, and monoanion to dianion to be 2.2, 4.4, and 6.6,
respectively. The neutral form exists thereby as an equilibrium mixture of the cyclic
lactone and the open carboxy form [14d,e]. Little information is available about 5-
aminofluorescein (7), since its use in fluorescence immunoassays is mainly mentioned
in patents. We determined the pK values of the methyl ester 9 of 5-aminofluorescein to
be 1.6, 3.1, and 6.6, showing that this molecule forms a dication at pH 0. At pH 2, the
monocation exists, and, at pH 5, the neutral form is present. Finally, at pH 8, the
monoanion exhibits the typical strong increase in extinction coefficient at 492 nm to
82500 l ¥ molÀ1 ¥ cmÀ1 (Fig. 1) and correlates expectedly with the dianion of fluorescein.
An analogous pKa determination of 5-aminofluorescein (7) is problematic since in the
pH range 3 6, overlapping pK values do not allow a clear separation.
The adenosine conjugates were prepared from 2',3',5'-tri-O-acetyl-N6-(phenoxy-
carbonyl)adenosine (10) with 5-aminofluorescein (7) and its methyl ester 9 to give 11
and 13, respectively, in very good yields (Scheme 1). To facilitate workup, the educt 10
should be applied in an excess of 1.3 1.5 equiv. to get complete reaction of the dyestuff
which has chromatographic properties similar to those of the conjugate. Deacetylation
of 11 works well with methanolic ammonia to give 12, whereas the cleavage of the
acetyl groups of 13 required K2CO3/MeOH to give the methyl ester 14. The NMR

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Scheme 1
spectra of the adenosine fluorescein conjugates show nicely separated signals of the
nucleoside and of the dyestuff (Fig. 2) and allow easy assignment of the protons. The
presence of 2 OH protons (10.1 ppm) of the xanthene moiety of 11 illustrates that this
compound exists in the cyclic lactone form. Also, the signals of the NH functions of the
urea moiety (12.2 and 10.5 ppm) are quite characteristic. Typical for this type of
compounds are also the only hardly separated HÀC(2) and HÀC(8) protons of the
purine ring.
The UV/VIS spectra depend on the molecular species present at the related pH. In
the normal pH range, 13 shows 4 pKa values at 0.96 (dication > monocation), 2.48
(monocation > neutral form), 6.57 (neutral form > monoanion), and 11.9 (mono-
anion > dianion). We assume that the dication is protonated at the N(1) atom of the
purine ring and the carbonyl function of the xanthene moiety. The monocation is,

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Fig. 1. UV-Absorbtion spectra of the dication (pH 0), the monocation (pH 2), the neutral form (pH 5), and the
monoanion (pH 8) of 9
according to the partially overlapping pKa in strong acid, a mixture of two species with
preference for the xanthene protonated form. The long-wavelength-absorption band of
the cations is found at 440nm and is shifted characteristically to a double maximum at
466 and 494 nm with lower extinction for the neutral species (see Fig. 3,a). Monoanion
formation involving ionization at the xanthene OH group is associated with an
enormous increase in extinction coefficient to 86000 l ¥ molÀ1 ¥ cmÀ1 at 494 nm and
appearance of strong fluorescence. The dianion formation is related to deprotonation
of the urea function, since N6-(N-phenylcarbamoyl)adenosine [11] has, as a more-
simple analog, a pKa of 12.2.
A more-complex situation is present in compound 11, since here the neutral species
exists preferentially in the cyclic lactone form as established by the low extinction at
440nm. At pH 5.5, the lactone ring has opened up to form a monoanion at the carboxy
group characterized by the double maximum at 450and 470nm. Further deprotonation

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Fig. 2. NMR Spectra of 11 and 13 in (D6)DMSO. H-2, H-8, N6H: protons of the nucleobase; H-1', H-2', H-3', H-
4', H-5', H-5'': protons of the sugar moiety; H3, H4, H6: protons of the isobenzofuran or benzoic acid moiety
(arbitrary numberings); H1'', H2'', H4'', H5'', H7'', H8'': protons of the xanthene moiety; integrations in
brackets.

Fig. 3. UV Spectra of a) the monocation (pH 0), the neutral form (pH 2), the monoanion (pH 5.5), and the dianion (pH 9) of 11 and b) of the monocation (pH 1), the
neutral form (pH 5), and the monoanion (pH 9) of 13

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Fig. 4. pH-Dependent fluorescence spectra of 11
at the xanthene moiety gives the strongly fluorescent dianion with a high extinction
coefficient (Fig. 3,b). The emission properties of 11 reflect also the structural features,
since the neutral species shows very weak fluorescence that increases systematically
with higher pH (Fig. 4).
Cytidine carbamates are less investigated and known only in the form of N4-fmoc
[16] and N4-npeoc [17] protecting groups (fmoc ˆ (9H-fluoren-9-ylmethoxy)carbonyl,
npeoc ˆ [2-(4-nitrophenyl)ethoxy]carbonyl). Also, very few N4-urea derivatives of
cytosine and cytidine have been prepared by reaction with isocyanates [18]. Treatment
of 5'-O-(dimethoxytrityl)-N4-{[2-(4-nitrophenyl)ethoxy]carbonyl}cytidine with ammo-
nia in H2O/MeOH proceeded only by b-elimination of the protecting group [19],
whereas the N4-(phenoxycarbonyl)cytidine proved to be a versatile starting material
for N4-urea formation [11]. The 2',3',5'-tri-O-acetyl-N4-(phenoxycarbonyl)cytidine (15)

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reacted with 5-aminofluorescein (7) methyl ester 9 of 5-aminofluorescein in pyridine at
708 in good yields to the corresponding conjugates 16 and 17, respectively (Scheme 2).
The 1H-NMR- and UV data are in good agreement with the findings in the adenosine
series. Compound 16 exists as neutral species mainly in the cyclic lactone form, but its
UV spectrum is best characterized as a dianion at pH 9. The various molecular forms of
17 are better separated spectrophotometrically, as seen from the pKa values 0.40, 2.79,
6.13, and 11.33. The less-basic position in the dication is the N(3) ring atom followed by
the xanthene moiety. The neutral form at pH 4.5 has a relatively low extinction which
raises very high on conversion to the monoanion at pH 9. The further deprotonation of
the urea function can be recognized from the spectral change in the UV region at ca.
310nm, whereas the characteristic long-wavelength band at 496 nm does not change.
N2-Urea derivatives in the guanine/guanosine series have also not been systemati-
cally investigated, and the few examples have been derived from the reaction of
guanine [18b] or guanosine [18e][20][21] with various isocyanates. Ammonolysis of
carbamates have been only occassionally described [20]. We started our investigations
in this respect from 3',5'-di-O-acetyl-2'-deoxy-O6-[2-(4-nitrophenyl)ethyl]guanosine,
which is a reasonably soluble guanosine derivative prone to substitution reaction.
Phenyl carbonochloridate reacted with 18 in pyridine at room temperature in good
yield to the corresponding N2-(phenoxycarbonyl) derivate 19, which could be further
converted to the deoxyguanosine conjugate 20 (Scheme 3). Its structure was
1
established by H-NMR and UV spectra, similarly as for the adenosine and cytidine
analogs, as well as by elemental analysis.
To allow the use of labelled adenosine derivatives in oligonucleotide synthesis, we
developed a new strategy starting at the 3'-end with 2'-deoxy-N6,3'-O-bis-{[2-(4-
nitrophenyl)ethoxy]carbonyl}adenosine (21) [19] and condensation with 2'-deoxy-5'-
O-(monomethoxytrityl)- (22) and 2'-deoxy-5'-O-(dimethoxytrityl)-N6-[(9H-fluoren-9-
ylmethoxy)carbonyl]adenosine 3'-[2-(4-nitrophenyl)ethyl diisopropylphosphorami-
dite] (23), which were derived from N6-fmoc-protected 2'-deoxyadenosine 24 [22]
first by monomethoxytritylation ( ! 25) and dimethoxytritylation ( ! 26), respectively,
and followed by phosphitylation with 2-(4-nitrophenyl)ethyl diisopropylphosphorami-
dochloridite to give the dimers 27 and 28 in 70 80% yield ( Scheme 4). Their
detritylations led to the protected adenylyladenosine 29, and their treatment with Et3N
in MeCN cleaved selectively the fmoc protecting group to form 30 and 31, respectively.
The introduction of the phenoxycarbonyl function onto the 6-amino group of 30 and 31
with 1-(phenoxycarbonyl)-1H-tetrazole was problematic since partial detritylation
took place, and isolation of the products 32 and 33, respectively, was difficult and gave
only 65% yield when protic solvents like MeOH were omitted on chromatography.
To overcome these difficulties, N6-fmoc-protected 2'-deoxyadenosine 24 was
protected at the 5'-OH group by the npeoc group ( ! 34) and then phosphitylated to
give 35. The condensation of 21 with 35 gave the dimer 36, which lost the fmoc group on
treatment with Et3N yielding 37. The 6-amino group of 37 was then acylated by 5-(4-
nitrophenyl)-1-(phenoxycarbonyl)-1H-tetrazole to form the fully protected dimer 38.
The introduction of the fluorophore was achieved by reaction of 33 or 38 with 5-
aminofluorescein (7) to give the conjugates 39 and 40, respectively. Compound 40 was
then deprotected in one step by treatment with DBU (1,8-diazabicyclo[5.4.0]undec-7-
ene) in pyridine and subsequently purified by FP (fast-performance) liquid chroma-

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Scheme 4
tography (DEAE-Sephadex; (Et3NH)HCO3 buffer (TBK)). The resulting triethylam-
monium salt was finally transformed into its more-stable sodium salt 41.
The syntheses of the corresponding trimers 42 and 43 worked in an analogous
manner by treatment of the dimer 29 with either 2'-deoxy-5'-O-(monomethoxytrityl)-
N6-[2-(4-nitrophenyl)ethoxy]carbonyl}- (44) [22] or 2'-deoxy-N6,5'-O-bis-{[2-(4-nitro-
phenyl)ethoxy]carbonyl}adenosine 3'-[2-(4-nitrophenyl)ethyl diisopropylphosphora-
midite] (45) (Scheme 5), which was prepared from its precursor 46 [23] (cf. Scheme 4).
Deprotection of the fmoc group of 42 and 43 by Et3N led in 94% yield to 47 and 48,
respectively. The anticipated acylation with 1-(phenoxycarbonyl)-5-(4-nitrophenyl)-
1H-tetrazole was, again, unsuccessful with 47 but gave an excellent yield with 48 to form
49.

Helvetica Chimica Acta Vol. 86 (2003)
2309
Scheme 5
Treatment of 49 with 5-aminofluorescein (7) gave the fluorescence-labelled trimer
50, however, in only 22% isolated yield, showing that the labelling experiments proceed
with decreasing yields going from the monomer 11 via the dimer 40 to the trimer
conjugate 50. Complete deprotection of 50 was achieved by DBU treatment, which
cleaved in one step all npe and npeoc groups; DEAE-Sephadex purification and
conversion of the resulting triethylammonium salt gave the sodium salt 51 in 60% yield
(from 50), which exhibited a very clean HPLC analysis (Fig. 5).
Characterization of the various dimers and trimers was done by 1H-NMR and UV
1
spectra as well as by elemental analysis. The H-NMR spectra present quite complex
patterns of which several characteristic signals can be assigned unambiguously.

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Fig. 5. HPLC of 51
Since the above-described results have not been satisfactory in every respect, we
decided to synthesize fluorescein-labelled 5'-O-(dimethoxytrityl protected 3'-phos-
phoramidites for use in a conventional DNA synthesizer. For this purpose, the
xanthene moiety also had to be protected to avoid side reactions. The 2',3',5'-tri-O-
acetyl-N6-carbamoyladenosine fluorescein (methyl ester) conjugate 13 was first
treated as a model substance with various anhydrides to protect the OH group. The
resulting compounds 52 54 (Scheme 6), which are formally vinylogous anhydrides,
turned out to be too reactive for further use. Compound 52 could be seen only on TLC,
but isolation in pure form was not possible. The next attempt was an esterification
reaction of 13 with 2-(4-nitrophenyl)ethanol under Mitsunobu [24] conditions, which
led to a mixture of two compounds of which the npe-protected 3-hydroxyxanthenyl
derivative 55 was formed only in 8%, whereas the main reaction product 56 was
additionally substituted at the urea O-atom. The structural assignment of the second
npe group at this O-atom is based on the 1H-NMR chemical shift of a t for a CH2CH2O
moiety appearing at lower field than expected for a CH2CH2N group.
In the 2'-deoxyadenosine series, the 3',5'-O-(1,1,3,3-tetraisopropyldisiloxane-1,3-
diyl) derivative 57 was chosen as the starting material. Phenoxycarbonylation to 58
worked well, and subsequent treatment with 5-aminofluorescein (7) and methyl ester 9
of 5-aminofluorescein led to the conjugates 59 and 60, respectively, in very good yields.
The Mitsunobu reaction of 59 with 2-(4-nitrophenyl)ethanol afforded a threefold
substitution to give 61 in 90% yield. Desilylation to 62 was achieved by Bu4NF in
AcOH/THF. The transformations into the 5'-O-(dimethoxytrityl) derivative 63 and the
corresponding 3'-[2-(nitrophenyl)ethyl diisopropylphosphoramidite] 64 were per-
formed routinely. The fully protected building block 64 was reacted with compound
21 to the dimer 65, which was detritylated to 66 and then condensed with 45 to give the
fully protected trimer 67. A new surprise was encountered on DBU treatment, expected
to remove all blocking groups. HPLC Analysis showed, on comparison with 51, only
20% conversion to 51, whereas the main peak was due to a more-lipophilic substance
that still contained a protecting group and presumably has the isourea structure 68.
To find out which function is relatively stable towards DBU, compound 63 was
treated under the same reaction conditions as 67: the O-[2-(4-nitrophenyl)ethyl]isour-
ea 69 (Scheme 6) was isolated in 81% yield. The cleavage of the npe group from the
isourea moiety under more-drastic conditions with 2000 equiv. of DBU was followed by
HPLC. Even after 48 h, still some starting material 69 was present, indicating that

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Scheme 6

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Helvetica Chimica Acta Vol. 86 (2003)
building block 64 is not suitable for oligonucleotide synthesis in a machine-aided
approach.
To overcome this problem, we concentrated again on the Mitsunobu reaction of 11
and 59 by changing the reaction conditions to get preferentially disubstitution at the
fluorescein moiety and less trisubstitution. It was found that the educt should be mixed
in dioxane with 2-(4-nitrophenyl)ethanol and diethyl azodicarboxylate first, and then
triphenylphosphine should be added in small portions controlling the progress of the
reaction by TLC after each addition and waiting for 5 min. Compound 11 gave thus a
mixture of 48% of the desired product 70 and 38% of 56 (Scheme 6). The analogous
reaction of 11 with 59 applying 2.1 to 2.3 equiv. of triphenylphosphine led to the desired
disubstituted product 72 in 64%, whereas the trisubstituted 61 was formed in only 16%
yield. Desilylation of 72 gave 73 in 92% yield and subsequent dimethoxytritylation led
in 76% yield to 74. The anticipated labelled building block 75 for oligonucleotide
synthesis was prepared by phosphitylation in the usual manner, and condensation with
21 led to the dimer 77, which was again detritylated to 78 in 83% yield and used for the
next condensation with the phosphoramidite 45 to give the fully protected dApdAfludA
trimer 79 (Scheme 7). The DBU cleavage removed all protecting groups in one step,
and, after DEAE-Sephadex purification and conversion into the sodium salt, 80 was
isolated in 60% yield and established by physical means to be identical to compound 51
prepared by a different route.
The trimer 51 (ˆ 80) was also synthesized on an ABI-DNA synthesizer 392 starting
with a modified CPG-solid support loaded with 2'-deoxy-5'-O-(dimethoxytrityl)-N6-
{[2-(4-nitrophenyl)ethoxy]carbonyl}adenosine 3'-succinate in the usual manner. For
the next cycle, the 2-cyanoethyl phosphoramidite 76 was found to be more reactive than
75 and giving higher yields. Proceeding with the 2'-deoxy-5'-O-(dimethoxytrityl)-N6-
{[2-(4-nitrophenyl)ethoxy]carbonyl}adenosine 3'-(2-cyanoethyl diisopropylphosphora-
midite) [22] gave the trimer, which was treated first with CHCl2COOH to remove the
dimethoxytrityl group, then with DBU to cleave the ce, npe, and npeoc groups before
detaching from the solid-support by ammonia. This sample was identical to both the
preparatively synthesized materials 51 and 80 as shown by HPLC. The analogous trimer
d(ApApAflu) carrying the fluorophore at the 3'-end required first the synthesis of the 3'-
succinate 81, which was coupled to the CPG solid support; the following two
conventional cycles yielded a high-quality product. The analogous combination with
dAflu led, to the trimer (dAflu)3 that showed, however, some impurities in the HPLC.
3. Oligonucleotide Synthesis.
To establish the usefulness of the fluorescein-
labelled dA as a new type of building block, several oligodeoxynucleotides carrying the
fluorophore in different positions were synthesized from the npe- and npeoc-protected
dA, dC, and dG phosphoramidites [22] (Table 1). First, we assembled the unlabeled 15-
mer sequence 82 and its complementary strain 83 as a reference. Then, the dAflu was
attached to the 5'-end ( ! 84), the 3'-end ( ! 86), the 3',5'-end ( ! 87), and the middle
( ! 85) of the chain. The complementary strains for hybridization studies carried on
both ends a 10-mer (see 88), a 20-mer (see 89), or a 30-mer-T (see 90) extension. The
‡
melting points (Table 1) were determined at a salt concentration of 0.12m (Na ) and at
pH 7.4. The unlabelled duplex 82 ¥ 83 showed a Tm of 59.68, and the attachment of one
dAflu unit at the 3'- or 5'-end forming the hybrids 83 ¥ 84 and 83 ¥ 86 caused a small

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decrease of 1 28, whereas labelling on both ends did not change the Tm of the standard.
Labelling in the middle of the chain gave, expectedly, a strong depression to 45.78 (83 ¥
85), which is also recognized in the duplexes 84 ¥ 88, 84 ¥ 89, and 84 ¥ 90, where the label
does not allow perfect H-bonding.
Table 1. Oligodeoxynucleotide Sequences and Tm of Hybridazationsa)
Sequence
Hybrid
Tm [8]
82
83
84
85
86
87
88
89
90
5'-d(TCC CAG TCA CGA CGT)-3'
5'-d(ACG TCG TGA CTG GGA)-3'
5'-d(AfluTCC CAG TCA CGA CGT)-3'
5'-d(TCC CAG TCAflu CGA CGT)-3'
5'-d(TCC CAG TCA CGA CGT-Aflu)-3'
5'-d(Aflu-TCC CAG TCA CGA CGT-Aflu)-3'
5'-d(T10ACG TCG TGA CTG GGA T10)-3'
5'-d(T20ACG TCG TGA CTG GGA T20)-3'
5'-d(T30ACG TCG TGA CTG GGA T30)-3'
82 ¥ 83
83 ¥ 84
83 ¥ 85
83 ¥ 86
83 ¥ 87
84 ¥ 88
84 ¥ 89
84 ¥ 90
59.6
58.5
45.7
57.6
59.8
54.6
54.3
54.0
a
‡
) Salt concentration: 0.12m (Na ).
4. Fluorescence Polarization. Fluorescence-polarization spectroscopy (FPS) is
used as a tool in biochemistry [25] and diagnostic [26] to detect interactions between
big molecules such as proteins or molecule aggregates present in membranes based
upon the mobility of the molecules. Fluorescing molecules excited by polarized light
show in viscous solvents a strong polarization of the emitted light. Small molecules that
rotate faster show, in general, almost complete fluorescence depolarization, whereas,
with big molecules, the grade of polarization is increased due to their restricted rotation
and mobility. Although nucleic acids belong to the group of large biomolecules,
relatively little is known about the detection of interactions of nucleic acids with other
nucleic acid components or other biomolecules by fluorescence polarization [27]. In
the first experiments, the relationship between the fluorescence polarization and the
molecular mass as well as the time scale of hybridization was studied (Table 2). The
addition of T10 did not have any influence on the observed results. The fluorescence
polarization was also dependent on the salt concentration and the pH of the solution
(Table 3). Higher salt concentrations stabilized the hybridization and increased the
viscosity of the solution, and a lower pH was consistent with stronger duplex stability.
Furthermore, the grade of fluorescence polarization was dependent on the size of
the target molecule, as demonstrated by the duplexes 83 ¥ 84, 84 ¥ 88, 84 ¥ 89, and 84 ¥ 90.
Table 2. Fluorescence Polarization: Dependence on Target Size and Hybridization Time in Buffer at pH 9
Substance
P
Substance
Time
P
Aflu (12)
0.012
0.018
0.041
0.085
0.040
83 ¥ 84 ‡ T10
83 ¥ 84 ‡ T10
83 ¥ 84 ‡ T10
83 ¥ 84 ‡ T10
83 ¥ 84 ‡ T10
10 min
20 min
45 min
90 min
20 h
0.036
0.053
0.066
0.069
0.083
d(AAfluA) (51)
16-mer 84
83 ¥ 84
T10 ‡ 84

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Table 3. Fluorescence Polarization: Dependence of pH, Salt Concentration, and Oligonucleotide Sequence
Sequence pH [NaCl]
P
Sequence pH [NaCl]
P
Sequence pH [NaCl]
P
84
84
84
84
84
84
84
84
84
84
7
7
7
7
7
9
9
9
9
9
0.076 84 ¥ 88
.1 0.083 84 ¥ 88
m .2 0.094 84 ¥ 88
m .4 0.108 84 ¥ 88
0.130 84 ¥ 88
0.052 84 ¥ 88
.1 0.063 84 ¥ 88
m .2 0.083 84 ¥ 88
m .4 0.100 84 ¥ 88
7
7
7
7
7
9
9
9
9
9
0.122 83 ¥ 84
.1 0.184 84 ¥ 88
m .2 0.194 84 ¥ 89
m .4 0.205 84 ¥ 90
7
7
7
7
0.06 m
0.06 m
0.06 m
0.06 m
0.106
0.154
0.167
0.170
0
0
0
m
0
0
0
m
1.0 m
1.0 m
0.223
0.115 83 ¥ 84
m .1 0.168 84 ¥ 88
m .2 0.175 84 ¥ 89
m .4 0.190 84 ¥ 90
9
9
9
9
0
0
0
0
m .2 0.120
m .2 0.175
m .2 0.191
m .2 0.190
0
0
0
m
0
0
0
1.0 m
0.129 84 ¥ 88
1.0 m
0.203
Experimental Part
General. Products were dried under high vacuum. All solvents used were of anhydrous grade. TLC:
precoated silica gel thin-layer sheets 60 F 254 from Merck. Flash chromatography (FC): silica gel Baker (30
60 mm); 0.2 0.3 bar; FPLC ˆ fast performance liquid chromatography; CC ˆ column chromatography. HPLC:
pump L 6000, autosampler AS 4000, UV detector L 4000, Merck-Hitachi; column RP 18, Licrocart, 125 Â 4 mm,
5 mm, Merck; elution: A ˆ 0.1m (Et3NH)OAc buffer pH 7, B ˆ 0.1m (Et3NH)OAc buffer/MeCN 1:1; gradient:
5% B (0 2 min), 5 40% B (2 30 min), 40 100% B (30 50 min), 100% B (50 60min); flow rate 1 ml/min.
M. p.: Gallenkamp melting-point apparatus; no corrections. UV/VIS: Perkin-Elmer Lambda 5; lmax in nm
(log e); the pKa determinations were performed spectrophotometrically [28]. Fluorescence spectra: Perkin-
1
Elmer LS 50. Fluorescence polarization: measured at Merck with Merck vitalabeclair . H-NMR: Bruker AC-
250; d in ppm rel. to SiMe4 or CDCl3 ((D6)DMSO) as internal standard; arbitrary numbering of the fluorescein
(flu) moiety (cf. 3 7); the same numbering is retained for the open form of fluorescein-derived substituents (cf.
8 and 9). 31P-NMR: Jeol JMN-GX400.
1. 3',6'-Di-O-acetyl-5-nitrofluorescein (ˆ 3',6'-Bis(acetyloxy)-5-nitrospiro[isobenzofuran-1(3H),9'-
[9H]xanthen]-3-one; 6). A mixture of 4-nitrophthalic acid (50 g, 0.24 mol) and resorcinol (53 g, 0.48 mol)
was heated in an open Erlenmeyer flask to 180 190 8 for 3 h with stirring, whereby the melt solidified to a dark
mass. The crude material was ground and then heated in 0.5n HCl (1 l) for 1 h under reflux. After cooling, the
precipitate was collected, dried, then boiled in Ac2O (110ml) for 1 h, and filtered hot through a glass frit. The
filtrate was kept several days in the icebox for crystallization. The separated solid was recrystallized first from
Ac2O (70ml) and finally from toluene (70ml): 17 g (28%) of 6. M.p. 215 2188. TLC (toluene/AcOEt 7:1): Rf
0.64. 1H-NMR ((D6)DMSO): 8.70( d, HÀC(6)); 8.55 (dd, HÀC(4)); 7.75 (d, HÀC(3)); 7.30( d, HÀC(4'),
HÀC(5')); 7.05 6.52 (m, HÀC(1'), HÀC(2'), HÀC(7'), HÀC(8')); 2.28 (s, 2 Ac).
2. 3',6'-Di-O-acetyl-4-nitrofluorescein (ˆ 3',6'-Bis(acetyloxy)-4-nitrospiro[isobenzofuran-1(3H),9'-
[9H]xanthen]-3-one; 5). The mother liquor of 6 was concentrated to a smaller volume to give a second
fraction of 6 (2 g). The filtrate was evaporated and the resulting syrup dissolved in hot toluene (200 ml). On
cooling, 5 separated as a crystalline solid, which was again recrystallized from toluene: 15 g (25%) of 5. M.p.
1918. TLC (toluene/AcOEt 7:1): Rf 0.51.
3. 5-Nitrofluorescein (ˆ 3',6'-Dihydroxy-5-nitrospiro[isobenzofuran-1(3H),9'-[9H]xanthen]-3-one; 4).
Compound 6 (10 g, 20.4 mmol) was heated in a mixture of 10% NaOH (100 ml) and MeOH (500 ml) till a
clear soln. was obtained (15 min). The warm soln. was diluted with H2O (150ml), acidified with AcOH (50ml),
heated again to boiling, and then slowly cooled and kept in the icebox overnight. The orange precipitate was
collected and dried: 7.3 g (95%) of 4. M.p. > 2508 (dec.). 1H-NMR ((D6)DMSO): 10.20 (br. s, 2 OH); 8.70
(d, HÀC(6)); 8.62 (dd, HÀC(4)); 7.65 (d, HÀC(3)); 6.80 6.50 ( m, HÀC(1'), HÀC(2'), HÀC(4'), HÀC(5'),
HÀC(7'), HÀC(8')).
4. 5-Aminofluorescein (ˆ 5-Amino-3',6'-dihydroxyspiro[isobenzofuran-1(3H),9'-[9H]xanthen]-3-one; 7).
A mixture of 5-nitrofluorescein (4; 1.1 g, 3 mmol), Na2S ¥ 9 H2O (2.6 g, 11 mmol), and NaSH (1.2 g, 21 mmol)
was heated in H2O (45 ml) for 24 h under reflux. After cooling, the mixture was acidified with AcOH and the
resulting dark precipitate collected. The solid was dissolved in 6% HCl soln. (50ml), insoluble sulfur and
charcoal were filtered off, and the filtrate was kept in the icebox overnight for crystallization. The precipitate

2316
Helvetica Chimica Acta Vol. 86 (2003)
was again dissolved in 0.5n NaOH (40ml) and then AcOH added to adjust the pH to 4. The precipitate was
collected and dried: 0.8 g (77%) of 7. UV (pH 9): 237 (4.63), 257 (4.29), 281 (4.12), 314 (3.78), 378 (3.70), 488
(4.87). 1H-NMR ((D6)DMSO): 10.05 (br. s, 2 OH); 7.00 6.80 (m, HÀC(3), HÀC(4), HÀC(6)); 6.65 6.50
(m, 6 arom. H (xan)); 5.70( s, NH2).
5. 2-(6-Hydroxy-3-oxo-3H-xanthen-9-yl)-5-nitrobenzoic Acid Methyl Ester (8). A mixture of 6 (1 g,
2.2 mmol), MeOH (30ml), and conc. H 2SO4 soln. (1 ml) was heated for 40h under reflux. The yellow soln. was
diluted with Et2O to crystallize slowly by standing overnight in the icebox. The precipitate (0.86 g, 80%)
consisted of the hydrogensulfate salt. Anal. calc. for C21H13NO7 ¥ H2SO4 (489.4): C 51.54, H 3.09, N 2.86; found:
C 52.35, H 3.40, N 2.53.
The hydrogensulfate salt (0.5 g) was suspended in MeOH (20 ml), then H2O (20ml) was added, the pH
adjusted to 5 by addition of little Na2HPO4 soln., and the mixture stirred for 15 min. The dark red precipitate
was collected and dried: 0.36 g (94%) of 8. 1H-NMR ((D6)DMSO): 8.90( d, HÀC(6)); 8.72 (dd, HÀC(4));
8.70 8.00 (br. s, OH); 7.82 (d, HÀC(3)); 7.10 6.80 ( m, HÀC(1'), HÀC(2'), HÀC(4'), HÀC(5'), HÀC(7'),
HÀC(8')); 3.65 (s, MeO).
6. 5-Amino-2-(6-hydroxy-3-oxo-3H-xanthen-9-yl)benzoic Acid Ethyl Ester (9). A mixture of 7 (3 g,
8.6 mmol), MeOH (60ml), and conc. H 2SO4 soln. (1 ml) was heated for 40h under reflux. The soln. was poured
on ice (300 g), and then, under stirring, a sat. NaHCO3 soln. was slowly added to adjust the pH to 6.5. An orange
precipitate separated, which was collected by centrifugation. The solid was washed twice with H2O, centrifuged,
and then heated in MeOH (100 ml) to boiling. After cooling, the solid was collected and dried under high
vacuum at 508: 2.2 g (70%) of 9. M.p. 2848 (dec.). TLC (CH2Cl2/MeOH 9 :1): Rf 0.28. UV (pH 9): 221 (4.63),
237 (4.70), 257 (4.41), 280 (4.15), 314 (3.94), 3.74 (3.76), 457 (4.40), 492 (4.91). pKa Values: 1.61, 3.09, 5.99.
1H-NMR ((D6)DMSO): 11.0(br. s, OH); 7.35 (d, HÀC(6)); 7.05 6.52 (m, HÀC(3), HÀC(4), 6 arom. H
(xan)); 5.90(br. s, NH2); 3.49 (s, MeO). Anal. calc. for C21H15NO5 (361.4): C 69.80, H 4.18, N 3.88; found: C
69.17, H 4.33, N 3.52.
7. 2',3',5'-Tri-O-acetyl-N6-[(fluorescein-5-ylamino)carbonyl]adenosine (ˆ2',3',5'-Tri-O-acetyl-N6-{[(3',6'-di-
hydroxy-3-oxospiro[isobenzofuran-1(3H),9'-[9H]xanthen]-5-yl)amino]carbonyl}adenosine; 11). A mixture of
2',3',5'-tri-O-acetyl-N6-(phenoxycarbonyl)adenosine (10) [11] (1 g, 2 mmol) and 7 (0.347 g, 1 mmol) was heated
in pyridine (10ml) to 70 8 for 1 h with stirring. After evaporation and co-evaporation with toluene, the residue
was dissolved in a little AcOEt and submitted to FC (silica gel, 11 Â 3.5 cm; toluene (100 ml), toluene/AcOEt
4 :1 (200 ml), toluene/AcOEt 1:1 (200 ml), toluene/AcOEt 1 :1 ‡ MeOH (10ml) (200ml). The product
fraction was concentrated to a smaller volume, whereby 11 separated from toluene. The solid was collected and
dried: 0.78 g (99%) of crude 11, which was pure enough for further reactions. The substance was recrystallized
from iPrOH: 0.61 g (79%) of 11. Colorless powder. M.p. 188 1908 (dec.). TLC (toluene/AcOEt/MeOH
5 :4 :1): Rf 0.16. UV (pH 9): 238 (4.69), 280 (4.62), 317 (3.96), 368 (sh., 3.70), 455 (4.35), 492 (4.91). pKa Values:
1.07, 1.71, 4.92, 6.55, 12.2. 1H-NMR ((D6)DMSO): 12.1 (s, NH); 10.5 (s, HÀN(6); 10.1 (s, 2OH); 8.74
(s, HÀC(2)); 8.70( s, HÀC(8)); 8.33 (dd, HÀC(6)(flu)); 7.93 (dd, HÀC(4)(flu)); 7.25 (d, HÀC(3)(flu)); 6.67
6.52 (m, 6 arom. H (xan)); 6.32 (d, HÀC(1')); 6.04 (t, HÀC(2')); 5.64 (t, HÀC(3')); 4.43 (m, 2 HÀC(5',
HÀC(5'')); 4.28 (m, HÀC(4')); 2.12, 2.04, 202 (3 s, 3 Ac). Anal. calc. for C37H30N6O13 (766.7): C 57.97, H 3.94, N
10.96; found: C 57.27, H 4.49, N 11.11.
8. N6-[(Fluorescein-5-ylamino)carbonyl]adenosine (ˆ N6-{[(3',6'-Dihydroxy-3-oxospiro[isobenzofuran-
1(3H),9'-[9H]xanthen]-5-yl)amino]carbonyl}adenosine; 12). A soln. of 11 (0.6 g, 0.78 mmol), K2CO3 (0.2 g),
and Bu4NBr (50mg) in MeOH (20ml) was stirred at r.t. for 3.5 h and then diluted with H 2O (100 ml) and
acidified with AcOH. The formed solid was collected by suction through a glass frit, washed with H2O, and
treated with EtOH to give a gelatinous precipitate, which was again collected and dried under high vacuum at
508: 0.39 g (78%) of 12. M.p. 2078 (dec.). TLC (iPrOH/conc. NH3 soln./H2O 7:1:2): Rf 0.46. UV (MeOH): 238
(4.67), 261 (sh., 4.44), 281 (4.61), 317 (3.95), 344 (sh., 4.36), 456 (sh., 4.36), 490(4.89). 1H-NMR ((D6)DMSO):
12.2 (s, NH); 10 .4 (br. s, HÀN(6), 2 OH); 8.70( s, HÀC(2), HÀC(8)); 8.30( dd, HÀC(6)(flu)); 7.90
(dd, HÀC(4)(flu)); 7.21 (d, HÀC(3)(flu)); 6.8 6.4 (m, 6 arom.
H
(xan)); 6.0( d, HÀC(1')); 5.7 5.0
(m, HÀC(2'), HÀC(3'), OH); 3.91 (m, HÀC(4')); 3.60( m, HÀC(5')). Anal. calc. for C31H24N6O10 ¥ 2 H2O
(640.6): C 55.03, H 4.17, N 12.42; found: C 55.46, H 4.44, N 12.21.
9. 2',3',5'-Tri-O-acetyl-N6-{{[4-(6-hydroxy-3-oxo-3H-xanthen-9-yl)-3-(methoxycarbonyl)phenyl]amino}car-
bonyl}adenosine (13). A mixture of 2',3',5'-tri-O-acetyl-N6-(phenoxycarbonyl)adenosine (10) [11] (2 g, 4 mmol)
and 9 (0.347 g, 1 mmol) was heated in abs. dioxane to 708 for 1.5 h with stirring. After cooling and standing
overnight, a precipitate separated and was collected and dried to give 1.5 g (96%) of almost pure material, which
was recrystallized from dioxane (60ml): 1.2 g (75%) of 13. M.p. 1908 (dec.). TLC (CH2Cl2/MeOH 9 :1): Rf 0.45.
UV (pH 9): 238 (4.73), 280(4.65), 317 (4.08), 356 (sh., 3.74), 460(4.40), 492 (4.91). p Ka Values: 0.96, 2.48, 6.57,

Helvetica Chimica Acta Vol. 86 (2003)
2317
11.9. 1H-NMR ((D6)DMSO): 12.1 (s, NH); 11.2 (br. s, OH); 10.5 (s, HÀN(6); 10.1; 8.75 (s, HÀC(2)); 8.71
(s, HÀC(8)); 8.54 (dd, HÀC(6)(flu)); 7.99 (dd, HÀC(4)(flu)); 7.45 (d, HÀC(3)(flu)); 6.89 6.52 (m, 6 arom. H
(xan); 6.33 (d, HÀC(1')); 6.05 (t, HÀC(2')); 5.64 (t, HÀC(3')); 4.44 (m, 2 HÀC(5')); 4.28 (m, HÀC(4')); 3.59
(s, MeO); 2.12, 2.04, 202 (3 s, 3 Ac).
10. N6-{{[4-(6-Hydroxy-3-oxo-3H-xanthen-9-yl)-3-(methoxycarbonyl)phenyl]amino}carbonyl}adenosine
(14). A soln. of 13 (0.6 g, 0.77 mmol), K2CO3 (0.2 g), and Bu4NBr (50mg) in MeOH (30ml) was stirred at
r.t. for 2.5 h, then diluted with H2O (100 ml), acidified with a few drops of AcOH, and concentrated to 20 ml.
The resulting precipitate was collected, then stirred in MeOH for 2 h, filtered again, and dried under high
vacuum: 0.38 g (73%) of 14. M.p. 208 2098. TLC (iPrOH/conc. NH3 soln./H2O 7:1 :2): Rf 0.57. UV (pH 9): 238
(4.72), 258 (sh., 4.48), 280(4.64), 316 (4.08), 348 (sh., 3.81), 460(sh., 4.40), 494 (4.91). 1H-NMR ((D6)DMSO):
12.1 (s, NH); 10 .4 (br. s, HÀN(6), OH); 8.70 (s, HÀC(2), HÀC(8)); 8.5 (dd, HÀC(6)(flu)); 8.00
(dd, HÀC(4)(flu)); 7.51 (d, HÀC(3)(flu)); 7.5 7.0( m, 6 arom.
H
(xan); 6.05 (d, HÀC(1')); 5.7 5.0
(m, HÀC(2'), HÀC(3', OH); 3.99 (m, HÀC(4')); 3.52 (m, 2 HÀC(5')). Anal. calc. for C32H26N6O10 ¥ H2O
(654.6): C 57.14, H 4.10, N 12.49; found: C 57.36, H 4.32, N 12.91.
11. 2',3',5'-Tri-O-acetyl-N4-(phenoxycarbonyl)cytidine (15). A mixture of 2',3',5'-tri-O-acetylcytidine
(0.74 g, 2 mmol) [29] and 1-(phenoxycarbonyl)-1H-tetrazole (2.5 g, 13 mmol) in dioxane (10ml) was stirred
at 408 for 30min. After evaporation, the residue was purified by FC (12 Â 2.5 cm; toluene (50ml), toluene/
acetone 9 :1 (200 ml), toluene/acetone 4 :2 (600 ml); 50-ml fractions). Fr. 13 16 gave 0.6 g (61%) of 15.
Colorless solid foam. TLC (toluene/acetone 1:1): Rf 0.5. UV (MeOH): 243 (4.28), 264 (3.89). 1H-NMR
((D6)DMSO): 11.4 (br. s, NH); 8.14 (d, HÀC(6)); 7.47 7.17 (m, Ph); 7.0 6 (d, HÀC(5)); 5.90( d, HÀC(1')); 5.49
(dd, HÀC(2')); 5.36 (t, HÀC(3')); 4.38 4.17 (m, HÀC(4'), 2 HÀC(5')); 2.07, 2.06 2.03 (3 s, 3 Ac). Anal. calc.
for C22H23N3O10 (489.4): C 53.99, H 4.74, N 8.59; found: C 54.08. H 4.71, N 9.02.
12. 2',3',5'-Tri-O-acetyl-N4-[(fluorescein-5-ylamino)carbonyl]cytidine (ˆ2',3',5'-Tri-O-acetyl-N4-{[(3',6'-di-
hydroxy-3-oxospiro[isobenzofuran-1(3H),9'-[9H]xanthen]-5-yl)amino]carbonyl}cytidine; 16). A mixture of 7
(0.52 g, 1.5 mmol) and 15 (1.2 g, 2.4 mmol) in pyridine (10ml) was heated to 70 8 for 3 h. Stirring was continued
overnight, the mixture evaporated and co-evaporated with toluene, and the residue dissolved in CH2Cl2/MeOH
and submitted to FC (13 Â 3.5 cm; CH2Cl2 (100 ml), CH2Cl2/MeOH 97:3 (200 ml), CH2Cl2/MeOH 95 :5
(100 ml), CH2Cl2/MeOH 93 :7 (200 ml), CH2Cl2/MeOH 9 :1 (300 ml; 50-ml fractions). Fr. 14 18 were
evaporated and co-evaporated with toluene and then the residue dried to give 1.1 g (97%) of crude material. A
sample (0.3 g) was heated in acetone (50 ml), the soln. filtered, and then Et2O (30ml) slowly added with
stirring. The yellow precipitate was collected and dried under high vacuum at 508: 0.26 g (85%) of pure 16. TLC
(CH2Cl2/MeOH 4 :1): Rf 0.63. UV (pH 9): 238 (4.73), 260 (sh., 4.40), 288 (4.89), 316 (4.14), 348 (sh., 3.88), 428
(sh., 3.70), 456 (sh., 4.38), 491 (4.91). 1H-NMR ((D6)DMSO): 11.6 (br. s, NH); 10.5 (s, NH); 10.1 (s, OH); 8.22
(d, HÀC(6)(flu)); 8.05 (d, HÀC(6)); 7.67 (m, HÀC(4)(flu)); 7.24 (m, HÀC(3)(flu), HÀC(5)); 6.67 6.48
(m, 6 arom. H (xan)); 5.91 (d, HÀC(1')); 5.50( dd, HÀC(2')); 5.37 (t, HÀC(3')); 4.37 4.19 (m, HÀC(4'),
2 HÀC(5')); 2.06, 2.04 (2 s, 3 Ac). Anal. calc. for C36H30N4O14 (742.7): C 58.22, H 4.07, N 7.54; found: C 57.59, H
4.25, N 7.46.
13. 2',3',5'-Tri-O-acetyl-N4-{{[4-(6-hydroxy-3-oxo-3H-xanthen-9-yl)-3-(methoxycarbonyl)phenyl]amino}-
carbonyl}cytidine (17). A mixture of 9 (0.2 g, 0.55 mmol) and 15 (0.5 g, 1 mmol) in pyridine (10 ml) was
heated to 708 for 2 h. The soln. was evaporated and co-evaporated with toluene, the residue treated with warm
dioxane by ultrasound, collected, and then dissolved in dioxane/MeOH 1:1 (50ml). The soln. was filtered hot
and MeOH carefully removed until a precipitate formed. After standing overnight, the solid was collected and
dried: 0.26 g (63%) of 17. Yellow powder. M.p. 188 1908 (dec.). TLC (CH2Cl2/MeOH 9 :1): Rf 0.45. UV (Ho
À
1.54): 226 (4.76), 249 (4.59), 310(4.54), 376 (3.91), 439 (4.74). UV (pH 1): 223 (4.75), 248 (4.60), 297 (4.44), 360
(sh., 3.64), 439 (4.72). UV (pH 4): 216 (4.67), 232 (4.68), 256 (sh., 4.46), 275 (4.46), 292 (sh., 4.42), 372 (3.99),
458 (4.42), 474 (4.41). UV (pH 8): 216 (4.71), 238 (4.77), 259 (sh., 4.46), 288 (4.47), 318 (sh., 4.19), 346 (sh.,
3.85), 434 (sh., 3.81), 464 (sh., 4.46), 496 (4.93). UV (pH 13): 219 (4.63), 239 (4.76), 258 (sh., 4.45), 288 (4.45),
309 (4.55), 313 (sh., 4.45), 365 (sh., 3.89), 434 (sh., 3.81), 464 (sh., 4.50), 494 (4.95). pKa Values: 0.40, 2.79, 6.13,
11.33. 1H-NMR ((D6)DMSO): 11.6 (br. s, NH); 11.1 (s, OH); 10.5 (s, NH); 8.34 (m, HÀC(6)(flu)); 8.08
(d, HÀC(6)); 7.88 (m, HÀC(4)(flu)); 7.43 (m, HÀC(3)(flu)); 6.87 6.52 (m, HÀC(5), 6 arom. H (xan); 5.92
(d, HÀC(1')); 5.50( dd, HÀC(2')); 5.37 (t, HÀC(3')); 4.37 4.22 (m, HÀC(4', 2 HÀC(5')); 3.59 (s, MeO); 2.07,
2.05 (2 s, 3 Ac). Anal. calc. for C37H32N4O14 (756.7): C 58.73, H 4.26, N 7.40; found: C 58.59, H 4.24, N 6.96.
14. 3',5'-Di-O-acetyl-2'-deoxy-O6-[2-(4-nitrophenyl)ethyl]guanosine (18). A mixture of 3',5'-di-O-acetyl-2'-
deoxyguanosine [30] (0.702 g, 2 mmol), PPh3 (0.84 g, 3.2 mmol), and 2-(4-nitrophenyl)ethanol (0.501 g,
3 mmol) in dry dioxane (40ml) was stirred for 5 min. Then, diethyl azodicarboxylate (0.558 g, 3.2 mmol) was
added and stirred vigorously for 1 h at r.t. ( ! clear soln.). The soln. was evaporated and the residue treated

2318
Helvetica Chimica Acta Vol. 86 (2003)
with Et2O to remove triphenylphosphine oxide. The resulting solid was recrystallized from MeOH: 0.78 g (78%)
of 18. Colorless crystals. M.p. 1248. 1H-NMR ((D6)DMSO): 8.17 (d, 2 H, o to NO2); 8.06 (s, HÀC(8)); 7.62
(d, 2 H,
m
to NO2); 6.52 (s, NH2); 6.22 (dd, HÀC(1')); 5.30( d, HÀC(3')); 4.66 (t, CH2); 4.30 4.16
(m, HÀC(4'), 2 HÀC(5')); 3.24 (t, CH2); 3.06 2.94 (m, HÀC(2')); 2.45 (m, 1 HÀC(2')); 2.07 (s, 1 Ac); 2.01
(s, 1 Ac). Anal. calc. for C22H24N6O8 (500.5): C 52.80, H 4.83, N 16.79; found: C 52.73, H 4.87, N 16.70.
15. 3',5'-Di-O-acetyl-2'-deoxy-O6-[2-(4-nitrophenyl)ethyl]-N2-(phenoxycarbonyl)guanosine (19). A soln. of
18 (0.751 g, 0.67 mmol) in abs. pyridine (10 ml) was cooled to 08, and then phenyl carbonochloridate (260 ml,
1 mmol) was added dropwise within 4 min. The mixture was kept 30min at 0 8 and another 30min at r.t., then
diluted with CH2Cl2 (70ml), and washed with sat. NaHCO 3 soln. The org. layer was dried (MgSO4), evaporated
and co-evaporated with toluene and the residue dissolved in CH2Cl2 and submitted to FC (silica gel, 2.5 Â 10cm;
toluene (20 ml), toluene/AcOEt 7 :3 (200 ml), toluene/AcOEt 3 :2 (200 ml), toluene/AcOEt 1 :1 (200 ml),
toluene/AcOEt 2 :3 (200 ml), and toluene/AcOEt/MeOH 5 :4 :1 (200 ml); 50-ml fractions). Fr. 6 19 gave
1
0.735 g (79%) of 19. Colorless solid. TLC (CH2Cl2/AcOEt/MeOH 10: 9 :1): Rf 0.45. H-NMR ((D6)DMSO):
10.95 (br. s, NH); 8.40( s, HÀC(8)); 8.16 (m, 2 H o to NO2); 7.60 7.20 ( m, Ph, 2 H m to NO2); 6.32
(t, HÀC(1')); 5.40( m, HÀC(3')); 4.74 (t, CH2CH2O); 4.36 (m, HÀC(4'), 2 HÀC(5')); 3.30( m, 1 HÀC(2'),
CH2CH2O); 2.49 (m, 1 HÀC(2')); 2.06, 1.95 (2 s, 2 Ac). Anal. calc. for C29H28N6O10 ¥ 0.5 toluene (666.6): C 58.26,
H 4.78, N 12.12; found: C 58.55, H 4.84, N 12.61.
16. 3',5'-Di-O-acetyl-2'-deoxy-N2-{[(3',6'-dihydroxy-3-oxospiro[isobenzofuran-1(3H),9'-[9H]xanthen]-5-
yl)amino]carbonyl}-O6-[2-(4-nitrophenyl)ethyl]guanosine (20). A mixture of 7 (0.431 g, 1.3 mmol) and 19
(1.2 g, 1.9 mmol) was heated in pyridine (10ml) to 70 8 for 2 h. After evaporation and co-evaporation with
toluene, the residue was dissolved in CH2Cl2/MeOH and purified by FC (silica gel (30g); CH 2Cl2 (250ml),
CH2Cl2/MeOH 49 :1 (200 ml), CH2Cl2/MeOH 95 :5 (200 ml), CH2Cl2/MeOH 9 :1 (50-ml fractions; 500 ml).
Fr. 10 15 gave a residue that was treated with MeOH (10ml). Then the solid was collected and dried: 0.87 g
(76%) of 20. TLC (toluene/AcOEt/MeOH 5 :4 :1): Rf 0.64. 1H-NMR ((D6)DMSO): 11.4 (br. s, NH); 10.3
(s, NH); 10.1 (s, 2 OH); 8.40( s, HÀC(8)); 8.32 (d, HÀC(6)); 8.11 (d, 2 H o to NO2); 7.75 (dd, HÀC(4)); 7.65
(d, 2 arom. H m to NO2); 7.19 (d, HÀC(3)(flu)); 6.67 6.53 (m, 6 arom. H (xan); 6.42 (t, HÀC(1')); 5.35
(m, HÀC(3')); 4.85 (t, CH2CH2O); 4.36 4.10( m, HÀC(4'), 2 HÀC(5')); 3.30( m, CH2CH2O); 3.13
(m, HÀC(2')); 2.60( dd, 1 HÀC(2')); 1.95, 1.90(2 s, 2 Ac). Anal. calc. for C43H35N7O14 ¥ 0.5 H2O (882.8): C
58.50, H 4.11, N 11.11; found: C 58.54, H 4.32, N 11.22.
17. 2'-Deoxy-N6,3'-O-bis{[2-(4-nitrophenyl)ethoxy]carbonyl}adenosine (21) [23].
18. 2'-Deoxy-N6-[(9H-fluoren-9-ylmethoxy)carbonyl]-5'-O-(monomethoxytrityl)adenosine 3'-[2-(4-Nitro-
phenyl)ethyl Diisopropylphosphoramidite] (22). A soln. of 2'-deoxy-N6-[(9H-fluoren-9-ylmethoxy)carbonyl]-
5'-O-(monomethoxytrityl)adenosine (24; 0.53 g, 0.7 mmol) in CH2Cl2 (10ml) and Et iPr2N (0.6 ml) was treated
under N2 with 2-(4-nitrophenyl)ethyl diisopropylphosphoramidochloridite (0.6 g, 1.8 mmol), and the mixture
stirred at r.t. for 1.5 h. The orange soln. was diluted with CHCl3 (300 ml), washed with phosphate buffer pH 7
(70ml), dried (MgSO 4), and evaporated, and the resulting brown oil dissolved in a little CH2Cl2 and purified by
CC (Alox (neutral; 60g), hexane (50ml), hexane/acetone 19 :1 (200ml), hexane/acetone 9 :1 (200ml),
hexane/acetone 17:3 (200 ml), hexane/acetone 15 :5 (200 ml), hexane/acetone 1:1 (200 ml; 50-ml fractions)).
Fr. 18 28 gave 0.4 g (55%) of 22. TLC (Alox, hexane/acetone 4 :1): Rf 0.45. UV (MeOH): 226 (4.45), 237
(4.39), 256 (4.59), 265 (4.67), 270(4.62), 288 (4.12), 298 (4.01). 1H-NMR (CDCl3): 8.71 (s, HÀC(2)); 8.47
(s, NH); 8.13 8.07 (m, HÀC(8), 2 H o to NO2); 7.78 7.63 (m, all fmoc, HÀC(1), HÀC(4), HÀC(5),
HÀC(8)); 7.43 7.16 (m, HÀC(2), HÀC(3), HÀC(6), HÀC(7) (all fmoc), 10arom. H, 2 H m to NO2, 2 H o to
MeO); 6.78 (d, 2 H m to MeO); 6.45 (d, HÀC(1')); 4.70( m, HÀC(3')); 4.62 (d, CH2ÀC(9)(fmoc)); 4.32
(m, HÀC(9), HÀC(4')(fmoc)): 3.90 3.71 ( m, CH2CH2O); 3.78 (s, MeO); 3.52 (m, Me2CH); 3.37
(m, 2 HÀC(5')); 3.00, 2.90 (2 t,CH2CH2O); 2.80 2.57 ( m, 2 HÀC(2')); 1.10( m, 2 Me2CH). Anal. calc. for
C59H60N7O9P (1042.2): C 68.00, H 5.80, N 9.41; found: C 66.97, H 6.02, N 9.02.
19. 2'-Deoxy-5'-O-(dimethoxytrityl)-N6-[(9H-fluoren-9-ylmethoxy)carbonyl]adenosine 3'-[2-(4-Nitrophen-
yl)ethyl 4-Diisopropylphosphoramidite] (23). As described for 22, with 2'-Deoxy-5'-O-(dimethoxytrityl)-[N6-
[(9H-(fluoren-9-ylmethoxy)carbonyl]adenosine (26; 3 g, 3.9 mmol), CH2Cl2 (20ml), Et iPr2N (3 ml) and 2-(4-
nitrophenyl)ethyl diisopropylphosphoramidochloridite (3 g, 9 mmol). CC (Alox (150g), hexane (100ml),
hexane/acetone 19 :1 (400 ml), hexane/acetone 9 :1 (400 ml), hexane/acetone 17:3 (400 ml), hexane/acetone
4 :1 (400 ml), hexane/acetone 3 :1 (400 ml), hexane/acetone 1:1 (200 ml); 50-ml fractions). Fr. 26 40 gave 2.9 g
(70%) of 23. TLC (Alox, hexane/acetone 4 :1): Rf 0.45. UV (MeOH): 226 (4.49), 238 (4.49), 254 (4.59), 265
(4.67), 270(4.64), 288 (4.13), 298 (4.0).
1H-NMR (CDCl3): 8.71 (s, HÀC(2)); 8.32 (s, NH); 8.13 8.07
(m, HÀC(8), 2 H o to NO2); 7.78 7.63 (m, HÀC(1), HÀC(4), HÀC(5), HÀC(8)(all fmoc)); 7.44 7.15
(m, HÀC(2), HÀC(3), HÀC(6), HÀC(7) (all fmoc), 2 H m to NO2, 4 H m to MeO); 6.74 (d, 4 H o to MeO);

Helvetica Chimica Acta Vol. 86 (2003)
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6.45 (d, HÀC(1')); 4.70( m, HÀC(3')); 4.62 (d, CH2ÀC(9)(fmoc)); 4.32 (m, HÀC(9), HÀC(4')): 3.90 3.71
(m, CH2CH2O); 3.76 (2 s, 2 MeO); 3.55 3.46 (m, Me2CH); 3.41 3.27 (m, 2 HÀC(5')); 3.00, 2.90 (2 t, CHe
CH2O); 2.80 2.57 ( m, 2 H, HÀC(2', 2'')); 1.10( m, 2 Me2 CH). Anal. calc. for C60H61N7O10P (1072.2): C 67.22, H
5.83, N 9.15; found: C 66.83, H 5.91, N 9.03.
20. 2'-Deoxy-N6-[(9H-fluoren-9-ylmethoxy)carbonyl]adenosine (24). According to [31]. TLC (silica gel,
toluene/AcOEt/MeOH 5: 4 :1): Rf 0.25. UV (MeOH): 226 (4.00), 254 (4.46), 264 (4.57), 274 (4.43), 288 (3.72),
299 (3.74). 1H-NMR ((D6)DMSO): 10.9 (s, NH); 8.67 (s, HÀC(2)); 8.65 (s, HÀC(8)); 7.90 7.30 ( m, 8 H
(fmoc)); 6.45 (d, HÀC(1')); 5.36 (d, OHÀC(3')); 5.03 (t, OHÀC(5')); 4.42 4.27 (m, HÀC(3'),
HÀC(9)(fmoc), CH2ÀC(9)); 3.88 (q, HÀC(4')): 3.67 3.49 (m, 2 HÀC(5')); 2.76 (m, HÀC(2')); 2.32
(m, 1 HÀC(2')). Anal. calc. for C25H23N5O5 (473.5): C 63.42, H 4.90, N 14.79; found: C 63.04, H 5.06, N 14.65.
21. 2'-Deoxy-N6-[(9H-fluoren-9-ylmethoxy)carbonyl]-5'-O-(monomethoxytrityl)adenosine (25) [31]. A
mixture of 24 (1.42 g, 3 mmol; co-evaporated with pyridine) and monomethoxytrityl chloride (1.2 g, 4 mmol)
in pyridine (50ml) was stirred at r.t. for 18 h. The soln. was concentrated to 10ml, diluted with AcOEt (200ml),
washed with phosphate buffer pH 7, dried (MgSO4), evaporated, and co-evaporated with toluene. The residue
was dissolved in a little CH2Cl2 and submitted to FC (silica gel (50g); toluene (100ml), toluene/AcOEt 1:1
(300 ml), toluene/AcOEt 1 :1 (295 ml) ‡ MeOH (5 ml), toluene/AcOEt 1:1 (290ml) ‡ MeOH (10ml); 50-ml
fractions. Fr. 13 27 gave 1.8 g (80%) of 25. Colorless solid foam. TLC (silica gel, toluene/AcOEt/MeOH
5 :4 :1): Rf 0.50. UV (MeOH): 227 (4.34), 236 (4.30), 254 (4.46), 265 (4.55), 271 (4.47), 287 (3.73), 299 (3.70).
1H-NMR ((D6)DMSO): 10.9 (s, NH); 8.57 (s, HÀC(2)); 8.56 (s, HÀC(8)); 7.90 7.83 ( m, HÀC(1), HÀC(4),
HÀC(5), HÀC(8)(all fmoc)); 7.44 7.09 (m, HÀC(2), HÀC(3), HÀC(6), HÀC(7)(all fmoc), 12 arom. H);
6.79 (d, 2 H
o
to MeO); 6.48 (d, HÀC(1')); 5.43 (d, OHÀC(3')); 4.52 (m, HÀC(3'); 4.42 4.30
(m, HÀC(9)(fmoc), CH2ÀC(9)(fmoc)); 4.04 (m, HÀC(4')): 3.69 (s, MeO); 3.19 (m, 2 HÀC(5')); 2.94
(m, HÀC(2')); 2.40( m, 1 HÀC(2')). Anal. calc. for C45H39N5O6 ¥ 0.5 toluene (491.3): C 73.56, H 5.47, N 8.84;
found: C 73.35, H 5.51, N 8.86.
22. 2'-Deoxy-N6-5'-O-(dimethoxytrityl)-[(9H-fluoren-9-ylmethoxy)carbonyl]adenosine (26). As described
for 25, with 24 (3.5 g, 7.4 mmol), dimethoxytrityl chloride (3 g, 9 mmol), and pyridine (80ml). Purification by
FC (silica gel (80 g); toluene (100 ml), toluene/AcOEt 1 :1 (500 ml), toluene/AcOEt 1:1 (490 ml) ‡ MeOH
(10ml), toluene/AcOEt 1:1 (480ml) ‡ MeOH (20 ml); 100-ml fractions). Fr. 12 18 gave 4.7 g (82%) of 26.
Yellowish solid foam. TLC (silica gel, toluene/AcOEt/MeOH 5 :4 :1): Rf 0.48. UV (MeOH): 226 (4.41), 237
(4.42), 254 (4.47), 265 (4.55), 270(4.48), 288 (3.73), 299 (3.71). 1H-NMR ((D6)DMSO): 10.9 (s, NH); 8.58
(s, HÀC(2)); 8.57 (s, HÀC(8)); 7.90 7.83 ( m, HÀC(1), HÀC(4), HÀC(5), HÀC(8)(all fmoc)); 7.44 7.09
(m, HÀC(2), HÀC(3), HÀC(6), HÀC(7)(all fmoc), 9 arom. H); 6.79 (m, 4 H o to MeO); 6.47 (d, HÀC(1'));
5.41 (d, OHÀC(3')); 4.52 (m, HÀC(3'); 4.41 4.27 (m, HÀC(9)(fmoc), CH2ÀC(9)(fmoc)); 4.02
(m, HÀC(4')): 3.69 (2 s, 2 MeO); 3.18 (m, 2 HÀC(5')); 2.94 (m, HÀC(2')); 2.40( m, 1 HÀC(2')). Anal. calc.
for C46H41N5O7 ¥ 0.5 toluene (821.4): C 72.33, H 5.52, N 8.52; found: C 72.71, H 5.61, N 8.33.
23. 2'-Deoxy-N6-[(9H-fluoren-9-ylmethoxy)carbonyl]-5'-O-(monomethoxytrityl)adenylyl-{3'-{OP-[2-(4-ni-
trophenyl)ethyl]}-5'}-2'-deoxy-N6,3'-O-bis{[2-(4-nitrophenyl)ethoxy]carbonyl}adenosine (27). To a mixture of
22 (0.5 g, 0.5 mmol) and 21 (0.32 g, 0.5 mmol) in MeCN (10 ml) and CH2Cl2 (3 ml) under Ar was added under
stirring 1H-tetrazole (0.21 g, 3 mmol). Stirring was continued for 1.5 h at r.t. Then I2 (0.5 g) in pyridine/CH2Cl2/
H2O 5 :1:1 (7 ml) was added and the mixture stirred for 15 min. After dilution with CHCl3 (300 ml), the soln.
was decolorized by washing with a Na2S2O3/NaCl soln., dried (MgSO4), and evaporated. The resulting oil was
purified by FC (silica gel (20 g), toluene (50 ml), toluene/acetone 9 :1 (100 ml), toluene/acetone 4 :1 (100 ml),
toluene/acetone 7 :3 (100 ml), toluene/acetone 3 :2 (100 ml), toluene/acetone 1:1 (200 ml), toluene/acetone
2 :3 (100 ml), toluene/acetone 3 :7 (100 ml; 50-ml fractions). Fr. 12 17 were evaporated and co-evaporated
with CH2Cl2: 0.55 g (70%) of 27. Solid foam. TLC (silica gel, toluene/AcOEt/MeOH 5 :4 :1): Rf 0.24. UV
(MeOH): 224 (4.56), 237 (4.52), 256 (4.80), 265 (4.90), 276 (4.79), 289 (4.41), 299 (4.22). 1H-NMR
((D6)DMSO): 10.9 (s, NH); 10.6 (s, NH); 8.58 8.45 (4 s, HÀC(2), HÀC(8)); 8.16 7.11 (m, 32 arom. H); 6.74
(m, 2 H o to MeO); 6.40( m, HÀC(1')); 5.35 (m, HÀC(3')); 5.15 (t, OHÀC(5')); 5.00 (m, 1 HÀC(3')); 4.41
4.21 (m, 12 H, CH2CH2O, HÀC(9)(fmoc), CH2, HÀC(4'), HÀC(5')); 4.06 (m, HÀC(4')); 3.49 (m, 2 H,
HÀC(5')); 3.15 2.87 (m, 8 H, CH2CH2O, HÀC(2')); 2.58 2.50( m, HÀC(2')). Anal. calc. for C81H72N13O21P ¥
H2O (1594.5): C 60.33, H 4.63, N 11.29; found: C 60.56, H 4.70, N 11.04.
24. 2'-Deoxy-5'-O-(dimethoxytrityl)-N6-[(9H-fluoren-9-ylmethoxy)carbonyl]adenylyl-{3'-{OP-[2-(4-nitro-
phenyl)ethyl]}-5'}-2'-deoxy-N6,3'-O-bis{[2-(4-nitrophenyl)ethoxy]carbonyl}adenosine (28). As described for
27, with 23 (2.9 g, 2.7 mmol), 21 (1.4 g, 2.2 mmol), MeCN (10ml), CH 2Cl2 (3 ml), 1H-tetrazole (0.92 g), and
I2 soln. (15 ml). After FC (silica gel (60 g); toluene (100 ml), toluene/acetone 9 :1 (200 ml), toluene/acetone 4 :1
(200 ml), toluene/acetone 7:3 (200 ml), toluene/acetone 3 :2 (400 ml), toluene/acetone 1:1 (200 ml), toluene/

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Helvetica Chimica Acta Vol. 86 (2003)
acetone 2 :3 (200 ml), MeOH (100 ml); 50-ml fractions). Fr. 21 32 were evaporated and co-evaporated with
CH2Cl2 and EtOH: 2.8 g (78%) of 28. Solid foam. TLC (silica gel, toluene/AcOEt/MeOH 5 :4 :1): Rf 0.31. UV
(MeOH): 238 (4.62), 256 (4.84), 266 (4.92), 272 (4.86), 289 (4.01), 299 (4.23). 1H-NMR ((D6)DMSO): 10.9
(s, NH); 10.6 (s, NH); 8.59 8.47 (4 s, 4 H, HÀC(2), HÀC(8)); 8.16 7.13 (m, 29 arom. H); 6.74 (m, 4 H o to
MeO); 6.41 (m, HÀC(1')); 5.31 (m, 1 HÀC(3')); 5.15 (m, HÀC(3')); 4.40 4.15 ( m, 13 H, CH2CH2O,
HÀC(9)(fmoc), CH2, HÀC(4'), HÀC(5')); 3.66 (s, 2 MeO); 3.18 2.89 (m, 10H, C H2CH2O, HÀC(2'),
HÀC(5')); 2.62 2.48 (m, 2 H, HÀC(2')). 31P-NMR ((D6)DMSO): À 1.38; À 1.45. Anal. calc. for C82H74N13O22P
(1624.6): C 60.63, H 4.59, N 11.21; found: C 60.12, H 4.70, N 11.03.
25. 2'-Deoxy-N6-[(9H-fluoren-9-ylmethoxy)carbonyl]adenylyl-{3'-{OP-[2-(4-nitrophenyl)ethyl]}-5'}-2'-de-
oxy-N6,3'-O-bis{[2-(4-nitrophenyl)ethoxy]carbonyl}adenosine (29). A soln. of 28 (1.0g, 0.62 mmol) in CH 2Cl2
(30ml) containing 1.5% of CF 3COOH was stirred at r.t. for 1 h, then diluted with CHCl3 (300 ml), and washed
several times with phosphate buffer pH 7. The org. layer was dried (MgSO4) and evaporated. The residue was
dissolved in a little CH2Cl2 and purified by FC (silica gel (30 g), toluene (100 ml), toluene/AcOEt 1 :1 (200 ml),
toluene/AcOEt 1:1 (95 ml) ‡ MeOH (5 ml), toluene/AcOEt 1:1 (90ml) ‡ MeOH (10ml); toluene/AcOEt
1:1 (160ml) ‡ MeOH (40 ml); 100-ml fractions). Fr. 7 and 8 were evaporated and co-evaporated with CH2Cl2:
0.78 g (96%) of 29. Solid. TLC (silica gel, toluene/AcOEt/MeOH 5 :4 :1): Rf 0.2. UV (CH2Cl2): 266 (4.89), 272
(sh., 4.83), 289 (sh., 4.39), 299 (4.21). 1H-NMR ((D6)DMSO): 10.9 (s, NH); 10.6 (s, NH); 8.63 8.59
(m, 4 HÀC(2), HÀC(8)); 8.18 7.29 (m, 20arom. H); 6.40( m, 2 H, HÀC(1')); 5.35 (m, 1 H, HÀC(3')); 5.16
(m, 1 H, HÀC(3')); 5.00 (m, 1 H, HÀC(3')); 4.41 4.21 (m, 12 H, CH2CH2O, HÀC(9)(fmoc), CH2, HÀC(4'),
HÀC(5')); 4.06 (m, 1 H, HÀC(4')); 3.50( m, 2 H, HÀC(5')); 3.15 2.87 (m, 10H, C H2CH2O, HÀC(2'),
HÀC(5')); 2.58 2.50( m, 2 H, HÀC(2')). 31P-NMR ((D6)DMSO): À 1.45. Anal. calc. for C61H56N13O20P
(1322.2): C 55.42, H 4.27, N 13.77; found: C 55.94, H 4.65, N 13.45.
26. 2'-Deoxy-5'-O-(monomethoxytrityl)adenylyl-{3'-{OP-[2-(4-nitrophenyl)ethyl]}-5'}-2'-deoxy-N6,3'-O-
bis{[2-(4-nitrophenyl)ethoxy]carbonyl}adenosine (30). A soln. of 27 (0.46 g, 0.29 mmol) in MeCN (8 ml) and
Et3N (2 ml) was stirred at r.t. for 2.5 h. After evaporation, the residue was dissolved in little CH2Cl2 and
submitted to FC (silica gel (15 g); toluene (50ml), toluene/AcOEt 1:1 (150ml), toluene/AcOEt 1:1 (95 ml)
‡
MeOH (5 ml), toluene/AcOEt 1:1 (95 ml) ‡ MeOH (5 ml), toluene/AcOEt 1:1 (90ml) ‡ MeOH (10ml),
toluene/AcOEt 1:1 (85 ml) ‡ MeOH (15 ml), toluene/AcOEt 1:1 (80ml) ‡ MeOH (20ml); 40-ml fractions).
Fr. 8 12 were evaporated and co-evaporated with CH2Cl2: 0.36 g (91%) of 29. Solid foam. TLC (silica gel,
toluene/AcOEt/MeOH 5 :4 :1): Rf 0.53. UV (MeOH): 240 (4.57), 266 (4.79). 1H-NMR ((D6)DMSO): 10.6
(s, NH); 8.59 (2 s, 2 H, HÀC(2)); 8.20 8.11 ( m, 5 H, HÀC(8), H o to NO2);); 8.02 (m, 3 H, HÀC(8), H o to
NO2); 7.56 (m, 4 H m to NO2); 7.42 7.12 (m, 20H, arom. H, NH 2); 6.75 (d, 2 H, H o to MeO); 6.40( t, 2 H,
HÀC(1')); 6.30( t, 1 H, HÀC(1')); 5.30( m, 1 H, HÀC(3')); 5.10( m, 1 H, HÀC(3')); 4.36 4.13 (m, 10 H,
CH2CH2O, HÀC(4'), HÀC(5')); 3.67 (s, MeO); 3.17 2.87 (m, 10H, C H2CH2O, HÀC(2'), HÀC(5')); 2.50
(m, 2 H, HÀC(2')); 2.28 (s, Me (toluene)). Anal. calc. for C66H62N13O19P ¥ 0.5 toluene (1418.4): C 58.86, H 4.69,
N 12.84; found: C 58.87, H 4.82, N 12.91.
27. 2'-Deoxy-5'-O-(dimethoxytrityl)adenylyl-{3'-{OP-[2-(4-nitrophenyl)ethyl]}-5'}-2'-deoxy-N6,3'-O-bis{[2-
(4-nitrophenyl)ethoxy]carbonyl}adenosine (31). As described for 30, with 28 (0.74 g, 0.46 mmol), in MeCN
(20ml), and Et 3N (5 ml). After FC (silica gel (20g); CH 2Cl2 (100 ml), CH2Cl2/MeOH 95 :5 (100 ml), CH2Cl2/
MeOH 9 :1 (100 ml), CH2Cl2/MeOH 85 :15 (100 ml); 50-ml fractions). Fr. 5 7 gave 0.58 g (91%) of 31. TLC
(silica gel, toluene/AcOEt/MeOH 5 :4 :1): Rf 0.53. UV (MeOH): 240 (4.57), 266 (4.79). 1H-NMR
((D6)DMSO): 8.80( s, NH); 8.66 (2 s, 2 H, HÀC(2)); 8.19 7.83 (m, 8 H, HÀC(8), H o to NO2);) 7.38 7.13
(m, 15 H, arom. H, H m to NO2, H m to MeO); 6.75 (m, 4 H, H o to MeO); 6.40( m, 2 H, HÀC(1')); 5.91
(s, NH2); 5.32 3.18 (m, 2 H, HÀC(3')); 4.51 4.17 (m, 10 H, CH2CH2O, HÀC(4'), HÀC(5')); 3.72
(2 s, 2 MeO); 3.33 (m, 2 H, HÀC(5')); 3.11 2.87 (m, 8 H, CH2CH2O, HÀC(2')); 2.60( m, 2 H, HÀC(2')).
Anal. calc. for C67H64N13O20P ¥ H2O (1420.3): C 56.66, H 4.68, N 12.82; found: C 56.50, H 4.64, N 12.81.
28. 2'-Deoxy-5'-O-(monomethoxytrityl)-N6-(phenoxycarbonyl)adenylyl-{3'-{OP-[2-(4-nitrophenyl)ethyl]}-
5'}-2'-deoxy-N6,3'-O-bis{[2-(4-nitrophenyl)ethoxy]carbonyl}adenosine (32). To a soln. of 30 (0.1 g, 0.071 mmol)
in dioxane (2 ml), 1-(phenoxycarbonyl)-1H-tetrazole (0.27 g, 1.4 mmol) was added and the mixture stirred for
14 h at 408. After evaporation, the residue was dissolved in CH2Cl2 (5 ml) and submitted to FC (silica gel (10g);
toluene (30 ml), toluene/acetone 3 :1 (100 ml), toluene/acetone 1:1 (100 ml), toluene/acetone 1:3 (100 ml); 30-
ml fractions). Fr. 6 8 gave 70mg (65%) of 33. TLC (silica gel, toluene/acetone 1:1): Rf 0.23. The substance
decomposed on storage.
29. 2'-Deoxy-5'-O-(dimethoxytrityl)-N6-(phenoxycarbonyl)adenylyl-{3'-{OP-[2-(4-nitrophenyl)ethyl]}-5'}-
2'-deoxy-N6,3'-O-bis{[2-(4-nitrophenyl)ethoxy]carbonyl}adenosine (33). As described for 32, with 31 (0.1 g,

Helvetica Chimica Acta Vol. 86 (2003)
2321
0.07 mmol) and 1-(phenoxycarbonyl)-1H-tetrazole (0.27 g, 1.4 mmol). TLC (silica gel, toluene/acetone 1:1): Rf
0.25. The substance decomposed on storage.
30. 2'-Deoxy-N6-[(9H-fluoren-9-ylmethoxy)carbonyl]-5'-O-{[2-(4-nitrophenyl)ethoxy]carbonyl}adenosine
(34). Compound 24 (0.95 g, 2 mmol) was co-evaporated twice with dry pyridine (10 ml). The residue was
dissolved in pyridine (20ml), the soln. cooled to À 408, 2-(4-nitrophenyl)ethyl carbonochloridate (0.6 g,
2.6 mmol) in CH2Cl2 (10ml) added, and the mixture stirred for 30min at À 308, and for 30min at À 108. Then
another 0.15 g of the reagent was added and stirring continued for 3 h at r.t. The mixture was diluted with
CH2Cl2 (300 ml) and washed several times with phosphate buffer pH 7. The org. phase was dried (Na2SO4),
evaporated, and co-evaporated with toluene (3 Â 10ml) and the residue purified by FC (silica gel (40g);
toluene (50ml), toluene/AcOEt 1:1 (300ml), toluene/AcOEt 1:1 (290ml) ‡ MeOH (10ml), toluene/AcOEt
1:1 (280ml) ‡ MeOH (20ml), toluene/AcOEt 1:1 (100ml) ‡ MeOH (100 ml); 100-ml fractions). Fr. 8 10
were evaporated, and the residue was treated with Et2O by ultrasound to give, after drying under high vacuum,
1.05 g (78%) of 34. TLC (silica gel, toluene/AcOEt/MeOH 5 :4 :1): Rf 0.27. UV (MeOH): 226 (4.11), 256
(4.52), 265 (4.59), 287 (4.03), 298 (3.93). 1H-NMR ((D6)DMSO): 10.9 (s, NH); 8.65 (s, HÀC(2)); 8.60
(s, HÀC(8)); 8.15 (d, 2 H, H o to NO2); 7.90 7.81 ( m, HÀC(1), HÀC(4), HÀC(5), HÀC(8)(all fmoc)); 7.51
7.32 ( m, HÀC(2), HÀC(3), HÀC(6), HÀC(7)(all fmoc), 2 H m to NO2); 6.46 (m, HÀC(1')); 5.55
(d, OHÀC(3')); 4.52 3.99 (m, HÀC(9)(fmoc), CH2, HÀC(3'), HÀC(4'), 2 HÀC(5'), CH2CH2O); 3.01
(m, CH2CH2O); 2.94 (m, HÀC(2')); 2.40( m, HÀC(2')). Anal. calc. for C34H30N6O9 (660.6): C 61.26, H 4.54, N
12.61; found: C 61.95, H 4.72, N 12.58.
31. 2'-Deoxy-N6-[(9H-fluoren-9-yloxy)carbonyl]-5'-O-{[2-(4-nitrophenyl)ethoxy]carbonyl}adenosine 3'-[2-
(4-Nitrophenyl)ethyl Diisopropylphosphoramidite] (35). To a soln. of 34 (0.4 g, 0.6 mmol) in CH2Cl2 (10ml)
and EtiPr2N (0.5 ml), 2-(4-nitrophenyl)ethyl diisopropylphosphoramidochloridite (0.4 g, 1.2 mmol) was added
under Ar and then stirred for 70min at r.t. The mixture was diluted with CHCl 3 (200 ml) and extracted with
phosphate buffer pH 7 (70ml). The aq. phase was washed again with CHCl 3 and the combined org. layer dried
(MgSO4) and evaporated in vacuo followed by high vacuum to remove EtiPr2N. The residue was dissolved in a
little CH2Cl2 and submitted to FC (Alox (70g): hexane (50ml), hexane/acetone 7:3 (200ml), hexane/acetone
2 :3 (200 ml), acetone (200 ml), acetone/MeOH 95 :5 (400 ml); 50-ml fractions). Fr. 17 21 were evaporated
and co-evaporated with CH2Cl2: 0.35 g (60%) of 35. TLC (Alox, hexane/acetone 4 :1): Rf 0.35. 1H-NMR
((D6)DMSO): 10.9 (s, NH); 8.73 (s, HÀC(2)); 8.29 (s, NH); 8.18 8.04, 8.15 (m, HÀC(8), 4 H o to NO2));
7.77 7.65 (m, HÀC(1), HÀC(4), HÀC(5), HÀC(8)(all fmoc)); 7.42 7.24 (m, HÀC(2), HÀ(3), HÀC(6),
HÀC(7)(all fmoc), 4 H m to NO2); 6.43 (m, HÀC(1')); 4.60( m, CH2ÀC(9)(fmoc), HÀC(3')); 4.38
(m, HÀC(9)(fmoc), HÀC(4'), 2 CH2CH2O); 3.89 3.76 (m, 2 HÀC(5')); 3.52 (m, 2 Me2CH); 3.08 2.97
(m, 2 CH2CH2O); 2.85 2.61 (m, 2 HÀC(2')); 1.13 (m, 2 Me2CH). Anal. calc. for C48H51N8O12P (962.9): C
59.87, H 5.34, N 11.64; found: C 58.98, H 5.48, N 10.80.
32. 2'-Deoxy-N6-[(9H-fluoren-9-ylmethoxy)carbonyl]-5'-O-{[2-(4-nitrophenyl)ethoxy]carbonyl}adenylyl-
{3'-{OP-[2-(4-nitrophenyl)ethyl]}-5'}-2'-deoxy-N6,3'-O-bis{[2-(4-nitrophenyl)ethoxy]carbonyl}adenosine (36).
A mixture of 21 (0.528 g, 0.83 mmol) and 1H-tetrazole (0.2 g) was stirred in MeCN (5 ml) for 5 min and
then evaporated. A soln. of 35 (1.6 g, 1.7 mmol) in CH2Cl2 (10ml) was added and then stirred at r.t. for 2 h.
Subsequent oxidation with I2 (0.2 g) in pyridine/CH2Cl2/H2O 5 :1:1 (3 ml) by stirring for 15 min gave a brown
mixture that was diluted with CHCl3 (300 ml) and then decolorized by washing with a Na2S2O3/NaCl. soln. The
org. layer was dried (MgSO4) and evaporated and the obtained oil purified by FC (silica gel (50g); toluene
(50ml), toluene/AcOEt 1 :1 (200ml), toluene/AcOEt 1:1 (190ml) ‡ MeOH (10ml), toluene/AcOEt 1:1
(185 ml) ‡ MeOH (15 ml), toluene/AcOEt 1:1 (180ml) ‡ MeOH (20ml), toluene/AcOEt 1 :1 (190ml)
‡
MeOH (10ml), toluene/AcOEt 1:1 (180ml) ‡ MeOH (10ml) and toluene/AcOEt 1:1 (190ml) ‡ MeOH
(20ml); 50-ml fractions). Fr. 16 20 were evaporated and the residue was treated with Et2O and then the solid
dried in vacuo: 0.78 g of 36 (63%). TLC (silica gel, toluene/AcOEt/MeOH 5 :4 :1): Rf 0.24. UV (MeOH): 227
(4.37), 265 (4.94), 286 (4.51), 298 (4.27). 1H-NMR ((D6)DMSO): 10.9 (s, NH); 10.9 (s, NH); 8.58 8.45
(4 s, 4 H, HÀC(2), HÀC(8)); 8.16 7.29 (m, 24 arom. H); 6.40( m, 2 H, HÀC(1')); 5.30( m, 1 H, HÀC(3')); 5.12
(m, 1 H, HÀC(3')); 4.40 4.14 ( m, 17 H, CH2CH2O, HÀC(9)(fmoc), CH2, HÀC(4'), HÀC(5')); 3.15 2.87
(m, 10H, C H2CH2O, HÀC(2')); 2.58 2.50( m, 2 H, HÀC(2')). Anal. calc. for C70H63N14O24P (1515.3): C 55.49,
H 4.19, N 12.84; found: C 55.20, H 4.33, N 12.99.
33. 2'-Deoxy-5'-O-{[2-(4-nitrophenyl)ethoxy]carbonyl}adenylyl-{3'-{OP-[2-(4-nitrophenyl)ethyl]}-5'}-2'-de-
oxy-N6,3'-O-bis{[2-(4-nitrophenyl)ethoxy]carbonyl}adenosine (37). A soln. of 36 (0.8 g, 0.54 mmol) in MeCN
(20ml), CH 2Cl2 (20ml), and Et 3N (10ml) was stirred at r.t. for 2 h. After evaporation, the residue was dissolved
in CH2Cl2 and submitted to FC (silica gel; CH2Cl2 (100 ml), CH2Cl2/MeOH 98 :2 (200 ml), CH2Cl2/MeOH 96 :4
(200 ml), CH2Cl2/MeOH 94 :6 (200 ml), CH2Cl2/MeOH 1:1 (100 ml); 100-ml fractions). Fr. 6 and 7 were

2322
Helvetica Chimica Acta Vol. 86 (2003)
evaporated, and the residue was treated with Et2O and dried: 0.57 g (87%) of 37. TLC (silica gel, toluene/
AcOEt/MeOH 5 :4 :1): Rf 0.24. UV (MeOH): 265 (4.80). 1H-NMR ((D6)DMSO): 10.9 (s, NH); 10.6 (s, NH);
8.59 (s, 2 H, HÀC(2)); 8.23 8.02 (m, 10H, H ÀC(8), H o to NO2); 7.31 (br. s, 2 H, NH2); 6.43 (t, 1 H, HÀC(1'));
6.31 (t, 1 H, HÀC(1')); 5.34 (m, 1 H, HÀC(3')); 5.07 (m, 1 H, HÀC(3')); 4.51 4.17 (m, 14 H, CH2CH2O,
HÀC(4'), HÀC(5')); 3.11 2.88 (m, 10H, C H2CH2O, HÀC(2')); 2.60( m, 2 H, HÀC(2')). Anal. calc. for
C55H53N14O22P (1293.1): C 51.09, H 4.13, N 15.17; found: C 50.87, H 4.28, N 15.31.
34. 2'-Deoxy-5'-O-{[2-(4-nitrophenyl)ethoxy]carbonyl}-N6-(phenoxycarbonyl)adenylyl-{3'-{OP-[2-(4-nitro-
phenyl)ethyl]}-5'}-2'-deoxy-N6,3'-O-bis{[2-(4-nitrophenyl)ethoxy]carbonyl}adenosine (38). To a soln. of 37
(0.13 g, 0.1 mmol) in dry dioxane (5 ml) was added 5-(4-nitrophenyl)-1-(phenoxycarbonyl)-1H-tetrazole
(0.16 g, 0.16 mmol) and then stirred at 408 for 20h. After evaporation, the residue was dissolved in little CH 2Cl2
and purified by FC (silica gel (15 g); CH2Cl2 (25 ml), CH2Cl2/MeOH 98 :2 (100 ml), CH2Cl2/MeOH 9 :3
(200 ml), CH2Cl2/MeOH 9 :3 (200 ml); 25-ml fractions). Fr. 7 11 were evaporated, and the residue was treated
with Et2O and then the solid collected and dried: 0.14 g (93%) of 38. TLC (silica gel, CH2Cl2/MeOH 9 :1): Rf
0.68. UV (MeOH): 267 (4.80), 296 (sh., 4.22). 1H-NMR ((D6)DMSO): 11.0( s, NH); 10.6 (s, NH); 8.61 (m, 4 H,
HÀC(2), HÀC(8)); 8.17 8.02 (m, 8 H o to NO2); 7.60 7.40( m, 10 H m to NO2, Ho (Ph)); 7.25 (m, 3 arom. H);
6.40( m, 2 H, HÀC(1')); 5.33 (m, 1 H, HÀC(3')); 5.11 (m, 1 H, HÀC(3')); 4.40 4.21 ( m, 14 H, CH2CH2O,
HÀC(4'), HÀC(5')); 3.09 2.89 (m, 10H, C H2CH2O, HÀC(2')); 2.63 2.50( m, 2 H, HÀC(2')). Anal. calc. for
C62H57N14O24P (1413.2): C 52.70, H 4.07, N 13.88; found: C 51.94, H 4.21, N 14.46.
35. 2'-Deoxy-N6-{[(3',6'-dihydroxy-3-oxospiro[isobenzofuran-1(3H),9'-[9H]xanthen]-5-yl)amino]carbon-
yl}-5'-O-(dimethoxytrityl)adenylyl-{3'-{OP-[2-(4-nitrophenyl)ethyl]}-5'}-2'-deoxy-N6,3'-O-bis{[2-(4-nitrophenyl)-
ethoxy]carbonyl}adenosine (39). A mixture of 33 (0.1 g, 0.066 mmol) and 7 (0.068 g, 0.2 mmol) in pyridine
(2 ml) was heated to 658 for 1.5 h with stirring. The soln. was evaporated and co-evaporated with toluene and the
residue dissolved in a little CH2Cl2/EtOH and submitted to FC (silica gel (20g); toluene (25 g), toluene/AcOEt
1:1 (100 ml), toluene/AcOEt 1:1 (95 ml) ‡ MeOH (5 ml), toluene/AcOEt 1 :1 (90ml) ‡ MeOH (10ml),
toluene/AcOEt 1:1 (85 ml) ‡ MeOH (15 ml), toluene/AcOEt 1:1 (80ml) ‡ MeOH (20ml), toluene/AcOEt
1:1 (75 ml) ‡ MeOH (25 ml), toluene/AcOEt 1 :1 (70ml) ‡ MeOH (30ml), toluene/AcOEt 1:1 (60ml)
‡
MeOH (40ml), toluene/AcOEt 1:1 (120ml) ‡ MeOH (80ml); 25-ml fractions). Fr. 26 37 were evaporated,
and the residue was treated with a little MeOH: 0.08 g (68%) of 39. TLC (silica gel, CH2Cl2/MeOH 9 :1): Rf
0.60 0.65. 1H-NMR ((D6)DMSO): 12.2 (s, NH); 10.25 10.20 (s, 4 H, NH, OH); 8.60 6.50( m, 38 H, arom. H,
HÀC(2), HÀC(8) (4 H), npe/npeoc (12 H), (MeO)2Tr (13 H), flu (9 H)); 6.75 (m, 4 H o to MeO); 6.40
(m, 2 H, HÀC(1')); 5.32 5.03 (m, 2 H, HÀC(3')); 4.40 4.11 ( m, 10H, CH 2CH2O, HÀC(4'), HÀC(5')); 3.75
(2 s, 2 MeO); 3.33 2.88 (m, 10H, C H2CH2O, HÀC(5'), HÀC(2')); 2.60( m, 2 H, HÀC(2')). Anal. calc. for
C88H75N14O26P ¥ 2 H2O (1811.7): C 58.34, H 4.39, N 10.82; found: C 58.23, H 4.40, N 10.65.
36. 2'-Deoxy-N6-{[(3',6'-dihydroxy-3-oxospiro[isobenzofuran-1(3H),9-[9H]xanthen]-5-yl)amino]carbon-
yl}-5'-O-{[2-(4-nitrophenyl)ethoxy]carbonyl}adenylyl-{3'-{OP-[2-(4-nitrophenyl)ethyl]}-5'}-2'-deoxy-N6,3'-O-
bis{[2-(4-nitrophenyl)ethoxy]carbonyl}adenosine (40). A mixture of dry 7 (0.2 g, 0.59 mmol) and 38 (0.1 g,
0.07 mmol) in abs. pyridine (2 ml) was heated to 708 for 45 min. After evaporation and co-evaporation with
toluene, the residue was treated with MeOH (3 ml). The solid was washed with MeOH to remove excess of the
dye and then dissolved in CH2Cl2 for purification by FC (silica gel (25 g); CH2Cl2/MeOH 98 :2 (100 ml),
CH2Cl2/MeOH 97:3 (100 ml), CH2Cl2/MeOH 95 :5 (100 ml), CH2Cl2/MeOH 94 :6 (100 ml), CH2Cl2/MeOH
9 :1 (100 ml), CH2Cl2/MeOH 4 :1 (100 ml), and CH2Cl2/MeOH 1:1 (100 ml); 50-ml fractions). Fr. 10 13 were
evaporated, and the residue was treated with MeOH, filtered, and dried: 0.06 g (51%) of 40. TLC (silica gel,
CH2Cl2/MeOH 9 :1): Rf 0.48 UV (MeOH/CH2Cl2 1 :1): 224 (4.89), 227 (sh., 4.87), 271 (sh., 4.94), 275 (4.95), 347
(3.53), 424 (sh., 3.59), 455 (3.91), 483 (sh., 3.96). 1H-NMR ((D6)DMSO): 12.1 (s, NH); 10 .6 (s, 3 H, NH, OH);
10.4 (s, NH); 8.65 8.55 (m, 4 H, HÀC(2), HÀC(8)); 8.30( s, HÀC(6)(flu)); 8.12 8.02 (m, 8 H o to NO2); 7.91
(d, HÀC(4)(flu)); 7.60 7.43 ( m, 8 H m to NO2); 7.22 (d, HÀC(3)(flu)); 6.64 6.37 (m, 8 H, xan, HÀC(1')); 5.33
(m, 1 H, HÀC(3')); 5.13 (m, 1 H, HÀC(3')); 4.34 4.19 (m, 14 H, CH2CH2O, HÀC(4'), HÀC(5')); 3.11 2.93
(m, 10H, C H2CH2O, HÀC(5'), HÀC(2')); 2.53 (m, 2 H, HÀC(2')). Anal. calc. for C76H65N15O28P ¥ 2 H2O
(1703.4): C 53.58, H 4.08, N 12.33; found: C 53.35, H 4.00, N 12.25.
37. 2'-Deoxy-N6-{[(3',6'-dihydroxy-3-oxospiro[isobenzofuran-1(3H),9-[9H]xanthen]-5-yl)amino]carbonyl}-
adenylyl-(3'-5')-2'-deoxyadenosine Sodium Salt (41). A soln. of 40 (50 mg, 0.03 mmol) in 0.5m DBU in pyridine
(10ml) was stirred at r.t. for 4 h. After dilution with H 2O (50ml) and neutralization with AcOH (1 ml), the
mixture was extracted with CHCl3 (100 ml). Phase separation was achieved by addition of conc. NH3 soln.
(10ml). The aq. layer was again extracted with CHCl 3 and finally evaporated. The residue was purified by FPLC
(DEAE-Sephadex A25, column 50 Â 2 cm; gradient of 100% H2O to 100% TBK buffer (1m, pH 8) within 32 h).
The fluorescent fractions were eluted with 80 90% buffer concentration, evaporated, and co-evaporated with

Helvetica Chimica Acta Vol. 86 (2003)
2323
H2O. The resulting triethylammonium salt was dissolved in MeOH (5 ml) and treated with NaI (0.1 g) in
acetone to give 41, which was collected and dried: 20mg (67%) of 41. Yellow powder. UV (pH 9): 264 (4.33),
280(sh., 4.33), 492 (4.62).
38. 2'-Deoxy-5'-O-(monomethoxytrityl)-N6-{[2-(4-nitrophenyl)ethoxy]carbonyl}adenylyl-{3'-{OP-[2-(4-ni-
trophenyl)ethyl]}-5'}-2'-deoxy-N6-[(9H)-fluoren-9-ylmethoxy)carbonyl]adenylyl-{3'-{OP-[2-(4-nitrophenyl)eth-
yl]}-5'}-2'-deoxy-N6,3'-O-bis{[2-(4-nitrophenyl)ethoxy]carbonyl}adenosine (42).
A soln. of 29 (0.53 g,
0.4 mmol) and 44 (0.842 g, 0.8 mmol) in dry MeCN (10 ml) and CH2Cl2 (10ml) was treated under N 2 with
1H-tetrazole (0.17 g) by stirring at r.t. for 2 h. Then a soln. of I2 (0.5 g) in pyridine/CH2Cl2/H2O 5 :1:1 (7 ml) was
added and stirred for 15 min for oxidation. The mixture was diluted with CHCl3, extracted with a Na2S2O3/NaCl
soln. until decolorazition was achieved, the org. layer dried (MgSO4), evaporated, and co-evaporated with
toluene, and the resulting syrup dissolved in little CH2Cl2 and purified by FC (silica gel (25 g); toluene (50ml),
toluene/AcOEt 1 :1 (100 ml), toluene/AcOEt 1:1 (95 ml) ‡ MeOH (5 ml), toluene/AcOEt 1:1 (90ml) ‡
MeOH (10ml), toluene/AcOEt 1:1 (85 ml) ‡ MeOH (15 ml), toluene/AcOEt 1:1 (80ml) ‡ MeOH (20ml),
toluene/AcOEt 1:1 (160ml) ‡ MeOH (40ml); 50-ml fractions. Fr. 8 12 were evaporated and co-evaporated
with CH2Cl2: 0.87 g (96%) of 42. Colorless solid foam. TLC (silica gel, toluene/AcOEt/MeOH 5 :4 :1): Rf 0.2.
UV (CH2Cl2): 258 (sh., 4.97), 265 (5.04), 274 (sh., 4.97), 299 (sh., 4.39). 1H-NMR ((D6)DMSO): 10.9 (s, NH);
10.6 (s, NH); 8.58 8.41 (m, 6 H, HÀC(2), HÀC(8)); 8.15 7.97 (m, 14 H, H o to NO2, HÀC(1), HÀC(4),
HÀC(5), HÀC(8)(all fmoc)); 7.61 7.09 (m, 26 H, arom. H, H m to NO2, HÀC(2), HÀC(3), HÀC(6),
HÀC(7)(all fmoc)); 7.91 (d, HÀC(4)(flu)); 7.72 (m, 2 H o to MeO)); 6.40( m, 3 H, HÀC(1')); 5.33 (m, 1 H,
HÀC(3')); 5.13 (m, 2 H, HÀC(3')); 4.39 4.14 (m, 20H, CH 2CH2O, HÀC(4'), HÀC(5'), HÀC(9)(fmoc),
CH2); 3.64 (s, MeO); 3.17 2.88 (m, 15 H, CH2CH2O, HÀC(5'), HÀC(2')); 2.62 2.48 (m, 3 H, HÀC(2')). Anal.
calc. for C108H98N20O32P2 ¥ H2O (2250.1): C 57.19, H 4.44, N 12.35; found: C 56.97, H 4.61, N 12.28.
39. 2'-Deoxy-N6,5'-O-bis{[2-(4-nitrophenyl)ethoxy]carbonyl}adenylyl-{3'-{OP-[2-(4-nitrophenyl)ethyl]}-5'}-
2'-deoxy-N6-[(9H-fluoren-9-ylmethoxy)carbonyl]adenylyl-{3'-{OP-[2-(4-nitrophenyl)ethyl]}-5'}-2'-deoxy-N6,3'-
O-bis{[2-(4-nitrophenyl)ethoxy]carbonyl}adenosine (43). As described for 42, with 29 (0.6 g, 0.45 mmol), 45
(0.85 g, 0.91 mmol), 1H-tetrazole (0.19 g, 2.8 mmol), MeCN (10 ml), and CH2Cl2 (10ml), followed by I 2
oxidation. After FC (silica gel (30g); toluene/AcOEt 1:1 (400ml), toluene/AcOEt 1:1 (195 ml) ‡ MeOH
(5 ml), toluene/AcOEt 1:1 (190ml) ‡ MeOH (10ml), toluene/AcOEt 1:1 (180ml) ‡ MeOH (20ml), toluene/
AcOEt 1:1 (340ml) ‡ MeOH (60ml), toluene/AcOEt 1 :1 (160ml) ‡ MeOH (40 ml); 100-ml fractions).
Fr. 10 15 were evaporated and co-evaporated with AcOEt: 0.92 g (93%) of 43. Solid foam. TLC (silica gel,
toluene/AcOEt/MeOH 5 :4 :1): Rf 0.11. UV (CH2Cl2): 266 (5.06), 271 (sh., 4.97), 291 (4.61), 298 (sh., 4.43).
1H-NMR ((D6)DMSO): 10.9 (s, NH); 10.6 (s, 2 H, NH); 8.56 (m, 6 H, HÀC(2), HÀC(8)); 8.15 7.12 (m, 32 H,
arom. H, H o to NO2, HÀC(1) to HÀC(8)(fmoc)); 6.40( m, 3 H, HÀC(1')); 5.33 (m, 1 H, HÀC(3')); 5.15
(m, 1 H, HÀC(3')); 5.08 (m, 1 H, HÀC(3')); 4.40 4.15 ( m, 24 H, CH2CH2O, HÀC(4'), HÀC(5'),
HÀC(9)(fmoc), CH2); 3.18 2.94 (m, 15 H, CH2CH2O, HÀC(5'), HÀC(2')); 2.63 2.50( m, 3 H, HÀC(2')).
Anal. calc. for C97H89N21O35P2 (2170.9): C 53.67, H 4.13, N 13.55; found: C 52.96, H 4.13, N 13.08.
40. 2'-Deoxy-5'-O-(monomethoxytrityl)-N6-{[2-(4-nitrophenyl)ethoxy]carbonyl}adenosine 3'-[2-(4-Nitro-
phenyl)ethyl Diisopropylphosphoramidite] (44) [22].
41. 2'-Deoxy-N6,5'-O-bis{[2-(4-nitrophenyl)ethoxy]carbonyl}adenosine 3'-[2-(4-Nitrophenyl)ethyl Diiso-
propylphosphoramidite] (45). A soln. of 46 (1.2 g, 1.9 mmol) [23] in CH2Cl2 (10ml) and Et iPr2N (1.2 ml) was
treated under N2 with 2-(4-nitrophenyl)ethyl diisopropylphosphoramidochloridite (1.2 g, 4 mmol) with stirring
at r.t. for 1.5 h. After dilution with CHCl3 (300 ml), the mixture was extracted with sat. NaHCO3 soln. (70ml),
the aq. phase extracted with CHCl3, the combined org. phase dried (MgSO4) and evaporated finally under high
vacuum to remove EtiPr2N. Purification was achieved by FC (silica gel (30g), prepared with hexane ‡ 1% Et3N;
hexane ‡ 1% Et3N (100 ml), hexane ‡ 1% Et3N/AcOEt 2 :1 (100 ml), hexane ‡ 1% Et3N/AcOEt 1:1 (100 ml),
hexane ‡ 1% Et3N/AcOEt 1:2 (100 ml), AcOEt ‡ 1% Et3N (200 ml), AcOEt/acetone 1:1 (200 ml); 50-ml
fractions). Fr. 10 14 were evaporated and co-evaporated with CH2Cl2: 1.42 g (81%) of 45. Solid foam. TLC
(silica gel, hexane/AcOEt 1 :2 ‡ 1% Et3N): Rf 0.25. UV (MeOH): 267 (4.63), 272 (sh., 4.61). 1H-NMR (CDCl3):
8.73, 8.72 (2 s, HÀC(2)); 8.37 (s, NH); 8.13 8.03 (m, HÀC(8), 6 H o to NO2)); 7.48 7.36 (m, 6 H m to NO2);
6.44 (2 t, HÀC(1')); 4.58 4.15 (m, 3 CH2CH2O, HÀC(3'), HÀC(4')); 3.96 2.99 (m, 2 CMe2CH, 3 CH2CH2O,
2 HÀC(5')); 2.80 2.60( m, 2 HÀC(2')); 1.10( m, 2 Me2CH). Anal. calc. for C42H48N9O14P (933.9): C 54.02, H
5.18, N 13.50; found: C 53.88, H 5.50, N 12.99.
42. 2'-Deoxy-N6,5'-O-bis{[2-(4-nitrophenyl)ethoxy]carbonyl}adenosine (46) [23].
43. 2'-Deoxy-5'-O-(monomethoxytrityl)-N6-{[2-(4-nitrophenyl)ethoxy]carbonyl}adenylyl-{3'-{OP-[2-(4-ni-
trophenyl)ethyl]}-5'}-2'-deoxyadenylyl-{3'-{OP-[2-(4-nitrophenyl)ethyl]}-5'}-2'-deoxy-N6,3'-O-bis{[2-(4-nitro-
phenyl)ethoxy]carbonyl}adenosine (47). A soln. of 42 (0.75 g, 0.33 mmol) in MeCN/CH2Cl2 1:1 (40ml) was

2324
Helvetica Chimica Acta Vol. 86 (2003)
treated with Et3N (10ml) and stirred at r.t. for 2.5 h. After evaporation and co-evaporation with EtOH, the
residue was dissolved in a little CH2Cl2 for purification by FC (silica gel (20g); CH 2Cl2 (40ml), CH 2Cl2/MeOH
95 :5 (200 ml), CH2Cl2/MeOH 9 :1 (200 ml); 40-ml fractions). Fr. 6 9 were evaporated, and the residue was
treated with a little EtOH by ultrasound and then the precipitate collected: 0.62 g (93%) of 47. TLC (silica gel,
CH2Cl2/MeOH 9 :1): Rf 0.53. UV (CHCl3): 238 (sh., 4.61), 266 (4.94), 275 (sh., 4.87). 1H-NMR ((D6)DMSO):
10.6 (s, 2 H, NH); 8.58 8.41 (m, 4 H, 3 HÀC(2), 1 HÀC(8)); 8.24 (s, 1 H, HÀC(8)); 8.16 7.97 (m, 11 H, H o to
NO2, HÀC(8)); 7.61 7.09 (m, 24 H, H m to NO2, arom. H, NH2); 6.71 (m, 2 H o to MeO); 6.43 6.32 (m, 3 H,
HÀC(1')); 5.32 (m, 1 H, HÀC(3')); 5.11 (m, 2 H, HÀC(3')); 4.37 4.21 (m, 17 H, CH2CH2O, HÀC(4'),
HÀC(5')); 3.64 (s, MeO); 3.16 2.88 (m, 15 H, CH2CH2O, HÀC(5')), HÀC(2')); 2.62 2.50( m, 3 H, HÀC(2')).
Anal. calc. for C93H88N20O30P2 ¥ 2 H2O (2063.8): C 54.12, H 4.49, N 13.64; found: C 53.48, H 4.51, N 13.40.
44. 2'-Deoxy-N6,5'-O-bis{[2-(4-nitrophenyl)ethoxy]carbonyl}adenylyl-{3'-{OP-[2-(4-nitrophenyl)ethyl]}-5'}-
2'-deoxyadenylyl-{3'-{OP-[2-(4-nitrophenyl)ethyl]}-5'}-2'-deoxy-N6,3'-O-bis{[2-(4-nitrophenyl)ethoxy]carbonyl}-
adenosine (48). A soln. of 43 (0.6 g, 0.28 mmol) in MeCN/CH2Cl2 1:1 and Et3N (10ml) was stirred at r.t. for 4 h.
The mixture was evaporated and the residue dissolved in CH2Cl2 for FC (silica gel (30g); CH 2Cl2 (50ml),
CH2Cl2/MeOH 98 :2 (200 ml), CH2Cl2/MeOH 96 :4 (400 ml), CH2Cl2/MeOH 95 :5 (200 ml), CH2Cl2/MeOH
9 :1 (200 ml); 50-ml fractions) Fr. 10 19 were evaporated. The residue was treated with CH2Cl2 (25 ml) and the
soln. filtered and then treated with EtOH until the soln. became turbid. The CH2Cl2 was removed in vacuo at r.t.
whereby 48 separated at the glaswall. The EtOH was decanted and the residue treated with Et2O: 0.51 g (94%)
of 48. Colorless solid. TLC (silica gel, CH2Cl2/MeOH 9 :1): Rf 0.55. UV (CHCl3): 266 (4.95), 270(sh., 4.93).
1H-NMR ((D6)DMSO): 10.6 (s, 2 H, NH); 8.55 (m, 4 H, 3 HÀC(2), 1 HÀC(8)); 8.27 (s, 1 H, HÀC(8)); 8.16
8.00 (m, 13 H, H o to NO2, HÀC(8)); 7.60 7.40 ( m, 12 H, H m to NO2); 7.32 (br. s, 2 H, NH2); 6.43 6.36
(m, 3 H, HÀC(1')); 5.33 (m, 1 H, HÀC(3')); 5.11 (m, 2 H, HÀC(3')); 4.40 4.21 ( m, 21 H, CH2CH2O, HÀC(4'),
HÀC(5')); 3.09 2.96 (m, 15 H, CH2CH2O, HÀC(5'), HÀC(2')); 2.63 2.50( m, 3 H, HÀC(2')). 31P-NMR
((D6)DMSO): À 1.41, À 1.48, À 1.56. Anal. calc. for C82H79N21O33P2 (1948.6): C 50.54, H 4.09, N 15.10; found: C
50.55, H 4.27, N 15.09.
45. 2'-Deoxy-N6,5'-O-bis{[2-(4-nitrophenyl)ethoxy]carbonyl}adenylyl-{3'-{OP-[2-(4-nitrophenyl)ethyl]}-5'}-
2'-deoxy-N6-(phenoxycarbonyl)adenylyl-{3'-{OP-[2-(4-nitrophenyl)ethyl]}-5'}-2'-deoxy-N6,3'-O-bis{[2-(4-nitro-
phenyl)ethoxy]carbonyl}adenosine (49). To a soln. of 48 (0.3 g, 0.15 mmol) in dry dioxane (5 ml), 5-(4-
nitrophenyl)-1-(phenoxycarbonyl)-1H-tetrazole (0.2 g, 0.6 mmol) was added and then stirred at 408 for 48 h.
The mixture was evaporated and the residue dissolved in little CH2Cl2 and purified by FC (silica gel (7 g);
CH2Cl2 (40ml), CH 2Cl2/MeOH 97:3 (100 ml), CH2Cl2/MeOH 95 :5 (200 ml); 20-ml fractions). Fr. 7 10 were
treated with some AcOEt and then concentrated to a small volume whereby a precipitate separated on the
glasswall. The yellowish residual AcOEt was decanted and then the solid treated with Et2O: 0.29 g (93%) of
colorless 49. TLC (silica gel, CH2Cl2/MeOH 9 :1): Rf 0.63, 0.68. UV (CHCl3): 267 (4.99), 272 (sh., 4.95).
1H-NMR ((D6)DMSO): 11.1 (s, 2 H, NH); 10.6 (m, 2 H, NH); 8.63 8.51 (m, 6 H, HÀC(2), HÀC(8)); 8.15
7.99 (m, 12 H o to NO2); 7.60 7.39 ( m, 14 H, H m to NO2, Ho of (Ph)); 7.28 (m, 3 H, Hm, Hp (Ph)); 6.40( m, 3 H,
HÀC(1')); 5.33 (m, 1 H, HÀC(3')); 5.11 (m, 2 H, HÀC(3')); 4.40 4.21 ( m, 21 H, CH2CH2O, HÀC(4'),
HÀC(5')); 3.09 2.95 (m, 15 H, CH2CH2O, HÀC(5'), HÀC(2')); 2.56 (m, 3 H, HÀC(2')). 31P-NMR
((D6)DMSO): À 1.34; À 1.42. Anal. calc. for C89H83N21O35P2 (2068.7): C 51.67, H 4.04, N 14.22; found: C
51.09, H 4.09, N 13.97.
46. 2'-Deoxy-N6,5'-O-bis{[2-(4-nitrophenyl)ethoxy]carbonyl}adenylyl-{3'-{OP-[2-(4-nitrophenyl)ethyl]}-5'}-
2'-deoxy-N6-{[3',6'-dihydroxy-3-oxospiro[isobenzofuran-1(3H),9-[9H]xanthen]-5-yl)amino]carbonyl}adenyl-
yl-{3'-{OP-[2-(4-nitrophenyl)ethyl]}-5'}-2'-deoxy-N6,3'-O-bis{[2-(4-nitrophenyl)ethoxy]carbonyl}adenosine (50).
A mixture of 49 (0.15 g, 0.073 mmol) and 7 (0.051 g, 0.146 mmol) in pyridine (2 ml) was heated to 60 708 for
2 h with stirring. The soln. was evaporated and co-evaporated with toluene. The residue was dissolved in a little
CH2Cl2 and submitted to FC (silica gel (20g); CH 2Cl2/MeOH 99 :1 (50ml), CH 2Cl2/MeOH 98 :2 (100 ml),
CH2Cl2/MeOH 95 :5 (100 ml), CH2Cl2/MeOH 9 :1 (100 ml), CH2Cl2/MeOH 1:1 (100 ml); 30-ml fractions).
Fr. 11 13 were evaporated, and the residue was treated with Et2O: 0.075 g (44%) of 50. Yellow powder. TLC
(silica gel, CH2Cl2/MeOH 9 :1): Rf 0.39. 1H-NMR ((D6)DMSO): 12.1 (s, 1 H, NH); 10.4 (m, 5 H, NH, OH);
8.63 8.51 (m, 6 H, HÀC(2), HÀC(8)); 8.30( s, HÀC(6)(flu)); 8.15 8.02 (m, 12 H
o to NO2); 7.88
(s, HÀC(4)(flu)); 7.60 7.43 ( m, 12 H m to NO2); 7.22 (s, HÀC(3)(flu)); 6.64 6.42 (m, 9 H, xan, HÀC(1'));
5.33 (m, 1 H, HÀC(3')); 5.13 (m, 2 H, HÀC(3')); 4.34 4.22 (m, 21 H, CH2CH2O, HÀC(4'), HÀC(5')); 3.05
2.93 (m, 15 H, CH2CH2O, HÀC(5'), HÀC(2')); 2.53 (m, 3 H, HÀC(2')). 31P-NMR ((D6)DMSO): À 1.36; À
1.40. Anal. calc. for C103H90N22O39P2 ¥ H2O (2339.9): C 52.87, H 3.96, N 13.17; found: C 52.66, H 4.04, N 13.02.
47. 2'-Deoxyadenylyl-(3'-5')-2'-deoxy-N6-{[(3',6'-dihydroxy-3-oxospiro[isobenzofuran-1(3H),9-[9H]xan-
then]-5-yl)amino]carbonyl}adenylyl-(3'-5')-2'-deoxyadenosine (51 (ˆ 80)). Compound 50 (90 mg, 0.039 mmol)

Helvetica Chimica Acta Vol. 86 (2003)
2325
was dissolved in 0.5m DBU in pyridine (10ml) and stirred at r.t. for 3 h. A Na 2HPO4 soln. was added and then
the mixture extracted with CH2Cl2 (3 Â 30ml). The aq. phase was treated with conc. NH 3 soln. (5 ml), the
strongly fluorescent soln. extracted again with CH2Cl2, the aq. layer evaporated, and the residue purified by
FPLC (DEAE-Sephadex 25; gradient H2O/(Et3NH)HCO3 buffer (pH 7)). The product fraction was evaporated
and several times co-evaporated with H2O. The resulting residue was dissolved in a little MeOH and then
treated with NaI (0.2 g) in acetone (50 ml) under N2 for 20h. The formed orange precipitate was collected and
dried under high vacuum: 21 mg (60%) of 51.
Analogous treatment with 79 (80 mg, 0.03 mmol) gave 26 mg (65%) of 51. UV (buffer pH 9): 261 (4.55),
280(sh., 4.41), 492 (4.75). 1H-NMR ((D6)DMSO/H2O 1 :1): 8.7 8.05 (m, 7 H, HÀC(2), HÀC(8),
HÀC(6)(flu)); 7.70( d, 1 H, HÀC(4)(flu)); 7.10( d, HÀC(3)(flu)); 6.7 6.2 (m, 9 H, xan, HÀC(1')); 5.0 3.5
(m, 12 H, HÀC(3'), HÀC(4'), HÀC(5')); 2.7 2.3 (m, 6 H, HÀC(2')).
48. 2',3',5'-Tri-O-acetyl-N6-{{{4-[3-(acetyloxy)-6-hydroxy-9H-xanthen-9-yl]-3-(methoxycarbonyl)phenyl}-
amino}carbonyl}adenosine (52). Not isolated since the vinylogous AcO group turned out to be too reactive.
49. 2',3',5'-Tri-O-acetyl-N6-{{{4-{3-hydroxy-6-{[(4-methoxyphenyl)carbonyl]oxy}-9H-xanthen-9-yl}-3-
(methoxycarbonyl)phenyl}amino}carbonyl}adenosine (53). A soln. of 13 (0.1 g, 13 mmol) in dry pyridine
(5 ml) was treated with anisoyl chloride (20 ml) with stirring at r.t. for 5 min. After dilution with CH2Cl2 (30ml),
the mixture was extracted with 0.1m KH2PO4, the org. phase dried (MgSO4), evaporated, and co-evaporated
with toluene, the residue treated with a little EtOH, and the solid collected and dried: 0.1 g (85%) of 53. A
sample was purified by prep. TLC (CH2Cl2/MeOH 95 :5). TLC (silica gel, toluene/AcOEt/MeOH 5 :4 :1): Rf
0.39. UV (CH2Cl2): 260 (sh., 4.69), 276 (4.78), 338 (4.15), 352 (sh., 4.07), 402 (sh., 4.04), 431 (4.24), 453 (4.24),
486 (sh., 3.97). 1H-NMR ((D6)DMSO)2): 12.0( s, NH); 10.6 (s, HÀN(6)); 8.76 (s, HÀC(2)); 8.71 (s, HÀC(8));
8.60( d, HÀC(6)(flu)); 8.10( d, 2 H o to MeO); 8.04 (dd, HÀC(4)(flu)); 7.64 (d, HÀC(4)(xan)); 7.52
(d, HÀC(3)(flu)); 7.26 6.87 (m, 2 H
m
to MeO, HÀC(1), HÀC(2), HÀC(8)(all xan)); 6.45
(dd, HÀC(7)(xan)); 6.34 (d, HÀC(1')); 6.25 (d, HÀC(5)(xan)); 6.05 (t, HÀC(2')); 5.65 (t, HÀC(3')); 4.46
4.24 (m, HÀC(4'), 2 HÀC(5')); 3.86 (m, 1 MeO); 3.61 (s, 1 MeO); 2.12, 2.04, 2.02 (3 s, 3 Ac). Anal. calc. for
C46H38N6O15 ¥ 1.5 H2O (941.9): C 58.66, H 4.38, N 8.92; found: C 58.68, H 4.22, N 8.71.
50. 2',3',5'-Tri-O-acetyl-N6-{{{4-{3-hydroxy-6-{{[2-(4-nitrophenyl)ethoxy]carbonyl}oxy}-9H-xanthen-9-yl}-
3-(methoxycarbonyl)phenyl}amino}carbonyl}adenosine (54). A soln. of 13 (0.16 g, 0.2 mmol) in CH2Cl2
(5 ml) was treated with 3-methyl-1-{[2-(4-nitrophenyl)ethoxy]carbonyl}-1H-imidazolium chloride (0.2 g) by
stirring at r.t. for 3 h. The undissolved reagent was filtered off and the filtrate evaporated. The residue was
treated with EtOH (5 ml) and the orange solid collected and dried: 0.2 g (99%) of chromatographically pure 54.
TLC (silica gel, CH2Cl2/MeOH 95 :5): Rf 0.23. UV (CH2Cl2): 276 (4.74), 337 (4.08), 352 (sh., 3.99), 400 (sh.,
3.98), 429 (4.16), 452 (4.16), 482 (sh., 3.90). 1H-NMR ((D6)DMSO)2): 12.0( s, NH); 10.5 (s, HÀN(6)); 8.75
(s, HÀC(2)); 8.71 (s, HÀC(8)); 8.58 (d, HÀC(6)(flu)); 8.18 (d, 2 H o to NO2); 8.04 (dd, HÀC(4)(flu)); 7.60
7.48 (m, HÀC(3)(flu), HÀC(4)(xan), 2 H m to NO2); 7.16 6.90( m, HÀC(1), HÀC(2), HÀC(8)(all xan));
6.45 (dd, HÀC(7)(xan)); 6.33 (d, HÀC(1')); 6.25 (d, HÀC(5)(xan)); 6.05 (t, HÀC(2')); 5.64 (t, HÀC(3)); 4.55
(t, CH2CH2O); 4.46 4.23 (m, HÀC(4'), 2 HÀC(5')); 3.61 (s, MeO); 3.16 (t, CH2CH2O); 2.12, 2.04, 2.02
(3 s, 3 Ac). Anal. calc. for C47H39N7O17 (973.9): C 57.97, H 4.04, N 10.07; found: C 57.54, H 4.20, N 9.99.
51. 2',3',5'-Tri-O-acetyl-N6-{{{4-{3-hydroxy-6-[2-(4-nitrophenyl)ethoxy]-9H-xanthen-9-yl}-3-(methoxycar-
bonyl)phenyl}amino}carbonyl}adenosine (55). A mixture of 13 (0.1 g, 0.13 mmol), PPh3 (67 mg, 0.26 mmol)
and 2-(4-nitrophenyl)ethanol (0.106 g, 0.65 mmol) in dioxane (10 ml) was heated to 608 with stirring, and then
diethyl azodicarboxylate (40 ml) was added. After 1 h, more PPh3 (60mg) and diethyl azodicarboxylate (100ml)
were added, and stirring was continued for 2 h. The soln. was diluted with CH2Cl2 (30ml) and washed with
phosphate buffer (pH 7). The org. phase was dried (MgSO4) and evaporated and the orange oil separated by
prep. TLC (CH2Cl2/MeOH 95 :5): products 55 and 56. The slower moving product was eluted and, after
evaporation, treated with MeOH to give a solid: 0.07 g (51% ) of 55. TLC (silica gel, toluene/AcOEt/MeOH
5 :4 :1): Rf 0.27. UV (CH2Cl2): 231 (4.65), 256 (sh., 4.55), 277 (4.71), 307 (sh., 4.22), 335 (4.06), 404 (sh., 4.01),
436 (4.25), 458 (4.31), 488 (sh., 4.10). 1H-NMR ((D6)DMSO)2): 12.0( s, NH); 10.5 (s, HÀN(6); 8.75
(s, HÀC(2)); 8.71 (s, HÀC(8)); 8.54 (d, HÀC(6)(flu)); 8.17 (d, 2 H o to NO2); 8.00 (dd, HÀC(4)(flu)); 7.62
(m, 2 H m to NO2); 7.46 (m, HÀC(3)(flu)); 7.26 (m, HÀC(4)(xan)); 6.94 6.83 (m, HÀC(1), HÀC(2),
HÀC(8)(all xan)); 6.35 (m, HÀC(7)(xan), HÀC(1')); 6.25 (s, HÀC(5)(xan)); 6.02 (t, HÀC(2')); 5.64
(t, HÀC(3')); 4.45 4.26 (m, CH2CH2O, HÀC(4'), 2 HÀC(5')); 3.58 (s, MeO); 3.24 (m, CH2CH2O); 2.12,
2
)
For convenience and deviating from the systematic name, the OH or ˆO group is considered to be at C(6)
of the xanthene moiety.

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Helvetica Chimica Acta Vol. 86 (2003)
2.04, 2.02 (3 s, 3 Ac). Anal. calc. for C46H39N7O15 ¥ H2O (947.9): C 58.30, H 4.36, N 10.34; found: C 58.15, H 4.30,
N 9.97.
52. 2',3',5'-Tri-O-acetyl-N6-{{{4-{3-hydroxy-6-[2-(4-nitrophenyl)ethoxy]-9H-xanthen-9-yl}-3-(methoxycar-
bonyl)phenyl}amino}[2-(4-nitrophenyl)ethoxy]methylene}adenosine (56). The faster-moving product from
Exper. 51 was eluted and, after evaporation, treated with a little MeOH to give a precipitate that was dried:
10mg (8%) of 56. TLC (silica gel, toluene/AcOEt/MeOH 5 :4 :1): Rf 0.42. UV (CH2Cl2): 230(4.79), 260(sh.,
4.70), 284 (4.82), 338 (4.17), 356 (sh., 4.11), 404 (sh., 4.11), 434 (4.25), 437 (4.31), 458 (4.43), 488 (sh., 4.10).
1H-NMR ((D6)DMSO)2): 11.6 (s, NH); 8.78 (s, HÀC(2)); 8.75 (s, HÀC(8)); 8.45 (d, HÀC(6)(flu)); 8.19 8.08
(m, 4 H
o
to NO2); 7.94 (dd, HÀC(4)(flu)); 7.61 (m, 4 H
m
to NO2); 7.40( m, HÀC(3)(flu)); 7.25
(m, HÀC(4)(xan)); 6.92 6.83 (m, HÀC(1), HÀC(2), HÀC(8)(xan)); 6.35 (m, HÀC(7)(xan), HÀC(1'));
6.22 (d, HÀC(5)(xan)); 6.03 (t, HÀC(2')); 5.65 (t, HÀC(3')); 4.68 (m, 1 CH2CH2O); 4.43 4.23
(m, 1 CH2CH2O, HÀC(4'), 2 HÀC(5')); 3.56 (s, MeO); 3.23 (m, 2 CH2CH2O); 2.12, 2.06, 2.03 (3 s, 3 Ac).
Anal. calc. for C54H46N8O17 (1079.1): C 60.11, H 4.30, N 10.39; found: C 59.69, H 4.38, N 10.50.
53. 2'-Deoxy-3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)adenosine (57). Twice, 2'-deoxyadenosine (12.5 g,
50mmol) was co-evaporated with dry pyridine, then dissolved in pyridine (200ml), and treated with 1,1-
dichloro-1,1,3,3-tetraisopropyldisiloxane (17.3 ml, 55 mmol). After stirring at r.t. for 12 h, the mixture was
diluted with CH2Cl2 (300 ml) and extracted with NaHCO3 soln. (100 ml). The org. phase was dried (MgSO4),
evaporated, and co-evaporated with toluene, the residue dissolved in CH2Cl2, and the soln. evaporated: 24.5 g
(98%) of crude 57, which was used without further purification for the following experiments.
54. 2'-Deoxy-N6-(phenoxycarbonyl)-3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)adenosine (58). A mixture of
57 (5 g, 10mmol) and 1-(phenoxycarbonyl)-1 H-tetrazole (3.8 g, 20mmol) was stirred in dry dioxane (30ml) at
408 for 12 h. After evaporation, the residue was treated with toluene (50ml), the insoluble material filtered off,
and the filtrate concentrated to ca. 20ml and submitted to FC (silica gel; toluene/AcOEt 5 :1 (250ml), toluene/
AcOEt 2 :1 (600 ml); 100-ml fractions). Fr. 4 9 were evaporated, and the residue was dried under high vacuum:
4.5 g (77%) of 58. An anal. sample was prepared by recrystallization of 0.5 g from Et2O (20ml): 0.3 g of
colorless crystals. M.p. 106 1078. TLC (silica gel, toluene/AcOEt/MeOH 5 :4 :0.5): Rf 0.70. UV (MeOH): 253
1
(4.15), 267 (4.29), 274 (sh., 4.18). H-NMR ((D6)DMSO): 11.1 (s, NH); 8.60( s, HÀC(2)); 8.57 (s, HÀC(8));
7.47 7.22 (m, 5 arom. H); 6.38 (dd, HÀC(1')); 5.17 (q, HÀC(3')); 3.92 3.80( m, HÀC(4'), 2 HÀC(5')); 2.92
(m, 1 HÀC(2')); 2.60( m, 1 HÀC(2')); 1.03 (m, 2 iPr2Si). Anal. calc. for C29H43N5O6Si2 (613.9): C 56.74, H 7.06, N
11.41; found: C 56.67, H 7.10, N 11.56.
55. N6-{{[3-Carboxy-4-(6-hydroxy-3-oxo-3H-xanthen-9-yl)phenyl]amino}carbonyl}-2'-deoxy-3',5'-O-(tet-
raisopropyldisiloxane-1,3-diyl)adenosine (59). Separately, 7 (1.4 g, 4 mmol) and 58 (2 g, 4 mmol), were co-
evaporated with dry pyridine. Then 7 and 58 were dissolved together in dry pyridine (10ml) and heated to 60 8
for 1 h. After evaporation and co-evaporation with toluene, the mixture was dissolved in CH2Cl2/MeOH and
mixed with silica gel (10g). This solid was put onto a FC column (silica gel (60g); CH 2Cl2 (100 ml), CH2Cl2/
MeOH 98 :2 (200 ml), CH2Cl2/MeOH 97 :3 (200 ml), CH2Cl2/MeOH 96 :4 (200 ml), CH2Cl2/MeOH 95 :5
(200 ml), CH2Cl2/MeOH 9 :1 (200 ml), CH2Cl2/MeOH 4 :1 (100 ml); 100-ml fractions). Fr. 9 11 were
evaporated and co-evaporated with dioxane, and the residue was dried under high vacuum: 2.7 g (80%) of 59.
Colorless solid. TLC (silica gel, toluene/AcOEt/MeOH 5 :4 :1): Rf 0.44. UV (CH2Cl2): 228 (4.76), 277 (4.61),
281 (sh., 4.58), 310(sh., 3.37), 458 (2.90). 1H-NMR ((D6)DMSO)2): 12.1 (s, NH); 10.2 (s, NH, OH); 8.65
(s, HÀC(2)); 8.56 (s, HÀC(8)); 8.36 (d, HÀC(6)(flu)); 7.93 (dd, HÀC(4)(flu)); 7.25 (d, HÀC(3)(flu)); 6.67
6.52 (m, 6 arom. H (xan)); 6.37 (dd, HÀC(1')); 5.17 (q, HÀC(3')); 3.92 3.79 (m, HÀC(4'), 2 HÀC(5')); 2.90
(m, 1 HÀC(2')); 2.67 (m, 1 HÀC(2')); 1.05 (m, 2 iPr2Si). Anal. calc. for C43H50N6O10Si2 ¥ H2O (885.1): C 58.35, H
5.92, N 9.50; found: C 58.35, H 6.06, N 9.43.
56. 2'-Deoxy-N6-{{[4-(6-hydroxy-3-oxo-3H-xanthen-9-yl)-3-(methoxycarbonyl)phenyl]amino}carbonyl}-
3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)adenosine (60). A mixture of 9 (0.361 g, 1 mmol) and 58 (1 g,
1.6 mmol) in pyridine (10ml) was heated to 60 8 with stirring for 45 min. After evaporation and co-evaporation
with toluene, the residue was dissolved in a little CH2Cl2 and submitted to FC (silica gel (20g); CH 2Cl2 (150ml),
CH2Cl2/MeOH 98 :2 (100 ml), CH2Cl2/MeOH 96 :4 (100 ml), CH2Cl2/MeOH 94 :6 (100 ml), CH2Cl2/MeOH
9 :1 (200 ml); 50-ml fractions). Fr. 7 11 were evaporated, and the residue was treated with MeOH: 0.75 g
(85%) of 60. Orange powder. TLC (silica gel, toluene/AcOEt/MeOH 5 :4 :1): Rf 0.31. UV (CH2Cl2): 230(4.61),
1
253 (4.45), 277 (4.65), 307 (sh., 4.08), 341 (3.99), 404 (4.01), 433 (4.24), 454 (4.30), 484 (sh., 4.09). H-NMR
((D6)DMSO)2): 12.1 (s, NH); 11.2 (br. s, OH); 10.4 (s, NH); 8.65 (s, HÀC(2)); 8.57 (s, HÀC(8), HÀC(6)(flu));
8.03 (dd, HÀC(4)(flu)); 7.45 (d, HÀC(3)(flu)); 6.89 6.55 (m, 6 arom. H (xan)); 6.38 (dd, HÀC(1')); 5.17
(q, HÀC(3')); 3.94 3.80( m, HÀC(4'), 2 HÀC(5')); 2.90( m, 1 HÀC(2')); 2.62 (m, 1 HÀC(2')); 1.06 (m, 2 iPr2-
Si). Anal. calc. for C44H52N6O10Si2 (881.1): C 59.98, H 5.95, N 9.54; found: C 59.95, H 5.94, N 9.43.

Helvetica Chimica Acta Vol. 86 (2003)
2327
57. 2'-Deoxy-N6-{[2-(4-nitrophenyl)ethoxy]{{3-{[2-(4-nitrophenyl)ethoxy]carbonyl}-4-{6-[2-(4-nitrophenyl)-
ethoxy]-3-oxo-3H-xanthen-9-yl}phenyl}amino}methylene}-3',5'-O-(tetraisopropyldisiloxane-1,3-diyl)adenosine
(61). A mixture of 59 (2.7 g, 3.1 mmol), PPh3 (4.8 g, 18 mmol), and 2-(4-nitrophenyl)ethanol (4.1 g, 24 mmol) in
dry dioxane (50ml) was heated to 60 8 with stirring till a clear dark red soln. was obtained. Diethyl
azodicarboxylate (3 ml, 18 mmol) was added and heating at 608 continued for 1.5 h ( ! orange). The mixture
was diluted with CH2Cl2 (300 ml) and washed with phosphate buffer (pH 7; 100 ml). The org. phase was dried
(MgSO4) and evaporated, and the resulting oil dissolved in toluene (20ml) and submitted to FC (silica gel
(100 g), toluene (200 ml), toluene/AcOEt 4 :1 (500 ml), toluene/AcOEt 1:1 (500 ml), toluene/AcOEt 4 :1
(480ml) ‡ MeOH (20ml) and toluene/AcOEt 4 :1 (450ml) ‡ MeOH (50 ml); 100-ml fractions). Fr. 13 20
were evaporated and co-evaporated with EtOH, and the residue was treated with MeOH by ubltrasound. The
precipitate was stirred in MeOH for 1 h, collected, and dried under high vacuum: 3.7 g (90%) of 61. Orange
solid. TLC (silica gel, toluene/AcOEt/MeOH 5 :4 :1): Rf 0.69. UV (CH2Cl2): 230(4.71), 262 (sh., 4.71), 284
(4.80), 338 (4.10), 406 (sh., 4.06), 436 (4.30), 458 (4.35), 486 (sh., 4.14). 1H-NMR ((D6)DMSO)2): 11.7 (s, NH);
8.66 (s, HÀC(2)); 8.63 (s, HÀC(8)); 8.50( d, HÀC(6)(flu)); 8.19 8.01 (m, 6 H
o to NO2); 7.88
(dd, HÀC(4)(flu)); 7.64 7.55 (m, 4 H to NO2); 7.35 (m, 2 H to NO2 , HÀC(3)(flu)); 7.18
m
m
(d, HÀC(4)(xan)); 6.87 6.77 (m, HÀC(1), HÀC(2), HÀC(8)(all xan)); 6.42 6.33 (m, HÀC(1'),
HÀC(7)(xan)); 6.16 (d, HÀC(5)(xan)); 5.17 (q, HÀC(3')); 4.73 4.22 (m, 3 CH2CH2O); 3.92 3.83
(m, HÀC(4'), 2 HÀC(5')); 3.22 (m, 2 CH2CH2O); 2.82 (m, 1 CH2CH2O, 1 HÀC(2')); 2.65 (m, HÀC(2')); 1.05
(m, 2 iPr2Si). Anal. calc. for C67H71N9O16Si2 (1314.5): C 61.22, H 5.44, N 9.59; found: C 61.07, H 5.42, N 9.00.
58. 2'-Deoxy-N6-{[2-(4-nitrophenyl)ethoxy]{{3-{[2-(4-nitrophenyl)ethoxy]carbonyl}-4-{6-[2-(4-nitrophenyl)-
ethoxy]-3-oxo-3H-xanthen-9-yl}phenyl}amino}methylene}adenosine (62). To a soln. of 61 (1 g, 0.76 mmol) in
THF (10ml), AcOH (400 ml) and Bu4NF ¥ 3 H2O (0.718 g, 2.28 mmol) were added, and the mixture was stirred
at r.t. for 12 h. After dilution with CH2Cl2 (300 ml) and washing with NaHCO3 soln. (100 ml), the org. phase was
dried (MgSO4), and the resulting oil dissolved in CH2Cl2 (10ml) and submitted to FC (silica gel (20g); CH 2Cl2
(100 ml), CH2Cl2/MeOH 98 :2 (100 ml), CH2Cl2/MeOH 95 :5 (200 ml), CH2Cl2/MeOH 9 :1 (200 ml); 50-ml
fractions). Fr. 7 10 were evaporated and co-evaporated with EtOH, and the residue was treated in EtOH with
ultrasound: 0.68 g (84%) of 62. Orange solid. TLC (silica gel, CH2Cl2/MeOH 9 :1): Rf 0.35. UV (Ho À 4): 222
(4.80), 253 (4.70), 298 (4.65), 444 (4.71). UV (pH 0): 222 (4.80), 259 (4.68), 290 (4.70), 431 (sh., 4.45), 455
(4.59), 504 (sh., 3.56). UV (pH 3 13): 283 (4.69), 356 (4.03), 440 (sh., 4.21), 465 (4.33), 495 (4.23). Basic pKa:
À 1.54, 0.68. 1H-NMR ((D6)DMSO)2): 11.9 (s, NH); 8.76 (s, HÀC(2)); 8.74 (s, HÀC(8)); 8.50
(d, HÀC(6)(flu)); 8.19 8.01 (m, 6 H o to NO2); 7.88 (dd, HÀC(4)(flu)); 7.70( m, 4 H m to NO2); 7.38 7.31
(m, 2 H m to NO2, HÀC(3)(flu)); 7.17 (d, HÀC(4)(xan)); 6.84 (m, HÀC(1), HÀC(2), HÀC(8)(all xan)); 6.48
(t, HÀC(1')); 6.35 (dd, HÀC(7)(xan)); 6.16 (d, HÀC(5)(xan)); 5.38 (d, OHÀC(3')); 5.04 (t, OHÀC(5'));
4.75 4.22 (m, HÀC(3'), 3 CH2CH2O); 3.90( m, HÀC(4')); 3.56 (m, 2 HÀC(5')); 3.23 (m, 2 CH2CH2O); 2.76
(m, 1 CH2CH2O, 1 HÀC(2')); 2.47 (m, 1 HÀC(2')). Anal. calc. for C55H45N9O15 (1072.2): C 61.62, H 4.23, N
11.76; found: C 61.41, H 4.42, N 11.73.
59. 2'-Deoxy-5'-O-(dimethoxytrityl)-N6-{[2-(4-nitrophenyl)ethoxy]{{3-{[2-(4-nitrophenyl)ethoxy]carbon-
yl}-4-{6-[2-(4-nitrophenyl)ethoxy]-3-oxo-3H-xanthen-9-yl}phenyl}amino}methylene}adenosine (63). Compound
62 (10.7 g, 10mmol) was twice co-evaporated with pyridine (30ml), then dissolved in dry pyridine (100ml), and
treated with dimethoxytrityl chloride (4.1 g, 12 mmol) under stirring at r.t. for 3 h. The soln. was concentrated to
50ml, diluted with CH 2Cl2 (300 ml), and extracted with NaHCO3 soln. The org. phase was separated, dried
(MgSO4), evaporated, and co-evaporated with toluene. The residue was dissolved in CH2Cl2 (50ml) and
submitted to FC (silica gel (150 g); toluene/EtOH 4 :1 (200 ml), toluene/EtOH 1:1 (500 ml), toluene/EtOH 1:1
(195 ml) ‡ MeOH (5 ml), toluene/EtOH 1:1 (190ml) ‡ MeOH (10ml), toluene/EtOH 1:1 (185 ml) ‡ MeOH
(15 ml), toluene/EtOH 1 :1 (360ml) ‡ MeOH (40ml), toluene/EtOH 1:1 (450ml) ‡ MeOH (50ml), EtOH/
MeOH 4 :1 (300 ml); 100-ml fractions). Fr. 14 20 were evaporated and co-evaporated with EtOH, and the
residue was suspended in EtOH and treated with ultrasound. The mixture was stirred overnight and the
precipitate collected and dried: 12.0g (87%) of 63. M.p. starting at 1398 (dec.). TLC (silica gel, toluene/AcOEt/
MeOH 5 :4 :1): Rf 0.42. UV (CH2Cl2): 231 (4.82), 282 (4.79), 340 (4.08), 406 (sh., 4.03), 436 (4.27), 490 (sh.,
4.12). 1H-NMR ((D6)DMSO)2): 12.1 (s, NH); 8.70( s, HÀC(2)); 8.66 (s, HÀC(8)); 8.52 (d, HÀC(6)(flu));
8.18 8.01 (m, 6 H o to NO2); 7.88 (dd, HÀC(4)(flu)); 7.60( m, 4 H m to NO2); 7.38 7.13 (m, 2 H m to NO2,
HÀC(3)(flu), HÀC(4)(xan), 9 arom. H); 6.86 6.75 (m, HÀC(1), HÀC(2), HÀC(8)(all xan), 4 H m to
MeO); 6.51 (t, HÀC(1')); 6.34 (dd, HÀC(7)(xan)); 6.15 (d, HÀC(5)(xan)); 5.4 (d, OHÀC(3')); 4.75 4.22
(m, HÀC(3'), 3 CH2CH2O); 4.04 (m, HÀC(4')); 3.66 (2 s, 2 MeO); 3.23 (m, 2 CH2CH2O, 2 HÀC(5')); 2.76
(m, 1 CH2CH2O, 1 HÀC(2')); 2.45 (m, 1 HÀC(2')). Anal. calc. for C76H63N9O17 ¥ H2O (1392.4): C 65.56, H 4.71,
N 9.05; found: C 65.80, H 4.84, N 8.88.

2328
Helvetica Chimica Acta Vol. 86 (2003)
60. 2'-Deoxy-5'-O-(dimethoxytrityl)-N6-{[2-(4-nitrophenyl)ethoxy]{{3-{[2-(4-nitrophenyl)ethoxy]carbon-
yl}-4-{6-[2-(4-nitrophenyl)ethoxy]-3-oxo-3H-xanthen-9-yl}phenyl}amino}methylene}adenosine 3'-[2-(4-Nitro-
phenyl) Diisopropylphosphoramidite] (64). To a soln. of 63 (0.5 g, 0.36 mmol) in CH2Cl2 (7 ml) under N2
EtiPr2N (250 ml, 1.4 mmol) and 2-(4-nitrophenyl)ethyl diisopropylphosphoramidochloridite (0.242 g, 0.72 mmol)
were added and stirred at r.t. for 1 h. After dilution with CH2Cl2 (100 ml) and washing with NaHCO3 soln., the org.
phase was dried (MgSO4), and evaporated and the EtiPr2N removed under high vacuum. The residue was purified
by FC (Al2O3 (neutral, 75 g); CH2Cl2/AcOEt 1:1 (200 ml), AcOEt (200 ml), AcOEt (100 ml) ‡ MeOH (3 ml)
and AcOEt/MeOH 95 :5 (300 ml); 50-ml fractions). Fr. 9 16 were evaporated and co-evaporated with CH2Cl2:
0.48 g (79%) of 64. Solid foam. TLC (silica gel, toluene/AcOEt/MeOH 10:9 :1): Rf 0.57. 1H-NMR (CDCl3)2):
13.71 (s, NH); 8.64 (s, HÀC(2)); 8.43 (d, HÀC(6)(flu)); 8.33 (s, HÀC(8)); 8.20 8.01 ( m, 8 H o to NO2,
HÀC(4)(flu), HÀC(3)(flu)); 7.60 7.16 ( m, 21 H, 8 H m to NO2, arom. H); 6.92 6.50( m, HÀC(1), HÀC(2),
HÀC(4), HÀC(7), HÀC(8)(all xan), 4 H
m
to MeO, HÀC(1')); 6.39 (d, HÀC(5)(xan)); 4.99
(m, 1 CH2CH2O); 4.73 (m, HÀC(3')); 4.31 (m, 2 CH2CH2O); 3.73 (2 s, 2 MeO); 3.77 2.61 (m, 3 CH2CH2O,
HÀC(4'), 2 HÀC(5'), 2 HÀC(2'), 2 Me2CH); 1.1 1.0( m, 2 Me2CH). 31P-NMR (CDCl3): 148.3; 148.2. Anal.
calc. for C90H84N11O20 (1670.7): C 64.70, H 5.07, N 9.22; found: C 62.97, H 5.07, N 8.73.
61. 2'-Deoxy-5'-O-(dimethoxytrityl)-N6-{[2-(4-nitrophenyl)ethoxy]{{3-{[2-(4-nitrophenyl)ethoxy]carbonyl}-
4-{6-[2-(4-nitrophenyl)ethoxy]-3-oxo-3H-xanthen-9-yl}phenyl}amino}methylene}adenylyl-{3'-{OP-[2-(4-nitro-
phenyl)ethyl]}-5'}-2'-deoxy-N6,3'-O-bis{[2-(4-nitrophenyl)ethoxy]carbonyl}adenosine (65). To a mixture of 64
(0.63 g, 0.37 mmol) and 46 (0.12 g, 0.19 mmol) in MeCN (5 ml) and CH2Cl2 (5 ml) under N2, 1H-tetrazole
(0.1 g) was added and stirred for 30 min. Then a soln. of I2 (0.5 g) in pyridine/CH2Cl2/H2O 5 :1:1 (7 ml) was
added and stirred for 15 min for oxidation. After dilution with CH2Cl2 (200 ml) and decolorization with Na2S2O3
soln., the org. phase was dried (MgSO4), evaporated, and co-evaporated with toluene to remove pyridine. The
crude material was dissolved in CH2Cl2 and submitted to FC (silica gel (25 g); toluene (50ml), toluene/AcOEt
1:1 (100 ml), toluene/AcOEt 1:1 (97 ml) ‡ MeOH (3 ml), toluene/AcOEt 1:1 (190ml) ‡ MeOH (10ml),
toluene/AcOEt 1:1 (180ml) ‡ MeOH (20ml); 40-ml fractions). Fr. 12 15 were evaporated and co-evaporated
with EtOH. The residue was suspended in MeOH and exposed to ultrasound and the solid collected and dried
under high vacuum: 0.25 g of 65. Fr. 11 was rechromatographed to give another 0.06 g. Total yield 74%. TLC
(silica gel, toluene/AcOEt/MeOH 5 :4 :1): Rf 0.20. UV ((D6)DMSO): 234 (4.81), 274 (4.93), 342 (sh., 4.01), 412
(sh., 4.00), 434 (sh., 4.30), 458 (4.26), 490 (sh., 4.04). 1H-NMR (CDCl3)2): 12.1 (s, NH); 10.6 (br. s, NH); 8.61
8.52 (m, 5 H, HÀC(2), HÀC(8), HÀC(6)(flu)); 8.18 7.99 (m, 12 H o to NO2); 7.88 (m, HÀC(4)(flu)); 7.64
7.10( m, 23 H, H m to NO2, arom. H, HÀC(3)(flu), HÀC(4)(xan)); 6.86 6.71 (m, 7 H, HÀC(1), HÀC(2),
HÀC(8)(all xan),
H
m
to MeO, HÀC(1')); 6.44 6.32 (d, 3 H, HÀC(1'), HÀC(7)(xan)); 6.15
(d, HÀC(5)(xan)); 5.32 (m, HÀC(3')); 5.09 (m, HÀC(3')); 4.73 (m, 2 H, CH2CH2O); 4.44 4.19 (m, 14 H,
CH2CH2O, HÀC(4'), HÀC(5')); 3.64 (2 s, 2 MeO); 3.23 2.57 (m, 18 H, CH2CH2O, HÀC(5'), HÀC(2')). Anal.
calc. for C112H96N17O32P2 (2223.1): C 60.51, H 4.35, N 10.71; found: C 60.34, H 4.35, N 10.99.
62. 2'-Deoxy-N6-{[2-(4-nitrophenyl)ethoxy]{{3-{[2-(4-nitrophenyl)ethoxy]carbonyl}-4-{6-[2-(4-nitrophenyl)-
ethoxy]-3-oxo-3H-xanthen-9-yl}phenyl}amino}methylene}adenylyl-{3'-{OP-[2-(4-nitrophenyl)ethyl}-5'}-2'-de-
oxy-N6,3'-O-bis{[2-(4-nitrophenyl)ethoxy]carbonyl}adenosine (66). A soln. of 65 (1.1 g, 0.495 mmol) in 3%
Cl3CCOOH/CH2Cl2 (50ml) was stirred at r.t. for 30min. After washing with NaHCO 3 soln., the org. phase was
dried (MgSO4) and evaporated. The residue was dissolved in CH2Cl2 and purified by FC (silica gel (45 g);
toluene (50ml), toluene/AcOEt 1:1 (380ml) ‡ MeOH (20ml), toluene/AcOEt 1:1 (270ml) ‡ MeOH
(30ml), toluene/AcOEt 1:1 (255 ml) ‡ MeOH (45 ml), toluene/AcOEt/MeOH 1:1:1 (200 ml); 100-ml
fractions). Fr. 12 and 13 were evaporated and co-evaporated with EtOH, and the residue was treated with
MeOH and exposed to ultrasound: 0.73 g of 66. Fr. 11 and 14 were rechromatographed and gave a second crop
(0.15 g) for a total yield of 0.88 g (92%) of 66. TLC (silica gel, toluene/AcOEt/MeOH 5 :4 :1): Rf 0.37. UV
(CH2Cl2): 270 (sh., 4.90), 274 (4.98) 292 (sh., 4.86), 337 (4.10), 408 (sh., 4.02), 436 (sh., 4.24), 459 (4.30), 488 (sh.,
4.09). 1H-NMR ((D6)DMSO)2): 12.0( s, NH); 10.6 (br. s, NH); 8.72 8.50( m, 5 H, HÀC(2), HÀC(8),
HÀC(6)(flu)); 8.18 8.01 (m, 12 H o to NO2); 7.88 (m, HÀC(4)(flu)); 7.64 7.32 (m, 13 H, H m to NO2,
HÀC(3)(flu); 7.18 (m, HÀC(4)(xan)); 6.84 (m, HÀC(1), HÀC(2), HÀC(8)(all xan)); 6.42 6.33 (d, 3 H,
HÀC(1'), HÀC(7)(xan)); 6.15 (d, HÀC(5)(xan)); 5.34 (m, 1 H, HÀC(3')); 5.17 (t, 1 H, OHÀC(5')); 5.00
(m, 1 H, HÀC(3')); 4.75 (m, 2 H, CH2CH2O); 4.44 4.04 (m, 14 H, CH2CH2O, HÀC(4'), HÀC(5')); 3.51
(m, 2 H, HÀC(5')); 3.23 2.57 (m, 16 H, CH2CH2O, HÀC(5'), HÀC(2')). Anal. calc. for C91H78N17O30P2
(1920.7): C 56.91, H 4.09, N 12.40; found: C 56.35, H 4.15, N 12.99.
63. 2'-Deoxy-N6,5'-O-bis{[2-(4-nitrophenyl)ethoxy]carbonyl}adenylyl-{3'-{OP-[2-(4-nitrophenyl)ethyl]}-5'}-
2'-deoxy-N6-{[2-(4-nitrophenyl)ethoxy]{{3-{[2-(4-nitrophenyl)ethoxy]carbonyl}-4-{6-[2-(4-nitrophenyl)ethoxy]-
3-oxo-3H-xanthen-9-yl}phenyl}amino}methylene}adenylyl-{3'-{OP-[2-(4-nitrophenyl)ethyl]}-5'}-2'-deoxy-N6,3'-