Synthesis of oligonucleotides containing 5-carboxy-2′-deoxyuridine at defined sites

Full text of DOI:10.1021/jo960614f

J . Org. Chem. 1996, 61, 6075-6078
6075
order to avoid the formation of an amide function during
the deprotection step involving alkali treatment.10 The
ethyl group was chosen for the protection of the carboxylic
acid function with regard to the work of Bender11 on the
hydrolysis kinetic of benzoic acid esters in NaOH. This
author showed that the rate constant of hydrolysis of
ethyl benzoate was similar to that of the half-life 2-isobu-
tyryl-2′-deoxyguanosine8 (271 min) under the latter
conditions. The esterification of the carboxyl group of
CdU 2 was achieved without protecting the hydroxyl
functions of the sugar moiety using dicyclohexylcarbodi-
imide and 1-hydroxybenzotriazole in anhydrous etha-
nol.12 The expected ester 3 was isolated in 77% yield by
silica gel chromatography. The FAB-mass spectrometry
analysis of 3 in the positive mode showed a protonated
molecule at m/z ) 301 ( 0.1 Da. This is indicative of a
monoethylated product. Furthermore, the structure was
confirmed by NMR analyses. In particular, the 1H-NMR
spectrum exhibits two multiplets at 1.38 and 4.36 ppm
corresponding to the CH3 and the CH2 (ABX3 system) of
the protecting group of the carboxylic function, respec-
tively. It should be noted that the integrity of the base
moiety was inferred from the presence of a singlet at 8.96
ppm corresponding to the pyrimidine H6 proton. This
received further support from the presence of a signal at
164.27 ppm in the 13C-NMR spectrum that is character-
istic of the carboxyl group of an ester function.
Prior to the preparation of the phosphoramidite syn-
thon of CdU 5, the stability of the compound 3 was
checked at room temperature under two conditions.
These included incubation of 3 in 80% acetic acid and a
commercially available solution of iodine. These two
treatments correspond to the detritylation and the oxida-
tion of the internucleotide link steps, respectively. For
this purpose, aliquots of the reaction mixtures were taken
up at increasing periods of time and analyzed by RPLC
(system C), using thymidine as an internal standard. No
detectable degradation of 3 was observed after 24 h of
incubation. It should be noted that similar conclusions
were reached for CdU 2. Furthermore, 3 was quantita-
tively converted into the parent compound, CdU 2 (half-
life ) 200 min) upon treatment with 0.2 N NaOH/MeOH
for 16 h at room temperature.
Syn th esis of Oligon u cleotid es Con ta in in g
5-Ca r boxy-2′-d eoxyu r id in e a t Defin ed Sites
T. Berthod,† Y. Pe´tillot,‡ A. Guy,† J . Cadet,† and
D. Molko*,†
De´partement de Recherche Fondamentale sur la Matie`re
Condense´e/ SCIB, Le´sions des Acides Nucle´iques, CEA/
Grenoble, 17, Rue des Martyrs, 38054 Grenoble Cedex 9,
France, and Laboratoire de Spectrome´trie de Masse des
Prote´ines, Institut de Biologie Structurale (CEA/ CNRS), 41
Rue des Martyrs, 38027 Grenoble Cedex 1, France
Received April 2, 1996
5-Carboxy-2′-deoxyuridine (2) is an oxidized nucleoside
that may result from 2-methyl-1,4-naphthoquinone-
mediated (menadione) photosensitization of thymidine in
aerated aqueous solution.1 Furthermore, CdU 2 has been
detected as a decomposition product of thymidine upon
exposure to either 254 nm radiation or HO• radicals.1
Moreover, Thornburg and his co-workers reported that
5-carboxyuracil is generated through the catalyzed oxida-
tion of thymine residues by thymine hydroxylase in
DNA.2 In another respect, it has been shown that CdU
2 is an inhibitor of the de novo pyrimidine biosynthetic
pathway involving orotate phosphoribosyl transferase
and orotidine 5′-phosphate decarboxylase.3 It should be
noted that other methyl oxidation products of thymidine,
including 5-(hydroxymethyl)-2′-deoxyuridine4 and 5-formyl-
2′-deoxyuridine,5-7 have been recently inserted into
synthetic oligonucleotides.
Therefore, it would be important to delineate the
possible biological role of CdU 2 when present in DNA.
This could be achieved, at least partly, by using modified
oligonucleotides as substrates for replication and mu-
tagenesis studies. We report herein an efficient method
for the solid-phase synthesis of oligonucleotides contain-
ing CdU residues at selected sites.
5-Carboxy-2′-deoxyuridine (2) was synthesized by pho-
tosensitized oxidation of an aqueous solution of thymidine
(1) at 350 nm in the presence of 0.1 equiv of menadione
(Figure 1). After 16 h of irradiation, which led to the
complete degradation of thymidine (1), the crude mixture
was concentrated under reduced pressure. Then, CdU
2 was purified by preparative anion-exchange liquid
chromatography (system A).
The acidic function of CdU 2 has to be protected prior
to the chemical incorporation of 2 into DNA fragments.
In addition, the presence of the carboxylic group requires
that the concentrated aqueous solution of ammonia is
substituted by a solution of 0.2 N NaOH/MeOH,8,9 in
For the preparation of the phosphoramidite synthon,
the ethyl ester of CdU 3 was essentially treated as
thymidine. The 5′-O-(4,4′-dimethoxytrityl)- 4 and 3′-O-
(2-cyanoethyl)-N,N-diisopropylphosphoramidite 5 deriva-
tives were prepared by standard methods13,14 (Figure 1).
Two oligodeoxynucleotides, namely 5′-d(CCT CTA CXA
T) (decamer 6) and 5′-d(ACC CTG XAT T) (decamer 7)
were then prepared by phosphoramidite solid-phase
synthesis on a 1 mmol scale. All coupling yields, includ-
ing that with the CdU synthon 5, were higher than 97%.
Following the synthesis, the detritylated oligonucleotides
were deprotected in a solution of 0.2 N NaOH/MeOH (1:1
v:v) and then purified by RPLC. The separation was
achieved by using 25 mM triethylammonium buffer and
acetonitrile as the eluent (system D). The homogeneity
* To whom correspondence should be addressed. Phone: (33) 76-
88-99-07. Fax: (33) 76-88-50-90. E-mail: molko@drfmc.ceng.cea.fr.
† Le´sions des Acides Nucle´iques.
‡ Institut de Biologie Structurale.
(1) Berthod, T.; Cadet, J .; Molko, D. Manuscript in preparation.
(2) Thornburg, L. D.; Lai, M.-T.; Wishnock, J . S.; Stubbe, J . A.
Biochemistry 1993, 32, 14023.
(3) Clough, D. W.; Wigdahl, B. L.; Parkhurst, J . R. Antimicrob.
Agents Chemother. 1978, 14, 126.
(4) Sowers, L. C.; Beardsley, G. P. J . Org. Chem. 1993, 58, 1664.
(5) Matsuda, A.; Inada, M.; Nara, H.; Ohtsuka, E.; Ono, A. Bioorg.
Med. Chem. Lett. 1993, 3, 2751.
(6) Ono, A.; Okamoto, T.; Inada, M.; Nara, H.; Matsuda, A. Chem.
Pharm. Bull. 1994, 42, 2231.
(10) Sigmund, H.; Pfleiderer, W. Helv. Chim. Acta 1994, 77, 1267.
(11) Bender, M. L. J . Am. Chem. Soc. 1951, 73, 1626.
(12) Windridge, G. C.; J orgensen, E. C. J . Am. Chem. Soc. 1971,
93, 6318.
(7) Berthod, T.; Pe´tillot, Y.; Guy, A.; Cadet, J .; Forest, E.; Molko, D.
Nucleosides Nucleotides, in press.
(8) Ko¨ster, H.; Kulikowski, K.; Liese, T.; Heikens, W.; Kohli, V.
Tetrahedron 1981, 37, 363.
(13) Sinha, N. D.; Biernat, J .; Ko¨ster, H. Tetrahedron Lett. 1983,
24, 5843.
(14) Barone, A. D.; Tang, J .-Y.; Caruthers, M. H. Nucleic Acids Res.
1984, 12, 4051.
(9) Thuong, N. T.; Chassignol, M. Tetrahedron Lett. 1988, 29, 5905.
(15) Buchanan, J . G. Nature 1951, 168, 1901.
S0022-3263(96)00614-7 CCC: $12.00 © 1996 American Chemical Society

6076 J . Org. Chem., Vol. 61, No. 17, 1996
Notes
F igu r e 1. Synthetic reactions used for the preparation of the phosphoramidite synthon of CdU 2.
F igu r e 2. ESI-MS of the decamer 6 deprotected in 0.2 NaOH/MeOH (1:1, v/v) and purified by RPLC (system D).
tic acid, ethanol, and HPLC-grade methanol were obtained from
Carlo Erba (Milan, Italy). Dichloromethane, hexane, chloroform,
toluene, and NaOH were from SDS (France). Ammonium
formate was from BDM Laboratory Supplies, Poole (U.K.).
Buffers for high performance liquid chromatography (HPLC) and
capillary electrophoresis (CE) were prepared using water puri-
fied with a Milli-Q system (Milford, MA).
of the purified oligonucleotides was checked by HPLC on
an anion-exchange column (system E).
A fraction of the latter modified oligonucleotides was
analyzed by electrospray ionization mass spectrometry.
As shown in Figure 2, the mass spectra of the decamers
6 and 7 exhibited the expected molecular peaks at 2967.5
( 0.5 Da and 3008.0 ( 0.5 Da, respectively. Further-
more, the presence of intact CdU 2 in the synthesized
oligonucleotides was confirmed by the analysis of their
enzymatic digestion mixtures by Micellar Electrokinetic
Chromatography (system F). This was achieved by
comparison of the retention time (tR ) 5.43 min) and UV
spectroscopic features with those of the authentic CdU
2.
Con clu sion . The synthesis reported herein provides
a facile method for the preparation of oligonucleotides
containing 5-carboxy-2′-deoxyuridine (2) at specific posi-
tions. These modified DNA fragments are suitable to
study both the biochemical and conformational features
of CdU 2 when inserted into DNA fragments.
High -P er for m a n ce Liqu id Ch r om a togr a p h y Sep a r a -
tion s. CdU 2 was purified by preparative HPLC. Samples were
injected onto a preparative column filled with 70 g of Amicon
Matrex Silica PAE 300 (40 mm, Pore diameter 300 Å, particle
size 50 µm). The eluent was
a step gradient of 25 mM
triethylammonium acetate buffer at pH ) 7 (TEAA) in water,
at an elution pressure of 4 bars. Compounds were detected at
260 or 300 nm (System A).
HPLC was performed using an injector loop of 20 µL for the
analytical experiments and 500 µL for the purification steps.
Several chromatographic systems were used. System B: reversed-
phase high performance liquid chromatography in the ion
suppression mode (RPLC); column, Merck LiChrocart LiChro-
spher 100RP-18e (5 µm, 125 × 4 mm); eluent, 25 mM TEAA/
acetonitrile (96:4 v:v) at a flow rate of 1 mL/min. System C:
RPLC; column, Merck LiChrocart LiChrospher 100RP-18e (5 µm,
125 × 4 mm); eluent, 25 mM TEAA/methanol (95:5, v:v) at a
flow rate of 1 mL/min. System D: RPLC in the ion suppression
mode; column, Merck LiChrocart LiChrospher 100RP-18e (5 µm,
125 × 4 mm); eluent, a gradient of 0-12.5% of acetonitrile in
25 mM TEAA at a flow rate of 1 mL/min. System E: column,
anion-exchange Partisil P10 SAX 25F (5 µm, 250 × 4 mm);
eluent, a gradient of 20-70% of 0.2 M NaHPO4 buffer (pH 6.7)
containing 30% of acetonitrile in a mixture of H2O/acetonitrile
(70:30, v:v) at a flow rate of 1 mL/min.
Ma ter ia ls a n d Meth od s
Ch em ica ls. Thymidine (1) was purchased from Pharma-
Waldhof (Du¨sseldorf, Germany). 2-Methyl-1,4-naphthoquinone
(menadione) and boric acid were from Merck (Darmstadt, FR,
Germany). Triethylamine and sodium were obtained from
Prolabo (Paris, France). Dicyclohexylcarbodiimide and 1-hy-
droxybenzotriazole were from Fluka (Buchs, Switzerland). Ace-

Notes
k′ represented the capacity factor of the different products in
the conditions described previously, and tR is the abbreviation
for the retention time.
Micella r Electr ok in etic Ch r om a togr a p h y An a lysis. For
micellar electrokinetic chromatography (MEKC) analyses, an
untreated fused-silica capillary of 57 cm total length × 75 µm
i.d. (375 µm o.d.) was used with the detection window located
at a distance of 50 cm. The column temperature was maintained
at 25 °C during the analysis. The samples were hydrodynami-
cally injected during 1 s (∼5 nL). The separation was achieved
using a 50 mM sodium borate/25mM SDS buffer adjusted at pH
J . Org. Chem., Vol. 61, No. 17, 1996 6077
residue was dissolved in 14 mL of dry pyridine, and 4,4′-
dimethoxytrityl chloride (671 mg, 1.98 mmol) was added. The
mixture was left at room temperature for 2 h. The reaction was
checked for completion by TLC (CHCl3/CH3OH, 95:5). The
solution was cooled in an ice bath, and then ethanol (1 mL) was
added. After 10 min, the mixture was dissolved in 50 mL
solution of ethanol/toluene (3:1 v/v) and evaporated to almost
dryness. Two other coevaporations with the latter solution were
performed, and finally the residue was again coevaporated with
dichloromethane. The residue was dissolved in 40 mL of
dichloromethane and washed with 5% NaHCO3 (2 × 50 mL) and
water (2 × 50 mL). The organic layer was dried over Na2SO4
and evaporated to dryness. Then, the residue was coevaporated
twice with ethanol and finally with chloroform. The resulting
residue was then purified by flash chromatography on a Merck
Kieselgel silica gel column PF254 (60 g) with a step gradient of
0-5% ethanol in chloroform as the mobile phase. The appropri-
ate fractions were pooled and then concentrated to dryness
giving 408 mg (0.67 mmol) of 5′-O-(4,4′-dimethoxytrityl)-5-
(carboxyethyl)-2′-deoxyuridine (4, yield 41%). FAB-MS positive
mode: [MNa2]+ ) 647.7 ( 0.1 Da; [M + Na]+ ) 625.7 ( 0.1; [M
+ H]+ ) 602.7 ( 0.1 Da; [DMT]+ ) 303.4 ( 0.1 Da; [BH + Na]+
) 207.3 ( 0.1 Da; [BH + H]+ ) 185.3 ( 0.1 Da. 1H-NMR
(400.135 MHz; CD2Cl2) δ: 8.50 (s, 1H, H-6); 7.44-6.82 (m, 13
H, aromatic-H); 6.20 (t, J 1′,2′ ) 7.0 Hz, J 1′,2′′ ) 5.9 Hz, 1H, H-1′);
4.39 (m, J 2′,3′ ) 6.4 Hz, J 2′′,3′ ) 3.5 Hz, J 3′,4′ ) 3.5 Hz, 1H, H-3′);
4.06 (m, J 4′,5′ ) 4.2 Hz, J 4′,5′′ ) 4.2 Hz, 1H, H-4′); 3.99 and 3.86
10.0 ( 0.1 (system F) with
a 1 N NaOH solution. The
instrument was set at a fixed voltage of 25 kV leading to a
constant current of 78 µA. UV data (214 nm or diode array
detector) were collected at a rate of 10 points per s.
5-Ca r boxy-2′-d eoxyu r id in e (2). To 1 L of a 5 mM aqueous
solution of thymidine (1; 1.21 g) was added 13 mg of menadione
(0.5 mM, 0.1 equiv). The resulting solution was exposed to 16
350 nm black lamps (approximatively 4.5 W each with 90% of
the emitted light in the 350 nm range) in a Rayonet photochemi-
cal reactor at room temperature. A continuous flow of air
maintained the solution saturated with oxygen during the
irradiation. After the degradation of thymidine (1) was com-
pletely achieved as controlled by RPLC (system B: k′(dT) )
10.52; k′(CdU) ) 2.10), the reaction mixture was concentrated
under reduced pressure. The overall process was resumed 10
times in order to photooxidize a total of 12.1 g of thymidine (1).
CdU 2 was purified by preparative liquid chromatography
(system A), and the collected fractions were controlled by
analytical RPLC (system A). The appropriate fractions (20 <
k′ < 33) were pooled and then lyophilized. The resulting residue
was dissolved in water (100 mL), and the solution was desalted
through a column of Dowex 50 (H+) resin. The acidic fractions
that showed a positive 2′-deoxyribonucleoside test were pooled
and concentrated under reduced pressure and then lyophilized.
5-Carboxy-2′-deoxyuridine (2) (2.04 g, 7.5 mmol) was obtained
(yield 15%). UV (λmax; H2O) 272 nm (e ) 9714 M-1‚cm-1). FAB-
MS positive mode: [M + H]+ ) 273 ( 0.1 Da; [B + 2H]+ ) 157
( 0.1 Da; [dR]+ ) 117 ( 0.1 Da. ESI-MS negative mode: [M -
H]- ) 271 ( 0.1 Da. 1H-NMR (499.838 MHz; D2O) δ: 8.78 (s,
1H, H-6); 6.41 (t, J 1′,2′ ) 6.7 Hz, J 1′,2′′ ) 6.5 Hz, 1H, H-1′); 4.60
(m, J 2′,3′ ) 6.7 Hz, J 2′′,3′ ) 4.1 Hz, J 3′,4′ ) 3.8 Hz, 1H, H-3′); 4.17
(m, J 4′,5′ ) 3.5 Hz, J 4′,5′′ ) 5.1 Hz, 1H, H-4′); 3.96 and 3.89 (m,
(m, J
) 7.2 Hz, J
) 7.2 Hz, J a,b ) -10.81 Hz, 2H, CH2-
b-CH3
a-CH3
CH3); 3.77 (s, 6H, CH3O); 3.41 and 3.35 (m, J 5′,5′′ ) -10.5 Hz,
2H; H-5′ and H-5′′); 2.48 and 2.23 (m, J 2′,2′′ ) -13.8 Hz, 2H,
H-2′ and H-2′′); 1.06 (m, 3H, CH3CH2). 13C-NMR (100.62 MHz;
CD2Cl2) δ: 162.1 (COOEt); 158.92 (C-4); 158.9 (C-4, -4′ of DMT);
149.4 (C-2); 146.7 (C-6); 144.8 (C-1′′ of DMT); 135.8 and 135.7
(C-1, C-1′ of DMT); 130.4 and 130.2 (C-2, C-2′, C-6, C-6′ of DMT);
128.3-128.1 (C-2′′, C-3, C-5′′, C-6′′ of DMT); 127.1 (C-4′′ of DMT);
113.4-113.2 (C-3, C-3′, C-5, C-5′ of DMT); 105.7 (C-5); 87.5
((MeOPh)2PhCO); 86.8 (C-4′); 86.27 (C-1′); 72.3 (C-3′); 63.5 (C-
5′); 61.1 (CH2-CH3); 55.4 (CH3O); 41.2 (C-2′); 14.0 (CH3CH2).
5-Ca r boxy-2′-d eoxyu r id in e P h osp h or a m id ite Der iva tive
5. 5′-O-(4,4′-Dimethoxytrityl)-5-(carboxyethyl)-2′-deoxyuridine
(compound 4; 0.57 mmole) and diisopropylammonium tetrazolate
(0.28 mmol) were dissolved in dry dichloromethane (5 mL) and
evaporated to dryness. The operation was repeated twice. The
solid residue was then dissolved in dry dichloromethane (2.3 mL)
and kept under an argon atmosphere. Subsequently, (cyanoet-
hyl)bis(diisopropylamino)phosphine (200 µL, 0.628 mmol) was
added with a syringe through a rubber septum. The reaction
mixture was stirred during 35 min. The formation of the desired
product was checked by TLC. After dilution in ethyl acetate
(20 mL), the mixture was concentrated to dryness. The resulting
residue was taken up in ethyl acetate (10 mL) and washed
successively with 5% NaHCO3 (2 × 50 mL) and a saturated
solution of NaCl (2 × 50 mL). Then, the organic layer was
evaporated to dryness. The dry residue was purified by flash-
chromatography on a Merck Kieselgel silica gel column PF254
(30 g) with a step gradient of 0-5% AcOEt in dichloromethane/
triethylamine (99:1) as the mobile phase. The appropriate
fractions were pooled and then concentrated to dryness. The
product was dissolved in dry dichloromethane (1 mL) and
subsequently precipitated at -78 °C in hexane (40 mL). Filtra-
tion gave a white powder corresponding to the phosphoramidite
synthon of CdU 5. The building block was dried under reduced
pressure in a desiccator, and then stored under dry argon.
Under these conditions, 362 mg (0.45 mmole) of 5 were obtained
(yield 79%). FAB-MS positive mode: [M + K]+ ) 841.0 ( 0.1
Da; [M + Na]+ ) 825.8 ( 0.1 Da; [M + H]+ ) 803.8 ( 0.1 Da;
[DMT]+ ) 303.2 ( 0.1 Da. In addition, the exact mass measure-
ment of the pseudomolecular ion [M + Na]+ (m/z ) 825.3272; D
J 5′,5′′ ) -12.5 Hz, 2H; H-5′ and H-5′′); 2.55 and 2.51 (m, J 2′,2′′
)
-14.2 Hz, 2H, H-2′ and H-2′′). 13C-NMR (100.62 MHz; D2O) δ:
184.3 (COOH); 163.8 (C-4); 151.1 (C-2); 146.6 (C-6); 108.3 (C-5);
87.1 (C-4′); 86.2 (C-1′); 70.3 (C-3′); 61.1 (C-5′); 39.3 (C-2′).
5-Ca r boxyeth yl-2′-d eoxyu r id in e (3). CdU 2 (1.983 g, 7.28
mmol), 0.983 mg of 1-hydroxybenzotriazole (7.28 mmol), and 1.73
mL of tributylamine (7.28 mmol) were dissolved in 200 mL of
anhydrous ethanol that was previously desiccated with sodium
and then distilled. To the resulting solution was added 1.802 g
of dicyclohexylcarbodiimide (8.736 mmol), and the reaction was
kept overnight at room temperature. After a TLC control
(CHCl3/CH3OH; 80:20) of the reaction mixture, the solution was
evaporated under reduced pressure and then coevaporated twice
with chloroform. The resulting residue was then purified by
flash-chromatography on a Merck Kieselgel silica gel column
PF254 (200 g). The mobile phase was a step gradient of 0-5%
methanol in chloroform. The appropriate fractions were con-
centrated to dryness. 5-(Carboxyethyl)-2′-deoxyuridine (3) 1.685
g, 5.61 mmol) was obtained (yield 77%). FAB-MS positive
mode: [M + Na]+ ) 323.2 ( 0.1 Da; [M + H]+ ) 301.2 ( 0.1
Da; [BH + Na]+ ) 207.2 ( 0.1 Da; [B + 2H]+ ) 185.2 ( 0.1 Da.
1H-NMR (400.135 MHz; D2O) δ: 8.97 (s, 1H, H-6); 6.32 (t, J 1′,2′
) 5.4 Hz, J 1′,2′′ ) 6.7 Hz, 1H, H-1′); 4.55 (m, J 2′,3′ ) 6.6 Hz, J 2′′,3′
) 5.1 Hz and J 3′,4′ ) 4.4 Hz, 1H, H-3′); 4.38 and 4.35 (m, J a-CH
3
) 7.2 Hz, J b-CH ) 7.1 Hz, J a,b ) -10.7 Hz, 2H, CH2CH3); 4.15
3
(m, J 4′,5′ ) 3.2 Hz, J 4′,5′′ ) 4.4 Hz, 1H, H-4′); 3.97 and 3.86 (m,
) 3.7 ppm) was inferred from
analysis. This is indicative of an empirical formula of
42H51N4O10PNa. 1H-NMR (200.15 MHz; CD2Cl2) two diaster-
a high-resolution FAB-MS
J 5′,5′′ ) -12.6 Hz, 2H; H-5′ and H-5′′); 2.58 and 2.49 (m, J 2′,2′′
)
-14.3 Hz, 2H, H-2′ and H-2′′); 1.39 (m, 3H, CH3CH2). 13C-NMR
(100.614 MHz; D2O) δ: 164.3 (COOEt); 162.7 (C-4); 149.0 (C-6);
151.0 (C-2); 104.2 (C-5); 87.0 (C-4′); 86.5 (C-1′); 69.7 (C-3′); 62.0
(CH2-CH3); 60.4 (C-5′); 39.6 (C-2′); 13.4 (CH3CH2).
C
eomers δ: 8.52 and 8.47 (s, 1H, H-6); 7.43-6.79 (m, 13 H,
aromatic-H); 6.18 (t, 1H, H-1′); 4.45 (m, 1H, H-3′); 4.16 (m, 1H,
H-4′); 3.9 (m, 2H, CH2CH3); 3.64 (s, 6H, CH3O); 3.39 (m, 3H,
CHiPr and CH2OP); 3.28-3.23 (m, 2H; H-5′ and H-5′′); 2.55 (m,
1H, H-2′′); 2.41 (t, 2H, -CH2CN); 2.21 (m, 1H, H-2′); 1.41-1.03
(m, 15H, CH3iPr and CH3CH2). 31P-NMR (101.21 MHz; CD2-
Cl2) δ: 149.63 and 149.37 (two diastereomers).
5′-O-(4,4′-Dim eth oxytr ityl)-5-(ca r boxyeth yl)-2′-d eoxyu r i-
d in e (4). 5-(Carboxyethyl)-2′-deoxyuridine (3) (496 mg, 1.65
mmol) was dissolved in dry pyridine (10mL) and evaporated to
dryness. The operation was repeated twice. The resulting oily

6078 J . Org. Chem., Vol. 61, No. 17, 1996
Notes
Gen er a l Cou p lin g P r oced u r es for t h e Assem b ly of
Oligon u cleotid es. The preparation of oligonucleotides contain-
ing-CdU was performed on a DNA synthesizer using a 1 µmol
protected nucleoside functionnalized on a chemical modified CPG
polymer support. For optimal coupling efficiency, the flow rate
delivery of reagents was fixed at 2.3 mL/min. Synthesized
amidite 5 and commercially available amidites were used in 0.08
M dry acetonitrile solution. Standard 0.2 µmol scale synthesis
cycles were used. The coupling yield of the CdU unit 5 was 97%
estimated by quantitation of the dimethoxytrityl cation before
and after the CdU cycle.
Rem ova l of th e Oligom er fr om th e CP G Bea d s, Dep r o-
tection , a n d P u r ifica tion . The oligonucleotides bound to the
support were treated with 0.2 N NaOH/MeOH (1:1 v/v) solution.
Then, the collected fractions were neutralized with 1 M aqueous
acetic acid, and the resulting solutions were evaporated to
dryness. The decamers 6 and 7 were further purified by RPLC/
system D (tR(decamer 6) ) 40.2 min; tR(decamer 7) ) 41.51 min)
and the homogeneity of the purified decamers was checked by
HPLC/system E (tR(decamer 6) ) 51.4 min; tR(decamer 7) )
55.05 min).
added, and the resulting mixture was incubated at 37 °C for 1
h. Subsequently, 33 µL of Tris-HCl/MgCl2 buffer (pH ) 9) and
20 U of alkaline phosphatase were added, and the resulting
mixture was incubated for 2 h at 37 °C. After lyophilization,
the residues obtained were taken up in 1 mL of running buffer
and analyzed by MEKC/system F. The UV detector was set at
214 nm (tR(dC) ) 3.15 min; tR(dA) ) 3.29 min; tR(dT-1) ) 3.73
min; tR (Nuclease P1) ) 3.87 min; tR(dG) ) 4.06 min; tR(CdU-2)
) 5.43 min).
Ack n ow led gm en t. The contribution of C. Lebrun
(SCIB/CEA-Grenoble) to the mass spectrometry mea-
surements is greatly acknowledged. This work was
supported, in part, by the French Ministery of Science
and Research: ACC-SV no. 8-MESR 1995.
Su p p or tin g In for m a tion Ava ila ble: Copies of 1H-,13C-
NMR and FAB mass spectra of 2-4, UV spectrum of 2, 31P-
NMR and FAB mass spectra of 5, and electrospray ionization
mass spectrum of decamer 7 (15 pages). This material is
contained in libraries on microfiche, immediately follows this
article in microfilm version of the journal, and can be ordered
from the ACS; see any current masthead page for ordering
information.
Decamer 6 [5′-d(CCT CTA CXA T)-3′]: MW ) 2967.5 ( 0.5
Da. Decamer 7 [5′-d(ACC CTG XAT T)-3′]: MW ) 3008.0 (
0.5 Da.
En zym a tic Digestion of Deca m er s 6 a n d 7. 0.1 AUFS260nm
of purified decamer 6 and decamer 7 were taken up in 30 µL of
0.3 M sodium acetate buffer. Nuclease P1 (10 µL, 1 mg) was
J O960614F