Synthesis of 3'-S-(2-aminoethylthio)-3'-deoxythymidine-5'-triphosphates

Article abstract of DOI:10.1055/s-2000-6224

3'-S-(2-N-(t-Butyloxycarbonyl)aminoethylthio)-3'-deoxy-5'-O-(4,4'- dimethoxytrityl)thymidine (3) was synthesized by treating 5'-O-(4,4'- dimethoxytrityl)-2,3'-anhydrothymidine (2) with t-butyl N-(2- mercaptoethyl)carbamate (1) and DBU. Compound 3 was furt

Full text of DOI:10.1055/s-2000-6224

PAPER
149
Synthesis of 3´-S-(2-Aminoethylthio)-3´-deoxythymidine-5´-triphosphates
Christian Wojczewski, Joachim W. Engels*
Institut für Organische Chemie, Johann Wolfgang Goethe-Universität, Marie-Curie-Str. 11, D-60439 Frankfurt am Main, Germany
Fax: +49(69)79829148; E-mail: engels@ews1.org.chemie.uni-frankfurt.de
Received 16 July 1999
tion of the amino group failed. We avoided employing
amide protective groups since they are critical in the pres-
ence of thiolates or enolates. Hence, we chose the butyl-
oxycarbonyl group (Boc) which is stable towards most
Abstract:
3´-S-(2-N-(t-Butyloxycarbonyl)aminoethylthio)-3´-
deoxy-5´-O-(4,4´-dimethoxytrityl)thymidine (3) was synthesized
by treating 5´-O-(4,4´-dimethoxytrityl)-2,3´-anhydrothymidine (2)
with t-butyl N-(2-mercaptoethyl)carbamate (1) and DBU. Com-
pound 3 was further converted to 3´-S-(2-N-(t-butyloxycarbon- harsh nucleophilic and basic aqueous or non-aqueous re-
yl)aminoethylthio)-3´-deoxythymidine-5´-triphosphate (5) and 3´-
S-(2-aminoethylthio)-3´-deoxythymidine-5´-triphosphate (9). The
latter compound was labeled with a near-IR fluorescent dye.
action conditions and which can easily be removed with
trifluoroacetic acid.
5´-O-(4,4´-Dimethoxytrityl)-2,3´-anhydrothymidine (2)
Key words: fluorescent nucleotides, thioether, triphosphates, chro-
mophores, ring opening
was obtained as described before.15 Zuckermann de-
scribed the introduction of alkane dithiols of the type HS-
(CH2)n-SH into 5´-O-(4,4´-dimethoxytrityl)-2,3´-anhy-
drothymidine (2) employing sodium ethoxide in ethanol
Currently, there is widespread interest in nucleosides, in
as the basic medium.16 The reaction mixture was main-
particular nucleoside-5´-triphosphates that are covalently
tained at 75 °C for 2.5 hours. Although the educt was
coupled to fluorophores.1 Dependent on the application,
completely converted to only one major UV-absorbing
different positions at the ribose or base moiety within the
product as indicated by thin layer chromatography, the
nucleoside were preferred for introducing the label.
yield was only 58%. We also achieved this yield with hex-
Among the base modifications, the exocyclic amino
ane-1,6-dithiol as nucleophile. We could not observe a
groups2 or the C-5 position in the pyrimidines3 and the C-
considerable loss of product during the work up and isola-
7 or C-8 position in purines4 are usually the target for la-
tion procedure. Therefore, a great amount of the nucleo-
beling. Furthermore, a series of fluorescent base ana-
base seems to degrade under these reaction conditions.
logues like 2-aminopurine,5 ethenoadenosine,6 2'-
However, in our studies, application of this method with
t-butyl N-(2-mercaptoethyl)carbamate (1) as nucleophile
only resulted in starting material.
deoxyisoinosine,7 2-pyrimidinone8 and polyaromatic
hydrogencarbons9 exists.
Introducing a probe at the ribose residue of a nucleoside
was until now accomplished by conjugating the dye to the
Bera et al. demonstrated that introducing mercaptoethanol
by treating 5´-O-trityl-2,3´-anhydrothymidine with 1,8-
diazabicyclo[5.4.0]undec-7-ene (DBU) in DMF for 7 h at
60 °C or with 1,1,3,3-tetramethylguanidine resulted in a
2´-, 3´- or 5´-hydroxyl group through an ester or ether
bond.10 Alternatively, we and others have already report-
ed the syntheses of 3´-amino, amido-, isocyanato- and
very low yield of the corresponding 3´-thioether nucleo-
isothiocyanato- modified 2´,3´-dideoxynucleoside-5´-
side.17
triphosphates and their labeling with fluorescence dyes.11
We have found that the crucial parameters for opening an-
hydrothymidine with thiols are the reaction temperature
and the choice of the basic medium. At 50 °C, no product
However, Canard et al. have demonstrated that ester- and
amide-bonds adjacent to the 3´-carbon of nucleoside-5´-
triphosphates may unexpectedly be subjected to enzyme-
mediated hydrolysis and loss of the label.12 These results
are in accordance with our investigations on 3´-amido
tethered nucleoside triphosphates.13 In the context of our
studies on 3´-modified nucleoside-5´-triphosphates, we
decided to alter the type of link into a thioether function
which offers a more stable bond against enzymatic degra-
dation. Here, we describe an efficient synthetic route to
this novel class of 3´ thioether-thymidine-5´-triphos-
phates bearing an aminoethyl linker for fluorescent probe
coupling. An oxazine dye with spectroscopic properties
close to the cyanine dye, CY5 was conjugated.14
was observed no matter what kind of base was used. Only
when the reaction temperature was raised to 100 °C and
DBU was utilized as base, did 5´-O-(4,4´-dimethoxytri-
tyl)-2,3´-anhydrothymidine (2) readily react with t-butyl
N-(2-mercaptoethyl)carbamate (1), within 24 hours, to
produce the desired thioether 3´-S-(2-N-(t-butyloxycarbo-
nyl)aminoethylthio)-3´-deoxy-5´-O-(4,4´-dimethoxytri-
tyl)thymidine (3) in 80% yield.
Introducing the bifunctional linker into the nucleoside af-
forded an amino protecting group, which is stable under
various basic reaction conditions. Different attempts to in-
troduce cysteamine (2-aminoethanthiol) without protec-
Synthesis 2000, No. 1, 149–153 ISSN 0039-7881 © Thieme Stuttgart · New York

150
C. Wojczewski, J. W. Engels
PAPER
Compound 3 was detritylated in 80% aqueous acetic acid exchange HPLC by simultaneous UV- and fluorescence
at room temperature. Synthesis of 3´-S-(2-N-(t-butyloxy- detection and a gradient of aqueous 1M LiCl solution as
carbonyl)aminoethylthio)-3´-deoxythymidine-5´-triphos- eluent. Fractions containing product were subjected to
phate (5) was performed according to the procedure of size exclusion gel filtration on Sephadex G10. The fluo-
Ludwig and Eckstein.18 The purification was done by rescence spectrum revealed a bathochromic shift (4 nm) in
FPLC on DEAE-Sephadex with a gradient of 0.05-1M tri- the emission maximum for covalently attached JA242
ethylammonium bicarbonate pH 7.5. It is known that re- (667 nm) compared to free dye (663 nm). This corre-
peated co-evaporation or freeze drying of the collected sponds to the results found by Lieberwirth who attached
triphosphate fractions with ethanol until weight constancy JA242 covalently to amino-modified oligonucleotides.22
should remove triethylammonium bicarbonate complete-
ly. However, the yield determined by optical density mea-
surements is considerably lower than by weighing the
dried product. The difference in weight is due to the pres-
ence of further buffer. For obtaining the pure product, a
small amount was therefore purified by analytical anion
exchange HPLC followed by reverse phase HPLC. All
triphosphate yields were determined by their optical den-
sity in water.
Compound 5 proved to be inappropriate for dye-labeling
experiments, since the triphosphate unit was not stable un-
der deprotection conditions. With similar nucleotides, it
has been shown that treatment with concentrated trifluo-
roacetic acid for 2 min, instant freezing with liquid nitro-
gen and quenching with a great excess of diethyl ether
readily removed the Boc group without destroying the
triphosphate.11,19 However, in our studies, no nucleotide
could be isolated with compound 5 utilizing these reaction
conditions. Hence, we deprotected at the nucleoside level.
Treatment of 3´-S-(2-N-(t-butyloxycarbonyl)aminoethyl-
thio)-3´-deoxythymidine (4) with concentrated trifluoro-
acetic acid within 2 min gave 6 in 79% yield. It should be
mentioned that removal of both protecting groups of 3´-S-
(2-N-(t-butyloxycarbonyl)aminoethylthio)-3´-deoxy-5´-
O-(4,4´-dimethoxytrityl)thymidine (3) with trifluoroace-
tic acid in one step resulted in a lower yield and a more la-
borious work-up procedure. Selective reprotection of the
amino function was easily done by treating unprotected
nucleoside 6 with 9-fluorenylmethoxycarbonyl-N-hy-
droxysuccinimide (Fmoc-O-succinimide) and sodium bi-
(I) HS(CH2)2NHBoc, DBU, DMF, 24 h, 100 °C (80%); (II) 80% aq
HOAc, 1h, (91%); (III) (a) van Boom´s reagent (2-chloro-4H-1,3,2-
carbonate in water and acetone as described by Paquet.20
Subsequently,
3´-S-(2-N-(9-fluorenylmethoxycarbon-
benzodioxaphosphorin-4-one), dioxane, pyridine, DMF, 20 min, (b)
(Bu3NH)2P2O7, DMF, tributylamine, 30 min, (c) 1% I2 in pyridine/
H2O (98:2), 20 min, (d) 5% aq NaHSO3 (13%); (IV) concd TFA, 2
min (79%); (V) Fmoc-O-succinimide, H2O, acetone, NaHCO3, 3 h
(73%); (VI) (a) van Boom´s reagent, dioxane, pyridine, DMF, 20 min,
(b) (Bu3NH)2P2O7, DMF, tributylamine, 30 min, (c) 1% I2 in pyridine/
H2O (98:2), 20 min, (d) 5% aq NaHSO3 (22% including subsequent
yl)aminoethylthio)-3´-deoxythymidine-5´-triphosphate
(8) was synthesized. The Fmoc group was only partly
cleaved during the work-up procedure of the triphosphate.
5% of the protected product 8 and 17% of the unprotected
3´-S-(2-aminoethylthio)-3´-deoxythymidine-5´-triphos-
phate 9 were isolated. For a clean reaction, piperidine was product); (VII) piperidine, pyridine, DMF, 1 h (77%); (VIII) JA242,
DMF, dioxane, H2O, DIPEA, TSTU, 30 min
therefore added previous to the isolation procedure. Alter-
natively, remaining triphosphate 8 was deblocked with
20% piperidine in pyridine and DMF and finally the nu-
Compound 5 was chosen for enzymatic incorporation ex-
cleotide 9 was coupled with JA242 according to the meth-
periments. When 5 was substituted for ddTTP as termina-
od of Bannwarth et al.21 JA242 is a carboxyl
tor in a DNA sequencing experiment with Taq-DNA-
functionalized oxazine dye with spectroscopic character-
polymerase, we obtained a band pattern, which could not
istics similar to the commercially available cyanine dye
be correlated with the standard ddTTP pattern. Varying
CY5. The carboxyl group is activated as its N-hydroxy-
the concentration of 5 did not alter this result. No band
succinimidyl ester and coupled to the free amino group in
pattern was detected when Sequenase or Thermo-Seque-
a mixture of dioxane, DMF and water. The fluorescent nu-
nase was employed.
cleoside 5´-triphosphate was purified by analytical anion
Synthesis 2000, No. 1, 149–153 ISSN 0039-7881 © Thieme Stuttgart · New York

PAPER
Synthesis of 3’-S-(2-Aminoethylthio)-3’-deoxythymidine-5’-triphosphates
151
Solvents were of analytical grade and were dried and distilled or
purchased and stored over molecular sieve. t-Butyl N-(2-mercapto-
ethyl)carbamate was purchased at Fluka Chemie AG, Germany. Sil-
ica gel 60 F254 plates (Merck) were used for TLC. Preparative silica
gel TLC was done with covered glass plates (gypsum silica gel 60
PF254 Merck) on a Harrison Research 7924T Chromatotron. NMR
solvent signals were used for calibrating as follows: 1H NMR (250,
270 MHz, Bruker AM250, WH270): δ(DMSO-d6) = 2.50,
δ(CDCl3) = 7.26; 13C NMR (62.9, 67.9 MHz, Bruker AM250,
WH270): δ(DMSO-d6) = 39.5, δ(CDCl3) = 77.0; 31P (162 MHz,
Bruker AMX 400), external 85% phosphoric acid. Mass spectra
were recorded with a Fisons VG Platform II electrospray ionization
mass spectrometer. UV spectra and optical density were measured
on a Varian Cary 1. Optical densities were measured at 270 nm (mo-
lar absorptivity in water 8700 L mol-1 cm-1). Fluorescence spectra
were recorded on a Hitachi F-4500. Elemental analysis was per-
formed on Foss Heraeus CHN-O Rapid. FPLC was performed on a
Pharmacia instrument with LCC-500 controller, P-500 pumps, and
single path UV monitor UV-1. HPLC was performed on a Merck-
Hitachi with L-4250 UV-VIS detector and Shimadzu RF-535 fluo-
rescence monitor.
3´-S-(2-N-(t-Butyloxycarbonyl)aminoethylthio)-3´-deoxythymi-
dine (4)
A solution of 3 (0.90 g, 1.28 mmol) in aq HOAc (80%, 20 mL) was
stirred for 1 h at r.t., evaporated, then co-evaporated thrice with
H2O to remove residual acid. The residue was purified by prepara-
tive silica gel TLC (CH2Cl2:MeOH, 95:5) to give 4 (470 mg, 91%).
1H NMR (DMSO) δ = 11.25 (s, 1H, NH-3), 7.80 (s, 1H, H-6), 6.06
(dd, 1H, J = 4.4 Hz, H-1´), 3.76-3.40 (m, 4H, H-3´, H-4´, H-5´, H-
5´´), 3.12 (m, 2H, N-CH2), 2.64 (t, 2H, J = 6.8 Hz, S-CH2), 2.47-
2.20 (m, 2H, H-2´, H-2´´), 1.78 (s, 3H, C5-CH3), 1.38 (s, 9H, t-Bu-
CH3).
13C NMR (DMSO) δ = 163.8 (C4), 155.5 (NC(O)O), 150.4 (C2),
136.2 (C6), 109.0 (C5), 85.9 (C1´), 83.6 (C4´), 77.8 (C(CH3)3), 60.4
(C5´), 40.5 (C-3´), 40.4, 40.2 (C2´, N-CH2), (3 peaks covered by
DMSO), 30.6 (S-CH2), 28.2 (Boc-CH3), 12.3 (C5-CH3).
MS (ESI-): m/z = 400.3 (M− requires 400.5).
Anal: C17H27N3O6S (401.49). Calcd: C, 50.86; H 6.78; N 10.47.
Found: C, 50.70; H, 6.96; N, 10.21
3´-S-(2-N-(t-Butyloxycarbonyl)aminoethylthio)-3´-deoxythymi-
dine -5´-triphosphate (5) as triethylammonium salt
General Purification Procedures
Nucleoside-5´-triphosphates were synthesized according to Ludwig
and Eckstein18 as follows: 3´-S-(2-N-(t-Butyloxycarbonyl)amino-
ethylthio)-3´-deoxythymidine (4) (100 mg, 0.25 mmol) was dried
three times by co-evaporation with anhyd pyridine (0.5 mL) and an-
hyd DMF (2 mL). The residue was dried over P2O5 under vacuum
overnight. The nucleoside was dissolved in anhyd pyridine (0.5 mL)
and anhyd DMF (2 mL) and a freshly prepared solution of 1M 2-
chloro-4H-1,3,2-benzodioxaphosphorin-4-one in anhyd dioxane
(0.28 mL) was injected under Ar. After stirring for 20 min, a pre-
pared solution of 0.5 M (Bu3NH)2P2O7 in dry DMF was mixed well
with tributylamine (3:1) and 1.0 mL of this mixture was instantly
added. The reaction mixture was stirred further for 30 min. Oxida-
tion of phosphorus and ring opening was achieved by adding 5 mL
of 1% I2 in pyridine/ H2O (98:2). After 20 min, the remaining I2 was
reduced by adding a solution of 5% aq NaHSO3 drop by drop until
the brown colour changed to a light yellow. Solvents were removed
under reduced pressure without heating. H2O (5 ml) was poured
onto the residue and the solution containing the dissolved product
was separated from the remaining residue by filtration through a 0.2
µm Nalgene syringe filter. The solution was then purified by FPLC
(purification procedure 1), elution concentration = 25% B; analyti-
cal amounts of fractions containing triphosphate were further puri-
fied by anion exchange HPLC and finally desalted by reverse phase
HPLC (purification procedure 2 and 3), tR = 14.5 min. The yield was
determined by optical density measurement (31.1 µmol, 13%),
C17H30N3O15P3S (641.44).
FPLC: filtrated (0.2 µm Nalgene syringe filter) crude product in
H2O was applied onto a column filled with Pharmacia DEAE
Sephadex-A25 anion exchange gel; A = 0.05M TEAB (triethylam-
monium bicarbonate), pH 7.5, B = 1M TEAB, pH 7.5; flow rate 1
mL/min, 0-500 mL 0-50% B, 500-650 mL 50-100% B.
Anion exchange HPLC: Dionex NucleoPac PA-100 (4 x 250mm),
A = H2O/10% MeCN, B = 1M aq LiCl/10% MeCN, flow rate 1 mL/
min, 0-5 min 0% B, 5-20 min 0-25% B, 20-25 min 100% B.
Reverse phase HPLC: LiChrosphere RP-18 5 µm (3 x 125 mm),
A = 0.1M TEAA (triethylammonium acetate) pH 5.5, B = A+50%
MeCN, flow rate 1 mL/min, 0-10 min 5% B.
HPLC retention times (tR) refer to anion exchange HPLC (purifica-
tion procedure 2) and are given in min.
3´-S-(2-N-(t-Butyloxycarbonyl)aminoethylthio)-3´-deoxy-5´-O-
(4,4´-dimethoxytrityl)thymidine (3)
To a solution of t-butyl N-(2-mercaptoethyl)carbamate (1) (2 mL,
12 mmol) and DBU (1.4 mL, 9.5 mmol) in DMF (10 mL) 5´-O-
(4,4´-dimethoxytrityl)-2,3´-anhydrothymidine (2) (1.00 g, 1.9
mmol) was added. The reaction mixture was heated (bath tempera-
ture 100 °C) for 24 h, cooled to r.t., poured into NaHCO3 soln (50
mL) and extracted with EtOAc. The organic layer was washed with
H2O, dried (MgSO4) and evaporated. The residue was purified by
preparative silica gel TLC (CH2Cl2:MeOH, 98:2) to give 3 (1.07 g,
80%).
1H NMR (CDCl3): δ = 9.19 (s, 1H, NH-3), 7.75 (d, 1H, J = 1.2 Hz,
H-6), 7.43-6.84 (m, 13H, Ar-H), 6.17 (dd, 1H, J = 3.7 Hz, H-1´),
4.80 (t, 1H, J = 6.0 Hz, OCO-NH), 3.91 (m, 1H, H-4´), 3.79 (s, 6H,
O-CH3), 3.68-3.54 (m, 2H, H-3´, H-5´), 3.32 (dd, 1H, J = 2.7 Hz, H-
5´´), 3.26-3.15 (m, 2H, N-CH2), 2.54 (t, 2H, J = 6.0 Hz, S-CH2),
2.55-2.37 (m, 2H, H-2´, H-2´´), 1.46 (d, 3H, J = 0.9 Hz, C5-CH3),
1.42 (s, 9H, t-Bu-CH3).
1H NMR (D2O) δ = 7.82 (d, 1H, J = 1.2 Hz, H-6), 6.21 (dd, 1H, J =
4.7 Hz, H-1´), 4.37-4.22 (m, 2H, H-5´, H-5´´), 4.15 (m, 1H, H-4´),
3.59-3.53 (m, H-3´ partly overlapping with triethylammonium sig-
nal), 3.33 (m, 2H, N-CH2), 2.82 (t, 2H, J = 6.4 Hz, S-CH2), 2.60-
2.44 (m, 2H, H-2´, H-2´´), 1.94 (d, 3H, J = 1.0 Hz, C5-CH3), 1.41
(s, 9H, t-Bu-CH3).
13C NMR(D2O) δ = 166.6 (C4), 151.4 (C2), 137.3 (C6), 111.3 (C5),
84.9, 84.6 (C1´, C4´), 80.9 (C(CH3)3), 62.6 (C5´), 40.9 (C3´), 40.0,
39.1 (C2´, N-CH2), 29.9 (S-CH2), 28.0 (Boc-CH3), 11.8 (C5-CH3).
13C NMR(CDCl3) δ = 163.9 (C4), 158.6 (DMTr), 155.5 (NC(O)O),
150.2 (C2), 144.0 (DMTr), 135.4 (C6), 135.1 (DMTr), 129.9, 128.0,
127.9, 127.1, 113.1 (DMTr), 110.7 (C5), 86.5 (C-DMTr), 85.6, 84.5
(C1´, C4´), 79.5 (C(CH3)3), 61.7 (C5´), 55.1 (OCH3), 40.6 (C3´),
40.3, 39.8 (C2´, N-CH2), 31.8 (S-CH2), 28.2 (Boc-CH3), 11.8 (C5-
CH3).
31P NMR(D2O) δ = -22.2 (t, 1P, β-P), -10.8 (d, 1P, α-P), -9.2 (d, 1P,
γ-P).
MS (ESI-): m/z = 640.4 (M− requires 640.4).
MS (ESI-): m/z = 702.4 (M− requires 702.8).
3´-S-(2-Aminoethylthio)-3´-deoxythymidine (6)
Concd CF3CO2H (10 mL) was poured onto solid 4 (1.63 g, 4.06
mmol) and stirred for 2 min. The reaction was quenched by adding
Et2O (100 mL). The obtained white residue was filtered, washed
Anal: C38H45N3O8S (703.86). Calcd for C38H45N3O8S H2O: C,
63.23; H, 6.56; N, 5.82. Found: C, 63.55; H, 6.53; N, 5.81.
Synthesis 2000, No. 1, 149–153 ISSN 0039-7881 © Thieme Stuttgart · New York

152
C. Wojczewski, J. W. Engels
PAPER
with Et2O and dissolved in MeOH (10 ml) for purification by silica
gel chromatography (CH2Cl2:MeOH, 2:1) to give compound 6 as
the trifluoroacetic salt (1.33 g, 79%).
13C NMR (D2O): δ = 166.0 (C4), 158.1 (NC(O)O), 150.9 (C2),
143.7, 141.9 (Fmoc), 136.9 (C6), 128.1, 127.4, 124.9, 120.1
(Fmoc), 111.3 (C5), 84.9, 84.4 (C1´, C4´), 66.3 (Fmoc), 65.0 (C5´),
46.9 (Fmoc), 40.7 (C3´), 40.2, 39.8 (C2´, N-CH2), 27.8 (S-CH2),
12.8 (C5-CH3).
31P NMR (D2O): δ = -22.5 (t, 1P, β-P), -10.8 (d, 1P, α-P), -10.1 (d,
1P, γ-P).
tR (HPLC) = 9.2 min.
1H NMR (D2O) δ = 7.48 (s, 1H, H-6), 5.94 (dd, 1H, J = 4.4 Hz, H-
1´), 3.78-3.22 (m, 4H, H-5´, H-5´´, H-4´, H-3´), 3.03 (t, 2H, J = 6.6
Hz, N-CH2), 2.75 (t, 2H, J = 6.6 Hz, S-CH2), 2.45-2.21 (m, 2H, H-
2´, H-2´´), 1.65 (s, 3H, C5-CH3).
MS (ESI-): m/z = 762.3 (M− requires 762.5).
13C NMR (DMSO) δ = 163.8 (C4), 150.4 (C2), 136.2 (C6), 109.0
(C5), 85.7, 83.6 (C1´, C4´), 60.3 (C5´), 40.5 (C3´), 40.4, 40.1 (C2´,
N-CH2), 27.9 (S-CH2), 12.2 (C5-CH3).
MS (ESI-): m/z = 300.2 (M− requires 300.4), 414.2 (M TFA− re-
quires 414.4).
3´-S-(2-Aminoethylthio)-3´-deoxythymidine-5´-triphosphate
(9) as triethylammonium salt
A solution of 8 (16 mg, 13.7 µmol) in pyridine (5 mL), DMF (3 mL)
and piperidine (2 mL) was stirred for 1 h at r.t., evaporated, redis-
solved in H2O (5 mL) and filtered through a 0.2 µm Nalgene syringe
filter. The solution was then purified by FPLC (purification proce-
dure 1), elution concentration = 20% B; analytical amounts of frac-
tions containing triphosphate were further purified by anion
exchange HPLC and finally desalted by reverse phase HPLC (puri-
fication procedure 2 and 3); tR = 12.1 min. The yield was determined
by optical density measurement (10.6 µmol, 77%), C12H22N3O13P3S
(541.3).
1H NMR (D2O): δ = 7.81 (d, 1H, J = 1.1 Hz, H-6), 6.21 (dd, 1H, J
= 4.4 Hz, H-1´), 4.32 (m, 2H, H-5´, H-5´´), 4.19 (m, 1H, H-4´), 3.71
(m, 1H, H-3´), 3.27 (m, 2H, N-CH2), 3.02 (t, 2H, J = 6.8 Hz, S-CH2),
2.65 (m, 1H, H-2´), 2.46 (m, 1H, H-2´´), 1.94 (d, 3H, J = 1.1 Hz, C5-
CH3).
Anal: C12H19N3O4S (301.37). Calcd for C12H19N3O4S TFA: C,
40.48; H, 4.85; N, 10.12. Found: C, 40.10; H, 4.89; N, 9.63.
3´-S-(2-N-(9-Fluorenylmethoxycarbonyl)aminoethylthio)-3´-
deoxythymidine (7)
To a solution of 6 (1.31 g, 4.35 mmol) in H2O (60 mL) and acetone
(60 mL), NaHCO3 (364 mg, 4.36 mmol) and 9-fluorenylmethoxy-
carbonyl-N-hydroxysuccinimide (1.47 g, 4.36 mmol) was added.
The reaction mixture was stirred at for 3 h r.t. Further 9-fluorenyl-
methoxycarbonyl-N-hydroxysuccinimide (220 mg, 0.65 mmol) was
added since the reaction was incomplete as was evident from TLC.
After additional stirring for 1 h, the reaction mixture was adjusted
to pH 2 with concd HCl, acetone was removed by evaporation under
reduced pressure and the aq residue was extracted twice with
CH2Cl2 (100 mL). The combined organic layers were dried
(Na2SO4), evaporated and purified by silica gel chromatography
(CH2Cl2:MeOH, 95:5) giving compound 7 (1.66g, 73%).
1H NMR (CDCl3): δ = 9.28 (s, 1H, NH-3), 7.77-7.26 (m, 9H, H-6,
arom. fluorenyl), 6.03 (dd, 1H, J = 3.0 Hz, H-1´), 4.42 (d, 2H, J =
6.8 Hz, CH2 fluorenyl), 4.20 (t, 1H, J = 6.8 Hz, CH fluorenyl), 3.97
(m, 1H, H-5´), 3.80 (m, 2H, H-5´´, H-4´), 3.59-3.27 (m, 3H, H-3´,
N-CH2), 2.71 (t, 2H, J = 6.3 Hz, S-CH2), 2.59-2.50 (m, 1H, H-2´),
2.43-2.31 (m, 1H, H-2´´), 2.07 (s, 3H, C5-CH3).
13C NMR (D2O): δ = 166.5 (C4), 151.4 (C2), 137.3 (C6), 111.1
(C5), 84.7, 84.6 (C1´, C4´), 64.1 (C5´), 39.5 (C3´), 38.6, 38.3 (C2´,
N-CH2), 28.1 (S-CH2), 11.5 (C5-CH3).
31P NMR (D2O): δ = -21.9 (t, 1P, β-P), -10.8 (d, 1P, α-P), -7.8 (d,
1P, γ-P).
MS (ESI-): m/z = 540.2 (M− requires 540.3).
Labeling of 3´-S-(2-Aminoethylthio)-3´-deoxythymidine-5´-
triphosphate (9) with JA242, (10), isolated as lithium salt
Compound 9 (4.24 µmol) was dissolved in DMF (15 µl), dioxane
(35 µl) and H2O (15 µl). DIPEA (0.8 µl, 4.75 µmol), TSTU (1.3 mg,
4.3 µmol) and JA242 (1.56 mg, 3.5 µmol) were added and the solu-
tion was stirred under exclusion of light for 30 min at r.t., and fur-
ther over night at 4 °C. The solvent was removed (Speed Vac) and
the residue was dissolved in H2O (50 µl), vortexed well and centri-
fuged. The supernatant was separated and purified by anion ex-
change HPLC (purification procedure 2), tR = 13.0 min. The product
was desalted by size exclusion gel filtration on Sephadex G10 elut-
ing with 0.05M TEAB. UV-absorbing fractions were collected,
C39H54N6O15P3S (971.88).
13C NMR (CDCl3): δ = 164.1 (C4), 156.8 (NC(O)O), 150.4 (C2),
143.5, 141.3 (Fmoc), 136.7 (C6), 127.7, 127.0, 124.9, 120.0
(Fmoc), 110.4 (C5), 86.5, 85.6 (C1´, C4´), 66.9 (Fmoc), 60.7 (C5´),
47.1 (Fmoc), 40.8 (C3´), 40.2, 39.0 (C2´, N-CH2), 31.9 (S-CH2),
12.5 (C5-CH3).
MS (ESI+): m/z = 524.2 (M+ requires 524.6).
Anal: C27H29N3O6S (523.61). Calcd for C27H29N3O6S H2O: C,
59.87; H, 5.77; N, 7.76. Found: C, 60.25; H, 5.67; N, 7.91
3´-S-(2-N-(9-Fluorenylmethoxycarbonyl)aminoethylthio)-3´-
deoxythymidine-5´-triphosphate (8) as triethylammonium salt
Compound 8 was synthesized and purified following the same pro-
cedure as described for 5 with 3´-S-(2-N-(9-fluorenylmethoxycar-
bonyl)aminoethylthio)-3´-deoxythymidine (7) (0.19 mmol, 100
mg), anhyd pyridine (0.5 mL), anhyd DMF (2 mL), 1 M 2-chloro-
4H-1,3,2-benzodioxaphosphorin-4-one in anhyd dioxane (0.21
mL), 0.5 M (Bu3NH)2P2O7 in anhyd DMF/tributylamine (3:1) (0.76
mL), 1% I2 in pyridine/ H2O (98:2) (3.8 mL). Elution
concentration = 55% B; tR = 17.5 min. The yield was determined by
optical density measurement. Compound 8 (9.5 µmol, 5%),
C27H32N3O15P3S (763.56) and 9 (32.3 µmol, 17%).
1H NMR (D2O) δ: = 7.89-7.40 (m, 9H, H-6, arom. fluorenyl), 6.07
(d, 1H, J = 4.7, H-1´), 4.52 (m, 2H, H-5´, H5´´), 4.31-4.16 (m, 3H,
H-4´, CH2 fluorenyl), 4.04 (s, 1H, CH fluorenyl), 3.47-3.37 (m, 1H,
H-3´), 3.11-3.01 (m, 4H, N-CH2, S-CH2), 2.40-2.36 (m, 2H, H-2´,
H-2´´), 1.75 (s, 3H, C5-CH3).
UV (H2O): λabs-max1 = 664nm, λabs-max2 = 263nm.
Fluorescence (H2O): λem-max = 677nm.
MS (ESI+): m/z = 970.8 (M+), [C39H50N6O15P3S]H4 requires 971.9;
977.2 (M+), [C39H50N6O15P3S]H3Li requires 977.8;
983.3 (M+), [C39H50N6O15P3S]H2Li2 requires 983.8;
989.3 (M+), C39H50N6O15P3S]HLi3 requires 989.7;
995.2 (M+), [C39H50N6O15P3S] Li4 requires 995.6.
Acknowledgement
We would like to thank the Bundesministerium für Bildung und
Forschung (BEO0311088), the Verband der Chemischen Industrie
and the Deutsche Forschungsgemeinschaft (Graduiertenkolleg) for
financial support as well as Roche Diagnostics for cooperation and
financial support. Fluorescent dyes were kindly supplied by Prof.
Dr. K. H. Drexhage, Universität Siegen, Germany. Enzymatic in-
Synthesis 2000, No. 1, 149–153 ISSN 0039-7881 © Thieme Stuttgart · New York

PAPER
Synthesis of 3’-S-(2-Aminoethylthio)-3’-deoxythymidine-5’-triphosphates
153
corporation experiments were done by Dr. S. Klingel and Dr. G. Sa-
gner at Roche Diagnostics, Penzberg, Germany.
Aurup, H.; Tuschl, T., Benseler, F.; Ludwig, J.; Eckstein, F.
Nucleic Acids Res. 1994, 22, 20.
Yamana, K.; Aota, R.; Nakano, H. Tetrahedron Lett. 1995, 36,
8427.
Hovinen, J.; Azhayeva, E.; Azhayev, A.; Guzaev, A.;
Lönnberg, H. J. Chem. Soc., Perkin Trans. 1 1994, 211.
Sarfati, R. S.; Berthod, T.; Guerreiro, C.; Canard, B. J. Chem.
Soc., Perkin Trans. 1 1995, 1163.
Metzker, M. L.; Raghavachari, R.; Richards, S.; Jacutin, S. E.;
Civitello, A.; Burgess, K.; Gibbs, R. A. Nucleic Acids Res.
1994, 22, 4259.
References
(1) For a review, see Wojczewski, C.; Stolze, K.; Engels, J. W.
Synlett 1999,1667.
Jameson, D. M.; Eccleston, J. F. Methods Emzymol. 1997,
278, 363.
(2) Casale, R.; McLaughlin, L. W. J. Am Chem. Soc. 1990, 112,
5264.
(11) Stolze, K.; Koert, U.; Klingel, S.; Sagner, G.; Wartbichler, R.;
Engels, J. W. Helv. Chim. Acta 1999, 82,1311.
Wojczewski, C.; Faulstich, K.; Engels, J. W. Nucleosides
Nucleotides 1997, 16, 751.
Sigmund, H.; Maier, T.; Pfleiderer, W. Nucleosides
Nucleotides 1997, 16, 685.
Maier, T.; Pfleiderer, W. Nucleosides Nucleotides 1995, 14,
961.
Markiewicz, W. T.; Gröger, G.; Rösch, R.; Zebrowska, A.;
Markiewicz, M.; Klotz, M.; Hinz, M.; Godzina, P.; Seliger, H.
Nucleic Acids Res. 1997, 25, 3672.
Kroth, H.; Hertkorn, N.; Oesch, F.; Seidel, A. Polycyclic
Aromat. Compd. 1996, 11, 349.
Yamana, K.; Ohashi, Y.; Nunota, K.; Nakano, H. Tetrahedron
1997, 53, 4265.
Herrlein, M. K.; Konrad, R. E.; Engels, J. W.; Holletz, T.;
Cech, D. Helv. Chim. Acta 1994, 77, 586.
(12) Canard, B.; Cardona, B.; Sarfarti, R. S. Proc. Natl. Acad. Sci.
USA 1995, 92, 10859.
(13) Personal communication of Dr. G. Sagner at Roche
Diagnostics, Penzberg, Germany and unpublished results
about the application of 3´-amido-2´,3´-didesoxynucleoside-
5´-triphosphates for DNA sequencing.
(14) Müller, R.; Herten, D. P.; Lieberwirth, U.; Neumann, M.;
Sauer, M.; Schulz, A.; Siebert, S.; Drexhage, K. H.; Wolfrum,
J. Chem. Phys. Lett. 1997, 279, 282.
(15) Horwitz, J. P.; Chua, J.; Urbanski, J. A.; Noel, M. J. Org.
Chem. 1963, 28, 942.
Fox, J. J.; Miller, N. C. J. Org. Chem. 1963, 28, 936.
(16) Zuckermann, R.; Corey, D.; Schultz, P. Nucleic Acids Res.
1987, 15, 5305.
(17) Bera, S.; Sakthivel, K.; Pathak, T. Tetrahedron 1995, 51,
7857.
(18) Ludwig, J.; Eckstein, F. J. Org. Chem. 1989, 54, 631.
(19) Personal communication of Dr. S. Klingel at Roche
Diagnostics, Penzberg, Germany.
(20) Paquet, A. Can. J. Chem. 1982, 60, 976.
(21) Bannwarth, W.; Knorr, R. Tetrahedron Lett. 1991, 32, 1157.
(22) Lieberwirth, U.; Arden-Jacob, J.; Drexhage, K. H.; Herten, D.
P.; Müller, R.; Neumann, M.; Schulz, A.; Siebert, S.; Sagner,
G.; Klingel, S.; Sauer, M.; Wolfrum, J. Anal. Chem. 1998, 70,
4771.
(3) Prober, J. M.; Trainor, G. L.; Dam, R. J.; Hobbs, F.W.;
Robertson, C. W.; Zagursky, R. J.; Cocuzza, A. J.; Jensen, M.
A.; Baumeister, K. Science 1987, 238, 336.
(4) Lee, L. G.; Spurgeon, S. L.; Heiner, C. R.; Benson, S. C.;
Rosenblum, B. B.; Menchen, S. M.; Graham, R. J.;
Constantinescu, A.; Upadhya, K. G.; Cassel, J. M Nucleic
Acids Res. 1997, 25, 2816.
Sarfati, S. R.; Namane, A. Tetrahedron Lett. 1990, 31, 2581.
(5) Ward, D. C.; Reich, E.; Stryer, L. J. Biol. Chem. 1969, 244,
1228.
Bloom, L. B.; Otto, M. R.; Beechem, J. M.; Goodman, M. F.
Biochemistry 1993, 32, 11247.
(6) Secrist, J. A.; Barrio, J. R.; Leonhard, N. J. Science 1972, 175,
646.
Toulmé, J. J.; Hélene, C. Biochim. Biophys. Acta 1980, 606,
95.
(7) Seela, F.; Chen, Y. Nucleic Acids Res. 1995, 23, 2499.
Seela, F.; Chen, Y.; Bindig, U.; Kazimierczuk, Z. Helv. Chim.
Acta 1994, 77, 194.
(8) Adams, C. J.; Murray, J. B.; Arnold, J. R. P.; Stockley, P. G.
Tetrahedron Lett. 1994, 35, 1597.
(9) Ren, R. X.-F.; Chaudhuri, N. C.; Paris, P. L.; Rumney, S.;
Kool, E. T. J. Am. Chem. Soc. 1996, 118, 7671.
Schweitzer, B. A.; Kool, E. T. J. Am. Chem. Soc. 1995, 117,
1863.
(10) Fourrey, J. L.; Varenne, J.; Blonski, C.; Dousset, P.; Shire, D.
Tetrahedron Lett. 1987, 28, 5157.
Article Identifier:
1437-210X,E;2000,0,01,0149,0153,ftx,en;H05599SS.pdf
Yamana, K.; Nunota, K.; Nakano, H.; Sangen, O. Tetrahedron
Lett. 1994, 35, 2555.
Synthesis 2000, No. 1, 149–153 ISSN 0039-7881 © Thieme Stuttgart · New York