Synthesis of methylketone containing nucleoside triphosphates for RNA labelling

Article abstract of DOI:10.1016/S0040-4020(00)00577-9

The three nucleoside triphosphates 1, 2 and 3 bearing a linker with a terminal methylketone group were prepared for incorporation into RNA fragments and post-labelling by the fluorescein derivative 4 containing an aminooxy group. (C) 2000 Elsevier Science Ltd.

Full text of DOI:10.1016/S0040-4020(00)00577-9

TETRAHEDRON
Pergamon
Tetrahedron 56 (2000) 6501±6510
Synthesis of Methylketone Containing Nucleoside Triphosphates
for RNA Labelling
a
Emmanuelle Trevisiol, Eric Defrancq, Jean Lhomme, Ali Laayoun and Philippe Cros
a
a,
b
b
*
Â
a
Â
LEDSS, Chimie Bioorganique, UMR CNRS 5616, Universite Joseph Fourier, BP 53, 38041 Grenoble Cedex 9, France
b
 Â
BioMerieux, Laboratoire Sondes Nucleiques, Chemin de l'Orme, 69280 Marcy-l'Etoile, France
Received 17 April 2000; revised 9 June 2000; accepted 30 June 2000
AbstractÐThe three nucleoside triphosphates 1, 2 and 3 bearing a linker with a terminal methylketone group were prepared for incorpora-
tion into RNA fragments and post-labelling by the ¯uorescein derivative 4 containing an aminooxy group. q 2000 Elsevier Science Ltd. All
rights reserved.
Introduction
ribonucleotides.5 It has been recently reported for conju-
gation of peptides with oligosaccharides6 and to
immobilise oligonucleotides on solid supports.7 In our
previous post-ampli®cation labelling approach,4 the modi-
®ed nucleotide, uridine or adenosine triphosphate, carried
the aminooxy function and the ¯uorescent probe contained
the aldehydic function. Enzymatic incorporation of such
modi®ed triphosphates into an RNA fragment was found
effective and a remarkable ef®ciency of the labelling by
aldehydic ¯uorescein was reached. However, some draw-
backs appeared: the synthesis of the modi®ed triphosphates
required protection of the aminooxy group and at the ®nal
stage the acidic deprotection had to be carried out using
large excess of methoxyamine requiring therefore tedious
puri®cation. We thus considered the `reverse strategy' in
which the aminooxy moiety is supported by the ¯uorescent
label and the carbonyl by the triphosphate. The methyl-
ketone function was preferred to the aldehyde, on the
hypothesis that the aldehyde could be more susceptible to
interfere with the enzymatic process. In the present paper,
we report on the preparation of the uridine, adenosine and
cytidine triphosphate derivatives 1, 2 and 3, all of which
incorporating a linker with a terminal methylketone
group.8 We describe the preparation of the aminooxy
containing ¯uorescein derivative 4 and subsequent coupling
with the methylketone derivatives (see Fig. 1).
High density oligonucleotide probe arrays (DNA chips)
represent powerful new tools for the analysis of DNA and
RNA sequences.1 This technology can be applied to nucleic
acid sequence analysis such as pathogen identi®cation,
polymorphism, mutations detection, or mapping.1 The prin-
ciple of the technology is based on the hybridisation of the
labelled nucleic acid sample to be analysed, i.e. the target,
with a large set of oligonucleotides immobilised on the chip
at precise locations. The hybridisation pattern of the labelled
nucleic acid target gives primary structure informations.2
Quite generally, prior to analysis on the chip, the nucleic
acid target is ampli®ed and labelled. This is most frequently
achieved in one step by incubating a modi®ed ¯uorescent
nucleotide triphosphate, in addition to the natural, during
the PCR-type ampli®cation.3
In a recent paper,4 we proposed an alternative procedure for
labelling RNA targets, called `post-ampli®cation labelling'.
The approach ®rst involves enzymatic incorporation of a
nucleotide triphosphate previously modi®ed so as to carry
a reactive functional group X. The resulting ampli®ed RNA
sequences are then labelled by reaction with a ¯uorophore
functionalised by a Y group complementary to X. Severe
limitations exist as to the nature of X and Y. The conju-
gation reaction must be selective, quantitative and rapid to
allow for speci®c labelling and possible automation. The X
group must remain inert to the enzymatic conditions for
ef®cient incorporation. The aminooxy-carbonyl coupling
reaction meets these criteria of selectivity and ef®ciency.
We used it in the past to prepare ¯uorescent oligodeoxy-
Results and Discussion
Synthesis of the uridine triphosphate derivative 1
(Scheme 1)
To introduce the substituent at the C-5 position of uridine,
we selected the method described by Hobbs10 which we
previously used for preparing the aminooxy containing
triphosphate derivatives.4 Preparation of the protected
Keywords: ¯uorescence; labelling; ketone; nucleotides; phosphorylation.
* Corresponding author. Fax: 133-04-76-51-43-82;
e-mail: jean.lhomme@ujf-grenoble.fr
0040±4020/00/$ - see front matter q 2000 Elsevier Science Ltd. All rights reserved.
PII: S0040-4020(00)00577-9

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E. Trevisiol et al. / Tetrahedron 56 (2000) 6501±6510
6502
Figure 1. Structure of the modi®ed nucleosides triphosphates 1, 2, 3 and of ¯uorescein derivative 4.
Scheme 1.

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E. Trevisiol et al. / Tetrahedron 56 (2000) 6501±6510
6503
Scheme 2.
nucleoside 7 was accomplished as depicted in Scheme 1.
The alkynyl chain 6 was prepared in 80% yield by coupling
6-oxoheptanoic acid with propargylamine in the presence of
isobutylchloroformate. The alkynyl chain 6 was then intro-
duced on to the uridine ring by reaction of 5 with copper
iodide in the presence of triethylamine and tetrakis(tri-
phenylphosphine)palladium to afford the protected nucleo-
side 7 in 60% yield. Protection of the 20- and 30-hydroxyl
groups of the ribose moiety as an acetonide before intro-
duction of the alkynyl chain facilitated the puri®cation step.
We then determined the mildest acidic conditions required
for removal of the acetonide protecting group to be applied
in the ®nal stage of the syntheses. Treatment of 7 in 25%
aqueous tri¯uoroacetic acid at room temperature for 30 min
led to total removal of the acetonide. We thus prepared
the nucleoside 1 using Eckstein's procedure11 and acidic
hydrolysis. Reaction of the protected nucleoside 7 with
salicyl phosphorochloridite, subsequent reaction with
pyrophosphate and ®nal oxidation by iodine furnished the
intermediate protected nucleotide 9. The presence of the
acetonide group, of the chain at the C-5 position and of
the tributylammonium counterions rendered the nucleotide
lipophilic enough to allow for rapid and ef®cient puri®cation
by C18 reverse phase ¯ash chromatography. Deprotection
of the 20- and 30-hydroxyl groups was then achieved using
25% aqueous tri¯uoroacetic acid leading to the desired
nucleotide 1 as the sodium salt in 20% overall yield. The
troscopy and ESMS. The 31P NMR spectra showed in par-
ticular three signals at nearly 25.5, 210.5 and 221.3 ppm
corresponding, respectively, to the g, a and b phosphorus
atoms.
Synthesis of the adenosine triphosphate derivative 2
(Scheme 2)
To incorporate a chain containing the methyl ketone group
on the adenosine ring at the C-6 position, we used the key
intermediate 10 prepared by conventional route.4 Coupling
nucleoside 10 with 6-oxoheptanoic acid was performed
using isobutylchloroformate in strictly anhydrous con-
ditions leading to the protected nucleoside 11. Subsequent
treatment with tetrabutylammonium ¯uoride (TBAF)
removed the TBDMS protection and afforded the 20,30
protected nucleoside 12 in 85% overall yield from the key
intermediate 10. Triphosphorylation was then performed
using the same conditions as described above for the uridine
derivative 1. Nucleotide 2 was obtained as the sodium salt in
1
33% overall yield and characterised by H and 31P NMR
spectroscopy and ESMS.
Synthesis of the cytidine triphosphate derivative 3
(Scheme 3)
The methyl ketone chain at the C-4 position of cytidine was
introduced using the procedure developed by Czernecki and
1
structure of 1 was con®rmed by H and 31P NMR spec-

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E. Trevisiol et al. / Tetrahedron 56 (2000) 6501±6510
6504
Scheme 3.
Coll.12 This approach involves conversion of the hydroxyl
group at the C-4 position of uridine into a good leaving
group, i.e. a tosylate, followed by displacement with an
amino compound. Preparation of nucleotide 3 was accom-
plished by the route depicted in Scheme 3. The 50-hydroxyl
group was ®rst protected as the tert-butyldimethylsilyl ether
15. The C-4 position was then activated by treatment of 15
with tosyl chloride in basic conditions. The `activated'
nucleoside 16 was obtained in 76% overall yield from
commercial 20,30-isopropylidene uridine. The amine 17
was prepared in two steps from 5-oxohexanitrile as
described.13 Displacement of the tosylate in 16 was then
performed using 2 equiv. of the amine 17 to afford the
protected nucleoside 18 within 10 min at room temperature.
Finally, treatment with TBAF gave the desilylated
compound 19 in 40% overall yield from 20,30-isopropyl-
idene uridine. Nucleoside 19 was then transformed into
the triphosphate 3 using Eckstein's procedure and acidic
hydrolysis of the two protecting groups as described
above for the uridine derivative 1. The structure of nucleo-
side 3 was established by 1H and 31P NMR spectroscopy and
con®rmed by ESMS.
high polar character of 21. The ¯uorescein derivative 21 was
used directly without protection for coupling with the
protected O-(methoxycarbonyl)hydroxylamine 22 in the
presence of isobutylchloroformate to afford the protected
¯uorophore 23. No side reaction was observed in these
conditions. The Fmoc group was selected for protection of
the aminooxy function as it could be removed in mild alka-
line conditions, which did not affect the luminescent probe.
Compound 22 was prepared in quantitative yield by treat-
ment of commercial O-(carboxymethyl)hydroxylamine
hemihydrochloride with 9-¯uorenylmethylchloroformate.
Finally, the Fmoc protection of 23 was removed by basic
hydrolysis using piperidine in DMF to afford the ¯uoro-
phore 4 in 30% overall yield from FITC.
Aminooxy-methylketone coupling reaction (Scheme 5)
The ef®ciency of the coupling reaction was veri®ed by
reacting the ¯uorophore 4 with the unprotected nucleosides
8 and 13. The reactions were carried out at room tempera-
ture in water/acetonitrile solution using 2 equiv. of ¯uoro-
phore 4. The corresponding oxime ethers 24 and 25 were
thus obtained in 80±85% yield in isolated products. The
1
Preparation of the aminooxy ¯uorophore 4 (Scheme 4)
structure of each compound was determined by H NMR
using DQFcosy sequences and con®rmed by HRMS. The 1H
NMR spectra showed in particular two signals for the
oximic protons which clearly demonstrated that a dia-
stereoisomeric mixture of Z/E compounds was obtained.
The Z/E ratios, determined by 1H NMR for each compound
24 and 25 were, respectively, 60/40 and 70/30.
Compound 4 was prepared in a three step reaction from the
commercially available ¯uorescein isothiocyanate (FITC).
Reaction of FITC with an excess of 1,3-diaminopropane
gave compound 21 in 80% yield. Puri®cation had to be
performed using reverse phase chromatography due to the

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E. Trevisiol et al. / Tetrahedron 56 (2000) 6501±6510
6505
Scheme 4.
Scheme 5.

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E. Trevisiol et al. / Tetrahedron 56 (2000) 6501±6510
6506
tographies were done using silica gel (Merck 60, 200±
63 mm) or reverse phase silica gel (Merck-Lichroprep
RP18, 40±63 mm). Analytical HPLC were performed on a
Millipore-Waters equipment (two M-510 pumps, solvent
gradient M680) with a UV detector (M490 or diode array
990). Reverse phase analyses were performed using
m-bondapak C-18 column (Waters) with a methanol±
water pH 2.5 gradient, ¯ow 2 mL/min for 10 min. Anion
Exchange Chromatography used for the triphosphates
analysis was carried out using a Protein Pak DEAE 8HR
column (Waters, 8 mm, 10£100 mm) with Tris±HCl
20 mM, pH 7.6 for eluent A and Tris±HCl 20 mM, NaCl
0.5 M, pH 7.6 for eluent B with a ¯ow rate of 2 mL/min. The
gradient used was: 0±35% B in 40 min. Melting points were
measured on an Electrothermal Series IA9100 apparatus.
UV spectra were performed on a Perkin-Lambda 15UV/
VIS and Fourier Transform Infrared spectra on a Perkin±
Elmer Impact 400 spectrophotometer. NMR spectra were
recorded on Bruker AC200, Avance 300 and Varian Unity
plus 500 spectrometers. Spectra were referenced to the
residual proton solvent peaks. The DQFCOSY spectra
were run with 2048 points in t2, 400 points in t1 and 128
scans for each t1 values. The mass spectra were recorded on
a Delsi-Nermag R10-10 spectrometer. ES-MS analyses
were performed on a VG Platform II (Micromass) in the
negative ion mode. Elemental analyses were performed by
`Service Central de Microanalyse du CNRS' and HRMS by
Figure 2. Gel electrophoresis analysis of the transcription products result-
ing from incorporation of the modi®ed triphosphate 1 (100% 1; 0% UTP)
compared to incorporation of UTP. Visualisation was performed by UV
before (A) and after (B) ethidium bromide staining. Lines 1 and 3: modi®ed
transcripts resulting from incorporation of 1 and subsequent incubation
with ¯uorophore 4. Lines 2 and 4: natural transcripts resulting from incor-
poration of UTP and subsequent incubation with 4.
The incorporation of the modi®ed triphosphates 1, 2 and 3
by T7 RNA polymerase into a fragment of 16S ribosomal
RNA from Mycobacterium tuberculosis or into a fragment
of the reverse transcriptase gene of HIV was evaluated. Fig.
2 shows a typical gel electrophoresis analysis of the tran-
scription products resulting from incorporation of nucleo-
side triphosphate 1 into a fragment of 16S RNA from M.
tuberculosis. The presence of a ¯uorescent band (lines 1 and
3) having the same electrophoretic mobility as the tran-
scripts resulting from incubation with the unmodi®ed
triphosphate (line 4) indicates that the modi®ed triphosphate
1 has been incorporated. It also shows the ef®ciency of the
subsequent labelling with ¯uorophore 4. The selectivity of
the labelling of the transcription products by ¯uorophore 4
was con®rmed by the absence of ¯uorescent transcripts
using the unmodi®ed triphosphate (line 2).
Â
`Centre Regional de Mesures Physiques de l'Ouest'. In
several cases, correct elemental analysis could not be
obtained due to the polar and hygroscopic character of the
compounds.
20,30-Isopropylidene-5-iodouridine 5. To a mixture of
5-iodouridine (3 g, 8.1 mmol) and APTS (0.15 g,
0.8 mmol) in acetone (50 mL) was added dropwise under
argon, ethylorthoformate (2.7 mL, 16.2 mmol). The mixture
was stirred overnight at rt, then sodium hydrogenocarbonate
(0.680 g, 8.1 mmol) was added. The solution was stirred for
30 min, ®ltered and evaporated to afford crude compound 5.
Recrystallisation in EtOH/hexane solution (1/1, v/v) gave
compound 5 as a white powder (2.5 g, 75%). Mp 220±
Conclusion
1
2218C. H NMR (DMSO d6, 200 MHz): d ppmˆ1.27 and
We have prepared a new series of nucleotide triphosphates
which have been shown to be enzymatically incorporated
successfully into RNA fragments. The three nucleotides 1, 2
and 3 could be prepared in satisfactory yields.
0
00
1.47 (6H, 2s, 2 CH3 isoprop.), 3.57 (2H, m, C5 ,5 ±H), 4.08
0
(1H, m, C4 ±H), 4.73 (1H, dd, C3 ±H), 4.90 (1H, dd, C2 ±H),
0
0
0
0
5.14 (1H, t, C5 ±OH), 5.81 (1H, d, C1 ±H), 8.31 (1H, s, C6±
H), 11.72 (1H, br s, NH). 13C NMR (DMSO d6, 50 MHz): d
ppmˆ24.9 (CH3), 26.8 (CH3), 60.9 (CH), 69.3 (quat), 80.1
(CH), 83.7 (CH), 86.7 (CH), 91.1 (CH), 112.7 (quat), 145.9
(CH), 149.8 (quat), 160.4 (quat). MS (FAB (1), glycerol
matrix): m/eˆ411 [M1H]1.
Labelling of the modi®ed RNA transcripts by the ¯uor-
escein derivative 4 showed remarkable selectivity and
ef®ciency. These results show that the new activated nucleo-
tides and ¯uorescent probe can be useful in molecular diag-
nostic based on nucleic acid targets ampli®cation and DNA
chip technology.
N-(2-Propynyl)-6-oxoheptanamide 6. To a solution of
6-oxoheptanoic acid (2.75 g, 18 mmol) in dry THF
(40 mL) cooled at 08C were added successively under
argon N-methylmorpholine (2 mL, 18 mmol), isobutyl-
chloroformate (2.3 mL, 18 mmol) then after 15 min
propargylamine (1.25 mL, 18 mmol) and the reaction was
stirred for 30 min at rt. The solvent was evaporated and the
residue obtained was dissolved in CH2Cl2. The organic layer
was washed successively with aqueous NaOH (0.1N),
aqueous HCl (0.1 N), brine and dried (Na2SO4). The crude
product was puri®ed by silica gel column chromatography
(EtOAc) to give compound 6 as a white powder (2.6 g,
Experimental
General
All commercially available chemical reagents were used
without further puri®cation. The nucleoside 10 was
prepared as previously described.4 Analytical TLC was
performed on 0.2 mm silica 60 coated aluminium foils
with F-254 indicator (Merck). Prep. column chroma-

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E. Trevisiol et al. / Tetrahedron 56 (2000) 6501±6510
6507
80%). Mp 88±908C. 1H NMR (CDCl3, 200 MHz): d
ppmˆ1.57 (4H, m, CH2±CH2), 2.10 (3H, s, COCH3),
2.17±2.20 (3H, m, C C±H and NHCOCH2), 2.43 (2H, t,
COCH2), 4.00 (2H, dd, ±CH2), 6.09 (1H, br s, NH). 13C
NMR (CDCl3, 50 MHz): d ppmˆ23.0 (CH2), 24.7 (CH2),
28.8 (CH2), 29.8 (CH3), 35.7 (CH2), 43.1 (CH2), 71.0 (CH),
79.7 (quat), 172.5 (quat), 209.0 (quat). MS (CI): m/
eˆ182 [M1H]1. IR (KBr): 3273, 3077, 2930, 2873,
Nucleoside triphosphate 1. The protected nucleoside 7
(0.232 g, 0.5 mmol) was dissolved in anhydrous pyridine
and coevaporated twice to dryness in vacuo. To the resi-
due obtained, pyridine (0.5 mL), dioxan (1.5 mL) and a
solution of 2-chloro-4H-1,3,2-dioxaphosphorin-4-one in
dioxan (1 M, 0.65 mL, 0.65 mmol) were added under
argon. After 20 min, a solution of tributylammonium
pyrophosphate (0.5 M) in anhydrous DMF (1.60 mL,
0.80 mmol) and tri-n-butylamine (0.65 mL) were added.
The reaction was stirred for 30 min, then a solution of 1%
iodine (10 mL, 0.39 mmol) in pyridine/water (98/2, v/v)
was added. After 20 min, excess iodine was destroyed by
addition of a 5% aqueous NaHSO3 solution and after 10 min
the solution was evaporated to dryness. The residue was
dissolved in water (60 mL) and the aqueous layer was
washed with CH2Cl2 (20 mL). The crude product was puri-
®ed by C18 reverse phase column chromatography (H2O
then H2O/MeOH: 50/50, v/v) giving the intermediate
protected triphosphate 9 (0.16 mmol, 30%). tRˆ32 min.
31P NMR (D2O, 80 MHz): d ppmˆ222.40 (t, Pb),
2114, 1706, 1640, 1550 cm21
.
Anal. Calcd for
C10H15NO2, 0.25 H2O: C 64.67 H 8.41 N 7.54, found C
64.51 H 8.30 N 7.69.
Protected nucleoside 7. To a mixture of compound 5 (0.5 g,
1.22 mmol) and copper iodide (44 mg, 0.232 mmol) in
degassed DMF (5 mL) were added, under argon, triethyl-
amine (0.33 mL, 2.32 mmol) and alkyne
3.48 mmol). The reaction was stirred in the dark for
10 min then tetrakis(triphenylphosphine)palladium
6 (0.6 g,
(0.134 g, 0.116 mmol) was added. After stirring under
argon for 3 h, the solvent was evaporated and the crude
product was puri®ed by silica gel column chromatography
(EtOAc/MeOH: 95/5 then 90/10, v/v) to give nucleoside 7
as a white powder (0.34 g, 60%). Mp 138±1408C. 1H NMR
(DMSO d6, 200 MHz): d ppmˆ1.27 and 1.47 (6H, 2s, 2
CH3 isoprop.), 1.42±1.44 (4H, m, CH2±CH2±CH2±CH2),
2.05 (3H, s, COCH3), 2.06±2.10 (2H, m, NHCOCH2),
1
211.20 (d, Jˆ18 Hz, Pa), 29.80 (d, Jˆ20 Hz, Pg). H
NMR (D2O, 200 MHz): d ppmˆ1.22 and 1.42 (6H, 2s, 2
CH3 isoprop.), 1.36±1.45 (4H, m, CH2±CH2±CH2±CH2),
1.99 (3H, s, COCH3), 2.10 (2H, t, COCH2), 2.38 (2H, t,
0
COCH2), 3.98 (2H, s, ±CH2), 4.01±4.09 (2H, m, C5 ,5
00
±
0
H), 4.45 (1H, m, C4 ±H), 4.88 (2H, m, C2 ±H and C3 ±H),
0
0
0
2.40 (2H, t, COCH2), 3.56 (2H, m, C5 ,5 ±H), 4.04 (3H, m,
00
0
5.67 (1H, m, C1 ±H), 7.89 (1H, s, C6±H). The protected
0
C4 ±H and CH2NH), 4.74 (1H, dd, C3 ±H), 4.90 (1H, dd,
0
nucleotide 9 (0.045 mmol) was then dissolved in water
(15 mL) and a 50% aqueous solution of TFA (15 mL) was
added. The mixture was stirred for 15 min then the solvent
was evaporated. The residue obtained was dissolved in
water (10 mL) and the pH was adjusted at 8.5 by addition
of aqueous NaOH (0.1N). The crude product was puri®ed
using C18 reverse phase column chromatography by eluting
with H2O. After evaporation, the nucleotide 1 was obtained
as a white powder (0.03 mmol, 70%). 31P NMR (D2O,
80 MHz): d ppmˆ221.28 (t, Pb), 210.45 (d, Pa), 25.55
0
0
0
C2 ±H), 5.13 (1H, t, C5 ±OH), 5.81 (1H, d, C1 ±H), 8.11
(1H, s, C6±H), 8.30 (1H, t, CH2NH), 11.68 (1H, br s,
NH). 13C NMR (DMSO d6, 50 MHz): d ppmˆ22.7 (CH2),
24.4 (CH2), 24.9 (CH3), 26.8 (CH3), 28.3 (CH2), 29.5 (CH3),
34.7 (CH2), 42.2 (CH2), 60.9 (CH2), 73.7 (C C), 80.1 (CH),
83.6 (CH), 86.6 (CH), 89.7 (CuC), 91.1 (CH), 98.0 (quat),
112.7 (quat), 144.7 (CH), 149.2 (CvO), 161.4 (CvO),
171.5 (CvO), 208.2 (COCH3). MS (CI): m/eˆ464
[M1H]1. UV (MeOH): lmax(e)ˆ288 (13000). IR (KBr):
3494, 3330, 3060, 2930, 1722, 1640, 1560, 1453 cm21
.
(d, Pg). H NMR (D2O, 200 MHz): d ppmˆ1.39 (4H, m,
1
HRMS (FAB (1)) Calcd for C22H30N3O8: 464.2033,
found 464.2027.
CH2±CH2±CH2±CH2), 1.99 (3H, s, COCH3), 2.11 (2H, t,
COCH2), 2.38 (2H, t, COCH2), 3.98 (2H, s, ±CH2), 4.00±
0
4.10 (3H, m, C4 ±H and C5 ,5 ±H), 4.18 (1H, m, C3 ±H),
0
00
0
0
0
Deprotected nucleoside 8. The protected nucleoside 7
(0.2 g, 0.43 mmol) was dissolved in a 25% aqueous TFA
solution (10 mL) and the solution was stirred at rt for
30 min. The solvent was then evaporated and the residue
obtained was puri®ed by C18 reverse phase column
chromatography (H2O/MeOH: 50/50, v/v) to give
compound 8 as a white powder (0.143 g, 79%). Mp 138±
4.27 (1H, m, C2 ±H), 5.76 (1H, d, Jˆ4.6 Hz, C1 ±H), 7.97
(1H, s, C6±H). ES-MS (negative mode): m/eˆ662 [M2H]2,
684 [M2H1Na]2, 706 [M22H12Na]2.
Protected nucleoside 12. To a solution of 6-oxoheptanoic
acid (0.288 g, 2 mmol) in dry THF (5 mL) cooled at 08C
were added successively under argon N-methylmorpholine
(0.223 mL, 2 mmol), isobutylchloroformate (0.258 mL,
2 mmol) then after 15 min the nucleoside 10 (0.980 g,
2 mmol) and the reaction was stirred at rt for 2 h. The
solvent was then evaporated and the oily residue obtained
was dissolved in Et2O. The organic layer was washed with
aqueous NaOH (0.1N), brine, then dried (Na2SO4).
Evaporation of the solvent afforded the intermediate nucleo-
side 11 as an oily residue which was used without further
puri®cation in the next reaction. The crude product 11 was
dissolved in THF (10 mL) and a 1 M TBAF solution in THF
(3.35 mL, 3.35 mmol) was added and the reaction was
stirred for 1 h. The solvent was then evaporated and the
residue obtained was dissolved in CH2Cl2. The organic
layer was washed with brine, dried (Na2SO4) and evapo-
rated. The crude product was puri®ed by silica gel column
1
1408C. H NMR (DMSO d6, 300 MHz): d ppmˆ1.44 (4H,
m, CH2±CH2±CH2±CH2), 2.04 (3H, s, COCH3), 2.07 (2H,
m, NHCOCH2), 2.40 (2H, t, COCH2), 3.50±3.70 (2H, m,
0
C5 ,5 ±H), 3.85 (1H, m, C4 ±H), 3.96 (1H, m, C3 ±H), 4.00±
00
0
0
0
4.10 (3H, m, CH2NH and C2 ±H), 5.00 (1H, d, C3 ±OH),
0
0
5.12 (1H, t, C5 ±OH), 5.33 (1H, d, C2 ±OH), 5.74 (1H, d,
0
0
Jˆ5 Hz, C1 ±H), 8.19 (1H, s, C6±H), 8.24 (1H, t, NHCO).
13C NMR (DMSO d6, 50 MHz): d ppmˆ24.0 (CH2), 26.1
(CH2), 30.5 (CH2), 30.8 (CH3), 36.7 (CH2), 44.1 (CH2), 61.7
(CH2), 70.5 (CH), 74.8 (C C), 75.4 (CH), 85.7 (CH), 90.9
(CH), 91.4 (C C), 100.2 (quat), 146.1 (CH), 151.6 (quat),
165.3 (quat), 177.3 (quat), 212 (quat). MS (FAB (2),
glycerol matrix): m/eˆ422 [M2H]2. Anal. Calcd for
C19H25N3O8, 0.5 H2O: C 52.77 H 6.06 N 9.72, found C
53.01 H 6.06 N 9.88.

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E. Trevisiol et al. / Tetrahedron 56 (2000) 6501±6510
6508
chromatography (CH2Cl2 then CH2Cl2/MeOH: 95/5, v/v) to
give compound 12 as a white resin (0.860 g, 85%). 1H NMR
(CDCl3, 200 MHz): d ppmˆ1.34 and 1.61 (6H, 2s, 2 CH3
isoprop.), 1.50±1.70 (8H, m, 4 CH2), 2.10 (3H, s, COCH3),
2.15 (2H, m, COCH2), 2.43 (2H, t, COCH2), 3.27 (2H, m,
uridine (5 g, 17.6 mmol) in anhydrous pyridine (30 mL)
was added under argon tert-butyldimethylsilyl chloride
(3 g, 19.4 mmol). The solution was stirred for 6 h and the
solvent was then evaporated. The oily residue obtained was
dissolved in CH2Cl2 and the organic layer was washed with
brine and dried (Na2SO4). Evaporation of the solvent gave
0
CH2NH), 3.68 (2H, m, CH2NH), 3.92 (2H, m, C5 ,5 ±H),
00
1
0
4.50 (1H, m, C4 ±H), 5.07 (1H, m, C3 ±H), 5.17 (1H, m,
0
compound 15 as a white powder (5.9 g, 85%). H NMR
(CDCl3, 200 MHz): d ppmˆ0.04 (6H, s, Si(CH3)2), 0.85
0
0
0
C2 ±H), 5.81 (1H, d, Jˆ4.8 Hz, C1 ±H), 6.09 (1H, t, C5 ±
OH), 6.67 (1H, br m, NH), 7.76 (1H, s, C8±H), 8.27 (1H, s,
C2±H). 13C NMR (CDCl3, 50 MHz): d ppmˆ25.1 (CH2),
27.1 (CH2), 27.3 (CH3), 28.8 (CH2), 29.1 (CH2), 29.7 (CH3),
32.0 (CH3), 38.5 (CH2), 41.1 (CH2), 42.3 (CH2), 45.2 (CH2),
65.4 (CH2), 83.6 (CH), 84.9 (CH), 88.0 (CH), 96.3 (CH),
115.8 (quat), 141.4 (CH), 154.5 (CH), 157.2 (quat), 172
(NHCO), 209 (COCH3). MS (CI): m/eˆ505 [M1H]1. UV
(MeOH): lmax(e)ˆ267 (17000). IR (KBr): 3333, 2934,
(9H, s, Si±C(CH3)3), 1.30 and 1.54 (6H, 2s, 2 CH3 isoprop.),
0
3.85 (2H, m, C5 ,5 ±H), 4.25 (1H, m, C4 ±H), 4.66 (1H, dd,
00
0
0
0
C3 ±H), 4.71 (1H, dd, C2 ±H), 5.64 (1H, d, Jˆ7.2 Hz, C5-
0
H), 5.94 (1H, d, C1 ±H), 7.63 (1H, d, Jˆ7.2 Hz, C6±H), 9.65
(1H, s, NH). 13C NMR (CDCl3, 50 MHz): d ppmˆ25.6 and
25.5 (CH3), 18.2 (quat), 25.3 (CH3), 25.7 (CH3), 27.2
(CH3), 63.2 (CH2), 80.2 (CH), 85.2 (CH), 86.6 (CH), 91.8
(CH), 102.1 (CH), 113.9 (quat), 140.6 (CH), 150.2 (quat),
163.5 (quat). MS (FAB (2), glycerol matrix): m/eˆ397
[M2H]2.
2863, 1716, 1618 cm21
.
HRMS (CI) Calcd for
C24H37N6O6: 505.2775, found 505.2761.
Deprotected nucleoside 13. As described for 8, compound
13 was prepared by acidic treatment of 12 (yield: 65%). Mp
50-tert-Butyldimethylsilyl-20,30-O-isopropylidene-4-O-
tosyl-uridine 16. To a solution of the protected nucleoside
15 (2.4 g, 6 mmol) in acetonitrile (50 mL) were added
potassium carbonate (1 g, 7.2 mmol) and tosyl chloride
(1.26 g, 6.6 mmol). The solution was re¯uxed for 4 h then
®ltered and evaporated. The oily residue obtained was
dissolved in EtOAc and the organic layer was washed
with water, dried (Na2SO4) and evaporated to afford
1
74±758C. H NMR (DMSO d6, 300 MHz): d ppmˆ1.39±
1.46 (6H, m, 3 CH2), 1.57 (2H, m, CH2), 2.00 (2H, m,
COCH2), 2.03 (3H, s, COCH3), 2.38 (2H, t, COCH2), 3.04
(2H, m, CH2NH), 3.46 (2H, m, CH2NH), 3.60 (2H, m,
0
C5 ,5 ±H), 3.95 (1H, m, C4 ±H), 4.13 (1H, m, C3 ±H), 4.58
00
0
0
0
0
(1H, m, C2 ±H), 5.87 (1H, d, Jˆ6 Hz, C1 ±H), 7.73 (1H, t,
NH), 8.05 (1H, t, NH), 8.21 (1H, s, C8±H), 8.36 (1H, s, C2±
H). 13C NMR (DMSO d6, 75 MHz): d ppmˆ23.0 (CH2),
25.0 (CH2), 26.8 (CH2), 29.8 (CH3), 35.4 (CH2), 38.4
(CH2), 42.6 (CH2), 61.8 (CH2), 70.8 (CH), 73.7 (CH),
86.1 (CH), 88.1 (CH), 140.0 (CH), 154.3 (CH), 158.1
(quat), 158.5 (quat), 171.9 (NHCO), 208.5 (COCH3). MS
(FAB (1), glycerol matrix): m/eˆ465 [M1H]1. HRMS
(FAB (1)) Calcd for C21H33N6O6: 465.2462, found
465.2436.
1
compound 16 as a white powder (3 g, 90%). H NMR
(CDCl3, 200 MHz): d ppmˆ0.00 (6H, s, Si(CH3)2), 0.80
(9H, s, Si±C(CH3)3), 1.30 and 1.53 (6H, 2s, 2 CH3 isoprop.),
0
2.40 (3H, s, Tos±CH3), 3.80 (2H, m, C5 ,5 ±H), 4.37 (1H, m,
00
0
0
0
0
C4 ±H), 4.64 (2H, m, C3 ±H and C2 ±H), 5.84 (1H, s, C1 ±
H), 5.98 (1H, d, Jˆ7.2 Hz, C5±H), 7.32 (2H, d, ArH Tos),
8.02 (2H, d, ArH Tos), 8.15 (1H, d, Jˆ7.2 Hz, C6±H). 13C
NMR (CDCl3, 50 MHz): d ppmˆ25.7 and 25.6 (CH3),
18.1 (quat), 21.7 (CH3), 25.1 (CH3), 25.7 (CH3), 27.1
(CH3), 63.2 (CH2), 79.9 (CH), 86.1 (CH), 88.1 (CH), 94.2
(CH), 94.6 (CH), 113.6 (quat), 129.5 (CH), 129.8 (CH),
132.9 (quat), 145.9 (quat), 146.3 (CH), 153.6 (quat), 166.5
(quat).
Nucleoside triphosphate 2. Using the same procedure
described for 1, the nucleotide 2 was obtained from
protected nucleoside 12 as a white powder with a 33% over-
all yield. Protected nucleotide intermediate 14: tRˆ30 min.
31P NMR (D2O, 80 MHz): d ppmˆ222.22 (t, Pb), 210.50
(d, Pa), 29.85 (d, Pg). 1H NMR (D2O, 200 MHz): d ppmˆ
1.26 and 1.48 (6H, 2s, 2 CH3 isoprop.), 1.10±1.30 (4H, m,
CH2±CH2), 1.40±1.60 (4H, m, CH2±CH2), 1.91 (3H, s,
COCH3), 1.97 (2H, t, COCH2), 2.25 (3H, t, COCH2), 3.02
(2H, t, CH2NH), 3.37 (2H, m, CH2NH), 3.92±4.14 (2H, m,
Protected nucleoside 18. To a solution of nucleoside 16
(3 g, 5.4 mmol) in CH2Cl2 (100 mL) was added the amine
17 (1.94 g, 12.2 mmol) and the reaction was stirred under
argon for 10 min at rt. The solution was then washed succes-
sively with water, with a 5% aqueous HCl solution, with
brine then dried (Na2SO4) and evaporated. Compound 18
was puri®ed by silica gel column chromatography (EtOAc
then EtOAc/MeOH: 95/5, v/v) and was obtained as a white
powder (1.8 g, 62%). Mp 51±548C. 1H NMR (CDCl3,
200 MHz): d ppmˆ0.04 (6H, s, Si(CH3)2), 0.83 (9H, s,
Si±C(CH3)3), 1.22 (3H, s, CH3), 1.26 and 1.54 (6H, 2s, 2
CH3 isoprop.), 1.30±1.68 (6H, m, 3 CH2), 3.42 (2H, m,
0
C5 ,5 ±H), 4.49 (1H, m, C4 ±H), 5.05 (1H, dd, C3 ±H), 5.19
00
0
0
0
0
(1H, m, C2 ±H), 6.05 (1H, d, Jˆ4 Hz, C1 ±H), 8.01 (1H, s,
C2±H), 8.22 (1H, s, C8±H). Nucleotide triphosphate 2:
tRˆ30 min. 31P NMR (D2O, 80 MHz): d ppmˆ221.37 (t,
Pb), 210.38 (d, Pa), 25.40 (d, Pg). 1H NMR (D2O,
200 MHz): d ppmˆ1.28 (4H, m, CH2±CH2), 1.49 (4H, m,
CH2±CH2), 1.92 (3H, s, COCH3), 1.98 (2H, t, COCH2), 2.27
(2H, t, COCH2), 3.04 (2H, t, CH2NH), 3.40 (2H, m,
0
00
CH2NH), 3.70±3.90 (2H, m, C5 ,5 ±H), 3.84 (4H, m,
0
O±CH2CH2±O), 4.15 (1H, m, C4 ±H), 4.75 (2H, m, C3 ±
0
0
CH2NH), 3.92±4.14 (2H, m, C5 ,5 ±H), 4.19 (1H, m, C4 ±
00
0
0
H and C2 ±H), 5.57 (1H, d, Jˆ7.5 Hz, C5±H), 5.80 (1H, m,
C1 ±H), 7.42 (1H, d, Jˆ7.5 Hz, C6±H). 13C NMR (CDCl3,
0
H), 4.40±4.50 (2H, m, C3 ±H and C2 ±H), 5.92 (1H, d,
0
0
0
Jˆ5.9 Hz, C1 ±H), 8.05 (1H, s, C2±H), 8.32 (1H, s, C8±
50 MHz): d ppmˆ25.6 and 25.5 (CH3), 18.2 (quat), 21.4
(CH2), 23.6 (CH3), 25.3 (CH3), 25.8 (CH3), 27.2 (CH3), 29.1
(CH2), 38.6 (CH2), 40.7 (CH2), 63.3 (CH2), 64.5 (CH2), 80.1
(CH), 85.5 (CH), 87.5 (CH), 93.4 (CH), 95.1 (CH), 109.8
(quat), 113.4 (quat), 140.7 (CH), 155.9 (quat), 163.7 (quat).
MS (FAB (-), glycerol matrix): m/eˆ538 [M2H]2.
H). ES-MS (negative mode): m/eˆ703 [M2H]2, 725
[M22H1Na]2, 747 [M23H12Na]2.
50-tert-Butyldimethylsilyl-20,30-O-isopropylidene-uridine
15. To a solution of commercial 20,30-O-isopropylidene-

Â
E. Trevisiol et al. / Tetrahedron 56 (2000) 6501±6510
6509
Nucleoside 19. The 50-protected nucleoside 18 (1.1 g,
2.04 mmol) was dissolved in THF (30 mL) and a 1 M
TBAF solution in THF (2.24 mL, 2.24 mmol) was added.
The reaction was stirred for 30 min and the solvent was
evaporated. The oily residue obtained was dissolved in
CH2Cl2 and the organic layer was washed with brine,
dried (Na2SO4) and evaporated. Compound 19 was obtained
after puri®cation by silica gel column chromatography
(CH2Cl2 then CH2Cl2/MeOH: 95/5, v/v) as a white powder
(0.7 g, 80%). Mp 64±658C. 1H NMR (CDCl3, 200 MHz): d
ppmˆ1.23 (3H, s, CH3), 1.28 and 1.49 (6H, 2s, 2 CH3
isoprop.), 1.36±1.42 (2H, m, CH2), 1.50±1.56 (4H, m, 2
acid 22. To a 10% NaHCO3 aqueous solution (25 mL) of
commercially available O-(carboxymethyl)hydroxylamine
hemihydrochloride (0.847 g, 7.75 mmol) cooled at 08C,
was added dropwise under argon a solution of 9-¯uorenyl-
methylchloroformate (2 g, 7.75 mmol) in dioxan (20 mL).
The reaction mixture was stirred overnight at rt and then
evaporated. To the residue obtained was added H2O
(250 mL) and the aqueous layer was washed with Et2O
(200 mL). The pH of the aqueous layer was then adjusted
pHˆ3 by addition of aqueous HCl (1N) and extracted with
CH2Cl2 (3£250 mL). The organic layers were then dried
(Na2SO4) and evaporated to afford the acid 22 as a white
powder (2.4 g, 98%). Mp 125±1268C. 1H NMR (DMSO d6,
200 MHz): d ppmˆ4.23 (1H, m, CH2±CH), 4.27 (2H, s,
OCH2CO), 4.36 (2H, d, OCH2±CH), 7.31 (2H, t, 2 ArH),
7.40 (2H, t, 2 ArH), 7.70 (2H, d, Jˆ 6.9 Hz, 2 ArH), 7.86
(2H, d, Jˆ6.9 Hz, 2 ArH). 13C NMR (DMSO d6, 50 MHz):
d ppmˆ46.3 (CH), 65.9 (CH2), 71.9 (CH2), 119.9 (CH),
125.0, 126.9 and 127.5 (CH), 140.5 (quat), 143.4 (quat),
156.8 (quat), 169.9 (quat). MS (DCI): m/eˆ314 [M1H]1.
Anal. Calcd for C17H15NO5: C 65.17 H 4.83 N 4.47, found:
C 65.54 H 4.96 N 4.50.
0
CH2), 3.36 (2H, m, CH2NH), 3.75 (2H, m, C5 ,5 ±H), 3.85
00
0
(4H, m, O±CH2CH2±O), 4.23 (1H, m, C4 ±H), 5.05 (1H, m,
0
0
C3 ±H), 5.17 (1H, m, C2 ±H), 5.27 (1H, dd, C1 ±H), 5.69
0
0
(1H, d, Jˆ7.2 Hz, C5±H), 6.22 (1H, t, C5 ±OH), 7.16 (1H, d,
Jˆ7.2 Hz, C6±H). 13C NMR (CDCl3, 50 MHz): d ppmˆ
21.3 (CH2), 23.6 (CH3), 25.1 (CH3), 27.2 (CH3), 28.9
(CH2), 38.5 (CH2), 40.8 (CH2), 62.7 (CH2), 64.5 (CH2),
80.4 (CH), 83.5 (CH), 87.5 (CH), 95.9 (CH), 98.8 (CH),
109.8 (quat), 113.6 (quat), 143.2 (CH), 156.4 (quat), 164.1
(quat). MS (FAB (2), glycerol matrix): m/eˆ424 [M2H]2.
Anal. Calcd for C20H31N3O7: C 56.46 H 7.34 N 9.88, found:
C 56.00 H 7.61 N 9.79.
Protected ¯uorescein probe 23. To a solution of acid 22
(0.473 g, 1.5 mmol) in anhydrous DMF (10 mL) cooled at
08C were added successively under argon N-methylmorpho-
line (0.166 mL, 1.5 mmol), isobutylchloroformate (0.193
mL, 1.5 mmol) then after 15 min the compound 21
(0.350 g, 0.75 mmol) and the reaction mixture was stirred
for 1 h at rt. The solvent was then evaporated and the residue
obtained was puri®ed by silica gel column chromatography
(CH2Cl2/MeOH: 50/50, v/v) to afford compound 23 as an
Nucleotide triphosphate 3. The nucleotide 3 was obtained
as a white powder with an 30% overall yield from protected
nucleoside 19 using the same procedure as for obtention of
1. Protected nucleotide intermediate 20: tRˆ32 min. 31P
NMR (D2O, 80 MHz): d ppmˆ221.20 (t, Pb), 210.21
1
(d, Pa), 26.56 (m, Pg). H NMR (D2O, 200 MHz): d
ppmˆ1.15 (3H, s, CH3), 1.22 and 1.43 (6H, 2s, 2 CH3
isoprop.), 1.23±1.60 (6H, m, 3 CH2), 3.18 (2H, t,
CH2NH), 3.80 (4H, m, O±CH2CH2±O), 4.03 (2H, m,
1
orange powder (0.227 g, 40%). Mp 162±1658C. H NMR
(DMSO d6, 200 MHz): d ppmˆ1.71 (2H, m, CH2), 3.17
(2H, m, CH2NH), 3.49 (2H, m, CH2NH), 4.13 (2H, s,
CH2±ONH), 4.22 (1H, t, CH±CH2), 4.42 (2H, m, OCH2±
CH), 6.55±6.68 (6H, m, ArH Fluo), 7.10 (1H, d, ArH Fluo),
7.28 (2H, t, ArH Fmoc), 7.33 (1H, m, ArH Fluo), 7.37 (2H,
t, ArH Fmoc), 7.61 (2H, dd, Jˆ6.8 Hz, ArH Fmoc), 7.82
(2H, dd, Jˆ6.8 Hz, ArH Fmoc), 8.01 (2H, m, 2 NH), 8.21
(1H, s, ArH Fluo), 9.95, 10.10 and 10.72 (4H, s, NH and
OH). 13C NMR (DMSO d6, 50 MHz): d ppmˆ26.08 (CH2),
28.1 (CH2), 35.7 (CH2), 41.0 (CH2), 46.2 (CH), 66.1 (CH2),
74.5 (CH2), 83.0 (quat), 102.0 (quat), 109.5 (CH), 112.3
(quat), 119.9 (CH), 124.0 (quat), 124.8 (CH), 126.3 (quat),
126.9 (CH), 127.6 (CH), 128.8 (CH), 140.5 (quat), 143.1
(quat), 147 (quat), 151.6 (quat), 157.2 (quat), 159.0 (quat),
167.9 (quat), 168.5 (quat), 180.0 (quat). MS (FAB (1),
glycerol matrix): m/eˆ759 [M1H]1. HRMS (FAB (1))
Calcd for C41H34N4O9S: 759.2125, found 759.2128.
0
00
0
0
C5 ,5 ±H), 4.39 (1H, m, C4 ±H), 4.76 (1H, m, C3 ±H), 4.88
0
0
(1H, m, C2 ±H), 5.83±5.87 (2H, m, C1 ±H and C5±H), 7.53
(1H, d, Jˆ7.6 Hz, C6±H). Nucleotide 3: tRˆ30 min. 31P
NMR (D2O, 80 MHz): d ppmˆ222.55 (t, Pb), 210.75
1
(d, Pa), 210.01 (d, Pg). H NMR (D2O, 200 MHz): d
ppmˆ1.41 (4H, m, 2 CH2), 2.00 (3H, s, COCH3), 2.42
(2H, m, COCH2), 3.17 (2H, m, CH2NH), 4.04±4.07 (3H, m,
0
00
0
0
C5 ,5 ±H and C4 ±H), 4.14±4.22 (2H, m, C3 ±H and C2 ±H),
0
0
5.82±5.90 (2H, m, C1 ±H and C5±H), 7.64 (1H, d, Jˆ7.6 Hz,
C6±H). ES-MS (negative mode): m/eˆ580 [M2H]2.
5-[3-(3-Aminopropyl)thioureido]-2-(6-hydroxy-3-oxo-
3H-xanthen-9-yl) benzoic acid 21. To a solution of 1,3-
diaminopropane (0.5 mL, 6.9 mmol) in anhydrous DMF
(20 mL), was added dropwise under argon a solution of
FITC (0.5 g, 1.16 mmol) in dry DMF (7 mL). The reaction
mixture was stirred for 30 min and the solvent was then
evaporated. The oily residue obtained was puri®ed by C18
reverse phase column chromatography (H2O then H2O/
MeOH: 50/50, v/v) to give compound 21 as an orange
Fluoresceine probe 4. The protected probe 23 (0.116 g,
0.16 mmol) was dissolved in anhydrous DMF (3 mL) and
piperidine (0.025 mL, 0.24 mmol) was added. The solution
was stirred for 15 min at rt and the solvent was evaporated.
The residue obtained was puri®ed by C18 reverse phase
column chromatography to give compound 4 as an orange
1
powder (0.42 g, 80%). Mp 205±2078C (dec.). H NMR
(DMSO d6±AcOD, 200 MHz): d ppmˆ1.91 (2H, m,
CH2), 2.94 (2H, m, CH2N), 3.58 (2H, m, CH2N), 6.45±
6.57 (6H, m, 6 ArH), 7.01 (1H, d, Jˆ8.2 Hz, ArH), 7.61
(1H, d, Jˆ8.2 Hz, ArH), 8.02 (1H, s, ArH). MS (FAB (1),
glycerol matrix): m/eˆ464 [M1H]1. HRMS (FAB (1))
Calcd for C24H22N3O5S: 464.1280, found: 464.1279.
1
powder (0.078 g, 93%). Mp 188±1908C. H NMR (DMSO
d6, 500 MHz): d ppmˆ1.72 (2H, m, CH2), 3.17 (2H, m,
CH2NH), 3.39±3.48 (2H, m, CH2NH), 3.52 (2H, s,
ONH2), 3.94 (2H, s, CH2±ONH2), 6.53±6.63 (6H, m,
ArH), 7.16 (1H, d, ArH), 7.72 (1H, m, ArH), 7.86 (1H, t,
NH), 8.19 (1H, s, ArH). MS (FAB (2), glycerol matrix):
2-[(N-Fluorenemethyloxycarbonyl)-aminooxy]-acetic

Â
E. Trevisiol et al. / Tetrahedron 56 (2000) 6501±6510
6510
m/eˆ535 [M2H]2. HRMS (FAB (1)) Calcd for
at room temperature for 30 min. The labelled transcription
products were analysed by electrophoresis on a 6% urea/
polyacrylamide gel in Tris±borate buffer (pH 8.5) at 150 V
for 1 h.
C26H24N4O7S: 537.1444, found: 537.1445.
Oxime ether 24. To a solution of deprotected nucleoside
8 (5 mg, 0.01 mmol) in a 50% aqueous acetonitrile solution
(1 mL) was added a 50% aqueous acetonitrile solution
(2.5 mL) of ¯uorophore 4 (12 mg, 0.02 mmol). The
solution was stirred for 2 h at rt and X-treme resin was
then added. The mixture was stirred for 30 min and ®ltered.
The resin was washed several times with a 50% aqueous
acetonitrile solution and the solvent was then evaporated.
The crude product was puri®ed by C18 reverse phase
column chromatography (H2O/CH3CN: 80/20, v/v) to give
Acknowledgements
We are very grateful to Viviane Monnot-Thomet (Bio-
Â
Merieux S.A.) for her precious help in the biological experi-
ments. We thank J. Garcia for his contribution in the
analysis of NMR spectra and D. Ruf®eux (Biochimie C,
CHU Grenoble) for recording ESMS spectra.
1
compound 24 as an orange powder (9 mg, 81%). H NMR
(DMSO d6, 500 MHz, 2 diastereoisomers): d ppmˆ1.35±
1.52 (4H, m, CH2CH2±CH2±C(CH3)vN±O), 1.67 (2H, m,
CH2±CH2±NH±CvS), 1.76 and 1.84 (3H, 2s, CH2±
C(CH3)vN±O), 2.09 (2H, m, NHCOCH2), 2.34 and 2.39
(2H, 2t, CH2±C(CH3)vN±O), 3.16 (2H, m, NH±
CH2CH2CH2±NH±CvS), 3.51 (2H, m, CH2±NH±CvS),
References
1. (a) Ramsay, G. Nature Biotechnol. 1998, 16, 40±44. (b) Ginot,
F. Human Mut. 1997, 10, 1±10. (c) Chee, M.; Yang, R.; Hubbell,
E.; Berno, A.; Huang, X. C.; Stern, D.; Winkler, J.; Lockhart, D. J.;
Morris, M. S.; Fodor, S. P. A. Science 1996, 274, 610±614.
2. Lipshutz, R. J.; Morris, D.; Chee, M.; Hubbell, E.; Kozal, M. J.;
Shah, N.; Shen, N.; Yang, R.; Fodor, S. P. A. Biotechniques 1995,
19, 442±447.
0
3.54±3.65 (2H, m, C5 ,5 ±H), 3.83 (1H, m, C4 ±H), 3.94
00
0
(1H, m, C3 ±H), 4.02 (1H, m, C2 ±H), 4.05 (2H, d, 2CH2),
0
0
0
4.31 and 4.33 (2H, 2s, vN±O±CH2CO), 5.74 (1H, d, C1 ±
H), 6.54±6.66 (6H, m, 6 ArH), 7.16 (1H, d, ArH), 7.66±7.74
(2H, m, ArH), 8.20 (2H, s, ArH and C6±H), 8.29 (1H, t,
NHCO), 8.29 (1H, t, NHCO). MS (FAB (1), glycerol matrix):
m/eˆ942 [M1H]1, 965 [M1H1Na]1. HRMS (FAB(1))
Calcd for C45H48N7O14S: 942.2980, found 942.3016.
3. (a) Zhu, Z.; Chao, J.; Yu, H.; Waggoner, A. S. Nucleic Acids
Res. 1994, 22, 3418±3422. (b) Sarfati, S. R.; Namane, A.
Tetrahedron Lett. 1990, 31, 2581±2584. (c) Hazlett, T. L.;
Moore, K. J. M.; Lowe, P. N.; Jameson, D. M.; Eccleston, J. F.
Biochemistry 1993, 32, 13575±13583. (d) Cremo, C. R.; Neuron,
J. M.; Yount, R. G. Biochemistry 1990, 29, 3309±3319.
Oxime ether 25. The oxime ether 25 was obtained in the
same manner as compound 24 in a 82% yield. H NMR
1
Â
4. Trevisiol, E.; Defrancq, E.; Lhomme, J.; Laayoun, A.; Cros, P.
Eur. J. Org. Chem. 2000, 211±217.
Â
5. (a) Trevisiol, E.; Renard, A.; Defrancq, E.; Lhomme, J.
(DMSO d6, 500 MHz, 2 diastereoisomers): d ppmˆ1.36±
1.50 (6H, m, NH±CH2CH2±CH2 and NHCOCH2CH2±
CH2±) 1.67 (2H, quint., CH2±CH2±NH±CvS), 1.75 and
1.83 (3H, 2s, CH2±C(CH3)vN±O), 2.09 (2H, m, NHCO±
CH2), 2.32 and 2.37 (2H, 2t, CH2±C(CH3)vN±O), 3.02
(2H, m, HN6±CH2CH2±CH2CH2NHCO), 3.16 (2H, m,
NH±CH2CH2CH2±NH±CvS), 3.45 (2H, m, HN6CH2),
Tetrahedron Lett. 1997, 38, 8687±8690. (b) Boturyn, D.;
Defrancq, E.; Ducros, V.; Fontaine, C.; Lhomme, J. Nucleosides
Nucleotides 1997, 16, 2069±2077.
6. Peri, F.; Dumy, P.; Mutter, M. Tetrahedron 1998, 54, 12269±
12278.
7. Salo, H.; Virta, P.; Hakala, H.; Prakash, T. P.; Kawasaki, A. M.;
0
3.50 (2H, m, CH2±NH±CvS), 3.53±3.65 (2H, m, C5 ,5
00
±
0
H) 3.94 (1H, m, C4 ±H), 4.13 (1H, m, C3 ±H), 4.31 and 4.32
0
0
(2H, 2s, vN±O±CH2CO), 4.60 (1H, t, C2 ±H), 5.86 (1H, d,
0
C1 ±H), 6.53±6.66 (6H, m, ArH), 7.14 (1H, d, ArH), 7.66±
È
Manoharan, M.; Lonnberg, H. Bioconjugate Chem. 1999, 10, 815±
823.
7.74 (2H, m, ArH), 8.32 (1H, s, C2±H), 8.41 (1H, s, C8±H).
MS (FAB (1), glycerol matrix): m/eˆ983 [M1H]1, 1005
[M1Na]1. HRMS (FAB(1)) Calcd for C47H54N10O12S:
983.3722, found 983.3713.
8. Oligonucleotides containing a carbonyl residue for labelling by
amino containing probes have been reported.9
9. (a) Dechamps, M.; Sonveaux, E. Nucleosides Nucleotides 1998,
17, 697±709. (b) Francois, Ph.; Muzzin, Ph.; Dechamps, M.;
Sonveaux, E. New J. Chem. 1994, 18, 649±657. (c) Vasseur,
J.-J.; Peoc'h, D.; Rayner, B.; Imbach, J.-L. Nucleosides Nucleo-
tides 1991, 10, 107±117. (d) Proudnikov, D.; Mirzabekov, A.
Nucleic Acids Res. 1996, 24, 4535±4542.
Incorporation and labelling assay. The PCR target
containing the T7 promoter was prepared as described.14
This PCR target was used for generating the modi®ed
single-stranded RNA target by in vitro transcription.
Transcriptions were carried out as described previously14
by incubating the PCR target, T7 RNA polymerase and
equimolar concentrations of the nucleotides at 378C for
1 h. The ratios of modi®ed versus natural nucleotides were
varied in the range 0:100, 30:70, 70:30, 100:0. Labelling
was then achieved by adding an excess of a solution of
the ¯uorescein derivative 4 (10 equiv.) in DMF to the
crude transcription mixture. The reaction was performed
10. Hobbs, F. W. J. Org. Chem. 1989, 54, 3420±3422.
11. Ludwig, J.; Eckstein, F. J. Org. Chem. 1989, 54, 631±635.
Â
12. Czernecki, S.; Mulard, L.; Valery, J.-M.; CommencËon, A.
Can. J. Chem. 1993, 71, 413±416.
13. Ozaki, S.; Watanabe, Y.; Nagase, T.; Ike, Y.; Mori, H. Chem.
Pharm. Bull. 1986, 34, 893±896.
14. Troesch, A.; Nguyen, H.; Miyada, C. G.; Desvarenne, S.;
Gingeras, T. R.; Kaplan, P. M.; Cros, P.; Mabilat, C. J. Clin.
Microbiol. 1999, 37, 49±55.