Synthesis of 2′-amino-2′-deoxyuridine modified by 1,8-naphthalimide

Article abstract of DOI:10.1080/00397910600631924

A simple and efficient synthetic route of 2′-amino-2′- deoxyuridine was studied. 2′-Amino-2′-deoxyuridine was incorporated into 1,8-naphthalimides in the 2′-position of 2′-sugar via linking arms of different lengths. A convenient method for the synthesis of the conjugates was adopted. Copyright Taylor & Francis Group, LLC.

Full text of DOI:10.1080/00397910600631924

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Synthesis of 2′‐Amino‐2′‐deoxyuridine
Modified by 1,8‐Naphthalimide
Heting Li a , Zhiqin Jiang a , Xin Wang a & Chao Zheng a
a Department of Chemistry, Tongji University, Shanghai, China
Published online: 20 Aug 2006.
To cite this article: Heting Li , Zhiqin Jiang , Xin Wang & Chao Zheng (2006): Synthesis of
2′‐Amino‐2′‐deoxyuridine Modified by 1,8‐Naphthalimide, Synthetic Communications: An International
Journal for Rapid Communication of Synthetic Organic Chemistry, 36:14, 1933-1940
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Synthetic Communicationsw, 36: 1933–1940, 2006
Copyright # Taylor & Francis Group, LLC
ISSN 0039-7911 print/1532-2432 online
DOI: 10.1080/00397910600631924
Synthesis of 20-Amino-20-deoxyuridine
Modified by 1,8-Naphthalimide
Heting Li, Zhiqin Jiang, Xin Wang, and Chao Zheng
Department of Chemistry, Tongji University, Shanghai, China
Abstract: A simple and efficient synthetic route of 20-amino-20-deoxyuridine was
studied. 20-Amino-20-deoxyuridine was incorporated into 1,8-naphthalimides in the
20-position of 20-sugar via linking arms of different lengths. A convenient method
for the synthesis of the conjugates was adopted.
Keywords: 20-Amino-20-dexoyuridine, linker, modified oligonucleotides, 1,8-napthali-
mide, synthesis
INTRODUCTION
Oligonucleotides (ODNs) covalently linked to functional molecules are
widely used as inhibitors of specific gene expression and as probes for the
detection and biophysical studies of nucleosides.[1–4] In most cases, the
strategy to synthesize modified ODNs is accomplished by incorporating a
modified nucleoside or base.[5] The ODNs modified at their carbonhydrate
moiety have stimulated particular interest in recent years;[6] Mitsui et al.
have reported that 20-pentose position of ODNs is a suitable site for
covalent attachment of several fluorophors.[7] The nucleosides with amino
groups in the 20-pentose position are useful synthons to obtain the correspond-
ing phosphoamidite with functionality. Monomers such as the precursor of
ODNs can be utilized to synthesize versatile oligonucleotide conjugates.
Several variants of large-scale laboratory synthesis of 20-amino-20deoxyuridine
have been reported.[8,9] However, poor reproducibility, rigorous reaction
Received in Japan October 24, 2005
Address correspondence to Zhiqin Jiang, Department of Chemistry, Tongji
University, Shanghai 200092, China. Tel.: 86-2165983071; E-mail: zqjiang@mail.
tongji.edu.cn
1933

1934
H. Li et al.
conditions, and the use of uncommon compounds in the syntheses prompted us
to seek a simple and efficient method to prepare it.
A series of fluorescent derivatives of 1,8-naphthalimides and uridine have
been synthesized recently as potential DNA sequencing probes.[10] 1,8-
Naphthalimide was introduced into DNA by 20-amino-20-deoxyuridne
without significant loss of the duplex stability and its high fluorescence
quantum yield FF.[11] Application of this kind of compounds in molecular
biology and bioorganic chemistry has aroused great attention. It was
reported that 1,8-naphthalimide was usually incorporated into 20-amino-20-
deoxyuridine, but the reaction yields were very low.[12] In this context, modi-
fication of the changes of reaction conditions and coupling regent was empha-
sized to improve the yield of the title compounds. A simple and efficient
synthetic route was designed to successfully obtain 20-amino-20deoxyuridne
incorporating 1,8-naphthalimide.
RESULTS AND DISSCUSSION
20-Amino-20-deoxyuridine was a typical nucleoside,[9,13] As described in this
article, it was synthesized according to the modified process as depicted in
Scheme 1.
2, 20-O-Anhydro-1-(b-D-arabinofuranosyl) uracil 3 as a starting material of
20-amino-20-deoxyuridine was prepared easily from uridine 1 according to the
procedure of Sowa and Tsunoda.[14] The azide group was further incorporated
Scheme 1.

20-Amino-20-deoxyuridine
1935
into compound 3 at the 20-sugar position to give 20-azido-20-deoxyuridine 4. We
used hexamethylphosphoramide (HMPA) as solvent in this azide reaction. The
reaction time was greatly shortened from 36h to 1 h as compared with Vasil’eva
et al.’s method[15] in which the reaction was carried out in dimethylacetamide
(DMA) and dibenzo-18-crown-6 as phase transfer catalyst was utilized. After
workup of the resulting mixture, the residue was isolated by chromatography,
and a colloidal solid was obtained. The yield of this reaction is 65%. Without
further purification, the crude product 20azido-20deoxyuridine 4 was subjected
to hydrogenation with 10% Pd/C in methanol at room temperature to give
compound 5 in a high yield of 95%.
The conjugates of 1,8-naphthalimide-uridine 8a–8c were prepared from
N-substituted naphthalimide carboxy acids 7a–7c and 20-amino-20-deoxyuridine
(5) as depicted Scheme 2. Preparation of compounds 7a was carried out in the
literature[12] at very high temperature (above 2208C) because of the direct
reaction of 1,8-naphthalic anhydride and amino acid in the absence of solvent.
In our method, ethanol was adopted as solvent to simplify the reaction
condition. The reaction went smoothly, and the yields for 7a–7c were
good (63–83%). Other solvents were also tried in our experiment, including
an attempt to use toluene as solvent. Water produced in this reaction can be
separated from the reaction mixture by azeotropic distillation. Though that
would be helpful to the reaction, unfortunately 1,8-naphthalic anhydride 6 was
difficult to dissolve in toluene, which caused an uncompleted reaction.
Scheme 2.

1936
H. Li et al.
The final compounds 8a–8c were synthesized initially in DMF using
N,N-dicyclohexylcarbodiimide/1-hydroxybenzotriazole (DCC/HOBt) as the
condensation agent under a nitrogen atmosphere, but the product yields
were very low (20–30%) because of the by-product produced from the con-
densation of the starting acid with dimethylamine, which came from the
decomposition of DMF. This result made us seek a suitable solvent instead
of DMF. Several solvents were attempted for this purpose, and the best
result was to use the THF/CH2Cl2 mixture system as solvent.[16] In
addition, a more efficient coupling reagent, 1-(3-dimethylaminopropyl)-3-
ethylcarbodiimide/1-hydroxybenzotriazole (EDC/HOBt), was adopted for
the synthesis of the fluorescent conjugates 8a–8c. The target compounds
8a–8c were obtained in better yields (64–81%) from the reaction of 20-
amino-20-deoxyuridne and 1,8-naphthalimide 7a–7c in the solvent of THF/
CH2Cl2 mixture system using EDC/HOBt as the condensation reagent.
EXPERIMENTAL
IR spectra were obtained from NEXUS9121A0446 FTIR. UV/vis absorbtion
spectra were measured using an Agilent 8453-UV-VIS spectrometer.
1H NMR spectra were obtained on a Bruker Avant 500 M or Bruker 300 M
spectrometer. 13C NMR spectrum was record on a Bruker AM-300 spec-
trometer. Mass spectrum was determined on a V.G.ZAB-HS spectrometer
(EI 70ev). The melting points were determined on an XT4-100x apparatus
and are uncorrected.
20,30-O-Sulfinyl Uridine (2)
Uridine (1) (2 g, 8 mmol) was dissolved into a mixed solution of thionyl
chloride (2.5 ml) and acetonitrile (20 ml) with vigorously stirring, and then
the reaction mixture was maintained at 58C for about 4.5 h. The white precipi-
tates were formed after addition of water (0.8 g), filtrated, washed with cooled
methanol, and then dried in vacuum to give 20,30-O-sulfinyl uridine dihydrate
(2.5 g) in 85% yield. Mp 103–1048C (dec.), (ref. 104–1058C).[14]
2,20-O-Anhydro-1-(b-D-arabinofuranosyl) Uracil (3)
20,30-O-Sulfinyl uridine dihydrate (4.5 g, 13.58 mmol) and sodium acetate
(5.6 g, 67.97 mmol) were heated in N,N-dimethylformamide at about 858C;
the reaction was followed by TLC (CH2CI2/CH3OH 5 : 1), stirred for 4 h,
and then cooled. The precipitate was filtered off and washed with ml
DMF. The filtrate and washing were combined and evaporated. The crude
product was obtained and then recrystallized from methanol to give 3.3 g 2,

20-Amino-20-deoxyuridine
1937
20-O-anhydro-1-(b-D-arabinofuranosyl) uracil as a white solid in 94% yield.
Mp 233–2348C (dec.), (ref. 234–2368C).[14]
20Azido-20-deoxyuridine (4)
2,20-O-Anhydro-1-(b-D-arabinofuranosyl) uracil (4.5 g, 20 mmol) and sodium
azide (9.2 g, 141.54 mmol) were added in anhydrous hexamethylphoramide
(80 ml). The reaction mixture was rapidly stirred in a 1508C oil bath. After
0.5 h benzoic acid (2.44 g, 20 mmol) was added, heating continued for
15 min, during which time the solution became very dark. TLC (CH2-Cl2/
CH3OH 20 : 1) was used to follow the reaction. The solution was quickly
cooled, diluted with water (160 ml), and then extracted with chloroform
(200 ml). The chloroform extracts were back-extracted twice (80 ml each),
and the combined aqueous solution were extracted three times with chloro-
form. The final solution was evaporated under vacuum, and then the residue
was afforded and dissolved in a mixture of acetone (160 ml) and methanol
(60 ml), filtered, and evaporated to give oil. The oil was purified by column
chromatography (CH2Cl2/EtOAc 20 : 1) on a silica-gel column to give 3.5 g
of 20azido-20-deoxyuridine as a white colloidal solid in 65% yield.
20Amino-20-deoxyuridine (5)
A suspension of 20azido-20-deoxyuridine (3.5 g, 13.1 mmol) and 10%
palladium–carbon (1.7 g) in 150 ml of methanol was vigorously stirred in a
hydrogen atmosphere at room temperature. TLC (CH2Cl2/CH3OH 3 : 1)
was used to monitor the reaction. The reaction was stopped after 1 h; the
resulting mixture was filtered and washed three times with methanol; and
the combined solution was evaporated, leaving 3.0 g 20amino-20-deoxyuridine.
Yield was 95%. A sample recrystallized from ethanol had mp 196–1988C. 1H
NMR (300 MHz, DMSO-d6) d (ppm): 7.81 (d, J6.5 ¼ 8.24 Hz, 1H, H6), 5.66
(m, 1H, H10), 5.64 (d, 1H, H5), 5.38 (br s, 1H, NH), 5.06 (t, 1H), 3.85
(m, 1H, H30), 3.82 (m, 1H, H20), 3.71 (m, 2H, H50), 3.27 (m, 1H, H40); 13C
NMR (300 MHz, DMSO-d6): 163.30, 151.31, 140.99, 102.06, 88.16, 88.13,
71.51, 61.79, 51.72. ESI-MS (m/z, %): 243 (M).
1,8-Naphthalimide-N-acetic Acid (7a)
1,8-Naphthalic anhydride (6) (1.5 g, 7.58 mmol), 1.7 g (22.67 mmol) of
glycine, and 3.2 ml of triethylamine were dissolved with 150 ml of
anhydrous ethanol in a 250-ml flask. The mixture was refluxed for 3 h with
stirring. Then most of ethanol was evaporated, and 1 M of dilute hydrochloric
acid were added dropwise until precipitate formed. Filtration and

1938
H. Li et al.
recrystallization twice from 95% ethanol gave gray needle-like crystals (5)
1
1.53 g in 75% yield. Mp 274–2768C. H NMR (500 MHz, DMSO-d6) d
(ppm): 13.1 (s, 1H, COOH), 8.54 (m, 4H), 7.91 (t, J ¼ 7.8 Hz, 2H), 4.74
(s, 2H); ESI-MS (m/z, %): 254.2 (M þ H)þ. IR (KBr) n: 3446, 3285, 1607,
1317, 762, 671 cm21
;
UV-vis (H2O, lmax
,
1): 234 nm, 2.9 ꢀ
104 l mol21 cm21; 343 nm, 1.4 ꢀ 104 l mol21 cm21
.
1,8-Naphthalimide-N-butyric Acid (7b)
Compound (7b) was synthesized similar to the procedure for 7a. White solid
1
was obtained; the yield was 83%. Mp 186–1888C. H NMR (300 MHz,
DMSO-d6) d (ppm): 11.98 (s, 1H, COOH), 8.55 (m, 4H), 7.88 (t, J ¼ 7.7 Hz,
2H), 4.15 (t, Jbg ¼ 6.86 Hz, 2H, gH), 2.31 (t, 2H, aH), 1.91 (m, 2H, bH);
ESI-MS (m/z, %): 284.2 [(M þ H)þ, 80], 306.2 [(M þ Na)þ, 50)].
1,8-Naphthalimide-N-caproic Acid (7c)
Compound (7c) was synthesized similarly according to the procedure for 7a.
1
White solid was obtained; the yield was 63%. Mp 134–1368C. H NMR
(500 MHz, DMSO-d6) d (ppm): 12.0 (s, 1H, COOH), 8.49 (m, 4H), 7.88
(t, J ¼ 7.7 Hz, 2H), 4.04 (t, Jv,d ¼ 7.4 Hz, 2H, vH), 2.22(t, Jab ¼ 7.3 HZ,
2H, aH), 1.64 (m, 2H, dH), 1.55 (m, 2H, bH), 1.35 (m, 2H, gH); ESI-MS
(m/z, %): 312.2 [(M þ H)þ, 100], 334.2 [(M þ Na)þ, 45)] IR (KBr) n:
3456, 3478, 1702, 1649, 1238, 780 cm21; UV-vis (H2O, l max, 1): 234
nm, 3.8 ꢀ 104 1 mol21 cm21; 343 nm, 1: 1.3 ꢀ 104 1 mol21 cm21
.
20-[2-(1,8-Naphthalimide)acetamide]-20-deoxyuridine (8a)
To a solution of (7a) (76.5 mg; 0.3 mmol), 20-amino-20-deoxyuridine (5) (72.9
mg, 0.3 mmol), six drops of triethylamine and HOBt (81.07 mg, 0.6 mmol)
in CH2Cl2 (8 ml)/THF (5ml) were added EDC (115mg, 0.6 mmol). The
reaction mixture was stirred at room temperature overnight and monitored by
TLC (CH2Cl2/CH3OH ¼ 5/1, v/v), and the precipitates formed. The
solvents evaporated in vacuo, and then the residue was chromatographed on a
silica-gel column with eluent (CH2Cl2/CH3OH ¼ 40/1, v/v) to give a white
1
solid (92 mg in 64% yield). Mp 278–2808C (dec.). H NMR (300 MHz,
DMSO-d6) d (ppm): 8.45 (m, 4H), 8.29 (d, J5,6 ¼ 8.01 Hz, 1H, H6), 7.84
(m, 2H), 5.93 (d, J1 ,2 ¼ 8.01 Hz, 1H, H10), 5.58 (d, J6,5 ¼ 8.01 Hz, 1H, H5),
4.68 (m, 2H, Ha), 4.51 (m, 1H, H20), 4.08 (m, 1H, H30), 3.90 (m, 1H, H40),
3.55 (m, 2H, H50). ESI-MS (m/z, %): 481.2 [(M þ H)þ, 50)], 503.2
[(M þ Na)þ, 100)].
0
0

20-Amino-20-deoxyuridine
1939
20-[4-(1,8-Naphthalimide) butyramide]-20-deoxyuridine (8b)
Compound (8b) was prepared in the same way as 8a from, 1,8-naphthalimide-
N-butyric acid (7b) and 20-amino-20-deoxyuridine (5) in the solution of
CH2Cl2/THF. A yellow solid was obtained in 81% yield after column chrom-
atography on a silica-gel column. Mp 184–1868C. 1H NMR (300 MHz,
DMSO-d6) d (ppm): 8.45 (m,4H), 7.85 (m,3H, H300, H600, H6), 5.85 (m, 1H,
H10), 5.63 (m, 1H, H5), 4.45 (m, 1H, H20), 4.01 (t, Jb,g ¼ 6.3 Hz, 2H, gH),
3.96 (m, 1H, H30), 3.88(m, 1H, H40), 3.31 (m, 2H, H50), 2.23 (m, 2H, aH),
1.71 (m, 2H, bH). ESI-MS (m/z, %): 509.2 [(M þ H)þ, 100)], 531.2
[(M þ Na)þ, 75)].
20[6-(1,8-Naphthalimide) caproamide]-20-deoxyuridine (8c)
Compound (8c) was prepared in the same way as 8a from 1,8-naphthalimide-
N-caproic acid (7c) and 20-amino-20-deoxyuridine (5) in the solution of
CH2Cl2/THF. A white solid was afforded in 77% yield after column chrom-
1
atography on a silica-gel column. Mp 208–2108C; H NMR (300 MHz,
DMSO-d6) d (ppm): 8.50 (m, 2H, H200, H700), 7.39–8.00 (m, 5H, H300, H400,
H500, H600, H6), 5.85 (d, J1 ,2 ¼ 8.51 Hz, 1H, H10), 5.62 (m, 1H,.H5), 5.23
(t, 1H, H20), 4.49 (m, 1H, H30), 4.05 (m, 2H, vH), 3.91 (m, 1H, H40), 3.58
(m, 2H, H50), 2.08 (t, Ja,b ¼ 7.32 Hz, 2H, aH), 1.65 (m, 2H, dH), 1.48
(m, 2H, bH), 1.30 (m, 2H, gH); ESI-MS (m/z, %): 537.3 [(M þ H)þ, 100)],
559.3 [(M þ Na)þ, 70].
0
0
ACKNOWLEDGMENT
Authors are grateful to National Natural Science Foundation of China for the
financial support of this work (No. 20272042).
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