Synthesis and properties of fluorescent cycloSal nucleotides based on the pyrimidine nucleoside m5K and its 2′,3′-dideoxy analog dm5K

Article abstract of DOI:10.1002/ejoc.200500532

The synthesis of the fluorescent thymidine nucleoside analog 5-methyl-pyrimidin-2-one nucleoside 1 (m⁵K) and that of its 2′,3′-dideoxy derivative 2 (dm⁵K) are described. Moreover, the conversion of 1 and 2 into the corresponding 3-methyl-cycloSal- phosphate triesters 11 and 12 or an enzyme-cleavable prodrug 5-acetoxymethylpropionate-cycloSal-phosphate triester 13, and the fluorescence and hydrolysis properties of these new lipophilic triesters are reported. Finally, the suitability of these cycloSal pronucleotides as probes is demonstrated in a cell-extract hydrolysis experiment and a model study for cell uptake. Wiley-VCH Verlag GmbH & Co. KGaA, 2006.

Full text of DOI:10.1002/ejoc.200500532

FULL PAPER
DOI: 10.1002/ejoc.200500532
Synthesis and Properties of Fluorescent cycloSal Nucleotides Based on the
5
5
Pyrimidine Nucleoside m K and Its 2Ј,3Ј-Dideoxy Analog dm K
[a]
[a]
[a]
Henning J. Jessen, Wolfgang Fendrich, and Chris Meier*
Keywords: Pronucleotides / cycloSal triesters / Fluorescence / Nucleosides / Antiviral agents
The synthesis of the fluorescent thymidine nucleoside analog
ties of these new lipophilic triesters are reported. Finally, the
suitability of these cycloSal pronucleotides as probes is dem-
onstrated in a cell-extract hydrolysis experiment and a model
study for cell uptake.
5
5
-methyl-pyrimidin-2-one nucleoside 1 (m K) and that of its
5
2Ј,3Ј-dideoxy derivative 2 (dm K) are described. Moreover,
the conversion of 1 and 2 into the corresponding 3-methyl-
cycloSal-phosphate triesters 11 and 12 or an enzyme-cleav-
able prodrug 5-acetoxymethylpropionate-cycloSal-phos-
phate triester 13, and the fluorescence and hydrolysis proper-
(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim,
Germany, 2006)
Introduction
Nucleoside analogs are widely used as antiviral or antitu-
[
1]
mor agents. A severe limitation of these compounds is
that they often needed to be 5Ј-phosphorylated by host cell
enzymes into the 5Ј-triphosphates. The triphosphates act as
competitive inhibitors of DNA-polymerases, like the reverse
transcriptase of HIV, or they cause chain termination in
DNA-synthesis after incorporation.[ Often, the initial
phosphorylation into the nucleoside monophosphate (nu-
cleotide) is the metabolic limiting step. In some cases, the
nucleoside analog is entirely inactive in the nucleoside form,
but it is found to be highly active as the nucleoside triphos-
phate (e.g., 2Ј,3Ј-dideoxyuridine). However, applying nucle-
otides in chemotherapy has no benefit because nucleotides
are unable to penetrate the cell membrane and are sub-
strates of unspecific blood phosphatases. In order to over-
come this hurdle, lipophilic precursors have been developed
2]
Scheme 1.
choice. Despite the possibility of selecting specific fluores-
cence properties, attachment of a high-molecular-weight
fluorescent dye such as rhodamine, fluoresceine, or the dan-
syl group would change the size, lipophilicity, and/or hy-
drolysis properties of cycloSal compounds considerably. The
antiviral nucleoside analogs attached to the cycloSal moiety
are pyrimidine- and purine-type structures. Thus, a fluores-
cent analog that would be structurally as close as possible
to these nucleosides was needed. For the purine series, the
strong fluorescent 2-aminopurine 2Ј-deoxyriboside (2AP)
would be appropriate. However, as a surrogate for the pyr-
imidine-type nucleoside analogs, for example, 2Ј,3Ј-dideoxy-
thymidine (ddT) or 2Ј,3Ј-dideoxy-2Ј,3Ј-didehydrothymidine
[
3,4]
that release the nucleotides after membrane passage.
this context, we developed the cycloSal-pronucleotide ap-
In
[
5]
proach. It was shown with several antiviral active nucleo-
side analogs that the use of their corresponding cycloSal-
phosphate triesters led to improved biological activity due
to a successful bypass of phosphorylation limitations
[
6,7]
(Scheme 1).
Recently, the approach was extended by at-
(d4T), the highly fluorescent 5-methylpyrimidin-2-one nu-
taching an enzyme-cleavable group to the cycloSal moiety
5
cleoside 1 (m K), the deoxygenation product of thymidine,
[
8,9]
with the aim of trapping the compounds intracellularly.
[
10]
may be appropriate. Chemically, this compound is a thy-
midine analog, while functionally, it behaves like a cytidine
analog. The first synthesis of pyrimidin-2-one nucleoside
was carried out by direct reduction of the corresponding
thymidine or uridine derivative with sodium amalgam in
To study the intracellular fate of the cycloSal derivatives
and the subsequent cell uptake in more detail, we were
looking for a suitable probe. Radiolabeling of the cycloSal
nucleotides is very expensive and experimentally de-
manding; fluorescence labeling may thus be the method of
[
11]
low yields.
The approach has been modified sub-
5
sequently, and m K was successfully prepared by oxidation
of 4-hydrazinopyrimidinone nucleosides and rearrangement
of the intervening diazene with Ag O in refluxing aqueous
[
a] Institute of Organic Chemistry, University of Hamburg,
Martin-Luther-King-Platz 6, 20146 Hamburg, Germany
E-mail: chris.meier@chemie.uni-hamburg.de
2
924
© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Synthesis and Properties of Fluorescent cycloSal Nucleotides
FULL PAPER
dioxane or EtOH.[12] Holy published a method using 4-thio- give m K 1 in quantitative yield in the case of TBAF in
5
[
13]
[16]
pyrimidines and deactivated Raney-Ni.
Both methods THF.
5
led to m K in about 40–50% yield, although the yields were
This procedure has been applied to the synthesis of an
found to be better with ribonucleosides. However, all meth- analog of antivirally active 2Ј,3Ј-dideoxypyrimidine nucleo-
ods use metals, which are not desirable for syntheses of sides, the so far unknown 3Ј-deoxy-5-methyl-pyrimidine-2-
5
compounds to be used in biological systems because of pos- one nucleoside 2 (dm K) via intermediates 8–10 (Scheme 2).
5
sible toxic side effects. Recently, Singleton et al. published All steps proceeded with yields comparable to those of m K
a metal-free strategy by a mild, homogeneous oxidation (1). The comparable yields, particularly in the PTAD reac-
with N-phenyltriazolinedione (PTAD).[ However, in our tion step, are worth mentioning because Holy reported that
14]
5
hands this method led only to low yields of m K, most electron-acceptor groups should be introduced into the gly-
probably due to the labile triethylsilyl protecting groups con to stabilize the glycosidic bond in the reactions with
(TES) used for the 2Ј-deoxyribose.
metal oxides. Therefore, they introduced O-benzoyl groups.
Herein, we report on a slightly modified but reliable syn- Nevertheless, the yield of ribosylpyrimidin-2-one was 80%,
5
5
thetic protocol for the preparation of m K (1), the applica- whereas that of 2Ј-deoxyribosylpyrimidin-2-one (m K) was
[
12a]
5
tion of this protocol to the synthesis of the so far unknown 40–50%.
dideoxy analog dm K (2), the conversion of 1 and 2 into
5
5
Both, m K 1 and dm K 2 were subsequently converted
the cycloSal nucleotides, and the fluorescence and hydroly- into their 5Ј-cycloSal-phosphate triesters by the protocols
[
6]
sis properties of the latter compounds. Additionally, the described previously. The two 3-methyl-cycloSal-phos-
new fluorescent cycloSal-phosphate triesters were used as phate triesters 11 and 12 were obtained in 30–40% yield.
5
probes for cell-extract hydrolysis studies, demonstrating the In addition, one cycloSal-m KMP triester 13 bearing the
strength of the approach. Finally, a transport study with an esterase-cleavable 5-acetoxymethylpropionate (5-AM-propi-
“
aqueous phase-organic solvent-aqueous phase” system onate) group was prepared starting from the corresponding
[
9]
was used as a model for cell uptake.
chlorophosphite. This triester was obtained in a non-opti-
mized yield of 28%. After cell uptake, this compound
should be rapidly cleaved by esterases to acetic acid and
formaldehyde, releasing a highly polar carboxylate group,
which is negatively charged under physiological conditions.
As a consequence, the compound should be trapped intra-
cellularly.
Results and Discussion
Chemistry
In contrast to a previous report,[14] the more stable tert-
butyldimethylsilyl (TBDMS) group for the protection of the
5
glycon hydroxy groups was used in our m K synthesis pro-
Fluorescence Studies
tocol. Conversion of thymidine into 3Ј,5Ј-di-TBDMS-dT is
5
straightforward and proceeds in 90% yield. This compound
The absorption, excitation, and emission spectra for m K
5
was treated with P(O)Cl /triazole to give the 4-(triazol-1- (1) and dm K (2) were observed to be similar. The UV spec-
3
yl)-derivative 3 in quantitative yield. Compound 3 was tra showed maxima at λmax = 220 and 314 nm. Fluores-
treated with fresh anhydrous hydrazine (1  in THF), which cence excitation was achieved at 314 nm, while the fluores-
led to a substitution of the triazole ring to give the hydra- cence maximum was at 380 nm (Figure 1). Although the
zino intermediate 4.[ This material was immediately oxid- fluorescence of both pyrimidin-2-one nucleosides is 4 to 5-
ized with N-phenyltriazolinedione (PTAD, 1.2 equiv.) to fold weaker than that of the purine analog 2-aminopurine
15]
5
give 3Ј,5Ј-TBDMS-m K (5) in 60% yield (Scheme 2). It is 2Ј-deoxyriboside 14 (2AP), the advantage of these nucleo-
worth mentioning that the yields obtained in the last step sides is the red shift of the fluorescence band (λem
=
strongly depend on the quality of PTAD. The best results 380 nm) with respect to the emission band for 2AP (λem
were obtained when PTAD was prepared just prior to the = 352 nm). Thus, the fluorescence of the two nucleosides
reaction. In order to evaluate which of the two steps is the interferes only weakly with that of tryptophan (λem
yield-limiting step, we trapped the intermediate hydrazino 337 nm). Moreover, at the excitation wavelength (λex
=
=
derivative 4 by methylation. After substituting the triazol- 314 nm) neither tryptophan nor NAD(P)H are excited.
-yl ring by hydrazine, 4 equiv. methyl iodide was added, These spectral properties may have considerable advantages
1
leading to a stable double-methylation product 6 that was in studies carried out in biological media, for example, in
isolated in 88% yield. NMR spectroscopic analysis revealed cell extracts.
that both methyl groups were attached to the terminal
Interestingly, although the heterocycle was not modified,
amino group of compound 6. In addition, we isolated 4% the fluorescence intensity of the triesters 11 and 12 was
of unreacted 4-(triazol-1-yl)-thymidine 3 and the N,N-dimer found to be 40% lower than that of the nucleosides (Fig-
of 5-methyl-cytidine 7 in 8% yield. Consequently, the sub- ure 1, Table 1). It has been reported that the fluorescence
5
[10]
stitution reaction proceeded well, and the yield-limiting step of m K is strongly sensitive to its microenvironment.
was clearly the oxidation with PTAD. Finally, the TBDMS Here, an interaction of the pyrimidin-2-one ring with the
groups were cleaved with tetrabutylammonium fluoride cycloSal aromatic ring system led to a partial quenching of
(
TBAF) or triethylamine trihydrofluoride (NEt ·3HF) to the fluorescence. If this is true, the fluorescence should in-
3
Eur. J. Org. Chem. 2006, 924–931
© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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925

H. J. Jessen, W. Fendrich, C. Meier
FULL PAPER
Scheme 2.
crease during chemical hydrolysis because of nucleotide de- tection were used. In this context, it is worth mentioning
livery.
that the use of the fluorescent cycloSal triesters led to a
more than 1000-fold increase in sensitivity relative to UV
detection. The effect of this dramatic increase in sensitivity
Hydrolysis Studies
The fluorescent cycloSal triesters were hydrolyzed in on the detection of the hydrolysis products can be seen in
5 m phosphate buffer pH 7.3; UV- and fluorescence de- Figure 2.
2
926
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Synthesis and Properties of Fluorescent cycloSal Nucleotides
FULL PAPER
5
5
Figure 1. Fluorescence spectra of m K (1), dm K (2) and their pro-
5
nucleotide derivatives 3-Me-cycloSal-m KMP (11) and 3-Me-cy-
5
cloSal-dm KMP (12). Spectra were recorded in aqueous solution
and concentrations of 3.8 µ (11), 3.2 µ (1), 3.5 µ (12), and
4
.0 µ (2) with an excitation at 314 nm. At this wavelength, the
absorption of all compounds was identical.
Figure 2. Detection of the hydrolysis mixture of 3-Me-cycloSal-
5
dm KMP using either UV detection (a) or fluorescence detection
(
b): the sample was analyzed using an in-line setup of a UV detec-
Table 1. Chemical hydrolysis to yield the nucleotides and fluores-
tor and a fluorescence detector within an HPLC apparatus. R
2
t
:
cence properties of cycloSal triesters 11–13.[
a]
5
5
t
.3 min, dm KMP; R : 13.0 min, 3-Me-cycloSal-dm KMP.
[c]
[d]
t
1/2 (h)[b]
λ
ex
λ
em
Θ
re1
Θ
rel
1
1
6.8
n.a.
8.6
314
314
380
380
0.47
0.87
0.1
5
[g]
As expected, all triesters released the corresponding mo-
m K 1
0.19
[
e]
[g]
[g]
[g]
[g]
1
5
n.a.
314
314
n.a.
380
380
n.a.
0.57
1
n.a.
0.12
0.21
nophosphate derivatives of the two parent nucleoside ana-
logs. The half-lives at pH 7.3 were found to be in the same
stability range as the references 3-methyl-cycloSal-dTMP 15
or 3-methyl-cycloSal-ddTMP 16 (t1/2 8.6 h and 14.9 h,
respectively; Table 1).
1
2
15.4
5
[g]
dm K 2
1
1
n.a.
[
f]
[g]
[g]
[g]
[g]
6
3
14.9
2.0
n.a.
314
305
n.a.
380
355
n.a.
n.a.
0.14
1
0.65
[
g]
[g]
2
AP 14
n.a.
n.a.
Moreover, the expected increase of the total fluorescence
during hydrolysis due to the release of the nucleosides was
6 are also given for comparison. [b] Phosphate buffer (25 m, pH observed (Figure 3).
[
a] The corresponding data of the parent nucleosides 1, 2 and 2-
aminopurine 2Ј-deoxyriboside (2AP) 14 and cycloSal triesters 15,
1
7
.3, 37 °C). [c] Quantum yield relative to nucleoside 2. [d] Quantum
Chemical hydrolysis of 5-acetoxymethylpropionate-cy-
yield relative to 2AP 14. [e] 3-Me-cycloSal-dTMP 15. [f] 3-Me-cy-
cloSal-ddTMP 16. [g] Not available.
5
5
cloSal-m KMP (5-AM-propionate-cycloSal-m KMP) (13)
5
Figure 3. Hydrolysis study using 3-Me-cycloSal-dm KMP (12) in phosphate buffer, pH 7.3 using fluorescence detection.
Eur. J. Org. Chem. 2006, 924–931
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H. J. Jessen, W. Fendrich, C. Meier
FULL PAPER
had a half-life of 2.0 h, which is lower than previously ob- pH was set to 6.8 in both aqueous phases; and (ii) the pH
served for 5-alkyl-substituted cycloSal-2Ј-deoxynucleotides of the donor phase was 6.8 while that of the acceptor phase
(t1/2 = 4.1 h). Moreover, the fluorescence increased more was 8.7. In the first experiment, the expected formation of
than twofold on hydrolysis of the AM ester 13. an equilibrium between both phases was observed (data not
Fluorescence detection was used in the cell-extract hy- shown). However, in the second setup, an accumulation in
drolysis studies. Here, concentrations that were 500–1000- the acceptor phase was observed because the triester was
fold lower relative to UV-detection studies were used. As hydrolyzed to the nucleotide under these conditions. The
before, the increase in total fluorescence was observed again nucleotide formed was too polar to enter the organic phase
during the hydrolysis of the cycloSal triesters.
In cell extracts, 5-AM-propionate-cycloSal-m KMP (13)
again (Figure 4).
5
5
For 5-AM-propionate-cycloSal-m KMP (13) a different
was rapidly cleaved at the acylal site first and subsequently setup was used: The donor phase was again phosphate
5
hydrolyzed to give the nucleotide m KMP. These findings buffer, pH 6.8; however, the acceptor phase was phosphate
were confirmed in experiments in which pig liver esterase buffer, pH 7.3, containing PLE. As expected, an accumu-
(
PLE) was used in aqueous phosphate buffer, pH 7.3. The lation of fluorescent material in the acceptor phase was de-
acylal site was cleaved completely within 5 minutes to give tected (Figure 5).
the propionate. The compounds found in the acceptor phase by HPLC
Finally, fluorescence detection has been used in model were 5-propionate-cycloSal-m KMP and its hydrolysis
5
5
5
studies for cellular uptake. In these experiments, the mi- product m KMP. No 5-AM-propionate-cycloSal-m KMP
gration of the triesters from an aqueous donor phase to an (13) was detected because of its very high susceptibility to
aqueous acceptor phase via an organic phase (CH Cl ) was enzyme degradation. This experiment supports the idea of
2
2
monitored. Two different experimental setups of this U- a possible intracellular lock-in or trapping of the cycloSal-
tube experiment with triesters 11 and 12 were used: (i) the phosphate triesters.
Conclusions
A reliable synthetic procedure for the synthesis of two
fluorescent thymidine analogs and their conversion into the
corresponding cycloSal-phosphate triesters is described.
Hydrolysis data and the fluorescence properties of the cy-
cloSal-phosphate triesters proved that these compounds are
ideal equivalents for cycloSal-pyrimidine monophosphates
bearing antiviral nucleoside analogs like ddT or d4T, which
may be used as probes of important physiological processes.
The new phosphate triesters were used in cell-extract hy-
drolysis studies in which the products were detected by fluo-
rescence as well as in a first model study for a cell-uptake
process. These studies proved that an accumulation in the
acceptor phase could be achieved by pH difference and by
Figure 4. Diffusion experiment of 5-AM-propionate-cycloSal-
5
dm K 13 from a donor phase (pH 6.8) to an acceptor phase (pH
8.7).
5
Figure 5. Enzyme-driven transport of 5-AM-propionate-cycloSal-dm KMP 13 from an aqueous donor phase into an aqueous acceptor
phase containing PLE.
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Synthesis and Properties of Fluorescent cycloSal Nucleotides
FULL PAPER
enzyme-driven trapping. Further studies on the use of these arising from the mixed stereochemistry at the phosphate center.
The resulting lyophilized triesters did not give useful microanalyti-
fluorescent probes, e.g. in cell incubation studies, are con-
cal data, most probably because of incomplete combustion of the
tinuing in our laboratories.
compounds or varying amounts of water. However, they were
found to be pure by rigorous HPLC analysis (gradient of 5–100%
3 2
CH CN in H O within 25 min, flow 0.5 mL/min), high-field multi-
nuclear NMR spectroscopy and mass spectroscopy.
Experimental Section
All experiments involving water-sensitive compounds were con-
ducted under scrupulously dry conditions (under argon). Solvents:
Preparation of the Triazolo Compounds 3 and 8
3
To a suspension of 1,2,4-triazole (100 mmol) in anhydrous CH CN
2 2
Anhydrous dichloromethane (CH Cl ), tetrahydrofuran (THF),
3
(75 mL) at 0 °C was slowly added P(O)Cl (23 mmol) and then tri-
and acetonitrile (CH CN) were obtained in Sure/Seal bottles from
3
ethylamine (100 mmol). After 30 min at 0 °C, 3Ј,5Ј-O-bis(tert-bu-
tyldimethylsilyl)-2Ј-deoxythymidine or 5Ј-O-tert-butyldimethylsilyl-
Fluka and stored over 4 Å molecular sieves; ethyl acetate, dichloro-
methane, and methanol employed in chromatography were distilled
prior to use. N,N-Diisopropylethylamine (DIPEA) was distilled
from Na prior to use. Solvents for HPLC were obtained from
3
2Ј,3Ј-dideoxythymidine (6.50 mmol) in CH CN (40 mL) was added
dropwise over 30 min. The reaction mixture was stirred for a fur-
ther 20 h. It was then filtered, evaporated, and diluted with EtOAc.
3
Merck (CH CN, HPLC grade). Ion-pairing buffer solution was
2 3
The organic phase was washed with sat. Na CO and with brine.
prepared by mixing tetrabutylammonium hydroxide (6.6 mL) with
water (1000 mL). The pH value was adjusted to 3.8 by adding con-
centrated phosphoric acid (buffer I). Buffer I (60 mL) was diluted
with water (1000 mL) to form buffer II.
It was dried with Na SO and concentrated under reduced pressure.
Purification was carried out using a Chromatotron with CH Cl
2 2
and a MeOH-gradient (0–2% v/v).
2
4
3
Ј,5Ј-Bis(O-tert-butyldimethylsilyl)-4-(1,2,4-1H-triazol-1-yl)-thymi-
1
1
NMR spectra were recorded with a Bruker AMX 400 ( H NMR,
dine (3): Yield: 98% (colorless crystals; m.p. 104–107 °C). H NMR
(500 MHz, CDCl ): δ = 9.28 (s, 1 H), 8.24 (d, JH,H = 0.5 Hz, 1
3
00 MHz; 13C NMR, 101 MHz; P NMR, 162 MHz) or Bruker
31
4
4
1
13
31
3
3
DMX 500 ( H NMR, 500 MHz; C NMR, 123 MHz; P NMR, H), 8.10 (s, 1 H), 6.28 (dd, JH,H = 6.3, JH,H = 6.3 Hz, 1 H), 4.39
(ddd, JH,H = 6.6, JH,H = 3.6, 3JH,H = 3.6 Hz, 1 H), 4.06 (ddd,
3
3
2
02 MHz) spectrometer, with CDCl
3
or DMSO as internal stan-
1
13
3
J =
H,H = 2.8, 3JH,H = 3.4, 3JH,H = 3.4 Hz, 1 H), 3.96 (dd, JH,H
2
dard for H and C NMR spectroscopy and with H
3
PO
4
as exter-
nal standard for 31P NMR spectroscopy. All 1H and C NMR
13
3
2
3
11.5, JH,H = 2.5 Hz, 1 H), 3.79 (dd, JH,H = 11.5, JH,H = 2.5 Hz,
3
3
3
chemical shifts (δ) are quoted in parts per million (ppm) downfield
1 H), 2.64 (ddd, JH,H = 13.6, JH,H = 6.1, JH,H = 3.7 Hz, 1 H),
2.44 (d, JH,H = 0.7 Hz, 3 H), 2.07 (ddd, JH,H = 13.4, JH,H = 6.2,
H,H = 6.2 Hz, 1 H), 0.91 (s, 9 H, tBu), 0.90 (s, 9 H), 0.12 (s, 3 H,
-Si), 0.11 (s, 3 H), 0.08 (s, 3 H), 0.07 (s, 3 H) ppm. C NMR
3
3
3
from tetramethylsilane; (CD
reference. 31P NMR chemical shifts are quoted in ppm by using
PO as external reference. All spectra were recorded at room
3
)(CD
2
H)SO was set at δ
H
= 2.49 as a
3
J
1
3
H
3
4
CH
3
13
31
temperature, and C and P NMR spectra were recorded in the
proton-decoupled mode. Mass spectra were obtained with a Finni-
gan electrospray MAT 95 Trap XL (ESI) or a VG Analytical VG/
(100 MHz, CDCl ): δ = 158.1, 153.9, 153.4, 146.6, 145.1, 105.2,
3
88.8, 87.8, 71.6, 62.6, 42.6, 25.9, 25.7, 18.4, 18.0, 17.3, –4.6, –4.9,
+
–5.3, –5.4 ppm. MS (HR-FAB): calcd. 522.293 [M + H ]; found
70–250 F spectrometer [FAB, (double-focusing), matrix: m-ni-
522.294.
trobenzyl alcohol]. FAB high-resolution (HR) mass spectra were
recorded by using a VG Analytical 70–250S spectrometer with the
MCA method and polyethylene glycol as support. UV spectra were
recorded with a Varian Cary 1E UV/Vis spectrometer. Infrared
spectra were recorded with a Perkin–Elmer 1600 Series FT-IR or
a ATI Mattson Genesis Series FT-IR spectrometer; samples were
prepared in KBr pellets. Fluorescence spectra were recorded with a
Fluorolog 3 instrument; Jobin Yvon. Solvent: water (dist.); cuvette:
5
Ј-O-tert-Butyldimethylsilyl-4-(1,2,4-1H-triazol-1-yl)-2Ј,3Ј-dideoxy-
1
thymidine (8): Yield: 96 % (colorless oil). H NMR (500 MHz,
CDCl ): δ = 9.28 (s, 1 H), 8.39 (s, 1 H), 8.10 (s, 1 H), 6.08 (dd,
3
3
3
J
H,H = 3.2, JH,H = 6.6 Hz, 1 H), 4.30–4.25 (m, 1 H), 4.12 (dd,
2JH,H = 11.7, JH,H = 2.5 Hz, 1 H), 3.77 (dd, JH,H = 11.7, JH,H
3
2
3
=
2
.5 Hz, 1 H), 2.68–2.58 (m, 1 H), 2.45 (s, 3 H), 2.22–2.15 (m, 1 H)
.99–1.88 (m, 2 H), 0.93 (s, 9 H), 0.13 (s, 3 H), 0.12 (s, 3 H) ppm.
1
13
C NMR (100 MHz, CDCl
3
): δ = 155.1, 153.3, 147.1, 145.5, 145.0,
1
×1 cm; excitation wavelength: 314 nm; spectral bandwidth: 5 nm;
1
04.8, 88.5, 83.0, 63.9, 33.6, 26.0, 24.3, 18.6, 17.3, –5.2, –5.3 ppm.
stepwidth: 1 nm; temp.: 25 °C. In the HPLC system, a Merck Hita-
chi fluorescence detector -7480 was used. Chromatography: All
preparative TLCs were performed with a Chromatotron (Harrison
Research 7924) using glass plates coated with 1- or 2-mm layers of
Merck 60 PF254 silica gel containing a fluorescent indicator and
UV detection at 254 nm. Column chromatography: Merck silica gel
+
MS (HR-FAB): calcd. 392.2118 [M + H ]; found 392.2119.
Conversion of 3 and 8 into the Hydrazino Compounds and PTAD
Reduction (One-Pot Reaction)
3Ј,5Ј-Bis(O-tert-butyldimethylsilyl)-4-(1,2,4-triazol-1-yl)-thymidine
(
3) (2.53 mmol) or 5Ј-O-tert-butyldimethylsilyl-4-(1,2,4-triazol-1-
yl)-2Ј,3Ј-dideoxythymidine (8) (2.53 mmol) was dissolved in anhy-
drous dioxane (35 mL) and degassed. The solution was cooled until
dioxane began to solidify. After removal from the ice bath, anhy-
drous hydrazine/THF solution (5.0 mL, 1 , 5.00 mmol) was added
at once. The solution was stirred for 1 h at room temp. and was
then reduced to 5 mL. Fresh, anhydrous dioxane (35 mL) was
added and the solution was warmed to 60 °C. A solution of N-
phenyltriazolidinedione (PTAD; 3.70 mmol) in anhydrous dioxane
6
0, 230–400 mesh was used. TLC: analytical thin layer chromatog-
raphy was performed on Merck precoated aluminum plates 60 F254
with a 0.2 mm layer of silica gel containing a fluorescence indicator;
sugar-containing compounds were visualized with the sugar spray
reagent (0.5 mL 4-methoxybenzaldehyde, 9 mL ethanol, 0.5 mL
concentrated sulfuric acid, and 0.1 mL glacial acetic acid) by heat-
ing with a fan or a hot plate. Analytical HPLC: Merck-Hitachi
HPLC system (-7000) equipped with a LiChroCART 125–3 col-
umn containing reverse-phase silica gel Lichrospher 100 RP 18
(
30 mL) was added dropwise, and the mixture was stirred for one
additional hour. The solvent was then removed under reduced pres-
sure. Purification was carried out with a Chromatotron with
(
endcapped, 5 µ) (Merck, Darmstadt, Germany). HPLC method:
–25 min water or ion pairing (buffer/CH CN gradient 5–100%);
isocratic CH CN/H O, 5:95 v/v 25–35 min, flow: 0.5 mL/min; UV
0
3
CH
2
Cl
2
and a MeOH-gradient (0–2% v/v).
3
2
1
detection at 250 nm or fluorescence detection at 380 nm. The cy-
cloSal compounds 11–13 were isolated as mixtures of diastereomers
3Ј,5Ј-Bis(O-tert-butyldimethylsilyl)-N -(β-
D
-2Ј-deoxyribosyl)-5-
1
methyl-pyrimidin-2-one (5): Yield: 57 % (yellow oil). H NMR
Eur. J. Org. Chem. 2006, 924–931
© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
929

H. J. Jessen, W. Fendrich, C. Meier
FULL PAPER
(
400 MHz, CDCl
3
): δ = 8.44 (d, 4
J
H,H = 3.3 Hz, 1 H), 8.04 (dd,
to –20 °C, tBuOOH (0.13 mL) was added (70 % in n-decane,
0.81 mmol). After stirring for 1 h at room temp., the solvent was
removed under reduced pressure. For triester 13, the reaction mix-
ture was diluted with CH Cl , extracted twice with brine and di-
2 2
rectly purified. Purification of the residue was carried out with a
Chromatotron by using EtOAc and a MeOH-gradient (0–5% v/v)
2 2
and repeated purification by using CH Cl and a MeOH-gradient
(0–3% v/v). The products were lyophilized to yield white foams.
4
4
3
3
JH,H = 3.3, JH,H = 0.8 Hz, 1 H), 6.23 (dd, JH,H = 6.4, JH,H
=
=
3
3
6
2
1
=
3
.4 Hz, 1 H), 4.38–4.34 (m, 1 H), 4.01 (ddd, JH,H = 3.4, JH,H
.5, JH,H = 3.6 Hz, 1 H), 3.93 (dd, JH,H = 11.5, 3JH,H = 2.7 Hz,
3
2
2
3
3
H), 3.77 (dd, JH,H = 11.5, JH,H = 2.7 Hz, 1 H), 2.61 (ddd, JH,H
3
2
4
4.0, JH,H = 6.4, JH,H = 13.5 Hz, 1 H), 2.10 (d, JH,H = 0.8 Hz,
3
3
2
H), 2.03 (ddd, JH,H = 6.4, JH,H = 6.4, JH,H = 13.5 Hz, 1 H),
.91 (s, 9 H), 0.90 (s, 9 H), 0.11 (s, 3 H), 0.10 (s, 3 H), 0.07 (s, 3
0
13
H), 0.06 (s, 3 H) ppm. C NMR (100 MHz, CDCl
1
1
3
): δ = 167.4,
55.1, 140.8, 112.4, 88.4, 87.4, 71.4, 62.4, 42.5, 25.9, 25.7, 18.4,
8.0, 14.5, –4.6, –4.9, –5.4, –5.6 ppm. MS (FAB): calcd. 455.3 [M
1
3
-Methyl-cycloSal-N -(β-
D
-2Ј-deoxyribosyl)-5-methyl-pyrimidin-2-
5
one-5Ј-monophosphate (3-Me-cycloSal-m KMP) (11): Yield: 40%
(colorless, hygroscopic solid). H NMR (500 MHz, [D
1
+
6
]DMSO): δ
8.48 (d, JH,H = 3.3 Hz, 1 H, 4-H), 8.45 (d, JH,H = 3.3 Hz, 2 H,
-2Ј,3Ј-dideoxyribosyl)-5-methyl- 4-H), 8.00–7.96 (m, 2 H, 6-H), 7.30–7.24 (m, 2 H, 6-H ), 7.13–
+
H ]; found 455.0.
Ј-O-tert-Butyldimethylsilyl-N -(β-
pyrimidin-2-one (10): Yield: 48 % (yellow foam). H NMR 7.10 (m, 4 H, 5-Haryl, 4-Haryl), 6.11 (2×dd, J
4
4
=
1
5
D
aryl
1
3
= 6.6, 3JH,H
H,H
=
(
400 MHz, CDCl
3
): δ = 8.44 (d, 4
H,H = 3.0, JH,H = 0.8 Hz, 1 H), 6.03 (dd, JH,H = 3.3, JH,H
JH,H = 2.8 Hz, 1 H), 8.17 (dd,
6.6 Hz, 2 H, 1Ј-H), 5.55–5.37 (m, 6 H, 7-Hbenzyl, 3Ј-OH), 4.47–4.30
(m, 4 H, 5Ј-H), 4.26–4.19 (m, 2 H, 3Ј-H), 4.10–4.05 (m, 2 H, 4Ј-
H), 2.40–2.32 (m, 2 H, 2Ј-H), 2.21 (s, 3 H, Mearyl), 2.20 (s, 3 H,
4
4
3
3
J
=
=
2
3
6
2
2
1
.6 Hz, 1 H), 4.26–4.20 (m, 1 H), 4.10 (dd, JH,H = 11.7, JH,H
.3 Hz, 1 H), 3.74 (dd, JH,H = 11.7, JH,H = 2.3 Hz, 1 H), 2.65–
2
3
4
Mearyl) 2.08–1.99 (m, 2 H, 2Ј-H), 2.02 (d, JH,H = 0.5 Hz, 3 H,
4
4
13
.54 (m, 1 H), 2.16–2.08 (m, 1 H), 2.11 (d, JH,H = 0.6 Hz, 3 H), Mehet), 1.98 (d, JH,H = 0.5 Hz, 3 H, Mehet) ppm. C NMR
.95–1.86 (m, 2 H), 0.92 (s, 9 H), 0.12 (s, 3 H), 0.10 (s, 3 H) ppm.
6
(100 MHz, [D ]DMSO): δ = 2×168.0 (2×Chet-4), 157.1 (2×Chet-
1
3
C NMR (100 MHz, CDCl
3
): δ = 167.3, 155.3, 141.3, 112.2, 88.2,
2), 148.1 (2×Caryl-2), 141.4, 141.3 (2×Chet-6), 131.1 (2×Caryl-4),
127.1 (2×Caryl-3), 124.2 (2×Caryl-6), 2×123.8 (2×Caryl-5), 121.2,
121.1 (2×Caryl-1), 112.1 (2×Chet-5), 87.1, 87.0 (2×C-1Ј), 85.4, 85.3
8
2.7, 63.9, 33.5, 25.9, 24.3, 18.5, 14.6, –5.3 ppm. MS (HR-FAB):
+
calcd. 325.195 [M + H ]; found 325.193.
(
(
(
2 × C-3Ј), 70.0, 69.9 (2 × C-4Ј), 68.6, 68.5 (2 × C-7), 67.8, 67.7
2 × C-5Ј), 40.5 (2 × C-2Ј), 2 × 15.0 (2 × Mearyl), 13.8, 13.7
Deprotection of the Silylated Nucleosides 5 and 10
1
3
Ј,5Ј-Bis(O-tert-butyldimethylsilyl)-N -(β--2Ј-deoxyribosyl)-5-
31
1
2×Mehet) ppm. P NMR ( H-decoupled, 200 MHz, [D
6
]DMSO):
1
methyl-pyrimidin-2-one (5) or 5Ј-O-tert-butyldimethylsilyl-N -(β--
δ = –7.6, –7.7 ppm. UV (H
TOF) calcd. 431.1 [M + Na ]; found 431.1. R (HPLC) 11.95 min
t
and 12.19 min.
2
O): λmax = 314, 213 nm. MS (MALDI-
2
0
Ј,3Ј-dideoxyribosyl)-5-methylpyrimidin-2-one (10) (140 mg,
.43 mmol) was dissolved in THF (5 mL). TBAF/THF solution
+
(1.08 mL, 1 ; 2.0 equiv. in the case of 10 and 3.0 equiv. in the case
1
3
-Methyl-cycloSal-N -(β-
D
-2Ј,3Ј-dideoxyribosyl)-5-methyl-pyrimi-
of 5) was added and the reaction mixture was stirred for 20 min.
The solvent was removed under reduced pressure. Purification was
achieved with a Chromatotron by using EtOAc and a MeOH-gra-
dient (0–10% v/v) which yielded a yellow, sticky solid (2) or color-
less crystals (1).
5
din-2-one-5Ј-monophosphate (3-Me-cycloSal-dm KMP) (12): Yield:
2
=
2
=
1
8% (colorless, hygroscopic solid). H NMR (500 MHz, CDCl
3
): δ
4
4
8.43 (2×d, JH,H = 3.5 Hz, 2 H, 4-H), 8.08 (2×d, JH,H = 3.5 Hz,
3
3
H, 6-H), 7.22–7.15 (m, 2 H, 6-Haryl), 7.05 (dd, JH,H = 7.6, JH,H
3
7.6 Hz, 2 H, 5-Haryl), 6.94 (d, JH,H = 7.6 Hz, 4-Haryl), 6.05
1
5
N -(β-
D
-2Ј-Deoxyribosyl)-5-methyl-pyrimidin-2-one (m K) (1) was
3
3
(
2×dd, JH,H = 3.2, JH,H = 3.2 Hz, 2 H, 1Ј-H), 5.45–5.25 (m, 4
prepared as described above and isolated in quantitative yield. An-
2
H, 7-Hbenzyl), 4.60–4.11 (m, 6 H, 5Ј-H, 4Ј-H), 2.63 (2×dddd, JH,H
=
alytical data were identical with those published previously.[
12,13]
3
3
3
14.2, JH,H = 7.3, JH,H = 3.2, JH,H = 3.2 Hz, 2 H, 2Ј-H), 2.28,
5
2.25 (s, 6 H, Mearyl), 2.19–2.11 (m, 2 H, 2Ј-H), 2.06, 2.03 (s, 6 H,
Mehet), 2.11–2.00 (m, 2 H, 3Ј-H), 1.87–1.78 (m, 2 H, 3Ј-H) ppm.
Starting with di-TBDMS-protected m K 5 (500 mg, 1.1 mmol),
5
m K 1 (247 mg, 1.1 mmol, 100%) was isolated.
1
3
6
C NMR (100 MHz, [D ]DMSO): δ = 2 × 167.7 (2 × Chet-4),
1
5
N -(β-
D-2Ј,3Ј-Dideoxyribosyl)-5-methyl-pyrimidin-2-one (dm K)
2
1
1
5
×155.1 (2×Chet-2), 148.2 (2×Caryl-2), 141.3, 141.2 (2×Chet-6),
31.2, 131.1 (2 ×Caryl-4), 127.1 (2 × Caryl-3), 124.2 (2 ×Caryl-6),
(
10): Yield: 72% (light yellow solid, m.p. 139–140 °C). The spectra
+
1
did not show any traces of remaining (nBu)
4
N ions. H NMR
23.9, 123.8 (2×Caryl-5), 120.8 (2×Caryl-1), 112.0, 111.9 (2×Chet
-
3
(
500 MHz, [D ]DMSO): δ = 8.46 (s, 2 H), 5.91 (dd, JH,H = 2.5,
6
4
) 87.7, 87.6 (2 × C-1Ј), 2 × 79.8 (2 × C-4Ј, JC,P = 2.2 Hz), 69.0
3
3
J
H,H = 6.9 Hz, 1 H), 5.20 (t, JH,H = 5.4 Hz, 1 H), 4.17–4.12 (m,
2
(
2×C-7, JC,P = 6.1 Hz), 68.6 (2×C-5Ј), 32.3, 32.2 (2×C-2Ј), 24.5
2 × C-3Ј), 15.1, 15.0 (2 × Mearyl), 13.8, 13.7 (2 × Mehet) ppm.
2
3
3
1
3
2
H), 3.82 (ddd, JH,H = 12.3, JH,H = 3.2, JH,H = 5.6 Hz, 1 H),
.62 (ddd, JH,H = 12.3, 3JH,H = 3.4, 3JH,H = 4.9 Hz, 1 H), 2.46–
.38 (m, 1 H), 2.06 (s, 3 H), 1.99–1.93 (m, 1 H), 1.90–1.77 (m, 2
]DMSO): δ = 167.3, 154.2,
42.1, 111.8, 87.3, 83.1, 61.4, 33.1, 23.7, 14.3 ppm. UV (H O): λmax
311, 213 nm. IR (KBr): ν˜ = 3395, 2928, 1654, 1532, 1395, 1254,
(
2
31
1
P NMR ( H-decoupled, 200 MHz, [D
.5 ppm. UV (H O): λmax = 314, 271, 213 nm. HRMS (ESI ):
calcd. 415.10349; found 415.10344. R (HPLC) 13.17 min and
3.31 min.
6
]DMSO): δ = –7.4, –
+
7
2
1
3
H) ppm. C NMR (100 MHz, [D
1
=
1
6
t
2
1
-1
+
1
104, 797 cm . MS (HR-FAB): calcd. 211.108 [M + H ]; found
11.109.
5-(Acetoxymethylpropionate)-cycloSal-N -(β-D-2Ј-deoxyribosyl)-5-
methyl-pyrimidin-2-one-5Ј-monophosphate (5-AM-propionate-cy-
2
5
cloSal-m KMP) (13): Yield: 28 % (colorless, hygroscopic solid).
5
Preparation of the cycloSal-phosphate Triesters 11–13 from m K and
1
4
H NMR (500 MHz, [D
6
]DMSO): δ = 8.48 (d, JH,H = 3.2 Hz, 1
5
dm K
4
4
H, 4-H), 8.45 (d, JH,H = 3.2 Hz, 1 H, 4-H), 7.98 (dd, JH,H = 3.2,
4
H,H = 0.7 Hz, 1 H, 6-H), 7.95 (dd, 4JH,H = 3.2, JH,H = 0.8 Hz,
4
The nucleosides (0.72 mmol) were co-evaporated twice with pyri-
dine and dissolved in anhydrous THF (1 mL) and anhydrous DMF
J
4
1 H, 6-H), 7.27–7.22 (m, 2 H, 4-Haryl), 7.15 (2×d, JH,H = 2.3 Hz,
3
(1 mL). The reaction mixture was cooled to –20 °C. After addition
2 H, 6-Haryl), 7.05 (d, JH,H = 8.5 Hz, 1 H, 3-Haryl), 7.04 (d, 3JH,H
3
3
of DIPEA (0.54 mmol), 3-methyl- or 5-acetoxymethylpropionate-
saligenylchlorophosphite (0.30 mmol) was added dropwise. The re-
action mixture was warmed to room temp. for 1 h. After cooling
= 8.2 Hz, 1 H, 3-Haryl), 6.11 (dd, JH,H = 6.5, JH,H = 6.5 Hz, 1 H,
1Ј-H), 6.10 (dd, JH,H = 6.4, JH,H = 6.4 Hz, 1 H, 1Ј-H), 5.68, 5.67
(2×s, 4 H, 11-H), 5.52–5.38 (m, 6 H, 3Ј-OH, 7-Hbenzyl), 4.47–4.30
3
3
930
www.eurjoc.org
© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2006, 924–931

Synthesis and Properties of Fluorescent cycloSal Nucleotides
FULL PAPER
(
2
m, 4 H, 5Ј-H), 4.25–4.19 (m, 2 H, 3Ј-H), 4.09–4.05 (m, 2 H, 4Ј-H),
.84 (2×t, JH,H = 7.4 Hz, 4 H, 8-H), 2.70 (2×t, JH,H = 7.4 Hz, 4
tion D or A (1.0 µL) was dissolved in water (499 µL). The solution
(40 µL) was analyzed by RP-HPLC with fluorescence detection at
380 nm.
3
3
H, 9-H), 2.39–2.31 (m, 2 H, 2Ј-H), 2.10–1.98 (m, 2 H, 2Ј-H), 2.07
(
1
4
s, 3 H, Me), 2.06 (s, 3 H, Me), 2.02 (d, JH,H = 0.7 Hz, 6 H, Mehet),
.98 (d, 4 H,H = 0.8 Hz, 6 H, Mehet) ppm. 13C NMR (100 MHz,
]DMSO): δ = 173.7 (2×C-10), 171.2 (2×C-12), 168.0, 167.9
het-4), 154.3 (2×Chet-2), 148.5 (2×Caryl-2), 141.4, 141.3 (2×Chet
), 136.7 (2×Caryl-5), 129.8 (2×Caryl-4), 126.0, 125.9 (2×Caryl-6),
22.3 (2×Caryl-1), 118.5 (2×Caryl-3), 112.6, 112.5 (2×Chet-5), 87.1,
The pH-driven diffusion experiment was carried out with the same
experimental setup, except that no PLE was added to the acceptor
phase A. Instead, the pH of the phosphate buffer saline was set to
J
[D
6
(C
-
8.7.
6
1
8
6
7.0 (2×C-1Ј), 85.3 (2×C-3Ј), 82.9 (2×C-11), 69.9, 69.8 (2×C-4Ј), Acknowledgments
8.6 (2×C-7), 67.7 (2×C-5Ј), 40.4 (2×C-2Ј), 34.6 (2×C-9), 29.2
2×C-8), 20.6 (2×Me), 13.8, 13.7 (2×Mehet) ppm. 31P NMR ( H- Funding was provided by a grant from the Deutsche Forschungsge-
1
(
decoupled, 162 MHz, [D
6
]DMSO): δ = –9.31, –9.38 ppm. UV
meinschaft (CME-1161/2–4).
+
(H
2
O): λmax = 315, 271, 213 nm. HRMS (ESI ): calcd. 539.1431;
t
found 539.1434. R (HPLC) 12.8 min.
[
1] E. De Clercq, Nat. Rev. Drug Discov. 2002, 1, 13–25.
Kinetic Studies
[2] J. Balzarini, Pharm. World Sci. 1994, 16, 113–126.
[
[
3] C. Meier, Synlett 1998, 233–242.
(
a) Aqueous Buffers: DMSO stock solutions (11.4 µL, 50 m) of
the triesters were diluted in water or water/DMSO (300 µL, c =
.9 m). This solution (0.3 mL) was added to aqueous buffer
0.3 mL; 50 m phosphate buffer, pH 7.3 or 50 m phosphate
4] C. R. Wagner, V. V. Iyer, E. J. McIntee, Med. Res. Rev. 2000,
20, 417–451.
1
(
[
[
5] C. Meier, Mini-Rev. Med. Chem. 2002, 2, 219–234.
6] a) C. Meier, A. Meerbach, J. Balzarini, Front. Biosci. 2004, 9,
873–890; b) C. Meier, in Advances in Antiviral Drug Design
(Ed.: E. De Clercq), 2004, vol. 4, 147–213.
buffer, pH 6.8) containing an aqueous AZT solution (5 µL; AZT
used as internal standard) at 37 °C. The final concentrations were
0.96 m for the triesters and 24.8 m for the aqueous buffer. Ali-
[7] a) J. Balzarini, S. Aquaro, T. Knispel, C. Rampazzo, V. Bianchi,
C. F. Perno, E. De Clercq, C. Meier, Mol. Pharmacol. 2000, 58,
quots of the hydrolysis mixture (60 µL) were taken, and the hydrol-
ysis was stopped by freezing the samples in liquid air. After thaw-
ing, samples were analyzed by analytical HPLC [Merck LiChro-
CART column, LiChrospher 100 reverse-phase silica gel RP-18
endcapped (5 µm); UV detection at 310 nm]. The hydrolysis of the
compounds 5 and 6 was followed by integration of the decreasing
peak areas in the HPLC chromatograms. The rate constants (k)
were determined from the slope of the logarithmic degradation
curve. The half-lives (t1/2) were calculated by using the rate con-
stants k.
9
28–935; b) J. Balzarini, L. Naesens, S. Aquaro, T. Knispel, C.-
F. Perno, E. De Clercq, C. Meier, Mol. Pharmacol. 1999, 56,
1
354–1361.
[
8] a) C. Meier, M. Ruppel, D. Vukadinovic, J. Balzarini, Nucleos.
Nucleot. Nucl. 2004, 89–115.
9] C. Meier, H. Jessen, C. Ducho, D. Vukadinovic-Tenter, Eur. J.
Org. Chem., in press.
[
[10] P. Wu, T. M. Nordlund, B. Gildea, L. W. McLaughlin, Bio-
chemistry 1990, 29, 6508–6514.
[
11] S. G. Laland, G. S. Hannssen, Biochem. J. 1964, 90, 76–81.
12] a) D. Cech, A. Holy, Collect. Czech. Chem. Commun. 1977, 42,
[
(b) CEM Cell Extract: Stock solutions (1.5 m⁾ of the triesters in
2246–2260; b) B. Gildea, L. W. McLaughlin, Nucleic Acids Res.
DMSO were prepared. The stock solution (20 µL) was mixed with
cell extract (100 µL) and magnesium chloride solution (20 µL,
1989, 17, 2261–2281.
[13] A. Whightman, A. Holy, Collect. Czech. Chem. Commun. 1973,
38, 1381–1396.
[
70 m⁾. The hydrolysis process was stopped by addition of acidic
14] S. F. Singleton, F. Shan, M. W. Kanan, C. M. McIntosh, C. J.
methanol (300 µL) and storage for 5 min at 0 °C. The mixtures
were centrifuged at 13000 rpm for 10 min, filtered (Schleicher-
Schuell Spartan 13/30, 0.2 µm), and the supernatant was analyzed
as mentioned above.
Stearman, J. S. Helm, K. J. Webb, Org. Lett. 2001, 3, 3919–
3922.
[
15] V.-Z. Xu, Q. Zheng, P. F. Swann, J. Org. Chem. 1992, 57, 3839–
845.
[16] Moreover, after 5Ј-O-dimethoxytritylation into 5Ј-DMTr-m K
80% yield) and phosphitylation using β-cyanoethyl-bis(diiso-
3
5
(c) U-tube Experiments: DMSO stock solution (38.0 µL, 50 m)
(
was diluted with deionized water (962 µL, pH = 6.7, Donor solu-
tion D). Subsequently, phosphate-buffered saline (990 µL, pH =
propylamino)chlorophosphane/dicyanoimidazole (85% yield),
5
m K 1 was converted into its 3Ј-phosphoramidite that can be
7
.3, Accepter solution A) was mixed with a freshly prepared PLE
stock solution (10 µL, 275 U/mL). A U-tube (inner diameter:
.7 mm, width: 3.5 cm, length: 6.0 cm) was filled with CH Cl
2
used for automated oligonucleotide synthesis according to the
standard protocol on DNA-synthesizers. The yields obtained
here are considerably improved relative to those reported pre-
0
2
(
(
1.0 mL). The organic layer was stirred with a small magnetic bar
500 rpm). Both solutions D and A were poured simultaneously
[14]
viously.
Received: July 14, 2005
into the arms of the U-tube. Samples were taken as follows: solu-
Published Online: December 5, 2005
Eur. J. Org. Chem. 2006, 924–931
© 2006 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
931