Synthesis of 3'-thioamido-modified 3'-deoxythymidine 5'-triphosphates by regioselective thionation and their use as chain terminators in DNA sequencing

Article abstract of DOI:10.1002/1522-2675(20000607)83:6<1268::AID-HLCA1268>3.0.CO;2-S

The thioamide derivatives 3'-deoxy-5'-O-(4,4'-dimethoxytrityl)-3'-[(2- methyl-1-thioxopropyl)amino]thymidine (4a) and 3'-deoxy-5'-O-(4,4'- dimethoxytrityl)-3'-{{6-{[(9H-(fluoren-9-ylmethoxy)carbonyl]amino}-1- thioxohexyl}amino}thymidine (4b) were synthesi

Full text of DOI:10.1002/1522-2675(20000607)83:6<1268::AID-HLCA1268>3.0.CO;2-S

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Synthesis of 3'-Thioamido-Modified 3'-Deoxythymidine 5'-Triphosphates
by Regioselective Thionation and Their Use as Chain Terminators
in DNASequencing
by Christian Wojczewski, Karin Schwarzer, and Joachim W. Engels*
Institut für Organische Chemie, Johann Wolfgang Goethe-Universität, Marie-Curie-Str. 11,
60439 Frankfurt am Main, Germany (phone: (‡49)69-798 29146; fax: (‡49)69-798 29148;
e-mail: engels@ews1.org.chemie.uni-frankfurt.de)
Dedicated to Prof. Dr. F. Seela on the occasion of his 60th birthday
The thioamide derivatives 3'-deoxy-5'-O-(4,4'-dimethoxytrityl)-3'-[(2-methyl-1-thioxopropyl)amino]thy-
midine (4a) and 3'-deoxy-5'-O-(4,4'-dimethoxytrityl)-3'-{{6-{[(9H-(fluoren-9-ylmethoxy)carbonyl]amino}-1-
thioxohexyl}amino}thymidine (4b) were synthesized by regioselective thionation of the corresponding amides
3a and 3b with 2,4-bis(4-methoxyphenyl)-1,3,2,4-dithiadiphosphetane 2,4-disulfide (Lawessonꢀs reagent). The
addition of exact amounts of pyridine to the reaction mixture proved to be essential for an efficient
transformation. The thioamides were converted into the corresponding 5'-triphosphates 6a and 6b. Compound
6a was chosen for DNA sequencing experiments, and 6b was further labelled with fluorescein (! 8).
Introduction. ± Recently, the first complete sequence of a human chromosome has
been published [1]. Chromosome 22 is only one step in the huge Human Genome
Project, which, itself, is just one of several genome-sequencing projects that are
currently being carried out [2]. The Sanger procedure, invented in 1977, is still the
method of choice to determine the sequence of bases [3][4]. Although the develop-
ment of automated methods, combined with the dye-primer or dye-terminator
chemistry, has greatly accelerated the speed of sequencing, the need for high-
throughput and accurate DNA-sequencing techniques is still rapidly growing.
Dye-terminator sequencing is characterized by the enzymatic incorporation of
chain-terminating, fluorescent nucleotides. These terminators are attached to fluores-
cent dyes at their base moiety [5 ± 8]. Apart from this labelling strategy, there are other
potential positions within nucleosides that may be used for markers [9]. Different
groups have demonstrated that 3'-ether- and 3'-ester-functionalized nucleotides can act
as substrates for an enzyme-mediated DNA synthesis [10 ± 12]. We have shown that 3'-
amino- and 3'-amido-modified nucleoside 5'-triphosphates are excellent terminators
[13][14]. However, the ability of DNA polymerases to hydrolyze ester and amide
bonds at the 3'-position limits the use of these 3'-dye terminators for DNA sequencing
since the label is cleaved off during the incorporation process [15].
Therefore, we decided to alter the link between dye and sugar moiety to find a
linkage that is stable against enzymatic degradation. In this context, we have already
reported the synthesis of 3'-thioether- [16], 3'-alkyl- [17], 3'-urea-, and 3'-thiourea-
modified [18] 2',3'-dideoxynucleoside 5'-triphosphates.

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Here, we describe an efficient synthesis of 3'-thioamido-modified thymidines, a new
class of sugar-modified nucleosides, and the investigation of their substrate acceptance
in DNA-sequencing experiments.
Results and Discussion. ± The starting material 3'-azido-3'-deoxy-5'-O-(4,4'-
dimethoxytrityl)thymidine (1) was obtained as described before [19][20] and was
converted into the amino derivative 2 by hydrogenolytic reduction catalyzed by
PtO2 ´ H2O (Scheme 1). This procedure allowed the isolation of 2 in high yield without
further purification.
To our knowledge, no synthetic route to thioamido-tethered nucleosides has been
published until now. Although a broad spectrum of reactions exists for the synthesis of
thioamides [21][22], only the conversion of amides into thioamides seems to be
suitable for nucleoside chemistry. Therefore, we synthesized from 2 the readily
accessible amide 3a as a model compound for the investigation of the thionation
reaction. In a second synthetic approach, we chose linker 9 to introduce a free amino
group for dye labelling at position 3'. This amino function had to be protected to
prevent side reactions during the triphosphate synthesis. The protecting group ought to
be removable under basic conditions due to the acid lability of triphosphates.
Therefore, we chose the Fmoc group (Scheme 2), i.e., the linker 10 was introduced via
an amide coupling at the 3'-position of the sugar moiety, resulting in 3b.
Direct action of H2Sor P 2S5 on 3 would result in an undesired exchange of the two
carbonyl O-atoms at C(2) and C(4) of the base moiety. A more sophisticated thionation
reagent is 2,4-bis(4-methoxyphenyl)-1,3,2,4-dithiadiphosphetane 2,4-disulfide (Law-
essonꢀs reagent), which converts carbonyl groups into thiocarbonyl groups in high yield
[23 ± 25]. Unbranched, alkyl-substituted amides can be thionated by Lawessonꢀs
reagent at room temperature within minutes, whereas imides and compounds with
unsaturated residues require longer reaction times and/or elevated temperatures. Thus,
our aim was to utilize the different reaction conditions for a regioselective conversion
at the C(3') position. Wörner showed that 2',3',5'-tri-O-benzoyl-N2-benzoylguanosine
and -inosine were thionated at the nucleobase C(6)ˆO function when refluxed with
Lawessonꢀs reagent in pyridine for 6 h [26]. Therefore, 3a was reacted with Lawessonꢀs
reagent for 30 min without heating. Humidity had to be strictly eliminated to avoid
formation of phosphoric acids and thus removal of the 5'-hydroxy-protecting group.
Although no by-products were observed under these reaction conditions, the yield of
4a was rather poor (22%); 33% of the educt was recovered unchanged.
The synthesis of 4b under the same conditions resulted in even lower yields, caused
mainly by 5'-hydroxy deprotection. Therefore, we tested basic solvents as scavengers
for the phosphoric acids. Thionation of 3b (Fig. 1) in pure anh. pyridine instead of anh.
THF did not at all indicate any formation of 4b, even when the amount of Lawessonꢀs
reagent was drastically increased. Thus, the reaction of 3b with Lawessonꢀs reagent was
accomplished in mixtures of anh. THF/pyridine. We observed that lowering the amount
of pyridine gradually resulted in increased yield. Finally, the addition of 1 equiv. of
pyridine and 4 equiv. of Lawessonꢀs reagent in anh. THF as solvent yielded 4b in 50%;
almost 50% of the intact educt was recovered.
Both thioamides 4 were clearly identified by mass spectrometry and NMR
spectroscopy. The electrospray-ionization MS(ESI-MS) showed formation of only one

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Scheme 1
a) PtO2 ´ H2O, H2, MeOH. b) For 3a: (iPrCO)2O, pyridine; for 3b: 6-{[(9H-fluoren-9-ylmethoxy)carbonyl]-
i
amino}hexanoic acid (10), DMF, dioxane, H2O, Pr2NEt, TSTU. c) For 4a: Lawessonꢀs reagent, THF; for 4b:
Lawessonꢀs reagent, THF, pyridine. d) 80% aq. AcOH soln. e) 1. 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-
one, dioxane, pyridine, DMF; 2. (Bu3NH)2P2O7, DMF, tributylamine; 3. 1% I2 in pyridine/H2O 98 :2; 4. 5% aq.
NaHSO3 soln. f) piperidine, pyridine, DMF. g) FITC, DMF, aq. NaHCO3 soln. (pH 9.3).
product with an increased weight of 16 Da compared with the educt, representing the
exchange of an O-atom against a S-atom. The 13C-NMR spectrum revealed the identity
of the carbonyl group involved. The 13C-chemical shifts of C(2) and C(4) of the base
moiety remained unchanged, whereas the carbonyl signal at the 3'-terminal moved
downfield as expected (Table).

Helvetica Chimica Acta ± Vol. 83 (2000)
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Scheme 2
a) N-[(9H-Fluoren-9-ylmethoxy)carbonyl]succinimide, NaHCO3, H2O, acetone.
Fig. 1. 5'-O-(4,4'-dimethoxytrityl)-3'-{{6-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}-1-oxohexyl}amino}thy-
midine (3b)
Table. Electrospray-Ionization MS (ESI-MS) and 13C-NMR Carbonyl Signals of Compounds 3 and 4
m/z
d(C) [ppm]
C(2)
C(4)
CONHÀC(6'')
CXNHÀC(3')
3a (X ˆ O)
4a (X ˆ S)
3b (X ˆ O)
4b (X ˆ S)
612.7a)
628.3 a)
896.6b)
893.6 c)
150.8
150.7
150.8
150.7
164.1
164.2
163.9
164.2
±
±
177.4
213.2
173.2
206.4
156.5
156.4
‡
‡
c
a) ESI (neg.). b) ESI (pos.) and NH4 adduct, M 879.0. ) ESI (neg.), MÀ 895.1.
The thioamides 4a and 4b were deprotected in 80% aqueous AcOH solution at
room temperature, and the syntheses of the triphosphates 6a and 6b were achieved
according to the procedure of Ludwig and Eckstein [27]. The linker amino function of
6b was deprotected with 20% piperidine in pyridine and DMF at room temperature
(! 7) and subsequently coupled with fluorescein isothiocyanate (FITC) in aq. 0.1m
NaHCO3 (pH 9.3) and DMF at room temperature for 24 h (! 8).
Triphosphate 6a was substituted for ddTTP as a terminator in DNA-sequencing
experiments. No band pattern was detected with sequenase or with thermosequenase.
With Taq DNA polymerase, we obtained a band pattern over a range of several
hundred bases (Fig. 2, Lanes 7 ± 9). This pattern did not correlate with the standard
ddTTP sequence. It was, however, identical with the band pattern obtained with the 3'-
(alkylthio)-3'-deoxythymidine 5'-triphosphate 11 (Fig. 3), an alternatively 3'-modified

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Fig. 2. DNA Sequencing with IRD-41-labelled primer, M13 mp18 template DNA,
Taq DNA Polymerase, and 2'-deoxynucleoside 5'-triphosphates (dNTP) as
substrates. Fluorescence detection on a LI-COR DNA sequencer. Lanes A, C,
G, and T terminated with 2',3'-dideoxyadenosine 5'-triphosphate (ddATP),
ddCTP, ddGTP, or ddTTP, respectively; Lanes 1 ± 3 terminated with 11, lanes
7 ± 9 terminated with 6a. Terminator concentration: Lanes 1 and 7, 750 mm; Lanes
2 and 8, 3.75 mm; 3 and 9, 5.25 mm.

Helvetica Chimica Acta ± Vol. 83 (2000)
1273
Fig. 3. 3'-{{2-{[(tert-Butoxy)carbonyl]amino}ethyl}thio}-3'-deoxythymidine 5'-triphosphate (11)
nucleotide [16] (see Fig. 2, Lanes 1 ± 3). Although 6a and 11 are obviously not specific
terminators for the enzymatic DNA synthesis under the chosen conditions, the result
cannot be called nonspecific, since two structurally different nucleotides were
incorporated at exactly the same positions within the DNA. This excludes a random
process. Variation of the terminator concentration only resulted in a different
fragment-length distribution. The reason for this could be a conformationally driven
selection process.
Conclusion. ± We demonstrated that 3'-thioamido-modified nucleosides can
efficiently be synthesized from 3'-amido-tethered nucleosides by a regioselective
thionation strategy applying Lawessonꢀs reagent. Addition of 1 equiv. of pyridine to the
reaction mixture was necessary to prevent degradation of the nucleoside and ensure
good yields at the same time. Thioamide 6a was accepted as a substrate by the Taq
DNA polymerase. However, specific incorporation did not occur. Rather the band
pattern showed a distinct correlation with the 3'-thioether-tethered nucleoside
triphosphate 11.
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. DNA-Sequencing experiments were
performed by Dr. S. Klingel and Dr. G. Sagner at Roche Diagnostics, Penzberg, Germany.
Experimental Part
General. Solvents were of anal. grade and were dried and distilled or purchased and stored over molecular
sieves. Anal. TLC: Silica gel 60 F254 plates (Merck) for TLC. Prep. TLC: covered glass plates (gypsum silica gel
60 PF254 from Merck), Harrison Research 7924T Chromatotron. FPLC (fast protein liquid chromatography):
Pharmacia instrument with LCC-500 controller, P-500 pumps, and single-path UV monitor UV-1. HPLC:
Merck-Hitachi with L-4250-UV-VIS detector and Shimadzu RF-535 fluorescence monitor. DNA Sequencing:
LI-COR automated DNA sequencer with M13 mp18 as template, Taq DNA polymerase, 5'-fluorescent-labelled
primer, and 6a or 11 (0.75 mm, 3.75 mm, 5.25 mm) as substitute for ddTTP. UV Spectra and optical density:
Varian Cary 1; optical density measured at 270 nm (molar absorptivity in H2O, 8700 l molÀ1 cmÀ1). IR Spectra: nÄ
in cmÀ1 1H-NMR Spectra: Bruker AM250 (250 MHz) or WH270 (270 MHz); d in ppm; calibrating signals:
.
d((D6)DMSO) ˆ 2.50, d(CDCl3) ˆ 7.26. 13C-NMR Spectra: Bruker AM250 (62.9 MHz) or WH270 (67.9 MHz);
calibrating signals: d((D6)DMSO) 39.5, d(CDCl3) 77.0. 31P-NMR Spectra: Bruker AMX 400 (162 MHz);
calibrating signal: external 85% phosphoric acid. ESI-MS: Fisons-VG-Platform-II electrospray-ionization mass
spectrometer; in m/z.
General Purification Procedures. a) FPLC: filtered (0.2-mm Nalgene syringe filter) crude product in H2O
was applied to a column filled with Pharmacia-DEAE-Sephadex-A25 anion-exchange gel, 2.5 Â 15 cm, 48;

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Helvetica Chimica Acta ± Vol. 83 (2000)
A ˆ 0.05m (Et3NH)HCO3 (pH 7.5), B ˆ 0.5m (Et3NH)HCO3 (pH 7.5); flow rate 1 ml/min, 0 ! 100% B,
500 ml).
b) Anion-exchange HPLC: Synchropak AX 300, 6 mm (250  4.6 mm); A ˆ 0.05m KH2PO4/50% formamide
(pH 5.2), B ˆ 0.05m KH2PO4/1m (NH4)2SO4/50% formamide (pH 5.5); flow rate 1 ml/min, 0 ± 30 min 0 ! 100%
B. HPLC retention times tR [min] refer to anion-exchange HPLC.
c) Reversed-phase HPLC: LiChrosphere RP-18 5 mm (3  125 mm); A ˆ 0.1m (Et3NH)OAc (pH 5.5), B ˆ
A ‡ 50% MeCN; flow rate 1 ml/min, 0 ± 10 min 5% B.
3'-Amino-3'-deoxy-5'-O-(4,4'-dimethoxytrityl)thymidine (2). The 3'-azido-3'-deoxy-5'-O-(4,4'-dimethoxy-
trityl)thymidine (1; 2.5 g, 4.4 mmol) was filled in a heat-dried flask and dissolved in anh. MeOH (30 ml) under
Ar. The soln. was degassed repeatedly by alternating evacuation and flushing with Ar. A small amount (two
spatula tips) of PtO2 ´ H2O was added to the stirred soln., and H2 was injected for 1 h. The flask was fitted with a
septum and a H2-filled balloon and stirred overnight at 508. The mixture was then filtered over Celite, the Celite
washed with MeOH several times, and the combined filtrate evaporated: 2.21 g (93%) of pure 2. No further
purification was necessary. TLC (CH2Cl2/MeOH 95 :5): Rf 0.18. IR (KBr): 2931, 1694, 1607, 1509, 1445, 1252,
1179, 1033, 829. 1H-NMR ((D6)DMSO): 7.58 (d, HÀC(6)); 7.44 ± 7.18 (m, 9 arom. H); 6.83 (d, 4 arom. H); 6.25
(dd, HÀC(1')); 3.78 (s, 2 MeO); 3.77 ± 3.68 (m, HÀC(4'), HÀC(3')); 3.53 ± 3.32 (m, 2 HÀC(5')); 2.40 ± 2.15 (m,
2 HÀC(2')); 1.52 (s, MeÀC(5)). ESI-MS (neg.): 542.4 (MÀ, C31H32N3O6À ; calc. 542.6).
3'-Deoxy-5'-O-(4,4'-dimethoxytrityl)-3'-[(2-methyl-1-oxopropyl)amino]thymidine (3a).
A
soln. of
2
(400 mg, 0.74 mmol) in anh. pyridine (20 ml) and isobutyryl anhydride (2 ml, 8.5 mmol) was stirred under Ar
for 2 d and quenched with MeOH. The soln. was evaporated and the residue purified by prep. TLC (CH2Cl2/
1
MeOH 98 :2): 0.38 g (84%) of 3a. TLC (CH2Cl2/MeOH 9 :1): Rf 0.49. H-NMR (CDCl3): 7.65 (d, HÀC(6));
7.42 ± 6.79 (m, 13 arom. H); 6.43 (dd, HÀC(1')); 4.70 ± 4.65 (m, HÀC(3')); 4.02 (d, HÀC(4')); 3.78 (s, 2 MeO);
3.48 (s, 2 HÀC(5')); 2.60 ± 2.31 (m, 2 HÀC(2'), Me2CHCO); 1.37 (d, MeÀC(5)); 1.12 (d, Me2CHCO). 13C-NMR
(CDCl3): 177.4 (CONHÀC(3')); 164.1 (C(4)); 158.7 ((MeO)2Tr); 150.8 (C(2)); 144.4 ((MeO)2Tr); 135.7, 135.4
(C(6), (MeO)2Tr); 130.1, 128.2, 127.9, 127.1, 113.3 ((MeO)2Tr); 111.5 (C(5)); 87.0, 85.4, 84.6 (C(1'), C(4'),
(MeO)2Tr (C); 64.1 (C(5')); 55.2 (MeO); 51.0 (Me2CHCO); 38.0, 35.1 (C(3'), C(2')); 19.5, 18.8 (Me2CHCO);
11.6 (MeÀC(5)). ESI-MS (neg.): 612.7 (MÀ, C35H38N3O7À ; calc. 612.7).
3'-Deoxy-5'-O-(4,4'-dimethoxytrityl)-3'-[(2-methyl-1-thioxopropyl)amino]thymidine (4a). To a soln. of 3a
(200 mg, 0.33 mmol) in anh. THF (10 ml), Lawessonꢀs reagent (66 mg, 0.16 mmol) was added. After stirring at
r.t. for 30 min, sat. NaHCO3 soln. (20 ml) was added. The resulting white precipitate was filtered and washed
with H2O. The aq. phase was extracted with CH2Cl2 twice, the combined org. layer dried (Na2SO4) and
evaporated, and the residual solid purified by prep. TLC (CH2Cl2/MeOH 98 :2): 45 mg (22%) of 4a. TLC
(CH2Cl2/MeOH 9 :1): Rf 0.54. 1H-NMR (CDCl3): 7.76 (d, HÀC(6)); 7.46 ± 6.83 (m, 13 arom. H); 6.63 (t,
HÀC(1')); 5.31 (m, HÀC(3')); 4.28 (d, HÀC(4')); 3.91 (d, 1 HÀC(5')); 3.79 (s, 2 MeO); 3.42 (d, 1 HÀC(5'));
3.05 ± 2.97 (m, Me2CHCS); 2.53 ± 2.48 (m, 2 HÀC(2')); 1.42 (d, MeÀC(5)); 1.26 (d, 3 H, Me2CHCS); 1.23 (d,
3 H, Me2CHCS). 13C-NMR (CDCl3): 213.2 (CSNHÀC(3')); 164.2 (C(4)); 158.7 ((MeO)2Tr); 150.7 (C(2));
144.6 ((MeO)2Tr); 135.7, 135.5 (C(6), (MeO)2Tr); 130.1, 128.2, 128.0, 127.1, 113.3 ((MeO)2Tr); 111.7 (C(5));
87.2, 85.0 (C(1'), C(4')); 65.5 (C(5')); 57.0 (Me2CHCS); 55.3 (MeO); 42.9 (C(2')); 37.0 (C(3')); 23.1, 22.6
(Me2CHCS); 11.7 (MeÀC(5)). ESI-MS (neg.): 628.3 (MÀ, C35H38N3O6SÀ; calc. 628.8).
3'-Deoxy-3'-[(2-methyl-1-thioxopropyl)amino]thymidine (5a). A soln. of 4a (235 mg, 0.373 mmol) in 80%
AcOH/H2O (20 ml) was left stirring for 2 h. About 90% of the solvent was evaporated and the resulting oil co-
evaporated (3Â) with H2O to remove residual acid. The residue was purified by prep. TLC (CH2Cl2/MeOH
9 :1): 113 mg (93%). TLC (CH2Cl2/MeOH 8 :2): Rf 0.53. 1H-NMR ((D6)DMSO): 11.28 (s, HÀN(3)); 10.24 (d,
CSNHÀC(3')); 7.83 (d, HÀC(6)); 6.30 (t, HÀC(1')); 5.15 (t, OHÀC(5')); 4.92 ± 4.87 (m, HÀC(3')); 3.95 (q,
HÀC(4')); 3.69 (m, 2 HÀC(5')); 2.89 (sept., Me2CHCS); 2.39 ± 2.28 (m, 1 HÀC(2')); 2.20 (ddd, 1 HÀC(2'));
1.78 (s, MeÀC(5)); 1.12 (d, Me2CHCS). 13C-NMR ((D6)DMSO): 210.7 (CSNHÀC(3')); 163.7 (C(4)); 150.5
(C(2)); 136.0 (C(6)); 109.5 (C(5)); 85.0, 83.9 (C(1'), C(4')); 62.2 (C(5')); 55.6 (Me2CHCS); 41.2 (C(2')); 36.0
(C(3')); 22.9, 22.6 (Me2CHCS); 12.4 (MeÀC(5)). ESI-MS (neg.): 326.3 (MÀ, C14H20N3O4SÀ; calc. 326.4).
3'-Deoxy-3'-[(2-methyl-1-thioxopropyl)amino]thymidine 5'-Triphosphate (6a). Compound 5a (110 mg,
0.336 mmol) was dried by co-evaporation (3Â) with anh. pyridine (0.7 ml)/DMF (2.75 ml). The residue was
dried over P2O5 under vacuum overnight. The nucleoside was dissolved in anh. pyridine (0.7 ml)/DMF
(2.75 ml), a freshly prepared soln. of 1m 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one in anh. dioxane
(0.37 ml) was injected under Ar, and the mixture stirred for 20 min. Then a freshly prepared mixture (1.35 ml) of
0.5m (Bu3NH)2P2O7 in dry DMF/anh. Bu3N 3 :1 was instantly added. The mixture was stirred for further 30 min.
Oxidation of the P-moiety and ring opening was achieved by adding 6.7 ml of 1% I2 in pyridine/H2O 98 :2. After
20 min, remaining I2 was reduced by adding dropwise a 5% aq. NaHSO3 soln. until the brown color changed to

Helvetica Chimica Acta ± Vol. 83 (2000)
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light yellow. After evaporation without heating, H2O (5 ml) was poured onto the residue, and the soln.
containing the dissolved product was separated from the remaining residue by filtering through a 0.2 mm
Nalgene syringe filter. The soln. was evaporated without heating, the residual solid dissolved in H2O (5 ml), and
this soln. filtered through a 0.2 mm Nalgene syringe filter and purified by FPLC (purification procedure a),
elution concentration 0.26m (Et3NH)HCO3). The fractions containing product were lyophilized. The yield of 6a
was determined by optical-density measurement (32.9 mmol, 10%). TLC (iPrOH/NH3/H2O): Rf 0.08. 31P-NMR
(D2O): À23.3 (t, (b)); À11.8 (d, P(a)); À9.6 (d, P(g)).
6-{[(9H-Fluoren-9-ylmethoxy)carbonyl]amino}hexanoic Acid (10). N-[(9H-Fluoren-9-ylmethoxy)carbo-
nyl]succinimide (5 g, 14.82 mmol) and NaHCO3 (1.24 g, 14.82 mmol) were suspended in a soln. of 6-
aminohexanoic acid (9) (1.95 g, 14.82 mmol) in H2O/acetone 1:1 (100 ml). The mixture was stirred for 3 h (TLC
monitoring). Then the acetone was evaporated, and CH2Cl2 was added. The org. phase was washed with 0.1n
HCl and H2O, then dried (Na2SO4), and evaporated. Crystallization of the residue from CH2Cl2/hexane
1
afforded 10 (5.2 g, 99%). White solid. M.p. 117 ± 1208. TLC (CH2Cl2/MeOH 9 :1): Rf 0.13. H-NMR (CDCl3):
7.75 ± 7.78 (d, 2 H, Fmoc); 7.58 ± 7.61 (d, 2 H, Fmoc); 7.26 ± 7.43 (m, 4 H, Fmoc); 4.80 (br., NH); 4.41 ± 4.43 (d,
CH2OCONH); 4.20 ± 4.24 (m, HÀC(9) (Fmoc)); 3.07 ± 3.21 (m, CH2(6)); 2.33 ± 2.39 (t, CH2(2)); 1.37 ± 1.66 (m,
CH2(3), CH2(4), CH2(5)). 13C-NMR (CDCl3): 178.8 (C(1)); 156.34 (CH2OCONH); 143.8, 141.2, 127.5, 126.9,
124.9, 119.8 (C(Fmoc)); 66.4 (CH2OCONH); 47.1 (C(9) (Fmoc)); 40.6 (C(2)); 33.6 (C(6)); 29.4 (C(5)); 25.9
‡
‡
(C(3)); 24.1 (C(4)). ESI-MS (pos.): 354.1 (M , C21H24NO4 ; calc. 354.3).
3'-Deoxy-5'-O-(4,4'-dimethoxytrityl)-3'-{{6-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}-1-oxohexyl}amino}-
thymidine (3b). To a soln. of 10 (636 mg, 1.8 mmol) and 2 (1.17 g, 2.15 mmol) in DMF (5 ml), dioxane (15 ml),
i
and H2O (5 ml), Pr2NEt (0.4 ml, 2.4 mmol) and N,N,N',N'-tetramethyl-O-succinimidouronium tetrafluorobo-
rate (TSTU; 845 mg, 2.8 mmol) were added. After stirring at r.t. for 30 min (TLC monitoring), H2O was added.
The aq. phase was extracted with CH2Cl2 (4Â), the combined org. layer dried (MgSO4) and evaporated, and the
residue purified by prep. TLC (CH2Cl2/MeOH 98 :2): 585 mg (37%) of 3b. TLC (CH2Cl2/MeOH 9 :1): Rf 0.38.
1H-NMR (CDCl3): 9.90 (br., 1 NH); 7.99 (s, HÀC(6)); 7.71 ± 7.73 (d, 2 H, Fmoc); 6.78 ± 7.61 (m, 19 H, (MeO)2Tr,
Fmoc); 6.36 (t, HÀC(1')); 5.08 (br., CH2OCONH); 4.70 ± 4.71 (m, HÀC(3')); 4.34 ± 4.36 (m, CH2OCONH);
4.20 ± 4.22 (m, HÀC(9) (Fmoc)); 4.00 ± 4.01 (m, HÀC(4')); 3.79 (s, 2 MeO); 3.42 ± 3.43 (m, 2 HÀC(5')); 3.12 ±
3.14 (m, CH2(6'')); 2.15 ± 2.45 (m, 2 HÀC(2'), CH2(2'')); 1.25 ± 1.62 (m, CH2(3''), CH2(4''), CH2(5'')); 1.37 (s,
MeÀC(5)). 13C-NMR (CDCl3): 173.2 (CONHÀC(3')); 163.9 (C(4)); 158.6 ((MeO)2Tr); 156.5 (CH2OCONH);
150.8 (C(2)); 144.3 ((MeO)2Tr); 143.9, 141.2 (C(Fmoc)); 135.6, 135.4 (C(6), (MeO)2Tr); 130.1, 128.2, 127.9,
127.0 ((MeO)2Tr); 127.6, 126.9, 124.9, 119.9 (Fmoc)); 113.2 ((MeO)2Tr); 111.4 (C(5)); 87.0, 85.0, 84.4 (C(1'),
C(4'), (MeO)2Tr(C)); 66.5 (CH2OCONH); 64.1 (C(5')); 55.2 (MeO); 47.2 (C(9) (Fmoc)); 40.7 (C(2'')); 38.0,
35.9 (C(3'), C(2')); 31.4, 29.5, 26.2, 25.0 (C(6''), C(5''), C(4''), C(3'')); 11.6 (MeÀC(5)). ESI-MS (pos.): 896.6
‡
‡
(M , C52H54N4O9 ´ NH4 ; calc. 897.0).
3'-Deoxy-5'-O-(4,4'-dimethoxytrityl)-3'-{{6-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}-1-thioxohexyl}-
amino}thymidine (4b). To a soln. of 3b (1 g, 1.14 mmol) in anh. THF (70 ml) and anh. pyridine (92 ml,
1.14 mmol), Lawessonꢀs reagent (1.84 g, 4.56 ml) was added. After stirring at r.t. for 3 h, sat. NaHCO3 soln.
(100 ml) was added. The aq. phase was then extracted with CH2Cl2 (2Â), the combined org. layer dried
(MgSO4) and evaporated, and the residue purified by prep. TLC (CH2Cl2/MeOH 98 :2): 510 mg (50%) of 4b.
TLC (CH2Cl2/MeOH 9 :1): Rf 0.44. 1H-NMR (CDCl3): 10.19 (br., 1 NH); 9.22 (br., 1 NH); 6.72 ± 7.67 (m, 22 H,
HÀC(6), (MeO)2Tr, Fmoc); 6.49 (t, HÀC(1')); 5.42 (m, CH2OCONH); 4.83 (m, HÀC(3')); 4.27 ± 4.30 (m,
CH2OCONH); 4.21 (m, HÀC(9) (Fmoc)); 4.10 (m, HÀC(4')), 3.70 ± 3.78 (m, 1 HÀC(5')); 3.68 (s, 2 MeO);
3.31 ± 3.35 (m, 1 HÀC(5')); 3.06 ± 3.09 (m, CH2(6'')); 2.61 ± 2.66 (m, 2 HÀC(2')); 2.27 ± 2,42 (m, CH2(2'')); 1.27 ±
1.96 (m, CH2(3''), CH2(4''), CH2(5'')); 1.35 (s, MeÀC(5)). 13C-NMR (CDCl3): 206.4 (CSNHÀC(3)); 164.2
(C(4)); 158.4 ((MeO)2Tr); 156.4 (CH2OCONH); 150.7 (C(2)); 144.6 ((MeO)2Tr); 143.9, 141.3 (Fmoc); 135.6,
135.4 (C(6), (MeO)2Tr); 130.1, 128.1, 127.9, 127.05 ((MeO)2Tr); 127.6, 126.9, 125.0, 119.9 (Fmoc); 113.3
((MeO)2Tr); 111.5 (C(5)); 87.1, 85.8, 85.0 (C(1'), C(4'), (MeO)2Tr(C)); 66.5 (CH2OCONH); 65.3 (C(5')); 55.3
(MeO); 47.22 (C(9) (Fmoc)); 45.4 (C(2')); 40.7 (C(2'')); 38.6, 37.4 (C(3'), C(6'')); 29.5, 28.9, 25.8 (C(5''), C(3''),
C(4'')); 11.7 (MeÀC(5)). ESI-MS (neg.): 893.6 (MÀ, C52H53N4O8SÀ; calc. 894.1).
3'-Deoxy-3'-{{6-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}-1-thioxohexyl}amino}thymidine (5b). To a
soln. of 4b (140 mg, 0.156 mmol) in CH2Cl2 (2 ml), 80% AcOH/H2O (10 ml) was added. The mixture was stirred
at r.t. for 3 h (TLC monitoring) and then evaporated. The resulting oil was co-evaporated with H2O (3Â) and
hexane (1Â) to remove the residual acid before purifying. The residue was purified by prep. TLC (CH2Cl2/
MeOH 9 :1): 68 mg (74%) of 5b. TLC (CH2Cl2/MeOH 9 :1): Rf 0.37. 1H-NMR (CDCl3): 10.25 (s, 1 NH); 9.17 (d,
NHÀC(3')); 7.63 ± 7.69 (m, 3 H, HÀC(6), Fmoc); 7.47 (d, 2 H, Fmoc); 7.15 ± 7.31 (m, 4 H, Fmoc); 6.23 (t,
HÀC(1')); 5.15 (br., OHÀC(5')); 4.92 (m, HÀC(3')); 4.26 (m, CH2OCONH); 4.05 ± 4.11 (m, HÀC(9) (Fmoc));

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Helvetica Chimica Acta ± Vol. 83 (2000)
3.97 (m, HÀC(4')); 3.84 (m, 2 HÀC(5')); 3.02 ± 3.04 (m, CH2(6'')); 2.55 ± 2.58 (m, CH2(2'')); 2.18 ± 2.38 (m,
2 HÀC(2')); 1.78 (s, MeÀC(5)); 1.15 ± 1.68 (m, CH2(3''), CH2(4''), CH2(5'')). 13C-NMR (CDCl3): 206.4
(CSNHÀC(3')); 164.3 (C(4)); 156.6 (CH2OCONH); 150.8 (C(2)); 143.7, 141.1 (Fmoc); 136.0 (C(6)); 127.6,
126.9, 124.8, 119.9 (Fmoc); 111.0 (C(5)); 85.6, 84.7 (C(1'), C(4')); 66.6 (CH2OCONH); 62.4 (C(5')); 47.0 (C(9)
(Fmoc)); 45.6 (C(2')); 40.6 (C(2'')); 36.9 (C(3')); 29.0, 25.6 (C(5''), C(3''), C(4'')); 12.5 (MeÀC(5)). ESI-MS
(neg.): 591.5 (MÀ, C31H35N4O6SÀ; calc. 591.6).
3'-Deoxy-3'-{{6-{[(9H-fluoren-9-ylmethoxy)carbonyl]amino}-1-thioxohexyl}amino}thymidine 5'-Triphos-
phate (6b). As described for 6a, from 5b (150 mg, 0.253 mmol), after co-evaporation with anh. pyridine
(0.5 ml) and dissolution in anh. pyridine (0.5 ml)/DMF (2.55 ml), with 1m 2-chloro-4H-1,2,3-benzodioxaphos-
phorin-4-one in anh. dioxane (0.3 ml), 0.5m (Bu3NH)2P2O5 in dry DMF/Bu3N (3 :1 (1.07 ml), 5.4 ml of 1% I2 in
pyridine/H2O 98 :2), and FPLC (elution concentration 0.28m (Et3NH) HCO3): 6b (48 mmol, 19%). TLC
(iPrOH/NH3/H2O): Rf 0.15. 31P-NMR (D2O): À21.8 (t, P(b)); À10.8 (d, P(a)); À5.7 (d, P(g)). ESI-MS (neg.):
831.5 (MÀ, C31H38N4O15P3SÀ; calc. 831.7).
3'-[(6-Amino-1-thioxohexyl)amino]-3'-deoxythymidine 5'-Triphosphate (7). To a mixture of pyridine
(20 ml), DMF (12 ml), and piperidine (8 ml), 6b (48 mmol) was added. This soln. was stirred at r.t. for 1 h and
then evaporated without heating. The residual solid was dissolved in H2O (4 ml), filtered through a 0.2-mm
Nalgene syringe filter, and purified by FPLC (purification procedure a); elution concentration 0.14m
(Et3NH)HCO3). The fractions containing product were lyophilized. The yield of 7 was determined by
optical-density measurement (39 mmol, 81%). TLC (iPrOH/NH3/H2O): Rf 0.13.
1H-NMR (D2O): 7.86 (s, HÀC(6)); 6.26 (t, HÀC(1')); 4.95 ± 5.00 (m, HÀC(3')); 4.46 (m, HÀC(4')); 4.18 ±
4.28 (m, 2 HÀC(5')); 2.97 ± 3.15 (m, CH2(6'')); 2.66 ± 2.74 (m, CH2(2''), 1 HÀC(2')); 2.40 ± 2.47 (m, 1 HÀC(2'));
1.91 (s, MeÀC(5)); 1.33 ± 1.84 (m, CH2(3''), CH2(4''), CH2(5'')). 13C-NMR (D2O): 207.0 (CSNHÀC(3')); 166.5
(C(4)); 151.4 (C(2)); 137.6 (C(6)); 111.6 (C(5)); 85.3, 81.4 (C(1'), C(4')); 66.06 (C(5')); 42.54 (C(2')); 39.1
(C(2'')); 35.5 (C(3')); 28.1, 26.4, 24.3 (C(3''), C(4''), C(5'')); 11.5 (MeÀC(5)). 31P-NMR (D2O): À22.0 (t, P(b));
À10.6 (d, P(a)); À8.2 (d, P(g)). HPLC (purification procedures b and c): tR 4.8. ESI-MS (neg.): 609.2 (MÀ,
C11H33N4O13P3SÀ; calc. 609.4).
Reaction of 7 with Fluorescein Isothiocyanate: 3'-{{6-{{{[3-Carboxy-4-(6-hydroxy-3-oxo-3H-xanthen-9-
yl)phenyl]amino}thioxomethyl}amino}-1-thioxohexyl}amino}-3'-deoxythymidine 5'-Triphosphate (8). To a soln.
of 7 (10 mmol) in aq. NaHCO3 soln. (6 ml, pH 9.3), a soln. of fluorescein isothiocyanate (12 mg) in DMF (6 ml)
was added. The mixture was stirred for 24 h at r.t. The purification was done by HPLC (purification procedures
b and c): 8: HPLC: tR (8) 19.5; tR (fluorescein isothiocyanate) 12.5. ESI-MS (neg.): 998.3 (MÀ, C37H39N5O18P3SÀ2
;
calc. 998.8).
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Received February 29, 2000