Simple and stereoselective preparation of an 4-(aminomethyl)-1,2,3- triazolyl nucleoside phosphoramidite

Article abstract of DOI:10.1002/cbdv.201000047

A simple and stereoselective synthesis of a protected 4-(aminomethyl)-1-(2- deoxy-β-D-ribofuranosyl)-1,2,3-triazole cyanoethyl phosphoramidite was developed for the modification of synthetic oligonucleotides. The configuration of the 1,2,3-triazolyl moiet

Full text of DOI:10.1002/cbdv.201000047

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CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
Simple and Stereoselective Preparation of an 4-(Aminomethyl)-1,2,3-triazolyl
Nucleoside Phosphoramidite
by Natalia A. Kolganova, Vladimir L. Florentiev, Alexander V. Chudinov, Alexander S. Zasedatelev, and
Edward N. Timofeev*
Engelhardt Institute of Molecular Biology, 32 Vavilov St., Moscow 119991, Russia
(phone: þ7495-135-6591; fax: þ7495-135-1405; e-mail: edward@eimb.ru)
A simple and stereoselective synthesis of a protected 4-(aminomethyl)-1-(2-deoxy-b-d-ribofurano-
syl)-1,2,3-triazole cyanoethyl phosphoramidite was developed for the modification of synthetic
oligonucleotides. The configuration of the 1,2,3-triazolyl moiety with respect to the deoxyribose was
unambiguously determined in ROESY experiments. The aminomethyl group of the triazolyl nucleotide
was fully functional in labelling reactions. Furthermore, the hybridization behavior of 5’ triazole-
terminated oligonucleotide was similar to that of 5’ aminohexyl-terminated oligomer with the same
sequence. Internal modifications of the oligonucleotide strands resulted in significant decrease of duplex
stability.
Introduction. – Recently, several successful examples of azideꢀalkyne 1,3-dipolar
cycloadditions have been reported in nucleoside and oligonucleotide chemistry [1–4].
This relatively new method is promising for the regio- and stereoselective synthesis of
substituted triazole deoxynucleosides because of the easy access to protected b-azido-
deoxyribose 2 [5]. Triazolyl nucleoside derivatives have received much attention
because of their potential antiviral and antitumor properties [6–8]. In addition to this
therapeutic aspect, we were interested in a nucleoside analog containing an aminoalkyl
side chain that would not participate in WatsonꢀCrick base pairing. Several
applications in molecular biology require the presence of different functional, reactive,
linker, and reporter groups in a single oligonucleotide chain [9][10]. Amino modifiers
are the most commonly used building blocks to introduce a reactive amino group into
oligonucleotides for their post-synthetic modification or labelling [11]. There are two
types of amino modifiers to date, aliphatic amino modifiers with a flexible linker arm
[12–16], and nucleosidic-type amino modifiers [17][18]. Non-hybridizable aminoalkyl
nucleoside units have not been described so far. This type of amino modifier would
feature useful properties such as defined configuration, possibility for both internal and
terminal modification, standard DMTr protecting group at the 5’-end, and unchanged
sugar-phosphate backbone. In addition, for a short aminoalkyl arm, it would be possible
to place the group of interest inside the helical structure. This possibility is important
for applications based on the intercalation and stacking interactions [19][20].
In a recent study, a number of substituted 1,2,3-triazolyl nucleosides have been
synthesized by the Huisgen reaction [2]. The synthesis of aminoalkyl derivatives by this
method, however, has not been reported. Here, we report a simple and stereoselective
synthesis of a protected 4-(aminomethyl)-1,2,3-triazolyl deoxynucleoside and its
ꢀ 2011 Verlag Helvetica Chimica Acta AG, Zꢁrich

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phosphoramidite derivative, as well as the hybridization properties of triazole-modified
oligonucleotides.
Results and Discussion. – 1. Synthesis of Phosphoramidite 6 (Scheme). The
cycloaddition of propargylamine to 1-azido-2-deoxyribofuranosyl 3 is an attractive
route for the stereoselective preparation of the b-anomer of 4-(aminomethyl)-1,2,3-
triazolyl nucleoside [21]. We have optimized the synthetic route to reduce the number
of steps for the preparation of a protected nucleoside phosphoramidite. The
azideꢀalkyne cycloaddition was conducted with Fmoc-protected propargylamine and
the unprotected azido sugar 3 to provide the shortest route to the target phosphor-
amidite derivative 6. The unprotected azido compound 3 is readily soluble in organic
solvents, and this facilitated the choice of conditions for the cycloaddition reaction. On
the other hand, the presence of free OH groups at C(5’) and C(3’) of the triazolyl
nucleoside allow direct introduction of DMTr protecting group and subsequent
phosphitylation.
Scheme
a) LiN3, DMF. b) K2CO3, MeOH. c) Fmoc-protected propargyl amine, CuBr, CH2Cl2. d) DMTrCl
(¼ bis(4-methoxyphenyl)(phenyl)methyl chloride), Py. e) (iPr2N)2P(OCH2CH2CN), 1H-tetrazole, Py.
We prepared the protected 1-azido-2-deoxyribofuranosyl 2 from 2-deoxy-3,5-di-O-
(p-toluoyl)-a-d-erythro-pentofuranosyl chloride (1) and LiN3 in DMF at 08 [3][21].
The highest stereoselectivity observed in the formation of the azido derivative from the
protected a-chloride 1 was 85%, as evidenced by 1H-NMR. This selectivity is
comparable to the published procedure that is based on a p-chlorobenzoyl-deoxyribose
analog and CsN3 or KN3 in DMSO at room temperature (9 :1 anomer ratio) [5]. It was
found that the chromatographic separation of the a- and b-anomers is best performed
for DMTr derivatives of the triazolyl nucleoside due to the noticeable difference in Rf
values (0.49 vs. 0.55 in CH2Cl2/MeOH/hexane 9 :1:10). For this reason, we have used

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anomeric mixtures in the cycloaddition reaction and during the protection of the 5’-OH
group with DMTrCl.
The cycloaddition reaction was performed in CH2Cl2 in the presence of 0.15 equiv.
of EtNiPr2 and 1.6 equiv. of Me3PO3. Both the Fmoc-amine and the unprotected azido
sugar 3 are readily soluble in CH2Cl2. This allows for a smooth Huisgen coupling in the
presence of 0.4 equiv. of CuBr to yield 61% of the triazolyl nucleoside 4 after
purification.
The configuration of the triazolyl nucleoside at C(1’) was unambiguously
determined by ROESY experiments for derivative 5. 2D-ROESY Spectrum displayed
a strong HꢀC(1’)/HꢀC(4’) correlation, consistent with the b-configuration at the
glycosidic bond [2]. Additionally, analysis of the correlations between the H-atoms at
C(3’), C(2’), and C(1’) confirms b-configuration of the nucleoside. There are two
predominant sugar conformations of the pentafuranose ring, N and S [22], that do not
differ considerably from each other in terms of the distance between the H-atoms
Hpro-SꢀC(2’) and Hpro-SꢀC(3’). Therefore, it was possible to assign pro-S or pro-R
configuration for either of the H-atoms at C(2’). We followed the NOEs between the
H-atoms at C(3’) and C(2’) to identify the pro-S and pro-R H-atoms at C(2’) (Fig. 1). In
turn, following the NOEs between HꢀC(1’) and either one of the H-atoms at C(2’)
Fig. 1. Correlations between HꢀC(1’), HꢀC(2’), HꢀC(3’), and HꢀC(4’) in the ROESY spectrum of the
nucleoside 5. HaꢀC(2’) and HbꢀC(2’) were identified as pro-S and pro-R, resp.

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
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should be indicative of the nucleoside configuration. The larger value of the correlation
between HꢀC(1’) and the pro-R H-atom strongly supports the b-configuration of the
1,2,3-triazolyl nucleoside. This conclusion is consistent with the assumed SN2
mechanism of N3 substitution at C(1’).
The phosphoramidite derivative 6 was prepared by treatment of the protected
nucleoside 5 with 2-cyanoethyl N,N,N’,N’-tetraisopropylphosphoramidite in dry MeCN
in the presence of pyridinium tetrazolide [23].
2. Synthesis and Hybridization Properties of Modified Oligonucleotides. Modified
oligonucleotides ON1–ON3 (Table) were synthesized, and the effect of the modifi-
cation on the duplex stability was assessed. The standard reaction cycle of
oligonucleotide synthesis and deprotection procedures were used without modifica-
tions. The purification of modified oligomers was performed with 5’-DMTr-protected
oligonucleotides using reversed-phase (RP) HPLC. The final deprotection was
achieved using 2% aqueous CF3COOH. Both DMTr-protected and unprotected
oligomers showed a single peak in the RP-HPLC analysis. MALDI Mass spectra of
synthesized oligonucleotides confirmed incorporation of the modified residue. The
MALDI mass spectrum of the oligodeoxynucleotide ON1 is shown in Fig. 2.
Fig. 2. MALDI-TOF Spectra of the oligonucleotide ON1 (Mcalc ¼5486.7). Values Mexp/Mcalc for ON2 and
ON3 are 5153.2/5157.4 and 5065.1/5064.4, resp. Analysis was carried out in negative-ion mode with
hydroxypicolinic acid as a matrix.

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Table. Thermal Stability of DNA Duplexes
Tm [8]
DNA Duplexesa)
X¼A
G
C
T
5’- MATGCAGGTCCATGAGCT (ON1)
3’-AACXTACGTCCAGGTACTCGA
61.9
61.2
62.2
62.5
5’- HATGCAGGTCCATGAGCT (ON4)
3’-AACXTACGTCCAGGTACTCGA
62.3
49.2
62.3
49.3
62.0
47.0
62.0
48.4
5’-ATGCAGMTCCATGAGCT (ON2)
3’-TACGTCXAGGTACTCGA
5’-ATGCAGCTCCATGAGCT (ON5)
3’-TACGTCGAGGTACTCGA
61.6
34.9
5’-ATGCAMCTCCATMAGCT (ON3)
3’-TACGTCGAGGTACTCGA
a) M designates aminotriazole nucleotide, H designates 6-aminohexyl residue.
In the thermal denaturation studies, the triazole-modified oligonucleotide ON1 was
compared with amino-terminated oligonucleotide ON4. The complementary strand
contained one of four natural bases opposite to modified unit. Normalized UV
denaturation profiles of all eight duplexes virtually coincided and gave melting
temperatures in the range of 61.2–62.58 in 0.1m NaCl and 0.02m NaPO4 at pH 7.0. These
results clearly demonstrate that there is no difference between a terminal aminotriazole
and an aminohexyl linker with respect to the stability of duplexes. According to these
data, the nature of the nucleotide in the complementary strand opposite to the 5’
modification has no effect regardless of the modifying amine unit. This observation is in
good agreement with the poor hybridization properties of unsubstituted azole-based
nucleosides, which have been reported to considerably destabilize the DNA duplex
when placed in the middle of the sequence [24]. The ability to support stacking in the
double helix was observed only for nitrazole nucleobase analogs, such as a universal
nucleoside 1-(2-deoxy-b-d-ribofuranosyl)-3-nitropyrrole [25]. Protonation of the
amino group in the case of modified nucleotide in an aqueous environment should
account for even less favorable conditions for stacking or H-bonding as compared with
unsubstituted triazoles. The influence of a single positive charge on the duplex stability
is the same in ON1 and ON4, and is rather low if noticeable at all [26].
To estimate the effect of internal strand modifications on the duplex stability, we
incorporated one or two triazole units into the sequence (see ON2 and ON3). Thermal
denaturation of four duplexes with a single modification showed considerable decrease
in Tm values as compared with the reference duplex containing unmodified strand ON5
(Table). The average DTm was ꢀ13.18 (Fig. 3). Furthermore, two modified units in the
oligonucleotide ON3 induced a drop of Tm by 26.78. These results provide a convincing
proof of poor base-pairing properties of (aminomethyl)-triazolyl nucleoside.
The aminomethyl group of the triazolyl nucleotide was fully functional in labelling
reactions with N-hydroxysuccinimide esters of fluorescent dyes. Fluorescent conjugates
of modified oligonucleotide ON1 and the corresponding 5’-aminohexyl control ON4
with sulfonated derivatives of indocyanine dyes [27] were synthesized with similar

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
573
Fig. 3. Denaturation profiles of duplexes with ON5, ON2, and ON3 strand in 0.1m NaCl and 0.02m Na
phosphate at pH 7.0
efficiencies. The yields of labelled oligonucleotides were in the range of 60–80%
regardless of the terminus modifier. The presence of a fluorescent label in triazole-
terminated oligonucleotides SCy3-ON1 and SCy5-ON1 was confirmed by UV
spectroscopy, fluorescence, and MALDI-MS analysis. Fluorescently labelled oligomers
showed absorbance peaks at 551 and 645 nm, as well as emission peaks at 566 and
662 nm for SCy3 and SCy5 derivatives, respectively.
Finally, we examined hybridization behavior of the labelled oligonucleotide SCy3-
ON1 with a hydrogel-pad array [28] of four immobilized probes (5’-AGCTCATG-
GACCTGCATXCAA, where X¼A, G, C or T). Hybridization was carried out for
24 h at 258 in 0.1m NaCl and 0.02m Na phosphate at pH 7.0. Quantification of the
fluorescent signals from the gel-pads generally confirmed data from the thermal
denaturation studies and showed the close efficiency of hybridization of the modified
labelled target with all four immobilized probes (Fig. 4).
Fig. 4. Hybridization of SCy3-ON1 with four immobilized probes (5’-AGCTCATGGACCTGCATX-
CAA, where X¼A, G, C or T) in 0.1m NaCl and 0.02m Na phosphate at pH 7.0. a) Hybridization images.
b) Normalized average fluorescent signals from gel-pads.

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CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
Conclusions. – We proposed a simple and stereoselective synthesis of an (amino-
methyl)-triazolyl nucleoside phosphoramidite for the modification of synthetic
oligonucleotides. The synthetic scheme was optimized for the preparation of the
protected 4-(aminomethyl)-1-(2-deoxy-b-d-ribofuranosyl)-1,2,3-triazole cyanoethyl
phosphoramidite 6. The modification of oligonucleotides does not require modifica-
tions in the standard protocols of oligonucleotide synthesis and deprotection. Thermal
denaturation and hybridization studies of the 5’-modified oligonucleotide have
demonstrated that the modification has no effect on the duplex stability when
compared to oligonucleotide with a terminal 5’-aminohexyl group. The (aminomethyl)-
triazolyl nucleotide at the 5’ end of the oligonucleotide does not form base pairs and
does not discriminate between natural bases in the complementary strand. Internal
modifications of the oligonucleotide strand induce considerable decrease in thermal
stability of duplexes. The amino group of the modified nucleotide was shown to be
accessible for conjugation reactions such as fluorescent labelling.
We thank Dr. A. A. Stomakhin for recording the MALDI-TOF mass spectra. This work was
supported by the research grants No. 02740110080 and 02522112019 from the Russian Federal Agency of
Science and Innovations and by the research programme ꢂMolecular and Cell Biologyꢃ of the Russian
Academy of Sciences.
Experimental Part
General. Unless otherwise stated, all reagents were purchased from commercial sources and used
without purification. TLC: Kieselgel 260F (Merck) plates; solvent systems A and B refer to CH2Cl2/
MeOH/hexane 9 :1:30 and CH2Cl2/MeOH/hexane 9 :1:10, resp. UV Measurements and thermal
denaturation studies: Shimadzu UV-160A spectrophotometer. Fluorescent spectroscopy: Shimadzu RF-
5301PC spectrofluorophotometer. 1H- 13C-, and 31P-NMR: Bruker AMX 400 MHz instrument. MALDI-
MS: Bruker Reflex IV mass spectrometer in negative-ion mode with hydroxypicolinic acid as a matrix.
The microarray analysis was performed with a home-made portable biochip analyzer.
2-Deoxy-3,5-di-O-(p-toluoyl)-b-d-ribofuranosyl Azide (¼(2R,3S,5R)-5-Azido-2-{[(4-methylben-
zoyl)oxy]methyl}tetrahydrofuran-3-yl 4-Methylbenzoate; 2). Chloride 1 [29] (1.5 g, 4.42 mmol) was
added to an intensively stirred suspension of LiN3 (1.08 g, 22.06 mmol) in DMF (8 ml) at 08. The mixture
was stirred for 1 h at 08 and then 1 h at 258. It was diluted with Et2O (150 ml) and consequently washed
with 10% aq. NaHCO3 (100 ml) and H2O (100 ml). The org. layer was dried (Na2SO4) and evaporated in
vacuo to give 2 (1.2 g, 80%). Rf (A) 0.75, mixture of isomers. 1H-NMR (400 MHz, (D6)DMSO; b-
anomer): 7.91, 7.87 (2d, J¼8.4, 4 arom. o-H); 7.34, 7.33 (2d, J¼8.4, 4 arom. m-H); 5.88 (dd, J¼4.0, 5.6,
HꢀC(1)); 5.51–5.56 (m, HꢀC(3)); 4.40–4.57 (m, HꢀC(4), CH2(5)); 2.43–2.47 (m, HaꢀC(2)); 2.31–2.36
(m, HbꢀC(2)). 13C-NMR (100 MHz, CDCl3): 130.0; 129.9; 129.4; 129.3; 92.3; 82.9; 77.5; 77.2; 76.8; 75.1;
64.4; 38.9; 21.8. Anal. calc. for C21H21N3O5: C 63.79, H 5.35, N 10.63; found: C63.83, H 5.29, N 10.58.
2-Deoxy-b-d-ribofuranosyl Azide (¼(2R,3S,5R)-5-Azido-2-(hydroxymethyl)tetrahydrofuran-3-ol;
3). Azide 2 (0.63 g, 1.59 mmol) was added to a stirred suspension of K2CO3 (0.56 g) in MeOH (10 ml).
The mixture was stirred for 16 h at 558. Excess K2CO3 was neutralized with aq. HCl (36%), and the inorg.
salt was removed by filtration. The resulting soln. was concentrated under reduced pressure. The product
was purified by CC (SiO2; CH2Cl2/MeOH/hexane 9 :1:10): 0.19 g (73%) of 3. Rf (B) 0.25, mixture of
1
isomers. H-NMR (400 MHz, (D6)DMSO; b-anomer): 5.54 (dd, J¼4.4, 6.0, HꢀC(1)); 5.13 (d, J¼4.8,
HOꢀC(3)); 4.76 (t, J¼5.2, HOꢀC(5)); 4.11–4.16 (m, HꢀC(3)); 3.74–3.77 (m, HꢀC(4)); 3.37–3.48 (m,
CH2(5)); 1.92–2.04 (m, CH2(2)). 13C-NMR (100 MHz, CDCl3): 104.0; 91.3; 87.6; 84.9; 70.3; 70.0; 62.0;
61.3; 40.9; 40.3; 39.9; 39.7; 39.5; 39.3; 39.1. Anal. calc. for C5H9N3O3: C 37.74, H 5.70, N 26.40; found:
C37.80, H 5.65, N 26.48.
(9H-Fluoren-9-yl)methyl (Prop-2-yn-1-yl)carbamate. (9H-Fluoren-9-yl)methoxycarbonyl (Fmoc)
chloride (1.2 g, 4.65 mmol) in CH2Cl2 (10 ml) was added slowly to an intensively stirred aq. soln. (15 ml)
of Na2CO3 (2.6 g) and propargyl amine hydrochloride (0.57 g, 6.26 mmol) at 08. The mixture was stirred

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
575
for 2 h. The product was extracted with CH2Cl2 (2ꢁ50 ml), and the solvent was removed in vacuo. The
residue was crystallized from EtOH. Yield 1.4 g (73%). Rf (B) 0.67. 1H-NMR (400 MHz, (D6)DMSO):
7.89 (d, J¼7.6, HꢀC(4), HꢀC(5) of Fmoc); 7.69 (d, J¼7.6, HꢀC(1), HꢀC(8) of Fmoc); 7.42 (t, J¼7.6,
HꢀC(3), HꢀC(6) of Fmoc); 7.33 (t, J¼7.6, HꢀC(2), HꢀC(7) of Fmoc); 4.31 (d, J¼6.8, CH2 of Fmoc);
4.23 (t, J¼6.8, CH of Fmoc); 3.78 (dd, J¼2.4, 5.6, CH2NH); 3.08 (s, CH). 13C-NMR (100 MHz, CDCl3):
143.7; 140.7; 127.6; 127.0; 125.1; 120.0; 72.9; 65.6; 46.6; 39.9; 39.7; 39.5; 39.3; 39.1; 29.7. Anal. calc. for
C18H15NO2: C 77.96, H 5.45, N 5.05; found: C77.89, H 5.55, N 4.98.
1-(2-Deoxy-b-d-ribofuranosyl)-4-({[(9H-fluoren-9-yl)methoxycarbonyl]amino}methyl)-1H-1,2,3-
triazole (4). To a soln. of 3 (0.42 g, 2.68 mmol) and Fmoc-protected propargyl amine (1 g, 3.61 mmol) in
15 ml CH2Cl2 were added Me3PO3 (0.51 ml, 4.32 mmol), CuBr (0.15 g, 1.07 mmol), and EtNiPr2 (0.07 ml,
0.41 mmol). The mixture was stirred for 12 h at r.t. and then concentrated under reduced pressure. The
product was purified by CC (SiO2; CH2Cl2/MeOH/hexane 9 :1:10): 0.71 g (61%) of 4, mixture of isomers.
Rf (B) 0.15. An anal. sample of the b-anomer was purified by prep. TLC. 1H-NMR (400 MHz,
(D6)DMSO): 8.07 (s, HꢀC(5)); 7.88 (d, J¼7.6, HꢀC(4), HꢀC(5) of Fmoc); 7.69 (d, J¼7.6, HꢀC(1),
HꢀC(8) of Fmoc); 7.41 (t, J¼7.6, HꢀC(3), HꢀC(6) of Fmoc); 7.32 (t, J¼7.6, HꢀC(2), HꢀC(7) of Fmoc);
6.33 (t, J¼6.0, HꢀC(1’)); 5.30 (d, J¼4.4, HOꢀC(3’)); 4.83 (t, J¼5.6, HOꢀC(5’)); 4.36–4.39 (m,
HꢀC(3’)); 4.20–4.31 (m, CH2 and CH of Fmoc, CH2NH); 3.85–3.89 (m, HꢀC(4’)); 3.52 (dd, J¼4.8, 11.6,
HaꢀC(5’)); 3.43 (dd, J¼4.8, 11.6, HbꢀC(5’)); 2.55–2.61 (m, HaꢀC(2’)); 2.32–2.38 (m, HbꢀC(2’)).
13C-NMR (100 MHz, (D6)DMSO): 156.1; 143.8; 140.7; 127.6; 127.0; 125.1; 121.3; 120.0; 88.2; 87.9; 70.5;
65.5; 61.7; 46.7. Anal. calc. for C23H24N4O5: C 63.29, H 5.54, N 12.84; found: C63.34, H 5.61, N 12.78.
1-{5-O-[Bis(4-methoxyphenyl)(phenyl)methyl]-2-deoxy-b-d-ribofuranosyl}-4-({[(9 H-fluoren-9-yl)-
methoxycarbonyl]amino}methyl)-1H-1,2,3-triazole (5). A soln. of 4 (0.45 g, 1.03 mmol) in pyridine (5 ml)
was treated with DMTrCl (0.37 g, 1.08 mmol) at r.t. for 2 h. The mixture was then concentrated under
reduced pressure, diluted with CH2Cl2 (50 ml), and subsequently washed with 10% aq. NaHCO3 (50 ml)
and H2O (50 ml). The org. layer was dried (Na2SO4) and concentrated. The product was purified by CC
(SiO2; linear gradient from CH2Cl2/hexane 1:1 to CH2Cl2/MeOH/hexane 9 :1:10): 0.56 g (74%) of 5. Rf
(B) 0.49. 1H-NMR (400 MHz, (D6)DMSO): 8.58 (d, J¼4.4, HꢀC(5)); 7.87 (d, J¼7.6, HꢀC(4), HꢀC(5)
of Fmoc); 7.67 (d, J¼7.6, HꢀC(1), HꢀC(8) of Fmoc); 7.76–7.81, 7.16–7.42 (2m, 5 arom. of Ph, 4 arom. o-
H of DMTr, HꢀC(2), HꢀC(3), HꢀC(6), HꢀC(7) of Fmoc); 6.85, 6.84 (2d, J¼8.8, 4 arom. m-H of
DMTr); 6.36 (t, J¼5.6, HꢀC(1’)); 5.36 (d, J¼4.8, HOꢀC(3’)); 4.36–4.42 (m, HꢀC(3’)); 4.18–4.30 (m,
CH2, CH in Fmoc, CH2NH); 3.96–4.00 (m, HꢀC(4’)); 3.72 (s, 2 MeO); 3.05–3.13 (m, CH2(5’)); 2.63–
2.69 (m, HaꢀC(2’)); 2.34–2.40 (m, HbꢀC(2’)). 13C-NMR (100 MHz, (D6)DMSO): 158.0; 143.8; 140.7;
129.7; 129.6; 127.7; 127.6; 127.0; 126.5; 125.1; 120.0; 113.1; 87.3; 86.0; 70.5; 86.0; 70.5; 65.5; 55.0; 46.7; 40.2;
40.0; 39.8; 39.6; 39.3; 39.1; 38.9. Anal. calc. for C44H42N4O7: C 71.53, H 5.73, N 7.58; found: C71.60, H 5.63,
N 7.57.
1-(5-O-[Bis(4-methylphenyl)(phenyl)methyl]-3-O-{(2-cyanoethoxy)[di(propan-2-yl)amino]phos-
phinyl}-2-deoxy-b-d-ribofuranosyl)-4-({[(9H-fluoren-9-yl)methoxycarbonyl]amino}methyl)-1H-1,2,3-
triazole (6). Compound 6 was synthesized from 5 (0.44 g, mmol) according to the method described in
[7]. Yield 0.44 g (79%); mixture of diastereomers. Rf (B) 0.7. 1H-NMR (400 MHz, (D6)DMSO); selected
signals): 8.03 (d, J¼2.8, HꢀC(5)); 7.87 (d, J¼7.6, HꢀC(4), HꢀC(5) of Fmoc); 7.67 (d, J¼7.6, HꢀC(1),
HꢀC(8) of Fmoc); 7.76–7.81, 7.18–7.42 (m, 5 arom. H of Ph, 4 arom. o-H of DMTr, HꢀC(2), HꢀC(3),
HꢀC(6), HꢀC(7) of Fmoc); 6.81–6.85 (m, 4 arom. m-H of DMTr); 6.39; 6.41 (d, J¼5.6, HꢀC(1’)); 3.71,
3.72 (s, 2 MeO). 13C-NMR (100 MHz, (D6)DMSO): 158.0; 144.5; 143.8; 140.6; 135.4; 129.6; 127.7; 127.6;
127.5; 126.9; 126.5; 125.1; 122.1; 120.0; 113.0; 87.2; 85.5; 65.4; 54.9; 46.6; 42.6; 42.5; 40.1; 39.9; 39.7; 39.5;
39.3; 39.1; 38.9; 24.2; 22.5; 19.7. 31P-NMR (162 MHz, (D6)DMSO): 150.7; 150.2. Anal. calc. for
C53H59N6O8P: C 67.79, H 6.33, N 8.95; found: C67.88, H 6.25, N 8.87.
Oligodeoxynucleotides were synthesized with an ABI 3400 DNA Synthesizer (Applied Biosystems,
Forster City, CA, USA) according to standard manufacturer protocols. Purification of oligodeoxynu-
cleotides was performed using a Hypersil ODS column (5 mm; 4.6 mmꢁ250 mm), 0.05m TEAA
(pH 7.0), and a linear gradient of MeCN (10–50%, 30 min for DMTr-protected oligodeoxynucleotides,
and 0–25%, 30 min for fully deblocked oligodeoxynucleotides). The flow rate was 1 ml/min. Removal of
the 5’-O-DMTr group was achieved by treatment with 2% aq. CF3COOH for 1 min, followed by Et3N
neutralization and precipitation with 2% LiClO4 in acetone.

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CHEMISTRY & BIODIVERSITY – Vol. 8 (2011)
Absorbance vs. temp. profiles were determined with a Shimadzu UV160A spectrophotometer
equipped with a H2O-jacketed cell holder. Dry N2 was flushed through the cuvette chamber to prevent
moisture condensation at low temp. Melting experiments were carried out at 260 nm and with the
oligonucleotide concentration at 1 mm. The solns. were heated at a rate of 0.28/min. Tm Values were
calculated from DH and DS values. Thermodynamic parameters were obtained by fitting of experimental
curves using two-state intramolecular model with sloping base lines.
Labelling of aminooligonucleotides ON1 and ON2 with NHS esters of sulfonated fluorescent dyes
(SCy3 and SCy5) [11] was carried out in 0.1 m NaHCO3/Na2CO3 buffer (pH 9.5) at 48 for 16 h. HPLC
Purification of labelled oligonucleotides was carried out as mentioned above with a linear gradient of
MeCN (10–50%, 30 min). MALDI-MS Analysis: SCy3-ON1: m/z 6027.5; calc. 6020.3; SCy5-ON1: m/z
6049.3; calc. 6046.3.
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Received February 11, 2010