Synthesis of oligoribonucleotides containing 4-thiouridine using the convertible nucleoside approach and the 1-(2-fluorophenyl)-4-methoxypiperidin-4- yl group

Article abstract of DOI:10.1081/NCN-200034044

Oligoribonucleotides containing 4-thiouridine were prepared using the Fpmp group for protection of the 2′-OH. Two uridine derivatives with the 1,2,4-triazolyl and the 2-nitrophenyl groups at position 4 were used to obtain 4-thiouridine by postsynthetic su

Full text of DOI:10.1081/NCN-200034044

NUCLEOSIDES, NUCLEOTIDES & NUCLEIC ACIDS
Vol. 23, No. 11, pp. 1767–1777, 2004
Synthesis of Oligoribonucleotides Containing 4-Thiouridine
Using the Convertible Nucleoside Approach and
the 1-(2-Fluorophenyl)-4-Methoxypiperidin-4-yl Group
Anna Avin˜o´,1 Ramon Gu¨imil Garc´ıa,1,2 and Ramon Eritja1,
*
1Instituto de Biolog´ıa Molecular de Barcelona, Barcelona, Spain
2Mannheim, Germany
ABSTRACT
Oligoribonucleotides containing 4-thiouridine were prepared using the Fpmp group
for protection of the 2’-OH. Two uridine derivatives with the 1,2,4-triazolyl and the
2-nitrophenyl groups at position 4 were used to obtain 4-thiouridine by postsynthetic
substitution with sodium hydrogen sulfide. Both uridine derivatives allow the
preparation of the desired oligonucleotides in good yields.
Key Words: 4-Thiouridine; Oligonucleotides; RNA.
INTRODUCTION
Photochemical cross-linking can be used to characterize RNA-protein interactions
by trapping transient complexes, which can be difficult to isolate.[1–3] One of the most
common strategies used in photochemical photochemical cross-linking is to incorporate
photoreactive nucleotide analogs at defined positions. These photoreactive nucleotides
can be incorporated into the RNA chains by in vitro transcription and/or RNA
*
Correspondence: Ramon Eritja, Instituto de Biolog´ıa Molecular de Barcelona, C.S.I.C. Jordi
Girona 18-26, E-08034 Barcelona, Spain; Fax: 34-93-2045904; E-mail: recgma@cid.csic.es.
1767
DOI: 10.1081/NCN-200034044
1525-7770 (Print); 1532-2335 (Online)
www.dekker.com
Copyright D 2004 by Marcel Dekker, Inc.

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Avin˜o´ et al.
ligation.[1–3] Recent advances in chemical synthesis of RNA have opened up the
possibility of obtaining short RNA molecules containing some of these photoreactive
nucleosides by chemical synthesis. Specifically the synthesis of oligoribonucleotides
containing the photoreactive nucleosides 5-bromo-, 5-iodo- and 4-thiouridine has
been described.[4–8] For the preparation of 5-thiouridine oligoribonucleotides, the
S-cyanoethyl-protected 4-thiouridine phosphoramidite derivative[4,6,7] has been used.
Another possibility is to prepare an uridine derivative carrying a leaving group in
position 4. After the assembly of the desired sequence this derivative can be converted
to thiouridine by nucleophilic displacement with a thiol. This strategy, known as the
convertible nucleoside approach[9] or the postsynthetic modification approach,[10] has
been used to prepare oligoribonucleotides carrying N-alkyl-cytosine derivatives.[11] and
4-thiouridine.[5,8] In these studies, 2’-OH were protected as a tert-butyldimethylsilyl
(TBDMS) ethers.
We are interested in the preparation of oligoribonucleotides carrying photoreactive
nucleosides such as 4-thiouridine. In this paper, we describe the preparation and use of
two uridine derivatives for the synthesis of 4-thiouridine oligoribonucleotides. The 2’-
OH of the phosphoramidite derivatives is protected by the 1-(2-fluorophenyl)-4-
methoxypiperidin-4-yl (Fpmp) group.[12 – 14] In this way, Fpmp-protected RNA
intermediates can be isolated, facilitating the characterization of the products.
RESULTS AND DISCUSSION
Preparation of Oligonucleotides Carrying 4-Thiouridine Using the
1,2,4-Triazolyl Derivative of Uridine
Nucleoside derivatives carrying good leaving groups at specific positions can be
used to obtain oligonucleotides carrying modified bases.[4,5,8–11] The 1,2,4-triazolyl
group[5,15,16] was first selected to activate the position 4 of the uracil base in order to
obtain 4-thiouridine. To study the displacement conditions we prepared the 1,2,
4-triazolyl derivative of 2’,3’,5’-tri-O-acetyluridine. 2’,3’,5’-Tri-O-acetyluridine[17] (1,
Fig. 1) was reacted with a solution of phosphoryl tristriazolide[5,16] prepared by
Figure 1. Uridine derivatives 1–4.

Oligoribonucleotides Containing 4-Thiouridine
1769
mixing POCl3, 1,2,4-triazol and triethylamine in dry CH3CN. Compound 2 was then
treated with a 0.2 M solution of thiolacetic acid in CH3CN and with a 0.2 M solution
of NaSH in dimethylformamide (DMF). Both gave the desired 2’,3’,5’-tri-O-acetyl-4-
thiouridine (3, Fig. 1) but the reaction with NaSH was faster finishing after 3 hr at
room temperature.
Next, the commercially available uridine phosphoramidite carrying the Fpmp group
for the protection of the 2’-OH was reacted with the phosphoryl tristriazolide[5] solution
giving compound 4 (Fig. 1) in 85% yield after purification on silica gel.
Phosphoramidite 4 was reacted with thymidine linked to a controlled pore glass
(CPG) support to obtain after regular phosphite oxidation the dinucleotide sequence A:
5’ triUT 3’ (where triU is 4-(1,2,4-triazol-1-yl)-2-pyrimidon-1-yl-b-D-ribofuranoside).
The coupling reaction of phosphoramidite 4 proceeded in 95% yield, according to the
absorbance of the DMT group released after the coupling reaction. The resulting
support was then treated with a 0.2 M NaSH solution in DMF at room temperature for
3 hours, and, afterwards, with conc. NH3 for 1 hr at room temperature to release the
dinucleotide from the support and remove the cyanoethyl group. Only one peak was
observed, which was collected and found to have the expected molecular weight.
Oligoribonucleotide sequences B: 5’ triUG 3’ and C: 5’CUAGU(triU)CGAU 3’ were
assembled on a DNA synthesizer using Fpmp-protected nucleoside 2-cyanoethyl
phosphoramidites and solid supports. The resulting supports were treated with a 0.2 M
NaSH solution in DMF, and with conc. NH3. In these oligonucleotides ammonia
treatment must be longer than one hour in order to remove the protecting groups of
the bases. The effect of the ammonia treatment was studied on the dinucleotide B.
4S
Figure 2. HPLC purification of dinucleotide 5’
U
Fpmp
GFpmp 3’. Oligonucleotide support B: 5’
triUG 3’ was treated with a 0.2 M NaSH solution in DMF at room temperature for 3 hours and
the resulting supports were treated with concentrated ammonia a) at 50°C for 4 hours and b) at
50°C overnight.

1770
Avin˜o´ et al.
Overnight treatment at 50°C removed the thiol group of 4-thiouridine yielding the
dinucleotide carrying cytidine or uridine. The side product is more polar than the
desired oligonucleotide and it is easily separated by HPLC (see Fig. 2b). In this way,
the side product was isolated and characterized by mass spectrometry (MS,
electrospray, found1002.4 expected for CFpmpGFpmp 1002.8, expected for UFpmpGFpmp
1003.8). The formation of this side product is reduced by lowering the temperature
(24 hr, at room temperature) or the time of the ammonia treatment (4 h, 50°C, Fig. 2a).
Using a 4 h treatment at 50°C decamer C was obtained as the major product, and had
the expected molecular weight. Dinucleotide 5’ 4SUG 3’ was used to initiate the
transcription by T7 RNA polymerase. In this way, longer RNA transcripts with 4-
thiouridine at the 5’-end were obtained and used to study proteins involved in RNA
splicing, as described previously.[18]
We also found that samples with the (1,2,4-triazolyl) derivative should be
processed rapidly. After three months, both the phosphoramidite 4 and oligonucleotide
supports carrying the (1,2,4-triazolyl) derivative had lost the capacity to produce 4-
thiouridine. It is believed that the (1,2,4-triazolyl) derivative is hydrolyzed by humidity
to yield uridine even if the samples are maintained frozen. On the other hand, the 1,2,
4-triazolyl derivative of thymidine is much more stable. The presence of the methyl
group in thymidine may prevent hydrolysis by steric and electronic effects. For this
reason, a new uridine derivative was prepared.
Preparation of Oligonucleotides Carrying 4-Thiouridine Using the
2-Nitrophenyl Derivative of Uridine
The synthesis of the phosphoramidite derivative of 4-(2-nitrophenyl)uridine is
shown in Fig. 3. 3’,5’-O-Tetraisopropyldisiloxane-1,3-dilyl)-4-O-(2-nitrophenyl)uridine
(5) was prepared as described.[19] Compound 5 was reacted with 1-(2-fluorophenyl)-4-
methoxy-1,2,5,6 tetrahydropyridine[12] to give the 2’-Fpmp derivative 6 in 62% yield.
Removal of the silyl group with tetrabutylammonium fluoride produced compound 7 in
Figure 3. Uridine derivatives 5–9 carrying the 2-nitrophenyl group.

Oligoribonucleotides Containing 4-Thiouridine
1771
Figure 4. HPLC purification of oligonucleotide sequence E.
86% yield. 2’-Fpmp derivative 7 was reacted with dimethoxytrityl chloride and the
resulting compound 8 was reacted with 2-cyanoethoxy-N,N-diisopropylamino-chloro-
phosphine to obtain the desired phosphoramidite 9 in 91% yield.
Phosphoramidite 9 was reacted with thymidine linked to a controlled pore glass
(CPG) support to obtain the dinucleotide sequence A: 5’ npUT 3’ (being npU: 4-(2-
nitrophenyl)-uridine. The coupling reaction of phosphoramidite 9 proceeded in 94%
yield, similar to the Fpmp phosphoramidites of the natural nucleosides. The resulting
support was then treated with a 0.2 M NaSH solution, and with conc. NH3 at room
temperature. Only one peak was observed, which was collected and found to have
the expected molecular weight. Oligoribonucleotide sequence E: 5’ UAC(npU)GU 3’
was prepared using the appropriate Fpmp phosphoramidites. The support was treated
with a 0.2 M NaSH solution and the resulting support was then treated with conc.
ammonia as described above, producing the desired thiouridine oligonucleotide in good
yields (Fig. 4). This phosphoramidite maintained its properties for at least two years
when frozen.
CONCLUSION
We have shown that oligoribonucleotides containing 4-thiouridine can be prepared
from two different precursors: first, the 4-(1,2,4-triazolyl) derivative which can be
obtained from the readily available uridine phosphoramidite carrying the Fpmp group at
position 2’ but which has a limited shelf-life; second, the 4-(2-nitrophenyl) derivative,
which requires a longer synthetic scheme but, once prepared, presents a longer shelf-
life. These derivatives may be also used for N-15 labelling and preparation of
oligonucleotides carrying N-alkyl-C derivatives similarly as described with the
4-chlorophenyl uridine derivative.[11]
EXPERIMENTAL SECTION
General Procedures
Solvents, including those of HPLC grade, were from SDS and E. Merck. Reagents
were from Aldrich and Fluka and were used without further purification. Analytical

1772
Avin˜o´ et al.
TLC was run on aluminium sheets coated with silica gel 60 F254 from Merck. Silica gel
column chromatography was performed with Chromatogel 60 A C.C. (40–60 microns,
230–400 mesh, SDS). Fpmp-protected phosphoramidites and 1-(2-fluorophenyl)piper-
idin-4-one were obtained from Cruachem Ltd (Scotland). 1-(2-Fluorophenyl)-4-methoxy-
1,2,5,6 tetrahydropyridine was prepared from 1-(2-fluorophenyl)piperidin-4-one as
described.[12] 2´,3’,5’-tri-O-acetyluridine (1) was prepared by the reaction of uridine with
acetic anhydride in pyridine.[17] 3’,5’-O-Tetraisopropyldisiloxane-1,3-dilyl)-4-O-(2-nitro-
phenyl)uridine (5) was prepared as described with minor modifications.[19] 1H-NMR
(250 MHz), 13C-NMR (63 MHz) and 31P-NMR (75 MHz) spectra were recorded on a
Bruker AM-250. HPLC chromatography was performed on an HPLC Waters equipped
with an UV detector at 260 nm.
Standard Work-Up
All reactions were carried out in oven-dried glassware, under a nitrogen or argon
atmosphere, unless specified otherwise. Before use, starting materials were dried by
evaporation with the dry solvent that will be used for the reaction. Once the reaction
was completed, solutions were concentrated to dryness and the residues were dissolved
in CH2Cl2 or CHCl3 and washed with 5% aqueous NaHCO3 and saturated aqueous
NaCl. The organic phase was dried (Na2SO4) and the solvent was removed.
2’,3’,5’-O-Triacetyl-4-(1,2,4-Triazol-1-yl)-2-Pyrimidon-1-yl-b-D-
Ribofuranoside (2)
To an ice-cooled stirred suspension of 1,2,4-triazole (1,5 g, 21.7 mmol) in dry
CH3CN (30 ml) 0.42 ml (4.2 mmol) of POCl3 were added, followed by 3.27 ml
(23.4 mmol) of dry Et3N. After 30 min a solution of 2´,3’,5’-tri-O-acetyluridine[17] (1)
(2 mmol) in dry CH3CN was added over a period of 15 min and stirred for 2 h. The
reaction was stopped by addition of 25 ml of a saturated NaHCO3 aqueous solution and,
50 ml of ethyl acetate were added. The organic phase was washed with brine, dried over
Na2SO4 and concentrated to dryness. The resulting product (0.85 g unpurified with
Et3N) was used in the next step without further purification. TLC (10% MeOH in
CH2Cl2) one single fluorescent spot of Rf = 0.7. UV (max, MeOH) 243, 302 nm.
2’,3’,5’-Tri-O-Acetyl-4-Thiouridine (3)
Reaction with Thiolacetic Acid
Compound 2 (0.24 mmol) was treated with 0.142 ml (2 mmol) of thiolacetic acid
dissolved in 10 ml of CH3CN. After one day of magnetic stirring at room temperature
conversion to 2’,3’,5’-triacetyl thiouridine was judged to be around 50% by TLC (10%
CH3OH in CH2Cl2, starting material Rf = 0.7 fluorescent, final product Rf = 0.8 non
fluorescent). After 2 days the reaction was practically finished. The reaction mixture
was concentrated to dryness and treated using the standard work-up described above.
The product was purified by column chromatography. Yield: 58% (53 mg, 0.14 mmol).
Physical and spectroscopic data agreed with literature values.[20]

Oligoribonucleotides Containing 4-Thiouridine
1773
Reaction with Sodium Hydrogen Sulfide
2’,3’,5’-O-Triacetyl-4-(1,2,4-triazolyl)uridine (0.24 mmol) was treated with
148.2 mg (2 mmol) of sodium hydrogensulfide in 10 ml DMF. After 3 hr of magnetic
stirring the conversion was judged to be completed by TLC (see above). The reaction
mixture was concentrated to dryness and treated using the standard work-up described
above. The product was purified by column chromatography. Yield: 62 % (60 mg,
0.15 mmol. Physical and spectroscopic data agreed with literature values.[20]
5’-O-Dimethoxytrityl-2’-O-[1-(2-Fluorophenyl)-4-Methoxypiperidin-
4-yl]-4-(1,2,4-Triazolyl)Uridine 3’-O-[2-Cyanoethyl-N,
N-(Diisopropylamino)] Phosphoramidite (4)
The protocol described by Shah et al.[5] was used for the preparation of this
phosphoramidite. To an ice-cooled suspension of 1,2,4-Triazole (1.04 g, 15.5 mmol) in
dry CH3CN (20 ml) was added 0.3 ml of POCl3 was added, followed by 2.25 ml of
dry Et3N. After 30 min a solution of the DMT-U(Fpmp) phosphoramidite (188 mg,
0.25 mmol) in dry CH3CN was added over a period of 15 min and stirring was
continued for 2 hr. After standard work-up, the product was purified by column
chromatography (ethyl acetate/ CH2Cl2 2:3 + 1% triethylamine). Yield 170 mg, 85%.
31P-NMR (CDCl3): 151.3, 150.2 (two diastereoisomers) (Table 1).
3’,5’-O-(Tetraisopropyldisiloxane-1,3-Diyl)-2’-O-[1-(2-Fluorophenyl)-
4-Methoxypiperidin-4-yl]-4-O-(2-Nitrophenyl)Uridine (6)
3’,5’-O-(Tetraisopropyldisiloxane-1,3-diyl)-4-O-(2-nitrophenyl)uridine[18] (2.43 g,
4 mmol) and 1-(2-fluorophenyl)-4-methoxy 1,2,5,6-tetrahydropyridine (4.95 g, 24 mmol)
Table 1. 13C-NMR chemical shifts of compounds 1–8 (Cl3CD).
C4
C2
C6
C5
C1’
C4’
C2’
C3’
C5’
Other
1
2
3
4
5
6
7
8
163.2 150.1 136.5 102.8 87.2 79.4 72.3 69.7 62.8 Ac
169.3 153.2 142.5 94.4 89.7 79.1 72.7 66.7 61.7 Ac, tri
190.3 147.8 133.5 113.9 86.1 80.0 72.9 70.0 62.7 Ac
170.7 154.7 143.2
170.1 153.5 145.4
170.5 154.4 144.8
170.6 155.5 147.3
170.3 154.9 145.5
94.8 87.9 83.6 74.3 72.6 60.2 Tri, DMT, CE, iPr
93.7 91.8 81.0 73.5 68.2 59.9 Np
93.8 90.6 81.3 73.0 67.0 59.2 Np, Fpmp
95.8 89.8 86.0 73.0 71.0 61.6 Np, Fpmp,
94.8 88.0 83.7 74.4 70.2 62.4 Np, Fpmp, DMT
Signals from protecting groups: Ac (acetyl) : 168.5–169.5, 19.5–19.8; tri (1,2,4-triazolyl): 145.6;
DMT (dimethoxytrityl) : 158.5, 144.0, 135.0, 129.9, 127.9, 127.8, 127.0, 113.1, 87.0, 55.0; CE (2-
cyanoethyl): 116.2 and 115.8; 58.9 and 58.6; 20.8 and 20.6 (2 diasteroisomers); iPr (isopropyl):
43.1, 24.5; Np (2-nitrophenyl): 144.6, 141.2, 134.7, 126.5, 125.7, 125.2; Fpmp (1-(2-fluorophenyl)-
4-methoxypiperidin-4-yl): 157.3 and 153.4, 139.8 and 139.6, 124.1, 122.1, 119.2, 115.8 and 115.5,
100.5, 48.3, 47.8, 47.5, 34.0, 32.5.

1774
Avin˜o´ et al.
were dissolved in dry THF (40 ml). Mesitylenesulfonic acid dihydrate (0.25 g, 1.05 mmol)
was dried by coevaporation of dry CH3CN (2 Â 20 ml) under reduced pressure. The
dried mesitylenesulfonic acid was dissolved in dry CH3CN (10 ml) and added to the
THF solution. The reaction mixture was stirred overnight under anhydrous conditions,
whereupon TLC showed an almost complete reaction. Triethylamine was added and
after 5 minutes, THF was evaporated and the residue was dissolved in CH2Cl2. The
solution was treated with the standard work-up described above. The residue was
purified on silica gel using a 0–4% CH3OH gradient in CH2Cl2. Yield: 2.04 g
(2.5 mmol) 62%. Anal. Calcd for C39H55FN4O10Si2: C 57.47, H 6.80, N 6.87. Found C
57.05, H 6.67, N 6.61%. MS (FAB+) m/z: calculated for C39H55FN4O10Si2, 815; found
814.8 [M+].
2’-O-[1-(2-Fluorophenyl)-4-Methoxypiperidin-4-yl]-4-O-
(2-Nitrophenyl)Uridine (7)
Compound 6 (2.04g, 2.5 mmol) was dissolved in dry THF (50 ml) and 1.1 M
tetrabutylammonium fluoride in THF (5.5 ml) was added with stirring. Silica gel TLC
in ethanol/ CH2Cl2 (5: 95, v/v) showed a complete reaction after 5 minutes with a spot
of Rf.0.32. The reaction mixture was quenched with 16 ml of pyridine/methanol/water
(3:1:1), and the solution was poured into stirred Dowex 50Wx4 (200 mesh, pyridinium
form) resin suspended in 100 ml of pyridine/methanol/water (3:1:1). The mixture was
stirred for 20 min, the resin filtered off and washed with the same mixture (3 Â 50 ml).
The combined filtrates and washings were evaporated to dryness in vacuo. The residue
was purified by column chromatography on silica gel eluting with a gradient from
0 to 15% ethanol in CH2Cl2. Yield: 1.24 g (2.16 mmol) 86%. Anal. Calcd for
C27H30FN4O9: C 56.54, H 5.27, N 9.77. Found C 56.28, H 5.01, N 9.54%. MS (FAB+)
m/z: calculated for C27H30FN4O9, 573.5; found 573.9 [M+].
5’-O-Dimethoxytrityl-2’-O-[1-(2-Fluorophenyl)-4-
Methoxypiperidin-4-yl]-4-O-(2-Nitrophenyl)Uridine (8)
Compound 7 (1.24g, 2.16 mmol) was dried by evaporation of pyridine (3 Â 1,5 ml)
in vacuo. Anhydrous pyridine (30 ml) and 4,4’-dimethoxytrityl chloride (0.9 g
2.65 mmol) were added with stirring and exclusion of moisture. Silica gel TLC in
triethylamine/ethanol/CH2Cl2 (1:10:89, v/v/v) showed a complete reaction after 1.5 h
with a new spot of Rf. 0.65. The reaction was quenched by addition of methanol (5 ml)
and the solvent was removed in vacuo. The residual oil was dissolved in ethyl acetate
(50 ml) and the solution was washed as described above in the standard work-up.
Residual pyridine was removed by evaporation of toluene in vacuo. The crude product
was purified by column chromatography. The column was packed using a 1% triethyl-
amine solution in CH2Cl2 and the product was eluted with a 0–10% methanol gradient in
CH2Cl2. Compound 8 was obtained as a white foam. Yield: 1.69 g (1.93 mmol) 89%. Anal.
Calcd for C48H48FN4O11.2/3 CH2Cl2: C 62.76, H 5.34, N 6.02. Found C 62.40, H 5.21, N
6.04%. MS (FAB+) m/z: calculated for C48H48FN4O11, 875.9; found 874.9 [M+],
896.7 [M + Na+].

Oligoribonucleotides Containing 4-Thiouridine
5’-O-Dimethoxytrityl-2’-O-[1-(2-Fluorophenyl)-4-
1775
Methoxypiperidin-4-yl]-4-O-(2-Nitrophenyl)Uridine
3’-O-[2-Cyanoethyl-N,N-(Diisopropylamino)]
Phosphoramidite (9)
Compound 8 (1g, 1.14 mmol) was dried by evaporation of dry CH3CN (3 Â 10 ml)
in vacuo. The residual foam was dissolved in dry 1,2-dichloroethane (30 ml) containing
N,N-diisopropylethylamine (0.6 ml, 3.42 mmol) and the solution (under argon) was
cooled in an ice bath. 2-Cyanoethoxy-N,N-diisopropylamino-chlorophosphine (0.4 ml,
1.71 mmol) was added drop wise with stirring during approx. 1 min. The mixture was
kept 10 min at 0°C and was then stirred for 1 h at room temperature. TLC in
triethylamine/ CH2Cl2 showed a complete reaction with a product spot of Rf. 0.55.
Dichloromethane (50 ml) was added and the solution was washed as described above in
the standard work-up. The product was purified by column chromatography on silica
gel using CH2Cl2/ n-hexane (5:2) containing 2% triethylamine as eluant. Yield: 1.12 g
(1.04 mmol) 91%. 31P-NMR (Cl3CD) dP: 150.8 and 149.7 ppm. Anal. Calcd for
C57H65FN6O12P: C 63.62, H 6.09, N 7.81. Found C 63.25, H 6.13, N 7.69%.MS
(FAB+) m/z: calculated for C57H65FN6O12P, 1076.1; found 1097.1 [M + Na+].
Oligonucleotide Synthesis and Purification
Sequences A: 5’ triUT 3’, B: 5’ triUG 3’, C: 5’CUAGU(triU)CGAU 3’, D: 5’ npUT 3’,
and E: 5’ UAC(npU)GU 3’ were assembled using 35 mg (1 mmol) of the appropriate
supports and Fpmp-protected nucleoside 2-cyanoethyl phosphoramidites on a DNA
synthesizer (Applied Biosystems Mod. 394). Standard 1 mmol scale RNA synthesis
cycles were used. Coupling efficiencies were higher than 96%. The last DMT group
was removed before deprotection (DMT off) except in sequences C and E, in which it
was not removed (DMT on) so as to facilitate HPLC purification.
After the assembly of the sequences, supports were treated with a 0.2 M NaSH
solution in DMF (2 ml) at room temperature for 3 hours. The supports were washed
with DMF and CH3CN and dried. The resulting supports were treated with
concentrated ammonia at 50°C for 4 hours. The resulting Fpmp oligonucleotides were
purified by reverse-phase HPLC. HPLC conditions were as follows: Column : Nucleosil
120C18 (200 Â 4 mm), flow rate 1 ml/min, a 30 min linear gradient from 10 to 55%
CH3CN over 20 mM aqueous triethylammonium acetate. Overall (synthesis and
purification) yields were: sequence A (1 mmol scale): 5 OD, sequence B (4 mmol scale):
41 OD, sequence C (1 mmol scale): 19 OD, sequence D (1 mmol scale): 4 OD and D:
(1 mmol scale): 11 OD. All oligonucleotides have an UV maximum near 320 nm in
addition to the maximum near 260 nm. Oligonucleotides were analyzed by mass
4S
spectrometry (electrospray). 5’
U
T 3’: found 771.4 (M) calculated for
Fpmp
4S
C31H39FN5O13PS 771.5; 5’
43H52F2N9O14PS 1019.8; 5’ DMT-CFpmpUFpmpAFpmpGFpmpUFpmp
CFpmpGFpmpAFpmpUFpmp 3’: found 5511.1 (M) calculated for C227H275F10N44O83P9S
U
GFpmp 3’: found 1019.0 (M) calculated for
Fpmp
4S
C
U
Fpmp
4S
5448.1; 5’ DMT-UFpmpAFpmpCFpmp
for C149H173F6N25O49P5S 3398.3. The Fpmp and DMT groups were removed by
U
Fpmp
GFpmpUFpmp 3’: found 3399.3 (M) calculated
treatment of the samples with 0.5 ml of 10 mM aqueous HCl (pH 2.5–3) at room

1776
Avin˜o´ et al.
temperature overnight.[13,14] The samples were neutralized by adding 25 ml of sterile
2M Tris acetate pH 7 followed by ethanol precipitation of RNA with 3 volumes of
ethanol and 1/10 of volume of sterile 3M sodium acetate. Mass spectrometry (MALDI-
TOF). 5’ 4SUT 3’: found 586.4 (M + Na–H)À calculated for C19H25N4O12PS 564.3; 5’
4SUG 3’: found 627.5 (M + Na–H)À calculated for C19H24N7O12PS 605.3.
ABBREVIATIONS
A260 absorbance at 260 nm, controlled pore glass (CPG)
DMF N,N-dimethylformamide
DMT dimethoxytrityl
Fpmp 1-(2-fluorophenyl)-4-methoxypiperidin-4-yl
OD
optical density units at 260 nm
THF tetrahydrofuran
npU
4SU
triU
4-O-(2-nitrophenyl)uridine
4-thiouridine
4-(1,2,4-triazol-1-yl)-2-pyrimidon-1-yl-b-D-ribofuranoside
ACKNOWLEDGMENTS
We thank Drs. Matthias Mann, Gitte Neubauer, Matthias Wilm and Ole Jensen for
obtaining mass spectra. Anna Avin˜o´ was recipient of a EMBO short-term fellowship.
This work was supported by the Direccio´n General de Investigacio´n Cient´ıfica y
Te´cnica (grant BQU2003-00397), and the Generalitat de Catalunya (2001-SGR-0049).
REFERENCES
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Received April 19, 2004
Accepted July 2, 2004