The spontaneous rearrangement of 2,4-dinitrophenyl substituent in ribonucleosides under neutral conditions

Article abstract of DOI:10.1080/15257770.2010.507233

In the cytidine and adenosine derivatives an isomerization of a 2,4-dinitrophenyl group between the 2'-and 3'-positions of the ribose was observed under neutral conditions. Moreover, it was shown that isomerization of the 2,4-dinitrophenyl group in condit

Full text of DOI:10.1080/15257770.2010.507233

Nucleosides, Nucleotides and Nucleic Acids, 29:684–697, 2010
Copyright ꢀC Taylor and Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770.2010.507233
THE SPONTANEOUS REARRANGEMENT
OF 2,4-DINITROPHENYL SUBSTITUENT IN RIBONUCLEOSIDES
UNDER NEUTRAL CONDITIONS
Anna Olejnik, Zofia Gdaniec, Elzbieta Kierzek, and Ryszard Kierzek
Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland
In the cytidine and adenosine derivatives an isomerization of a 2,4-dinitrophenyl group be-
2
tween the 2ꢀ- and 3ꢀ-positions of the ribose was observed under neutral conditions. Moreover, it was
shown that isomerization of the 2,4-dinitrophenyl group in conditions required to synthesize phos-
phoramidites and lability in aqueous ammonia make chemical synthesis of 2ꢀ-O-(2,4-dinitrophenyl)
oligonucleotides impossible.
Keywords 2,4-Dinitrophenyl; isomeration; ribonucleosides; Smiles rearrangement
INTRODUCTION
The 2,4-dinitrophenyl (DNP) group in RNA exhibits very interest-
ing biological properties. Recently, it was reported that siRNA, contain-
ing several 2ꢀ-O-(2,4-dinitrophenyl) groups, reveals enhanced efficiency
for inhibiting cancer cell growth and can easily diffuse into cells and
remain there for several days without detectable degradation.[1,2] It was
also proven that poly-2ꢀ-O-(2,4-dinitrophenyl)-RNA was not hydrolyzed by
RNase H and its presence increased membrane permeability.[3,4] The ap-
propriate 2ꢀ-O-(2,4-dinitrophenyl)-RNA was obtained in the reaction of 2,4-
dinitrofluorobenzene with RNA, and 2,4-dinitrophenyl substituents were in-
troduced randomly. These unique properties of the 2,4-dinitrophenyl group
were the reason of our interest in the synthesis of 5ꢀ-O-dimethoxytrityl-2ꢀ-O-
(2,4-dinitrophenyl)-N-acylribonucleoside-3ꢀ-O-phosphoramidites providing
the opportunity to synthesize oligoribonucleotides with 2,4-dinitrophenyl
modifications at definite positions.
Received 8 June 2010; accepted 1 July 2010.
This work was supported by the Ministry of Science and Higher Education, grants PBZ-MNiSW-
07/I/2007 (R. K.) and N N301 3383 33 (E. K.). R. K. is the recipient of a fellowship of the Foundation
for Polish Science.
Address correspondence to Ryszard Kierzek, Institute of Bioorganic Chemistry, Polish Academy of
Sciences, Noskowskiego 12/14, Poznan 61-704, Poland. E-mail: rkierzek@ibch.poznan.pl
684

Rearrangement of 2,4-Dinitrophenyl Substituent
RESULTS AND DISCUSSION
685
The increasing demand for synthetic ribonucleotides has stimulated a
search for effective routes to 2ꢀ-O-substituted ribonucleotides.[5,6] In the
sythesis of 3ꢀ-O-phosphoramidites building blocks, the di-tert-butylsilylene
group was used to simultaneously protect the 3ꢀ- and 5ꢀ-hydroxyls of the N-
acylated nucleosides. This protection step was followed by treatment with
2,4-dinitrofluorobenzene.[7] Next, the di-tert-butylsilylene protecting group
was removed from derivative 3 with fluoride, yielding derivatives 4 and 5,
which were subsequently treated with dimethoxytrityl chloride to get the
appropriately protected derivatives 6 and 7. The final products of the syn-
thesis appeared on thin layer chromotography (TLC) as an equimolar mix-
ture of two nucleoside derivatives 6 and 7 (Scheme 1A). In the case of
the cytidine derivative, both products were separated by silica gel column
chromatography.
SCHEME 1 The chemical synthesis of 5ꢀ-O-dimethoxytrityl-2ꢀ-O-(2,4-dinitrophenyl)-N-acylribonuc
leosides (A) and 5ꢀ-O-dimethoxytrityl-2ꢀ-O-(2,4-dinitrophenyl)-4N-acetylarabinocytidine (B).

686
A. Olejnik et al.
TABLE 1 The 1H and 13C NMR chemical shift of the sugar residues of the 2,4-dinitrophenyl derivatives
of cytdidine and arabinocytidine
1H NMR [ppm]
13C NMR [ppm]
Cytidine derivative
3ꢀ-O-DNP (7A) 2ꢀ-O-DNP (6A)
Solvent
CDCl3
Position
2ꢀ-O-DNP (6A)
3ꢀ-O-DNP (7A)
1ꢀ
2ꢀ
6.07
5.18
4.73
4.30
—
3.33
5.75
4.58
5.50
4.12
—
5.91
4.65
4.79
4.61
3.47
—
88.44
84.43
68.28
82.61
—
92.73
76.20
79.81
83.14
—
3ꢀ
4ꢀ
2ꢀ-OH
3ꢀ-OH
1ꢀ
—
—
DMSO-d6
5.86
4.65
5.18
4.37
6.02
—
89.28
82.31
67.72
81.58
—
92.73
72.54
76.20
79.81
—
2ꢀ
3ꢀ
4ꢀ
2ꢀ-OH
3ꢀ-OH
5.97
—
—
Arabinocytidine derivative
2ꢀ-O-DNP
3ꢀ-O-DNP
2ꢀ-O-DNP
3ꢀ-O-DNP
DMSO-d6
1ꢀ
2ꢀ
6.36
5.37
4.31
4.11
6.22
—
—
—
—
—
85.64
82.77
73.21
82.29
—
—
—
—
—
—
3ꢀ
4ꢀ
3ꢀ-OH
The structures of the products were determined by one-dimensional nu-
clear magnetic resonance (NMR) (1H and 13C NMR) and two-dimensional
NMR (1H-1H COSY and 1H-13C HSQC) spectroscopy. In the case of the cyti-
dine derivative, the spectroscopic analysis proved that a mixture of isomers
1
was obtained as products of the synthesis. The H-1H COSY spectra were
helpful in identifying and distinguishing the signals from both isomers. The
spectrum of the first isomer 6 proved that the 2,4-dinitrophenyl substituent
was located at the 2ꢀ-position because a correlation between the H-3ꢀ and the
3ꢀ-hydroxyl was observed. In the case of the second isomer 7, the H-2ꢀ coupled
with the 2ꢀ-hydroxyl which proved that the 2,4-dinitrophenyl was located at
the 3ꢀ-position. Table 1 presents 1H and 13C NMR chemical shifts of the sugar
residues of the 2,4-dinitrophenyl derivatives of cytidine, arabinocytidine and
adenosine.
Isomer 6A, with the 2,4-dinitrophenyl group located at the 2ꢀ-position of
the ribose ring, migrates faster on TLC. Surprisingly, the signal characteristic
of both isomers were also observed in the NMR spectra when anhydrous
DMSO-d6 was used as the solvent even when the TLC pure isomers 6A and
7A were used for the analysis. In contrast, in CDCl3 solution, only signals

Rearrangement of 2,4-Dinitrophenyl Substituent
687
Ac
Cyt
Ac
Cyt
Ac
Cyt
DMTrO
DMTrO
DMTrO
O
O
O
H+
_
O
O
O
OH
O
OH
O2N
NO
NO
2
NO2
NO
2
2
NO2
SCHEME 2 The proposed Smiles type rearrangement of 2,4-dinitrophenyl substituted ribonucleosides.
from one isomer were observed. These experiments suggested that the 2,4-
dinitrophenyl group migrated from the 2ꢀ-position to the 3ꢀ-position of ribose
and vice versa under neutral conditions during the NMR experiment in
DMSO-d6 (Scheme 2). The isomerizations of the 2,4-dinitrophenyl group in
the derivatives 6A and 7A were also confirmed by TLC analyses. Interestingly,
the NMR spectra of the derivatives 2A and 3A showed the presence of one
product only. However, after deprotection of the silyl group from derivative
3A, the signals characteristic of the presence of a mixture of the 2ꢀ- and
3ꢀ-isomers of 2,4-dinitrophenyl appeared in the NMR spectra. This suggests
that the presence of the vicinal 3ꢀ-hydroxyl was sufficient to initiate the 2,4-
dinitrophenyl isomerization.
The mechanism of the migration of the 2,4-dinitrophenyl group in the
ribonucleosides has not been described in the literature. However, the
migration of the 4-nitrophenyl group of glucopyranose in alkaline solu-
tions was reported.[8] The migration of this group was explained to be an
intramolecular nucleophilic aromatic substitution. Moreover, Klemer and
Kleefeldt observed the migration of the 2,4-dinitrophenyl group in the glu-
cose ring under mild alkaline conditions.[9] According to the authors, the
2,4-dinitrophenyl group migrated particularly easily when it was located at
the C-2ꢀ position of glucose. In that case, the rearrangement to the C-3ꢀ
position was observed even after dissolution in acetone.
Isomerization of some protecting groups in ribonucleosides is mostly
limited to acyl and (trifluoromethyl)sulfonyl esters.[10,11] Moreover, the
isomerization in ribonucleosides concerns silyl ethers, including tert-
butyldimethylsilyl.[12] Also extrimely fast migration of phosphotriester link-
ages in the oligoribonucleotides was observed.[13–15] Additionally, in the
literature isomerization of the 2,4,6-trinitrophenyl group between 2ꢀ- and
3ꢀ-hydroxyl of 5ꢀ-O-triphosphate adenosine was reported and this derivative
was used as a fluorescent analog to study ATP binding to heavy meromyosin
ATPase.[16] Based on spectrospocic data demonstrating the formation of

688
A. Olejnik et al.
Meisenheimer complex, the existence of 5ꢀ-O-triphosphate 2ꢀ,3ꢀ-O-(2,4,6-
trinitrocyclohexadienylidene) adenosine at neutral and basic conditions was
postulated.[17,18]
Migration of the 2,4-dinitrophenyl was also reported for aminoal-
cohols. Skarzewski and Skrowaczewska observed migration of the 2,4-
dinitrophenyl in aminoethanol.[19] The primary product of the synthe-
sis, the 2,4-dinitrophenyl ether of aminoethanol, rearranged to 2-(2,4-
dinitrophenylamino)-ethanol in the presence of strong base. A similar
rearrangement was reported when 2-(acetylamino)ethanol was treated with
2,4-dinitrofluorobenzene in the presence of sodium methoxide or potassium
tert-butoxide.[20]
One of the aims of our studies was to elucidate the factors influencing
the isomerization of the 2,4-dinitrophenyl substituent in ribonucleotides.
The assumption was that traces of water in DMSO-d6 might cause the mi-
gration of the 2,4-dinitrophenyl group. Therefore an NMR analysis of the
purified isomers 6A and 7A in DMSO-d6 with 0.1% and 1% water (v/v) was
carried out. After 48 hours the isomerization of the 2ꢀ-O-(2,4-dinitrophenyl)
derivative 6A results in equimolar quantities of both isomers, independent
of the amount of water. The half life times of the isomerization of the 2ꢀ-
O-(2,4-dinitrophenyl) derivative 6A were 1.9 and 1.6 hours with 0.1% and
1% water in DMSO-d6, respectively. However, isomerization of the 3ꢀ-O-(2,4-
dinitrophenyl) derivative 7A was much slower. After 48 hours only ca. 20%
of the 2ꢀ-isomer was observed, independent of the amount of water. In this
case the half lives of the isomerization of 7A were 6.6 and 4.9 hours with
0.1% and 1% water in DMSO-d6, respectively.
To check whether the 2ꢀ-O-(2,4-dinitrophenyl) nucleosides can be used
to synthesize the phosphoramidites and oligonucleotides, the stabilities of
both 6A and 7A isomers in acetonitrile tetrazole solution were also analyzed.
We observed isomerization of both compounds. This process was faster (t1/2
ca. 0.9 hours) than the previously described isomerization in DMSO-d6.
The isomerization were more favorable for the 3ꢀ-isomer. Moreover, it was
observed that the 2,4-dinitrophenyl group was quantitatively removed under
conditions commonly used for the deprotection of oligoribonucleosides
(aqueous ammonia, 16 hours at 55◦C).[21]
The most probable mechanism for the isomerization is a Smiles type of
rearrangement in which the aromatic ring migrates between the O-2ꢀ and
the O-3ꢀ atoms (Scheme 2).[22] The Smiles rearrangement is facilitated by
the activation of the aromatic ring via the electron-withdrawing nitro groups
in the ortho and para positions. We thought that the same rearrangement
could be possible for the compounds described herein. For the ribose ring
in the nucleoside, in both the 2ꢀ- and 3ꢀ-endo conformers, the distance
ꢀ
ꢀ
˚
between the 2 - and the 3 -oxygen is short enough (ca. 2.8 A) to allow the
2,4-dinitrophenyl moiety to migrate in a reversible way.

Rearrangement of 2,4-Dinitrophenyl Substituent
689
In order to check whether the isomerization of the 2,4-dinitrophenyl
group also takes place in other nucleosides, the synthesis of the adenosine
derivative was carried out under the same conditions as described for the
cytidine derivative (Scheme 1). Two isomers of the adenosine derivatives 6B
and 7B were obtained and identified by TLC and by 1H and 13C NMR (Table
1). Unfortunately, the separation of both isomers by column chromatogra-
phy was impossible, and a detailed analysis of the isomerization could not be
performed.
In order to gain insight into the migration of the 2,4-dinitrophenyl
moiety in nucleosides, the synthesis of 5ꢀ-O-dimethoxytrityl-2ꢀ-O-(2,4-
dinitrophenyl)-N4-acetylarabinocytidine was performed (Scheme 1B). The
TLC analysis of the final derivative 13 showed the presence of one product
only, and the analysis of the 1H-1H COSY and 1H-13C HSQC (in DMSO-d6
and CDCl3) spectra proved that the 2,4-dinitrophenyl group was located at
the 2ꢀ-position of the arabinocytidine derivative (Table 1). However, even
though there was an hydroxyl group at the 3ꢀ-position (the distance between
ꢀ
ꢀ
˚
the 2 - and the 3 - oxygen is 3.4–4.0 A), migration of the substituent was not
observed.
EXPERIMENTAL
General Information
Thin-layer chromatography were carried out on Merck 60 F254 or
silanized (RF) TLC plates with variuos mitxures of dichloromethane and
methanol (95:5 v/v (A) and 9:1 v/v (B)) or acetone and water (7:3 v/v (C)).
Nuclear magnetic resonance spectra were recorded in DMSO-d6 and CDCl3
on Bruker Avance spectrometer (1H at 400 MHz, 13C at 100 MHz) and val-
ues are given in parts per milion (δ) downfiled from TMS. All reagents and
solvents were purchased from commercial sources (Aldrich, Munich, Ger-
many; Fluka, Buchs, Switzerland; Merck, Darmstadt, Germany) and were
used without futher purification.
The Synthesis of 3ꢁ,5ꢁ-O-di-tert-butylsilyl-4N-acetylcytidine (2A)
To a solution of 4N-acetylcytidine (2.88 g, 10 mmol) in 100 ml of N,N-
dimethylformamide 2,6-lutidine (3.48 ml, 30 mmol) was added and cooled to
0◦C. To the stirring solution of di-tert-butylsilyl ditriflate (3.89 ml, 12 mmol)
was added dropwise and warmed up to room temperature. The reaction
progress was monitored by TLC. After 2 hours, the reaction was completed
and half of the solvent was evaporated under reduced pressure. To the reac-
tion mixture saturated aqueous solution of sodium bicarbonate was added
extracted three times with dichloromethane. The combined organic layers

690
A. Olejnik et al.
were dried over anhydrous sodium sulfate, filtered off, and concentrated. Pu-
rification was accomplished with silica gel column chromatography (0–4%
methanol in dichloromethane). Yield: 67% (2.94 g), Rf 0.33 (A).
1H NMR (DMSO-d6) δ 10.90 (s, 1H, NH); 8.01 (d, 1H, H-5); 7.23 (d, 1H,
H-6); 5.74 (d, 1H, OH); 5.68 (s, 1H, H-1ꢀ); 4.40–4.36 (m, 1H, H-2ꢀ); 4.17–4.14
(m, 1H, H-3ꢀ); 4.10–4.12 (d, 1H, H-4ꢀ); 4.05–3.94 (m, 2H, H-5ꢀ, H-5ꢀꢀ); 2.09
(s, 3H, CH3); 1.02 (s, 18H, CH3). 13C NMR (DMSO-d6) δ 171.07 (C O);
162.50, 154.25, 145.58, 95.69 (C-Cyt); 93.14 (C-1ꢀ); 75.46 (C-3ꢀ); 74.01 (C-2ꢀ);
72.82 (C-4ꢀ); 66.66 (C-5ꢀ); 27.31–27.00 (tBu2Si); 24.38 (CH3).
The Synthesis of 2ꢁ-O-(2,4-dinitrophenyl)-3ꢁ-5ꢁ-O-di-tert-butylsilyl-4N-
acetylcytidine (3A)
The solution of 3ꢀ,5ꢀ-O-di-tert-butylsilyl-4N-acetylcytidine (2.94 g, 6.72
mmol) in 30 ml of N,N-dimethylformamide was cooled to -30◦C and then
40% mineral oil emulsion of sodium hydride (0.62 g, 15.12 mmol) and 2,4-
dinitrofluorobenzene (2.53 ml, 20.16 mmol) were added. After completion
of the reaction (3 hours), saturated aqueous solution of sodium bicarbonate
was added and extracted three times with dichloromethane. The combined
organic layers were dried over anhydrous sodium sulfate, filtered off, and
concentrated in vacuum. The product was purified by silica gel column
chromatography (0–3% methanol in dichloromethane) to afford pure com-
pound. Yield: 51% (2.06 g). Rf 0.44 (A).
1H NMR (DMSO-d6) δ 10.98 (s, 1H, NH); 8.80 (d, 1H, H-DNP); 8.50–8.52
(dd, 1H, H-DNP); 8.13 (d, 1H, H-DNP); 7.77 (d, 1H, H-5); 7.27 (d, 1H, H-
6); 5.74 (d, 1H, OH); 5.68 (s, 1H, H-1ꢀ); 4.40–4.36 (m, 1H, H-2ꢀ); 4.23–4.18
(t, 1H, H-3ꢀ); 4.20–4.12 (d, 1H, H-4ꢀ); 4.05–3.94 (m, 2H, H-5ꢀ, H-5ꢀ’); 2.11
(s, 3H, CH3); 1.04 (s, 18H, CH3). 13C NMR (DMSO-d6) δ 171.19 (C O);
162.90, 154.92 (C-Cyt); 154.23 (C-DNP); 145.94 (C-Cyt.); 140.01, 138.46,
128.40, 121.00, 117.62 (C-DNP); 96.00 (C-Cyt); 90.97 (C-1ꢀ); 79.55 (C-3ꢀ);
75.02 (C-2ꢀ); 74.52 (C-4ꢀ); 66.44 (C-5ꢀ); 27.66–26.38 (tBu2Si); 24.41 (CH3).
The Synthesis of 2ꢁ-O-(2,4-dinitrophenyl)-4N-acetylcytidine (4A, 5A)
To
2ꢀ-O-(2,4-dinitrophenyl)-3ꢀ,5ꢀ-O-di-tert-butylsilyl-4N-acetyl-cytidine
(2.06 g, 3.40 mmol) 1 molar solution of triethylamine hydrofluoride (10.20
ml, 10.20 mmol) in pyridine was added. The reaction mixture was stirred
at room temperature for 2 hours and the solution saturated aqueous
sodium bicarbonate was added, and the aqueous layer was extracted three
times with dichloromethane. The combined organic layers were dried
over anhydrous sodium sulfate, filtered off, and concentrated. The crude
product was purified on silica gel column chromatography (0–4% methanol
in dichloromethane). Yield: 30% (0.44 g). Rf 0.14 (A).
1H NMR (DMSO-d6) δ 10.92 (s, 1H, NH); 8.76 (d, 1H, H-DNP); 8.52–8.48
(d, 1H, H-DNP); 8.45–8.43 (d, 1H, H-DNP); 7.81 (d, 1H, H-5); 7.24–7.21

Rearrangement of 2,4-Dinitrophenyl Substituent
691
(d, 1H, H-6); 5.92 (d, 1H, H-1ꢀ); 5.36 (t, 1H, H-3ꢀ); 4.61–4.58 (d, 1H, H-2ꢀ);
4.29–4.26 (m, 1H, H-4ꢀ); 3.82–3.66 (m, 2H, H-5ꢀ, H-5ꢀ’); 2.11 (s, 3H, CH3). 13C
NMR (DMSO-d6) δ 171.16 (C O); 162.65, 154.86 (C-Cyt); 145.57 (C-Cyt.);
140.01, 138.88, 128.88, 123.97, 121.24, 117.71 (C-DNP); 95.78 (C-Cyt); 90.19
(C-1ꢀ); 82.50 (C-3ꢀ); 77.93 (C-2ꢀ); 72.54 (C-4ꢀ); 67.00 (C-5ꢀ); 24.42 (CH3).
The Synthesis of 5ꢁ-O-dimethoxytrityl-2ꢁ-O-(2,4-dinitro-phenyl)-4N-
acetylcytidine (6A, 7A)
To a solution of 2ꢀ-O-(2,4-dinitrophenyl)-4N-acetycytidine (0.44 g, 0.95
mmol) in 6.30 ml of pyridine the dimethoxytrityl chloride (0.37 g, 1.09
mmol) was added. The reaction was carried out at room temperature. After
3 hours the reaction was completed and to the reaction mixture saturated so-
lution of aqueous sodium bicarbonate was added and extracted three times
with dichloromethane. The combined organic layers were dried over an-
hydrous sodium sulfate, filtered off, and the solvent was evaporated under
vacuum. The crude product was purified by silica gel column chromatogra-
phy (0–3% methanol in dichloromethane). Yield: 62% (0.45 g). Rf isomer
6A 0.35 (A), Rf isomer 7A 0.30 (A).
Isomer 6A. 1H NMR (CDCl3) δ 8.82 (s, 1H, NH); 8.75 (d, 1H, H-
DNP); 8.63 (d, 1H, H-DNP); 8.53–8.51 (dd, 1H, H-DNP); 7.45 (d, 1H, H-5);
7.38–7.28 (m, 10H, H-DMTr); 7.13 (d, 1H, H-6); 6.90 (d, 3H, H-DMTr);
6.07 (s, 1H, H-1ꢀ); 5.18 (d, 1H, H-2ꢀ); 4.73 (m, 1H, H-3ꢀ); 4.30 (d, 1H, H-4ꢀ);
3.75–3.66 (m, 2H, H-5ꢀ, H-5ꢀꢀ); 3.84 (s, 6H, OCH3); 3.33 (s, 1H, OH-3ꢀ); 2.24
(s, 3H, CH3). 13C NMR (CDCl3) δ 169.66 (C O); 162.74, 155.48, 145.09
(C-Cyt); 143.96, 141.52, 138.75 (C-DNP); 135.19, 134.91, 130.19, 130.10 (C-
DMTr); 128.16, (C-DNP); 127.30 (C-DMTr); 121.11, 118.94 (C-DNP); 113.39
(C-DMTr); 97.09 (C-Cyt); 88.44 (C-1ꢀ); 87.47 (C-DMTr); 84.43 (C-2ꢀ); 82.61
(C-4ꢀ); 68.28 (C-3ꢀ); 60.11 (C-5ꢀ); 24.98 (CH3).
Isomer 6A. 1H NMR (DMSO-d6) δ 10.96 (s, 1H, NH); 8.76 (d, 1H, H-
DNP); 8.52–8.49 (dd, 1H, H-DNP); 8.33–8.30 (d, 1H, H-DNP); 7.84–7.79 (d,
1H, H-5); 7.41–7.12 (m, 9H, H-DMTr); 7.06–7.05 (d, 1H, H-6); 6.91–6.79 (m,
4H, DMTr); 5.97 (s, 1H, OH-3ꢀ); 5.75 (d, 1H, H-1ꢀ); 5.50 (d, 1H, H-3ꢀ); 4.58
(m, 1H, H-2ꢀ); 4.12 (m, 1H, H-4ꢀ); 3.72 (s, 6H, OCH3); 3.40–3.39 (m, 2H, H-
5ꢀ, H-5ꢀꢀ); 2.11 (s, 3H, CH3). 13C NMR (DMSO-d6) δ 171.12 (C O); 162.69,
158.24, 155.71, 144.89 (C-Cyt); 140.76, 138.88, 135.40 (C-DNP); 135.13,
129.84, 128.78, 128.86 (C-DMTr); 128.00 (C-DNP); 127.79, 127.65, 125.95,
129.84 (C-DMTr); 121.31, 117.75 (C-DNP); 113.33 (C-DMTr); 95.58 (C-Cyt);
89.28 (C-1ꢀ); 86.00 (C-DMTr); 82.31 (C-2ꢀ); 81.58 (C-4ꢀ); 67.72 (C-3ꢀ); 62.15
(C-5ꢀ); 24.42 (CH3).
Isomer 7A. 1H NMR (CDCl3) δ 9.09 (s, 1H, NH); 8.72 (s, 1H, H-DNP);
8.27–8.23 (dd, 1H, DNP); 8.20 (d, 1H, H-DNP); 7.41–7.39 (d, 1H, H-5);
7.33–7.20 (m, 13H, H-DMTr); 6.82 (d, 1H, H-6); 5.91 (s, 1H, H-1ꢀ); 4.79 (t,
1H, H-3ꢀ); 4.65 (t, 1H, H-2ꢀ); 4.61 (d, 1H, H-4ꢀ); 3.82–3.76 (s, 6H, OCH3);
3.75–3.72 (m, 2H, H-5ꢀ, H-5ꢀꢀ); 3.47 (s, 1H, OH-2ꢀ); 2.25 (s, 3H, CH3).

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A. Olejnik et al.
13C NMR (CDCl3) δ 170.129 (C O); 162.66 (C-Cyt); 158.81, 156.47 (C-
DMTr); 155.87, 144.44 (C-Cyt); 143.85 (C-DMTr); 140.63, 138.93 (C-DNP);
134.93, 134.69 (C-DMTr); 129.96 (C-DNP); 128.69, 128.13, 127.86, 127.30
(C-DMTr); 121.78, 116.88 (C-DNP); 113.48 (C-DMTr); 97.02 (C-Cyt); 92.73
(C-1ꢀ); 83.14 (C-4ꢀ); 79.81 (C-3ꢀ); 76.20 (C-2ꢀ); 72.54; 62.13 (C-5ꢀ); 25.00
(CH3).
Isomer 7A. 1H NMR (DMSO-d6) δ 10.95 (s, 1H, NH); 8.77 (d, 1H, H-
DNP); 8.52–8.48 (dd, 1H, H-DNP); 8.33–8.29 (d, 1H, H-5); 7.83–7.79 (d, 1H,
H-DNP); 7.41–7.14 (m, 9H, H-DMTr); 7.07–7.04 (d, 1H, H-6); 6.91–6.79 (m,
4H, H-DMTr); 6.02 (d, 1H, OH-2ꢀ); 5.86 (d, 1H, H-1ꢀ); 5.18 (t, 1H, H-3ꢀ); 4.65
(m, 1H, H-2ꢀ); 4.37 (m, 1H, H-4ꢀ); 3.70 (s, 6H, OCH3); 3.55–3.52 (m, 2H, H-
5ꢀ, H-5ꢀ’); 2.12 (s, 3H, CH3). 13C NMR (DMSO-d6) δ 170.15, 171.11 (C O);
162.69, 162.59, 154.94, (C-Cyt); 154.35 (C-DNP); 145.28 (C-Cyt); 140.15,
140.07, 138.87, 138.64 (C-DNP); 143.85 (C-DMTr); 140.63, 138.93 (C-DNP);
134.93, 134.69 (C-DMTr) 129.96 (C-DNP); 128.69, 128.13, 127.86, 127.30
(C-DMTr); 121.78 116.88 (C-DNP); 113.48 (C-DMTr); 97.02 (C-Cyt); 92.73
(C-1ꢀ); 79.81 (C-4ꢀ); 76.20 (C-3ꢀ); 72.54 (C-2ꢀ); 65.71 (C-5ꢀ); 24.41 (CH3).
The Synthesis of 3ꢁ,5ꢁ-O-di-tert-butylsilyl-6N-benzoyladenosine (2B)
To a solution of 6N-benzoyladenosine (1.48 g, 4 mmol) in 40 ml of N,N-
dimethylformamide 2,6-lutidine (1.39 ml, 12 mmol) was added and cooled
to 0◦C. To the stirring solution of the di-tert-butylsilyl ditriflate (1.39 ml,
5.6 mmol) was added dropwise and the solution was warmed up to room
temperature. The reaction progress was monitored by TLC. After 2 hours
the reaction was completed and half of the solvent was removed at reduced
pressure. To the reaction mixture saturated aqueous solution of sodium
bicarbonate was added and extracted three times with dichloromethane.
The combined organic layers were dried over anhydrous sodium sulfate,
filtered off, and concentrated. Purification was accomplished with silica gel
column chromatography (0–3% methanol in dichloromethane). Rf 0.45 (A).
1H NMR (DMSO-d6) δ 11.20 (s, 1H, NH); 8.73 (s, 1H, H-2); 8.63 (s,
1H, H-8); 8.04–7.43 (m, 5H, H-arom.); 6.06 (s, 1H, OH); 5.89 (d, 1H, H-1ꢀ);
4.71–4.68 (t, 1H, H-3ꢀ); 4.61–4.59 (t, 1H, H-2ꢀ); 4.38–4.35 (m, 1H, H-4ꢀ);
4.07–3.99 (m, 2H, H-5ꢀ,H-5ꢀꢀ); 1.08 (s, 18H, CH3). 13C NMR (DMSO-d6) δ
162.48 (C O); 151.81, 143.55, 133.37 (C-Ade); 129.34–128.55 (C-arom.);
125.87, 120.14 (C-Ade); 90.64, 75.90, 74.13, 72.91, 66.99 (C-rib.); 27.71,
27.38, 27.09 (C-CH3).
The Synthesis of 2ꢁ-O-(2,4-dinitrophenyl)-3ꢁ,5ꢁ-O-di-tert-butylsilyl-6N-
benzoyladenosine (3B)
The solution of 3ꢀ,5ꢀ-O-di-tert-butylsilyl-6N-benzoyladenosine (1.48 g,
4 mmol) in 20 ml of N,N-dimethylformamide was cooled to −30◦C and

Rearrangement of 2,4-Dinitrophenyl Substituent
693
40% mineral oil emulsion of sodium hydride (0.37 g, 9 mmol) and 2,4-
dinitrofluorobenzene (1.50 ml, 12 mmol) were added. After 3 hours the
reaction was completed and saturated aqueous solution of sodium bicar-
bonate was added and extracted three times with dichloromethane. Then
combined organic layers were dried over anhydrous sodium sulfate, filtered
off, and concentrated in vacuum. The product was purified by silica gel col-
umn chromatography (0–3.5% methanol in dichloromethane). Yield: 56%
(1.53 g). Rf 0.13 (C).
1H NMR (DMSO-d6) δ 8.97 (s, 1H, NH); 8.84–8.79 (m, 2H, H-2; H-8);
8.59–8.25 (m, 3H, H-DNP); 7.94–7.38 (m, 5H, arom.); 6.35 (d, H1, OH);
6.04–5.99 (dd, H1, H-1ꢀ); 5.15–5.12 (t, 1H, H-3ꢀ); 4.95–4.92 (t, 1H, H-2ꢀ);
4.50–4.43 (m, 1H, H-4ꢀ); 4.15–4.00 (m, 2H, H-5ꢀ, H-5ꢀꢀ); 1.06–1.00 (m, 18H,
CH3). 13C NMR (DMSO-d6) δ 175.76 (C O); 162.45, 154.75, 148.14, 143.91
(C-Ade); 140.40–132.35 (C-DNP); 129.91–128.45 (C-arom.); 117.86 (C-Ade);
87.96, 80.00, 75.43, 75.16, 66.66 (C-ryb.); 27.43 (C-CH3).
The Synthesis of 2ꢁ-O-(2,4-dinitrophenyl)-6N-benzoyl-adenosine (4B, 5B)
To 2ꢀ - O - (2,4 - dinitrophenyl) - 3ꢀ,5ꢀ - O - di - tert - butylsilyl - 6N - benzoyl-
adenosine (1.52 g, 2.25 mmol) 1 molar solution of triethylamine hydroflu-
oride (6.75 ml, 6.75 mmol) in pyridine was added. The reaction mixture
was stirred at room temperature for 2 hours. Next the saturated aqueous
solution of sodium bicarbonate was added and extracted three times with
dichloromethane. The combined organic layers were dried over anhydrous
sodium sulfate, filtered off, and concentrated to obtain crude product. Rf
0.58 (B).
1H NMR (CDCl3) δ 9.00 (d, 1H, NH); 8.38–8.34 (m, 2H, H-2; H-8); 8.02,
7.71–7.68 (m, 3H, H-DNP); 7.49–7.24 (m, 5H, H-arom.); 5.83 (d, 1H, H-1ꢀ);
4.91–4.87 (t, 1H, H-2ꢀ); 4.40 (d, 1H, H-3ꢀ); 3.93 (m, 1H, H-4ꢀ); 3.76–3.72
(m, 2H, H-5ꢀ, H-5ꢀꢀ); 3.01 (s, 1H, OH). 13C NMR (CDCl3) δ 170.54 (C O);
152.31, 151.78 (C-Ade); 151.78 (C-DNP); 146.30, 144.39 (C-Ade); 140.39,
133.62, 132.80, 132.30 (C-DNP); 128.28 (C-DNP); 129.91–127.28 (C-arom.);
121.86 (C-Ade); 91.34, 87.63, 72.20, 62.97, 53.41 (C-rib.).
The Synthesis of 5ꢁ-O-dimethoxytrityl-2ꢁ-O-(2,4-dinitro-phenyl)-6N-
benzoyladenosine (6B, 7B)
To a solution of 2ꢀ-O-(2,4-dinitrophenyl)-6N-benzoyladenosine (1.52 g,
2.25 mmol) in 10.2 ml of pirydine the dimethoxytrityl chloride (0.84 g,
2.47 mmol) was added. The reaction was carried out at room tempera-
ture. After 3 hours the reaction was completed, then to the mixture sat-
urated aqueous solution of sodium bicarbonate was added and extracted
with dichloromethane. The combined organic layers were dried over an-
hydrous sodium sulfate, filtered off, and the solvent was evaporated under

694
A. Olejnik et al.
vacuum. The crude product was purified by silica gel column chromatogra-
phy (0–3.5% methanol in dichloromethane). Yield: 61% (1.15 g). Rf 0.38
(A).
1H NMR (DMSO-d6) δ 9.00–8.99 (dd, 1H, NH); 8.98–8.97 (dd, 1H, NH);
8.88–8.87 (t, 1H, H-2); 8.86–8.85 (t, 1H, H-2); 8.78 (dd, 1H, H-8); 8.77 (dd,
1H, H-8); 8.55 (s, 1H, H-DNP); 8.53 (s, 1H, H-DNP); 8.45–8.44 (t, 1H, H-
DNP); 8.42–8.41 (t, 1H, H-DNP); 8.36–8.34 (d, 1H, H-DNP); 8;19–8.18 (d,
1H, H-DNP); 7.75–7.33 (m, 10H, H-arom.); 7.22–7.12 (m, 18H, H-DMTr);
6.82–6.77 (m, 8H, H-DMTr); 6.14–6.13 (d, 1H, OH); 6.13–6.11 (d, 1H, OH);
6.00–5.98 (d, 1H, H-1ꢀ); 5.96–5.95 (d, 1H, H-1ꢀ); 5.39–5.36 (q, 2H, H-3ꢀ);
5.19–5.14 (q, 1H, H-2ꢀ); 5.08–5.04 (q, 1H, H-2ꢀ); 4.35–4.34 (m, 2H, H-4ꢀ);
3.72–3.70 (m, 4H, H-5ꢀ, H-5ꢀꢀ); 3.31 (s, 12H, OCH3). 13C NMR (DMSO-
d6) δ 175.55 (C O); 158.06 (C-Ade); 155.16–145.27 (C-DMTr); 144.70 (C-
Ade); 144.61–135.25 (C-DNP); 134.66 (C-Ade); 129.67–128.22 (C-arom.);
126.69 (C-Ade); 121.21–120.58 (C-DMTr); 117.14 (C-Ade); 87.91, 87.01,
85.93, 81.09, 72.06, 71.31, 62.96, 62.73 (C-rib.); 54.95 (C-OCH3).
The Synthesis of 4N-acetylarabinocytidine (9)
Arabinocytidine (0.37 g, 1.50 mmol) was dissolved in methanol (22.5 ml)
and heated under reflux. Next acetic anhydride (2.25 ml) was added to the
solution. After 3 hours of stirring the mixture was evaporated with toluene
to obtain the product.
1H NMR (DMSO-d6) δ 10.82 (s, 1H, NH); 8.06 (d, 1H, H-5); 7.18 -7.16
(d, 1H, H-6); 6.07 (s, 1H, OH); 5.48 (s, 1H, H-1ꢀ); 5.04 (s, 1H, H-2ꢀ); 3.92 (s,
1H, H-3ꢀ); 3.84–3.80 (m, 1H, H-4ꢀ); 3.61 (d, 2H. H-5ꢀ,5ꢀꢀ); 2.09 (s, 1H, CH3).
13C NMR (DMSO-d6) δ 170.89 (C O); 162.13, 154.46, 146.69 (C-Cyt); 94.18
(C-Cyt); 86.95, 85.73, 76.09, 63.84, 60.98 (C-rib.); 24.32 (C-CH3).
The Synthesis of 3ꢁ,5ꢁ-O-di-tert-butylsilyl-4N-acetylarabino-cytidine (10)
To a solution of 4N-acetylarabinocytidine (0.37 g, 1.3 mmol) in 15 ml
of N,N-dimethylformamide 2,6-lutidine (0.52 ml, 4.5 mmol) was added and
mixture was cooled to 0◦C. To the stirring solution di-tert-butylsilyl ditri-
flate (0.58 ml, 1.8 mmol) was added dropwise and warmed up to room
temperature. The reaction progress was monitored by TLC. After 2 hours
the reaction was completed and half of the solvent was removed at reduced
pressure. To the reaction mixture saturated aqueous solution of sodium bi-
carbonate was added and extracted three times with dichloromethane. The
combined organic layers were dried over anhydrous sodium, sulfate, filtered
off, and concentrated. Purification was accomplished with silica gel column
chromatography (0–3% methanol in dichloromethane). Yield: 40% (0.22 g).
1H NMR (DMSO-d6) δ 10.86 (s, 1H, NH); 7.95 (t, 1H, H-5); 7.23 (d, 1H,
H-6); 6.32–6.30 (d, 1H, OH); 5.83–5.82 (d, 1H, H-1ꢀ); 5.49 (s, 1H, H-2ꢀ);
4.36–4.28 (m, 1H, H-3ꢀ); 4.11–4.07 (m, 1H, H-.4ꢀ); 3.96–3.94 (m, 2H, H-5ꢀ,

Rearrangement of 2,4-Dinitrophenyl Substituent
695
5ꢀꢀ); 2.10 (s, 3H, CH3); 0.97 (s, 18H, CH3). 13C NMR (DMSO-d6) δ 171.07
(C O); 162.50, 154.25, 145.58, 95.69 (C-Cyt); 93.14 (C-1ꢀ); 75.46 (C-3ꢀ);
74.01 (C-2ꢀ); 72.82 (C-4ꢀ); 66.66 (C-5ꢀ); 27.31–27.00 (tBu2Si); 24.38 (CH3).
The Synthesis of the 2ꢁ-O-(2,4-dinitrophenyl)-3ꢁ,5ꢁ-O-di-tert-butylsilyl-4N-
acetylarabinocytidine (11)
The solution of 3ꢀ,5ꢀ-O-di-tert-butylsilyl-4N-acetylarabinocytidine (0.22 g,
0.5 mmol) in 3 ml of N,N-dimethylformamide was cooled to −30◦C and
than 40% mineral oil emulsion of sodium hydride (0.05 g, 1.12 mmol) and
2,4-dinitrofluorobenzene (0.18 ml, 1.5 mmol) were added. After completion
of the reaction (3 hours), saturated aqueous sodium bicarbonate solution
was added and extracted three times with dichloromethane Then combined
organic layers were dried over anhydrous sodium sulfate, filtered off, and
concentrated in vacuum. The product was purified by silica gel column
chromatography (0–3% methanol in dichloromethane). Yield: 70% (0.20 g).
1H NMR (DMSO-d6) δ 10.87 (s, 1H, NH); 8.68–8.77 (d, 1H, H-DNP);
8.60–8.54 (dd, 1H, H-DNP); 8.14–8.13 (d, 1H, H-DNP); 7.73–7.70 (d, 1H,
H-5); 7.31–7.20 (d, 1H, H-6); 6.66 (d, 1H, OH); 5.70–5.66 (d, 1H, H-1ꢀ);
5.49 (s, 1H, H-rib.); 4.44–4.38 (m, 1H, H-ryb.); 4.24–4.19 (m, 1H, H-ryb.);
4.09–3.95 (m, 1H, H-ryb.); 2.09 (s, 3H, CH3); 0.97 (s, 18H, CH3). 13C NMR
(DMSO-d6) δ 171.06 (C O); 162.57, 154.15, 146.55 (C-Cyt); 129.68, 129.02,
121.11, 117.19, 116.43 (C-DNP); 95.85 (C-Cyt); 89.44 (C-1ꢀ); 82.78 (C-3ꢀ);
79.17 (C-2ꢀ); 74.67 (C-4ꢀ); 66.23 (C-5ꢀ); 27.20–26.89 (tBu2Si); 21.94 (CH3).
The Synthesis of 2ꢁ-O-(2,4-dinitrophenyl)-4N-acetylarabino-cytidine (12)
To 2ꢀ-O-(2,4-dinitrophenyl)-3ꢀ,5ꢀ-O-di-tert-butylsilyl-6N-acetyl-arabinocy
tidine (0.20 g, 0.35 mmol) 1 molar solution of triethylamine hydrofluo-
ride (1.06 ml, 1.05 mmol) in pyridine was added. The reaction mixture was
stirred at room temperature for 2 hours and then to the solution saturated
aqueous sodium bicarbonate was added, and the aqueous layer was extracted
three times with dichloromethane. The combined organic layers were dried
over anhydrous sodium sulfate, filtered off, and concentrated. Yield: 100%
(0.20 g).
Synthesis of 5ꢁ-O-dimetoxytrityl-2ꢁ-O-(2,4-dinitrophenyl)-4N-
acetylarabinocytidine (13)
To a solution of 2ꢀ-O-(2,4-dinitrophenyl)-4N-acetyarabino-cytidine (0.20
g, 0.35 mmol) in 2.5 ml pyridine the dimethoxytrityl chloride (0.13 g, 0.40
mmol) was added. The reaction was carried out at room temperature. After
3 hours the reaction was completed, then to the mixture saturated aqueous
solution of sodium bicarbonate was added and the solution was extracted
three times with dichloromethane. The combined organic layers were dried
over anhydrous sodium sulfate, filtered off, and the solvent was evaporated

696
A. Olejnik et al.
under vacuum. The crude product was purified by silica gel column chro-
matography (0–3% methanol in dichloromethane). Yield: 85% (0.17 g).
1H NMR (DMSO-d6) δ 10.96 (s, 1H, NH); 8.68 (d, 1H, H-DNP); 8.53–8.51
(dd, 1H, H-DNP); 8.33–8.30 (d, 1H, H-DNP); 8.02–8.00 (d, 1H, H-DNP);
7.71–7.67 (d, 1H, H-5); 7.38–7.7.15 (m, 9H, DMTr); 7.15–7.13 (d, 1H, H-6);
6.89–6.82 (m, 4H, DMTr); 6.36 (d, 1H, H-1ꢀ); 6.22 (d, 1H, OH-3ꢀ); 5.37 (t, 1H,
H-2ꢀ); 4.31 (m, 1H, H-3ꢀ); 4.11 (m, 1H, H-4ꢀ); 3.40–3.25 (m, 2H, H-5ꢀ, H-5ꢀꢀ);
2.10 (s, 1H, CH3). 13C NMR (CDCl3) δ 170.96 (C O); 162.49, 158.09, 154.06,
149.58 (C-Cyt); 146.10, 140.43, 138.45 (C-DNP); 135.27, 129.68, 127.38 (C-
DMTr); 127.74, 121.33 (C-DNP); 116.81 (C- Cyt); 113.39 (C-DMTr); 95.23
(C-Cyt); 85.64 (C-1ꢀ); 82.77 (C-2ꢀ); 82.29 (C-4ꢀ); 73.21 (C-3ꢀ); 62.08 (C-5ꢀ);
24.35 (CH3).
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