Synthesis of 3'-sugar- and base-modified nucleotides and their application as potent chain terminators in DNA sequencing

Article abstract of DOI:10.1002/(SICI)1522-2675(19990908)82:9<1311::AID-HLCA1311>3.0.CO;2-Z

Two 3'-modified and three base-modified ddNTPs were synthesized and tested with several DNA polymerases for incorporation activity. Starting from 3'-azido-3'-deoxythymidine (AZT; 1), we were able to produce 3'-deoxy-3'- isocyanato-thymidine and 3'-deoxy-3'-isothiocyanatothymidine (3 and 4, resp.) in a rapid synthesis based on the solid-support approach (Scheme 1). These 3'-functionalities could be used to attach a spacer molecule via urea and thiourea groups, respectively. Since the thus-obtained tethered nucleotides 7 and 8 can be used to label with fluorescent dyes (cf. Scheme 5), they are convenient building blocks for practical applications in DNA sequencing. Furthermore, we synthesized, via 14 (Scheme 2), 17 (scheme 3), and 19 (Scheme 4), the N⁴-modified dideoxycytidine 5'-triphosphate dye derivatives 22, 23, and 24, respectively, with different lengths of linkers between the base residue and the dye (Scheme 5). Base-specific termination for the derivatives 22 and 24 was demonstrated (Fig. 2a and 2b).

Full text of DOI:10.1002/(SICI)1522-2675(19990908)82:9<1311::AID-HLCA1311>3.0.CO;2-Z

Helvetica Chimica Acta ± Vol. 82 (1999)
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Synthesis of 3'-Sugar- and Base-Modified Nucleotides and Their Application
as Potent Chain Terminators in DNA Sequencing
by Karen Stolze and Ulrich Koert
Institut für Organische und Bioorganische Chemie, Humboldt-Universität zu Berlin, Hessische Strasse 1 ± 2,
D-10115 Berlin
and Sven Klingel
Interactiva Biotechnologie GmbH, Sedanstrasse 10, D-89077 Ulm
and Gregor Sagner and Regina Wartbichler
Roche Diagnostics GmbH, Nonnenwald 2, D-82377 Penzberg
and Joachim W. Engels*
Institut für Organische Chemie, Johann Wolfgang Goethe-Universität Frankfurt, Marie-Curie-Str. 11, D-60439
Frankfurt
Dedicated to Prof. Karl Heinz Drexhage on the occasion of his 65th birthday
Two 3'-modified and three base-modified ddNTPs were synthesized and tested with several DNA
polymerases for incorporation activity. Starting from 3'-azido-3'-deoxythymidine (AZT; 1), we were able to
produce 3'-deoxy-3'-isocyanato-thymidine and 3'-deoxy-3'-isothiocyanatothymidine (3 and 4, resp.) in a rapid
synthesis based on the solid-support approach (Scheme 1). These 3'-functionalities could be used to attach a
spacer molecule via urea and thiourea groups, respectively. Since the thus-obtained tethered nucleotides 7 and 8
can be used to label with fluorescent dyes (cf. Scheme 5), they are convenient building blocks for practical
applications in DNA sequencing. Furthermore, we synthesized, via 14 (Scheme 2), 17 (Scheme 3), and 19
(Scheme 4), the N4-modified dideoxycytidine 5'-triphosphate dye derivatives 22, 23, and 24, respectively, with
different lengths of linkers between the base residue and the dye (Scheme 5). Base-specific termination for the
derivatives 22 and 24 was demonstrated (Fig. 2a and 2b).
1. Introduction. ± The Human Genome Project has greatly contributed to the need
for high-throughput DNA sequencing. Radioactive methods have largely been
replaced by automated non-isotopic systems using laser-induced fluorescence. Auto-
mated DNA sequencing refers to a variation of traditional Sanger sequencing [1].
Commercial systems use the dye-primer or dye-terminator approach. Several
companies (Applied Biosystems Inc. [2][3], Du Pont de Nemours & Co. [4],
Pharmacia LKB Biotechnology Inc. [5], and Hitachi [6]) have produced automated
DNA sequencers based on one of the two methods mentioned above. Usually,
dye-primer sequencing affords four separate extension/termination reactions where
each reaction contains four deoxynucleoside triphosphates (dNTPs) and one
dideoxynucleoside triphosphate (ddNTP). In the approach of Applied Biosystems
Inc., either four separate dye primers corresponding to each ddNTP or four differently
labeled dye terminators [7] are used. These dyes are detected and distinguished by

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analysis of their fluorescence emission spectra. Reading of four dyes in one lane
requires fluorophores with different emission maxima resulting from different
structures.
Dye-terminator sequencing is characterized by exclusive detection of correctly
terminated DNA fragments. There are three different approaches to link a marker to
the nucleotide: either at the sugar residue, or at the base moiety, or at the a-phosphate
group. To further increase the speed and efficiency of these systems, we intended to use
3'-sugar- as well as base-modified nucleotides linked to a fluorescent chromophore as
chain terminators.
2. Results and Discussion. ± 2.1. Synthesis of 3'-Sugar- and Base-Modified
Nucleotides. The 2'-deoxyribonucleoside 5'-triphosphates (dNTPs) modified at their
3'-hydroxy position can act as terminators of enzyme-directed DNA synthesis [8]. The
3'-amino-2',3'-dideoxynucleoside 5'-triphosphates are well accepted as substrates by
commonly used DNA polymerases [9]. However, linking of the dye via a 3'-
(carbonylamino) bond (carboxamide) is not suitable for most sequencing enzymes
because of their ability to hydrolyze these functions [10]. To obviate this property, we
intended to create chain terminators that allow dye labeling by modification of the 3'-
position of the sugar residue via new types of coupling strategies. Thus, we synthesized
3'-modified chain terminators in the thymidine series starting from 3'-azido-3'-
deoxythymidine (AZT; 1) (Scheme 1). Usually polymer-bound triphenylphosphine is
suitable for the reduction of azido-nucleosides into the corresponding amino-nucleo-
sides [11][12]. In this case, the generated phosphine imide is hydrolyzed by
concentrated aqueous ammonia. In a modified Wittig reaction, the phosphine imide
bond can also be cleaved by other nucleophilic reagents. For the synthesis of the
thymidine linker conjugates, AZT (1) was immobilized on polymer-bound triphenyl-
phosphine suspended in dioxane. The intermediate 2 was then treated either by passing
a stream of dry CO2 through the suspension or by addition of CS2 according to the
method of Molina et al. [13] and Zehl and Cech [14]. The recovered 3'-deoxy-3'-
isocyanato- and 3'-deoxy-3'-isothiocyanatothymidines (3 and 4, resp.), respectively,
were either isolated without by-products or immediately reacted with a monoprotected
hexane-1,6-diamine spacer to yield the urea or thiourea conjugates 5 and 6, which were
transformed to the 5'-triphosphates 7 and 8, respectively.
Template-dependent DNA polymerases accept as substrates various 2'-deoxynu-
cleoside 5'-triphosphates with modified base residues [15 ± 18]. As an alternative to the
above-mentioned sugar-labeling methods, we examined a concept where labeling in the
cytidine series is achieved by modification of the exocyclic amino group. There are
different approaches to introduce spacers to the NH2-group at C(4), which start from
either uridine or cytidine precursors: treatment of 4-N-(arylsulfonyl)cytosine residues
[19] or 5'-O-(dimethoxytrityl)-4-O-(2,6-dimethylphenyl)thymidine [20] with primary
amines [21] led to 4-N-(aminoalkyl)-substituted cytosine derivatives. Reaction of
cytosines with sodium hydrogen sulfite resulted in 5,6-dihydrocytosine-6-sulfonate
[22], which could readily be substituted at NH2 C(4) with a primary amine [23][24].
Treatment of cytidines with 1-(phenoxycarbonyl)-1H-tetrazole resulted in conversion
of the exocyclic NH2 group to the corresponding urethane, which could be used to
introduce an amino linker under formation of an urea bond [25]. Starting from uracil

Helvetica Chimica Acta ± Vol. 82 (1999)
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Scheme 1. Synthesis of 3'-Sugar-Modified 3'-Deoxythymidine 5'-Triphosphates 7 and 8
a) Polymer-bound triphenylphosphine, dioxane. b) CO2, dioxane. c) CS2, dioxane. d) N1-(Trifluoracetyl)-
protected hexane-1,6-diamine trifluoroacetate, (i-Pr)2EtN, pyridine. e) 1. 2-Chloro-4H-1,3,2-benzodioxapho-
sphorin-4-one, bis(tributylammonium) pyrophosphate, Bu3N, I2, pyridine, dioxane, DMF; 2. Conc. NH3 soln.
derivatives, the C(4) position could be activated with 1H-1,2,4-triazole as well as with
phosphoroxychloride [26] or 4-chlorophenyl phosphorodichloridate [27].
For the modification of the exocyclic NH2 group in the cytidine series, with a
substituted hexane spacer, we used a modified Sung [28] procedure (Scheme 2).
Starting from 2',3'-dideoxyuridine, selective protection of the 5'-OH group was carried
out with dimethoxytrityl chloride in pyridine ( ! 9). Treatment of compound 9 with
1H-1,2,3,4-tetrazole and 2-chlorophenyl phosphorodichloridate gave the 4-N-deamino-
4-C-tetrazoylcytidine derivative 10 which was not isolated but treated immediately with
a monoprotected hexane-1,6-diamine to give 4-N-{6-{[(tert-butoxy)carbonyl]amino}-
hexyl}-2',3'-dideoxy-5'-O-(dimethoxytrityl)cytidine (11). Subsequently, removal of the
dimethoxytrityl group was carried out in 80% AcOH/H2O for 45 min. Purification of
the crude product gave N4-{6-{[(tert-butoxy)carbonyl]amino}hexyl}-2',3'-dideoxycyti-
dine (12) in 35% overall yield, which was transformed to the 5'-triphosphates 13 and 14.
Introduction of the short spacer arm in the cytidine series was performed by heating
2',3'-dideoxycytidine and monoprotected propane-1,3-diamine in a hydrogen-sulfite-
containing solution in water at 558 for 5 h to give 4-N-{3-{[(tert-butoxy)carbonyl]ami-
no}propyl}-2',3'-dideoxycytidine (15), which was transformed to the 5'-triphosphates 16
and 17 (Scheme 3).
The target nucleosides 5, and 6 (Scheme 1), 12 (Scheme 2), and 15 (Scheme 3)
could be triphosphorylated at the 5'-hydroxy group according to the method of Ludwig
and Eckstein [29]. All triphosphates were purified by ion-exchange chromatography.
1
The purity of the products was established by HPLC and H, 13C-, and 31P-NMR
spectroscopy. Removal of the (tert-butoxy)carbonyl group from triphosphates 13 and
16 with CF3COOH in 2 min did not affect the N-glycosidic bond in the cytidine

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Scheme 2. Synthesis of N4-(6-Aminohexyl)-2',3'-dideoxycytidine 5'-Triphosphate (14)
a) 1H-1,2,3,4-Tetrazole, 2-chlorophenyl phosphorodichloridate, pyridine. b) N1-[(tert-Butoxy)carbonyl]-
protected hexane-1,6-diamine, dioxane. c) 80% AcOH/H2O. d) 1. 2-Chloro-4H-1,3,2-benzodioxaphosphorin-
4-one, bis(tributylammonium) pyrophosphate, Bu3N, I2, pyridine, dioxane, DMF; 2. CF3COOH
Scheme 3. Synthesis of N4-(6-Aminopropyl)-2',3'-dideoxycytidine 5'-Triphosphate (17)
a) NaOH, NaHSO3, N1-[(tert-butoxy)carbonyl]-protected propane-1,3-diamine, H2O. b) 1. 2-Chloro-4H-1,3,2-
benzodioxaphosphorin-4-one, bis(tributylammonium) pyrophosphate, Bu3N, I2, pyridine, dioxane, DMF;
2. CF3COOH.
derivatives. The trifluoroacetyl groups of the modified 3'-deoxythymidine 5'-triphos-
phates obtained from 5 and 6 were removed with concentrated ammonia. Removal of
the protecting groups generated a primary amino group at the linker arm, which
allowed reaction with various types of compounds, i.e., linkers and dyes with a
functional group reacting with a nucleophilic amino group.
There is a strong dependence of the spacer length between dye and nucleotide upon
quantum yields and absorption coefficients [30][31]. We, therefore, decided to
elongate the spacer arm of the N4-modified cytidine derivative 14 with a rigid 4-
(aminomethyl)cyclohexylcarbonyl moiety ( ! 18 and 19; Scheme 4).

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Scheme 4. Synthesis of an N4-Modified 2',3'-Dideoxycytidine 5'-Triphosphate with a Rigid Linker Arm
a) 4-{[(Trifluoroacetyl)amino]methyl}cyclohexanecarboxylic acid, TSTU (N,N,N',N'-tetramethyl-O-succinimi-
douronium tetrafluoroborate), (i-Pr)2EtN, dioxane, DMF, H2O. b) Conc. NH3 soln.
To minimize the electrophoretic-mobility shifts under sequencing gel-electro-
phoresis conditions, we intended to use newly developed multiplex dyes with high
structural similarities. These chromophores are characterized by the same excitation
wavelength, but distinguishable fluorescence lifetimes [32]. Detection in the near-IR
range simplifies the apparatus requirements and minimizes the background fluores-
cence. Since the multiplex dye JA242 carries a free carboxy group, coupling is
possible via a carboxamide linkage. Dye labeling of the modified nucleotides 7, 14, 17,
and 19 was performed as shown in Scheme 5. Activation was performed according to a
procedure described by Bannwarth and Knorr [33] with N,N,N',N'-tetramethyl-
O-succinimidouronium tetrafluoroborate (TSTU) ± a convenient coupling reagent
in the synthesis of peptides. The procedure could be performed easily as a one-pot
reaction within a few min. After preactivation of the dye in dioxane/DMF
under H2O-free conditions a solution of the nucleotide in borate buffer (0.1m,
pH 8.5) was added. All thus-formed labeled nucleotides 21 ± 24 were purified by
reversed-phase column chromatography (0.1m (Et3NH)OAc/MeCN) first and sub-
sequently by anion-exchange chromatography (Sepharose Q, 0 ± 1m LiCl-buffer
gradient). The labeled nucleotides could be precipitated as lithium salts from
acetone/EtOH. Determination of the fluorescence quantum yield of the JA242-labeled
nucleoside triphosphates 21, 22, and 23 showed values of more than 0.90 with respect to
the uncoupled dye (1.00), which indicates that coupling resulted in no significant
quenching. However, the quantum yield of the JA242-labeled nucleotide 24 amounted
to only 0.71.
2.2. Application of the Modified Nucleotides in Incorporation Reactions with
Terminal Deoxynucleotidyl Transferase. Terminal deoxynucleotidyl transferase from
calf thymus catalyzes a template-independent addition of deoxyribonucleoside
triphosphates to the 3'-OH ends of double- or single-stranded DNA. In addition to
the natural deoxynucleoside triphosphates, the enzyme also accepts a number of
modified dNTPs as substrates. In a simple test, we investigated the incorporation rates
of our 3'-sugar- and base-modified dNTPs. The chemically synthesized oligodeoxy-
ribonucleotide (0.4 OD) was incubated with a modified nucleoside triphosphate at 378
for 2 h. Since these nucleotide analogues (21 and 22) did not possess a 3'-hydroxy
group, their incorporation into the elongating DNA strand would stop the labeling
reaction. The incorporation assays were analyzed by gel electrophoresis in a denaturing
polyacrylamide matrix at 70 V for 3 h. The 3'-modified thymidine triphosphate 21 was

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Scheme 5. Labeling of the 5'-Triphosphates 7, 14, 17, and 19 with the Dye JA242
a) TSTU, (i-Pr)2EtN, DMF, dioxane, H2O.
not accepted as substrate by terminal deoxynucleotidyl transferase. In contrast, the
reaction with the N4-modified dideoxycytidine triphosphate 22 showed no remaining
unlabeled oligonucleotide (Fig. 1).
Fig. 1. Gel electrophoresis of the incorporation reaction of ddTTP 21 or ddCTP 22 into 5'-d(ACACC-
CAATTCTGAAAATGGA)-3' with terminal deoxynucleotidyl transferase: a) irradiation with white light;
b) UV irradiation; c) reaction of the oligonucleotide bands with ethidium bromide. Lane I, 21 (0.05 OD); Lane II,
22 (0.05 OD); Lane III, 21 (0.05 OD); Lane IV, 22 (0.2 OD); Lane V, unlabeled oligodeoxyribonucleotide
(0.2 OD); Lane VI: 21 (0.2 OD)

Helvetica Chimica Acta ± Vol. 82 (1999)
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2.3. Application of the N4-Modified Labeled ddCTPs in Sequencing Reactions. To
apply the N4-modified nucleotides in sequencing reactions, we had to test the ability of
these nucleotides to serve as potent chain terminators for standard DNA polymerases.
Their performance was tested by sequence analysis of the bacteriophage vector
M13mp18 with the M13/pUC sequencing primer and two enzymes: ThermoSequenase
and a new chimeric thermostable DNA polymerase, which had shown improved
incorporation of commercially available chromophore-labeled sequencing terminators
in previous experiments [34]. Best results were also achieved with ThermoSequenase
and the new chimeric thermostable DNA polymerase in our cytidine series. Whereas
the other enzymes needed 15 to 150 mm final concentrations of the terminator in the
sequencing reaction, chimeric DNA polymerase gave best results at 1.5 mm. All
experiments were performed under cycle-sequencing conditions including denaturating
steps at 958, demonstrating that the dye-labeled nucleotides have also a good stability at
higher temperatures. In two different approaches, the dye-labeled nucleotides 22 ± 24
were incubated with either a dye-labeled sequencing primer to test only the acceptance
of the terminator or with an unlabeled primer to detect the multiplex dye attached to
the terminator. As a reference, a sequencing reaction was carried out with conventional
ddNTPs (ddGTP, ddATP, dTTP, and ddCTP). The base-labeled nucleotides 22 and 24
were well accepted by the mentioned enzymes and showed perfect termination quality
(Fig. 2a and 2b). Similar results were obtained with the dideoxycytidine derivative 23
labeled with other dyes like Cy5, JA133, and JA169 [32] (data not shown)
First results indicate that the longer the linker the better the acceptance by the
tested enzymes. Whereas the readability of the sequence using the labeled ddCTP 23
with the shortest linker was only 600 base pairs, we were able to determine the
sequence of up to 800 base pairs using the labeled ddCTP 24 with the longest linker.
Regarding both acceptance and quantum yield, best sequencing patterns were shown
by the ddCTP derivative 22 with the hexaneamine spacer.
Conclusion. ± In the present work, we demonstrated that N4-dye-labeled
dideoxycytidine 5'-triphosphates were well accepted as substrates by common
sequencing enzymes. Among the tested enzymes, ThermoSequenase and a newly
developed chimeric DNA polymerase showed the best acceptance of base-modified
ddCTPs, which is comparable to that of unlabeled ddCTP. For the synthesis of 3'-sugar-
modified 3'-deoxythymidine 5'-triphosphates, a rapid method to transform AZT (1)
into the corresponding 3'-deoxy-3'-isocyanato- or 3'-deoxy-3'-isothiocyanatothymidine
3 and 4, respectively, by a modified Wittig reaction [14] was elaborated. The
triphenylphosphine imide 2, which was obtained from the azido nucleoside 1 using
polymer-bound triphenylphosphine, served as the key intermediate. Treatment of
phosphine imide 2 of 3'-deoxythymidine with either CO2 or CS2 and monoprotected
hexane-1,6-diamine led to the corresponding urea and thiourea nucleoside derivatives 5
and 6, respectively. These tethered nucleosides could be used to label with fluorescent
dyes.
We would like to thank J. Arden-Jacob and K.H. Drexhage (GHS Siegen) for providing the dye JA242, J.
Â
Schneider-Mergener (Charite, Berlin) for help with the MS analysis, M. Sauer (Universität Heidelberg) for the
determination of the quantum yields, and B. Meinelt (Humboldt-Universität, Berlin) for technical assistance.
The financial support of this work by the Bundesministerium für Bildung, Wissenschaft, Forschung und
Technologie (BEO 0311088) is gratefully acknowledged.

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Fig. 2. Sequencing with a) dye-labeled primers and b) unlabeled primers. G, A, T, and C are short forms of
ddGTP, ddATP, ddTTP, and ddCTP, resp. For ThermoSequenase, the optimal ratio of unlabeled conventional
terminators to dNTPs was 1:100. The optimal ratio of N4-labeled ddCTP to dCTP was in the range of 1:100 to
1:10 (corresponding to 1.5 ± 15 mm final N4-labeled ddCTP concentration), indicating a similar acceptance for
N4-labeled and unlabeled ddCTP (see text).
Experimental Part
General. Terminal deoxynucleotidyl transferase and the newly developed chimeric DNA polymerase were
purchased from Boehringer Mannheim GmbH, ThermoSequenase from Amersham, 2',3'-dideoxyuridine, 2',3'-
dideoxycytidine, and AZT (1) from Boehringer Mannheim GmbH, and tributylamine, 2-chloro-4H-1,3,2-
benzodioxaphosphorin-4-one, polymer-bound triphenylphosphine, dry pyridine, DMF, and dioxane from Fluka,
bis(tributylammonium) pyrophosphate was purchased from Sigma, 1H-1,2,3,4-tetrazole from Pharmacia LKB,
and 2-chlorophenyl phosphorodichloridate from Aldrich. DNA Synthesis was performed on a Gene Assembler
Plus (Pharmacia LKB), phosphoramidites were purchased from Pharmacia LKB. DNA-sequencing reactions
were analyzed on a LiCor 4000 L sequencer. Column chromatography (CC): DEAE-Sephadex, Sepharose Q
(Pharmacia LKB), and DEAE cellulose DE52 (Whatman) at 48. UV/VIS Spectra: Spekord UV 160 A
(Shimadzu); lmax in nm. Fluorescence spectra: LS100 (PTI); lmax in nm. NMR Spectra: Bruker-DPX-300 and
Bruker AMX-600; d in ppm rel. to Me3Si and H3PO4 as reference; J in Hz. MALDI-TOF-MS: Laser Tec-
Benchtop-II system (PerSeptive Biosystems). Elemental analysis: Elemental analyzer 240 (Perkin Elmer).
N-{6-{[(tert-Butoxy)carbonyl]amino}hexyl}-2,2,2-trifluoroacetamide. tert-Butyl (6-aminohexyl)carbamate
hydrochloride (1.1 g, 4.35 mmol) was suspended in 0.5m NaOH (50 ml) and stirred at r.t. for 1 h. After
extraction with Et2O (50 ml), the org. phase was evaporated to give tert-butyl (6-aminohexyl)carbamate. To a
chilled (ice bath) and rapidly stirred soln. of the latter and Et3N (0.60 ml) in abs. MeOH (9.90 ml), ethyl
trifluoracetate (0.65 ml) in MeOH (1.23 ml) was slowly added. The mixture was stirred at r.t. for 40 h. After
evaporation, the crude product was purified by CC (silica gel, 0 ! 10% MeOH/CHCl3): 1.24 g (91%). White
powder. 1H-NMR (CDCl3): 3.28 (m, CF3CONHCH2); 3.05 (m, BocNHCH2); 1.27 ± 1.58 (m, 4 CH2); 1.37
(s, 3 Me).

Helvetica Chimica Acta ± Vol. 82 (1999)
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N-(6-Aminohexyl)-2,2,2-trifluoroacetamide Trifluoroacetate. N-{6-{[(tert-Butoxy)carbonyl]amino}hexyl}-
2,2,2-trifluoracetamide (670 mg, 2.20 mmol) was dissolved in CF3COOH (5 ml) and stirred at r.t. for 20 min.
After addition of H2O (20 ml), the mixture was evaporated. Excess CF3COOH was removed by repeated co-
evaporation with H2O and EtOH, resp.: 700 mg (98%) of trifluoracetate. White powder. 1H-NMR (CD3OD):
3.17 (t, J ˆ 6.78, CF3CONHCH2); 2.80 (t, J ˆ 7.54, NH2CH2); 1.26 ± 1.57 (m, 4 CH2).
3'-Deoxy-3'-isothiocyanatothymidine (4). A soln. of 3'-azido-3'-deoxythymidine (1; 1.54 g, 5.76 mmol) in
anh. dioxane (10 ml) was evaporated (3Â). The residue was dried further over P4O10 in vacuo at r.t. for 5 h. In a
flask flushed with dried Ar (via a desiccator) and fitted with an Ar-filled balloon and a septum, polymer-bound
triphenylphosphine (9.5 g, 3 mmol PPh3/g polymer) was suspended in anh. dioxane (100 ml). The support was
allowed to swell by shaking for 15 min. Then 1 in dioxane (50 ml) was added. After loading, 5 equiv. of CS2 were
added by syringe. The suspension was gently shaken for another 6 h and then filtered. The residual solid was
washed with dioxane several times and the filtrate evaporated. CC (silica gel, 0 ! 5% MeOH/CHCl3) gave
1.22 g (75%) of 4. White foam. TLC (CHCl3/MeOH 9 : 1): Rf 0.52. M.p. 1138. 1H-NMR ((D6)DMSO): 7.61
(s, H C(6)); 6.26 (t, H C(1')); 4.52 (m, H C(3')); 4.19 (m, H C(4')); 3.49 ± 3.62 (m, 2 H C(5')); 2.30 ± 2.69
(m, 2 H C(2')); 1.81 (s, Me).
3'-Deoxy-3'-{{thioxo{{6-[(trifluoroacetyl)amino]hexyl}amino}methyl}amino}thymidine (6). To a soln. of 4
(200 mg, 0.71 mmol) in anh. pyridine (18 ml), (i-Pr)2EtN (110 mg, 146 ml, 1.2 equiv.) was added, followed by N-
(6-aminohexyl)-2,2,2-trifluoroacetamide trifluoroacetate (280mg, 1.2 equiv.) in anh. pyridine (18 ml). The soln.
was stirred at r.t. overnight. After evaporation, the residue was co-evaporated with toluene to remove traces of
pyridine and then purified by CC (silica gel, 0 ! 10% MeOH/CHCl3): 230 mg (65%) of 6. White foam. TLC
(CHCl3/MeOH 9 : 1): Rf 0.21. 1H-NMR ((D6)acetone: 7.93 (s, H C(6)); 6.21 (t, H C(1')); 4.45
(m, H C(3')); 3.86 ± 3.97 (m, H C(4'), 2 H C(5')); 3.32 (m, CF3CONHCH2); 3.49 (m, NHCSNHCH2);
2.33 ± 2.55 (m, 2 H C(2')); 1.84 (s, Me); 1.35 ± 1.61 (m, 4 CH2). 13C-NMR (CDCl3): 164.45 (s, C(4)); 158.03
(s,CˆO); 150.97 (s, C(2)); 136.31 (s, C(6)); 110.99 (s, C(5)); 84.44 (s, C(1')); 61.13 (s, C(5')); 26.48 ± 29.20
(4 CH2); 12.01 (s, Me).
3'-Deoxy-3'-{{{{6-[(trifluoroacetyl)amino]hexyl}amino}carbonyl}amino}thymidine (5). As described for 4
and 6, with 1 (1.54 g, 5.76 mmol), dioxane (3 Â 10 ml), and polymer-bound triphenylphosphine (9.5 g, 3 mmol
PPh3/g polymer) in anh. dioxane (100 ml). After addition of 1 in dioxane (50 ml), a stream of dry CO2 was
passed through the suspension. The resulting 3'-deoxy-3'-isocyanato-thymidine (3) was not isolated, but
immediately treated with N-(6-aminohexyl)-2,2,2-trifluoroacetamide trifluoroacetate. CC (silica gel, MeOH/
CHCl3) gave 1.5 g (55%) of 5. White foam. TLC (CHCl3/MeOH 9 : 1): Rf 0.20. M.p. 183 ± 1858. 1H-NMR
((D6)acetone): 6.14 (t, H C(1')); 4.36 (m, H C(3')); 3.71 ± 3.81 (m, H C(4'), 2 H C(5')); 3.32
(m, CF3CONHCH2); 3.14 (m, NHCONHCH2); 2.30 ± 2.36 (m, 2 H C(2')); 1.83 (s, Me); 1.29 ± 1.61
(m, CH2). 13C-NMR ((D6)acetone): 159.29 (s, NHCONH); 136.81 (s, C(6)); 110.40 (s, C(5)); 87.10 (s, C(4'));
84.53 (s, C(1')); 62.17 (s, C(5')); 50.41 (s, C(3')); 40.19 (t, CF3CONHCH2); 38.60 (s, NHCH2); 26.95 ± 31.01
(4 CH2); 12.60 (s, Me).
2',3'-Dideoxy-5'-O-(dimethoxytrityl)uridine (9). To a soln. of 2',3'-dideoxyuridine (2 g, 9.4 mmol) in anh.
pyridine (20 ml), 4-(dimethylamino)pyridine (58 mg), Et3N (1.85 ml), and dimethoxytrityl chloride (3.83 g)
were added. The mixture was left stirring 5 h (TLC monitoring). Then MeOH (4.3 ml) was added and, after
15 min, the mixture was diluted with H2O (40 ml) and extracted with Et2O (3 Â 120 ml). The combined org.
phase was evaporated and the residue purified by CC (silica gel, 0 ! 10% MeOH/CHCl3): 4.1 g (85%) of 9.
White foam. TLC (CHCl3/MeOH 9 : 1): Rf 0.71. M.p. 81 ± 858. 1H-NMR (CDCl3): 7.91 (d, H C(6)); 6.02
(m, H C(1')); 5.28 (d, J(5,6) ˆ 7.91,
H C(5)); 4.14 (m, H C(4')); 3.24 ± 3.48 (m, 2H C(5')); 2.34
(m, Ha C(2')); 1.87 ± 2.08 (m, Hb C(2'), H C(3')); 3.72 (s, 2 MeO); 6.75 ± 7.35 (m, 13 arom. H). 13C-NMR
(CDCl3): 163.59 (C(4)); 158.64 ((MeO)2Tr); 150.38 (C(2)); 144.44 ((MeO)2Tr); 140.54 (C(6)); 135.50
((MeO)2Tr); 135.33 ((MeO)2Tr); 130.12 ((MeO)2Tr); 128.14 ((MeO)2Tr); 127.96 ((MeO)2Tr);
127.07((MeO)2Tr); 113.23 ((MeO)2Tr); 101.51 (C(5)); 86.34 (C(1')); 81.15 (C(4')); 63.64 (C(5')); 55.25
(MeO); 33.42 (C(2')); 25.11 (C(3')). Anal. calc. for C30H30N2O6: C 70.02, H 5.88, N 5.44; found: C 69.58, H 5.81,
N 5.26.
4-N-{6-{[(tert-Butoxy)carbonyl]amino}hexyl}-2',3'-dideoxy-5'-O-(dimethoxytrityl)cytidine (11). A soln. of
9 (611 mg, 1.19 mmol) in pyridine was evaporated. After addition of 1H-1,2,3,4-tetrazole (582 mg, 8.31 mmol,
7 equiv.; sublimated and dried over P4O10), the mixture was dissolved in pyridine and evaporated again. The
rapidly stirred soln. of the residue in pyridine (3.9 ml) was treated at 08 by slow addition with 2-chlorophenyl
phosphorodichloridate (469 mg, 1.91 mmol) in anh. pyridine (770 ml). The mixture was left stirring overnight
(TLC monitoring). After cooling in an ice bath, MeOH (2 ml) was added and the mixture evaporated and dried
in vacuo. The residue was dissolved in CHCl3 and the soln. washed carefully with sat. NaHCO3 soln. and brine

1320
Helvetica Chimica Acta ± Vol. 82 (1999)
and evaporated. The obtained tetrazolyl derivative 10 was dissolved in dioxane (2 ml) and treated with tert-butyl
(6-aminohexyl)carbamate (2.8 mmol) at r.t. overnight. After completion of the reaction, the soln. was
evaporated and the residue dried in vacuo. Purification by CC (silica gel, 0 ! 10% MeOH/CHCl3), gave 320 mg
(38%) of 11. White foam. TLC (CHCl3/MeOH 9 : 1): Rf 0.76. UV/VIS (MeOH): 275. 1H-NMR (CDCl3): 7.93
(d, J(5,6) ˆ 7.53, H C(6)); 7.34 ± 6.76 (m, 13 H, (MeO)2Tr); 6.18 (t, H C(1')); 5.57 (d, H C(5)); 4.97 (br. t,
OH C(5')); 4.18 (m, H C(4')); 3.73 (s, 2 MeO); 3.51 ± 3.26 (m, 2 H C(5'), 2 NHCH2); 2.40 (m, Ha C(2'));
2.14 ± 1.93 (m, Hb C(2'), H C(3')).
4-N-{6-{[(tert-Butoxy)carbonyl]amino}hexyl}-2',3'-dideoxycytidine (12). A soln. of 11 (200 mg, 267 mmol)
in 80% AcOH/H2O (10 ml) was stirred at r.t. for 45 min. Then H2O (20 ml) was added, the mixture evaporated,
and the residue coevaporated with H2O several times. Purification of the crude product by CC (silica gel, 0 !
10% MeOH/CHCl3) gave 93 mg (85%) of 12. White foam. TLC (CHCl3/MeOH 9 : 1): Rf 0.40. UV/VIS
(MeOH): 273. 1H-NMR (CDCl3): 8.12, 7.66 (2d, H C(6)); 5.97 (m, H C(1')); 5.66 (m, H C(5)); 4.12
(m, H C(4')); 3.93 (m, Ha C(5')); 3.66 (m, Hb C(5')); 3.35, 3.11 (2t each, CH2N C(4)); 2.99 ± 3.07
(m, BocNHCH2); 2.27 ± 2.42 (m, Ha C(2')); 2.05 (m, Hb C(2')); 1.86 (m, 2 H C(3')); 1.45 ± 1.57 (m, 4 CH2,
Me3C). 13C-NMR (CDCl3): 176.28 (C(4)); 163.78 (C(2)); 143.39, 139.89 (both C(6)); 95.12, 89.85 (both C(5));
87.70 (C(1')); 82.35 (C(4')); 63.54 (C(5')); 40.30 (CH2N C(4)); 32.75 (C(2')); 29.87 (CH2); 29.02 (CH2);
28.42 (Me3C); 26.20 (CH2); 25.01 (C(3')). Anal. calc. for C20H34N4O5 : C 58.52, H 8.35, N 13.65; found: C 58.10,
H 8.31, N 12.35.
Dideoxyribonucleoside 5'-Triphosphates: General Procedure. Each nucleoside (100 mmol) was dissolved in
anh. pyridine (2 ml) and evaporated (3Â). The residue was dried further over P4O10 in vacuo at r.t. for 5 h. In a
flask flushed with dried Ar (via a desiccator) and fitted with an Ar-filled balloon, anhydrous pyridine (100 ml)
was added by syringe through the septum, followed by anh. dioxane (300 ml). To the well-stirred soln. of the
nucleoside, freshly prepared 1m 2-chloro-4H-1,2,3-dioxaphosphorin-4-one in anh. dioxane (110 ml, 110 mmol)
was added. A white precipitate was formed during the reaction. After 15 min, 0.5m bis(tributylammonium)
pyrophosphate in anh. DMF (300 ml) and Bu3N (100 ml) were injected quickly. The white precipitate dissolved
immediately. After 20 min, the cyclic intermediate was oxidized by adding freshly prepared 1% I2 soln. in
pyridine/H2O 98 : 2 (2 ml). After 15 min, the excess I2 was reduced by 5% NaHSO3 soln. in H2O (1 ml).
If not stated otherwise, the reaction soln. was evaporated and the residue dissolved in H2O (2 ml). The
resulting soln. of triphosphates with a CF3CO protecting group at the linker arm was treated with conc. NH3
soln. (20 ml) for 6 h (see 14 for the removal of the Boc group). The final product was purified by CC (DEAE-
Sephadex A-25, 2.5 Â 25 cm, 48, linear gradient of 0 ! 1m LiCl (3 ml/min) over 8.5 h; 30-ml fractions). The
product fractions were lyophilized. On addition of acetone/EtOH 3 : 1 (10 ml) to a soln. of the residue in H2O
(1 ml), a white precipitate of the nucleoside triphosphate was formed.
‡
3'-{{[(6-Aminohexyl)amino]carbonyl}amino}-3'-deoxythymidine 5'-(Tetralithium Triphosphate) (7 ´ 4 Li ):
Yield 60%. White powder. 1H-NMR (D2O): 6.14 (t, H C(1')); 3.99 ± 4.20 (m, H C(4'), 2 H C(5')); 2.18 ±
2.38 (m, 2 H C(2')); 1.80 (s, Me C(5)); 7.57 (s, H C(6)); 2.98 (m, NH2CH2); 2.85 (t, J ˆ 7.53, NHCH2);
1.18 ± 1.60 (m, 4 CH2); 31P-NMR (D2O): 7.60 (d, P(g)); 9.89 (d, P(a)); 20.98 (t, P(b)).
‡
3'-{{[(6-Aminohexyl)amino]thioxomethyl}amino}-3'-deoxythymidine 5'-(Tetralithium Triphosphate) (8´4 Li ):
Yield 50%. White powder. 1H-NMR (D2O): 6.14 (t, H C(1')); 3.99 ± 4.28 (m, H C(4'), 2 H C(5')); 2.42 ±
2.61 (m, Ha C(2')); 2.18 ± 2.32 (m, Hb C(2')); 2.85 (t, NHCH2); 1.25 ± 1.57 (m, 4 CH2); 7.55 (s, H C(6)). 31P-
NMR (D2O): 10.31 (d, P(g)); 10.87 (d, P(a)); 22.78 (t, P(b)).
‡
4-N-{6-{[(tert-Butoxy)carbonyl]amino}hexyl}-2',3'-dideoxycytidine 5'-(Tetralithium Triphosphate) (13´4 Li ):
Yield 52%. White powder. UV/VIS (H2O): 272. 1H-NMR (D2O): 5.95 (m, H C(1')); 4.24 (m, H C(4'));
4.11 ± 4.17 (m, Ha C(5')); 3.94 ± 4.02 (m, Hb C(5')); 3.18 (t, BocNHCH2); 2.90 (t, CH2N C(4)); 2.23 ± 2.35
(m, Ha C(2')); 1.74 ± 2.00 (m, Hb C(2'), H C(3')); 1.13 ± 1.46 (m, 2 CH2, Me3C); 7.70 (d, J(5,6) ˆ 7.54,
H
C(6)); 5.86 (d, H C(5)). 31P-NMR (D2O): 4.14 (d, P(g); 9.74 (d, P(a)); 19.44 (t, P(b)).
‡
4-N-(6-Aminohexyl)-2',3'-dideoxycytidine 5'-(Tetralithium Triphosphate) Trifluoroacetate (14 ´ 4 Li ´
‡
CF3COOH): For the removal of the Boc group, 13 ´ 4 Li in CF3COOH (1 ml) was stirred at r.t. for 2 min.
On addition of Et2O (10 ml) 14 ´ 4 Li ´ CF3COOH precipitated. The precipitate was filtered and washed with
‡
Et2O several times. Purification of the crude product was achieved by ion-exchange CC (Sephadex 0 ! 1m LiCl
‡
gradient): 95% of 14 ´ 4 Li ´ CF3COOH. White powder. UV/VIS (H2O): 282. 1H-NMR (600 MHz, D2O): 8.28
(d, J ˆ 8.12, 0.4 H, H C(6)); 8.04 (d, J ˆ 8.04, 0.6 H, H C(6)); 6.34 (d, 0.4 H, H C(5)); 6.14 (d, 0.6 H,
H
C(5)); 5.96 (m, H C(1')); 4.33 (m, H C(4')); 4.20 ± 4.26 (m, Ha C(5')); 3.99 ± 4.05 (m, H C(5')); 3.31 ±
3.37 (2t, CH2N C(4)); 2.87 ± 2.89 (dd, CH2NH2); 2.34 ± 2.41 (m, Ha C(2')); 2.08 ± 2.14 (m, Ha C(2')); 1.97 ±
2.02 (m, Ha C(3')); 1.88 ± 1.95 (m, Ha C(3')); 1.54 ± 1.62 (m, 2 CH2); 1.30 ± 1.32 (m, 2 CH2). 13C-NMR
(300 MHz, D2O): 157.25 (C(4)); 148.89 (C(2)); 142.53 (C(6)); 98.31, 95.72 (both C(5)); 87.81 (C(1')); 81.58

Helvetica Chimica Acta ± Vol. 82 (1999)
1321
(C(4')); 66.45 (C(5')); 43.25, 42.74 (both CH2N C(4)); 39.69 (CH2NH2); 27.19 (CH2); 26.86 (CH2); 25.65
(C(2')); 25.45 (CH2); 25.32 (CH2); 24.42, 24.14 (both C(3')). 31P-NMR (300 MHz, D2O): 9.36 (P(a), P(g));
21.47 (P(b)).
2',3'-Dideoxy-4-N-{6-{{{4-{[(trifluoroacetyl)amino]methyl}cyclohexyl}carbonyl}amino}hexyl}cytidine 5'-
‡
(Tetralithium Triphosphate) (18 ´ 4 Li ). To a soln. of 4-{[(trifluoroacetyl)amino]methyl}cyclohexanecarboxylic
acid (33 mg, 130 mmol) in DMF/dioxane 1 : 1 (1720 ml), (i-Pr)2EtN (27 ml, 154 mmol) and TSTU (47 mg,
154 mmol) were added. After 10 min, complete conversion to the corresponding N-hydroxysuccinimide-derived
‡
ester had occurred (TLC), and 1m 14 ´ 4 Li ´ CF3COOH in H2O (430 ml) was added. After 10 h, the mixture was
diluted with H2O and lyophilized. Purification of the crude product was performed by ion-exchange CC
(Sepharose Q), linear gradient of 0 ! 1m LiCl over 4.5 h. A clear soln. of the residue in H2O (1 ml) was added
‡
to acetone/EtOH 3 : 1 (10 ml) yielding a white precipitate: 65% of 18 ´ 4 Li . White powder. UV/VIS (H2O):
272. 1H-NMR (D2O): 7.72 (d, H C(6)); 5.99 (m, H C(1')); 5.90 (d, H C(5)); 4.22 ± 4.32 (m, H C(4'));
4.12 ± 4.21 (m, Ha C(5')); 3.95 ± 4.07 (m, Hb C(5')); 3.32 (m, H C(1)(Chx)); 3.21 (m, CH2N C(4)); 2.99 ±
3.11 (m, 2 CH2NHCO); 2.30 (m, Ha C(2'), H C(4)(Chx)); 1.87 ± 2.07 (m, Hb C(2), 2 H C(3')); 1.68
(m, 2 CH2); 1.47 (m, 2 CH2); 1.38 (t, CH2); 1.15 ± 1.30 (m, 3 CH2). 31P-NMR (D2O): 3.9 (P(g)); 9.3 (P(a));
19.1 (P(b)). 19F-NMR (D2O): 76 (CF3).
4-N-{6-{{[4-(Aminomethyl)cyclohexyl]carbonyl}amino}hexyl}cytidine 5'-(Tetralithium Triphosphate) (19 ´
‡
‡
4 Li ). A soln. of 18 ´ 4 Li in conc. NH3 soln. (2 ml) was stirred at r.t. for 6 h. After evaporation, the crude
‡
product was purified by ion-exchange CC (Sepharose Q, 0 ! 1m LiCl gradient): 85% of 19 ´ 4 Li . White
powder. 1H-NMR (D2O): 7.79, 7.72 (2d, H C(6)); 5.95 (m, H C(1')); 5.86 (d, H C(5)); 3.72 ± 4.30
(m, H C(4'), 2 H C(5')); 3.28 (m, H C(1)(Chx)); 3.19 (m, CH2N C(4)); 3.02 (t, CH2NHCO); 2.20 ± 2.40
(m, Ha C(2'), H C(4)(Chx), CH2NH2); 1.87 ± 2.10 (m, Hb C(2'), H C(3')); 1.62 ± 1.75 (m, 2 CH2); 1.30 ±
1.50 (m, 2 CH2); 1.21 (m, 4 CH2). 31P-NMR (D2O): 3.8 (d, P(g)); 9.3 (d, P(a)); 19.0 (t, P(b)).
Coupling with JA242-S Dye: General Procedure. The multiplex dye JA 242-S (67 mg, 150 mmol) (ˆ 1-(3-
carboxypropyl)-11-ethyl-3,4,8,9,10,11-hexahydro-8,10,10-trimethyl-2H-dipyrido[3,2-b: 2',3'-i]phenoxazinium in-
ner salt) was dissolved in DMF/dioxane 1 : 1 (2 ml). To this soln., (i-Pr)2EtN (77 ml, 450 mmol) and TSTU
(57 mg, 190 mmol) were added. After 10 min, complete conversion into the corresponding N-hydroxysuccin-
imide-derived ester had occurred (TLC), and the nucleoside 5'-triphosphates 7, 14, 17, or 19 (50 mmol) was
added. After 3h the mixture was diluted with H2O and lyophilized. The residue was purified by reversed-phase
CC (C18 silica gel; 0.1m (Et3NH)OAc/MeCN) followed by ion-exchange CC (Sepharose Q, linear gradient of
0 ! 1m LiCl over 4.5 h). The clear soln. of the residue in H2O was added to acetone/EtOH 3 : 1, yielding the blue
precipitate of the dye-labeled nucleoside triphosphate.
3'-Deoxy-3'-{{{{6-{[4-(11-ethyl-3,4,8,9,10,11-hexahydro-8,10,10-trimethyl-2H-dipyrido[3,2-b:2',3'-i]phen-
oxazin-1-ium-1-yl)-1-oxobutyl]amino}hexyl}amino}carbonyl}amino}thymidine 5'-(Tetralithium Triphosphate)
‡
(21 ´ 4 Li ): Yield 18%. Blue powder. UV/VIS (H2O): 668. Fluorescence (H2O): 683. 1H-NMR (MeOH):
7.60 (s, H C(6)); 7.49 (s, 1 arom. H); 7.39 (s, 1 arom. H); 6.96 (s, 1 arom. H); 6.78 (s, 1 arom. H); 6.12
(t, H C(1')); 4.07 ± 4.12 (m, 2 H C(5')); H C(4'), 2 H C(3')); 3.58 ± 3.65 (m, 2 CH2); 3.09 (t, NHCH2); 2.99
(t, NHCH2, m, CH); 2.86 (t, CH2); 2.23 ± 2.33 (m, 2 H C(2'), CH2); 1.92 ± 2.01 (m, 2 CH2); 1.80
(s, Me C(5)(Thy)) 1.19 ± 1.48 (m, 6CH2, 4 Me). 13C-NMR (MeOH): 175.08 (NHC(O)CH2); 166.08 (C(4));
161.04 (HNC(O)NH); 156.32, 155.85 (arom. C); 152.58 (C(2)); 150.49, 149.82 (arom. C); 138.49 (C(6)); 135.99
(arom. C); 135.24 (arom. C); 131.99 (arom. CH); 130.96 (arom. C); 129.45 (arom. CH); 112.28 (C(5)); 97.96,
96.98 (arom. CH); 86.23 (C(1')); 84.30 (C(4')); 67.40 (C(5')); 60.65 (quart. C); 53.87 (alicycl. CH2); 52.56
(C (3')); 52.28 (CH2); 45.81 (CH2); 42.78 (CH2); 40.89 (NHCH2); 40.66 (NHCH2); 39.52 (C(2')); 33.55 (CH2);
31.24 (CH2); 30.40 (CH2); 29.65 (CH); 28.74 (CH2); 28.43 (Me); 27.75 (CH2); 27.59 (CH2); 26.42 (Me); 23.51
(CH2); 22.15 (CH2); 19.68 (Me); 14.70 (Me); 12.90 (Me C(5)(Thy)). 31P-NMR (MeOH): 8.14 (d, P(g));
‡
9.62 (d, P(a)); 21.04 (t, P(b)). MALDI-TOF-MS: 1054.74 ([M ‡ H] ).
2',3'-Dideoxy-4-N-{6-{[4-(11-ethyl-3,4,8,9,10,11-hexahydro-8,10,10-trimethyl-2H-dipyrido[3,2-b:2',3'-i]phen-
‡
oxazin-1-ium-1-yl)-1-oxobutyl]amino}hexyl}cytidine 5'-(Trilithium Triphosphate) (22 ´ 3 Li ): Yield 15%. Blue
powder. TLC (i-PrOH/25% NH3 soln./H2O 7: 1 : 2): Rf 0.02. UV/VIS (H2O): 668. Fluorescence (H2O): 683.
1H-NMR (CD3OD): 7.93 (d, H C(6)); 7.49 (s, 1 arom. H); 7.38 (s, 1 arom. H); 6.94 (s, 1 arom. H); 6.76
(s, 1 arom. H); 6.02 (m, H C(1')); 5.90 (d, H C(5)); 4.03 ± 4.30 (m, 2 H C(5'), H C(4')); 3.62 (m, 2 CH2);
3.09 ± 3.16 (m, 2 NHCH2); 2.85 ± 2.97 (m, CH2, CH); 2.27 ± 2.32 (m, Ha C(2'), CH2); 1.84 ± 2.02 (m, Hb C(2'),
2 H C(3'), 2 CH2); 1.20 ± 1.49 (m, 24 H, CH2, Me). 13C-NMR (CD3OD): 174.30 (NHC(O)CH2); 159.88
(C(4)); 156.36, 155.89 (arom. C); 147.96 (C(2)); 142.89, 141.63 (both C(6)); 132.20 (arom. CH); 130.96
(arom. C); 129.68 (arom. CH); 97.86, 96.91 (arom. CH); 96.12 (C(5)); 88.50 (C(1')); 60.69 (quart. C); 53.84
(alicycl. CH2); 52.26 (CH2); 45.87 (CH2); 42.74 (CH2); 40.63 (NHCH2); (C(2')); 33.49 (CH2); 30.54 (CH2); 29.61

1322
Helvetica Chimica Acta ± Vol. 82 (1999)
(CH); 28.74 (CH2); 28.45 (Me); 27.78 (CH2); 27.62 (CH2); 26.38 (Me); 23.48 (CH2); 22.15 (CH2); 19.68 (Me);
14.65 (Me). 31P-NMR (D2O): 8.22 (d, P(g)); 9.34 (d, P(a)); 20.36 (t, P(b)). MALDI-TOF-MS: 982.96
‡
([M ‡ H] ).
2',3'-Dideoxy-4-N-{6-{{{4-{{[4-(11-ethyl-3,4,8,9,10,11-hexahydro-8,10,10-trimethyl-2H-dipyrido[3,2-b:2',3'-i]-
phenoxazin-1-ium-1-yl)-1-oxobutyl]amino}methyl}cyclohexyl}carbonyl}amino}hexyl}cytidine 5'-(Trilithium Tri-
‡
phosphate) (24 ´ 3 Li ): Yield 15%. Blue powder. TLC (i-PrOH/25% NH3 soln./H2O 7: 1 : 2): Rf 0.02. UV/VIS
(H2O): 668. Fluorescence (H2O): 683. 31P-NMR (D2O): 3.6 (d, P(g)); 9.4 (d, P(a)); 18.7 (t, P(b)).
‡
MALDI-TOF-MS: 1121.93 ([M ‡ H] ).
3'-End-Labeling Reaction with Terminal Deoxynucleotidyl Transferase. Oligonucleotide 5'-d(ACACC-
CAATTCTGAAAATGGA)-3' (0.4 OD, 1.535 nmol, 11.4 mg) was dissolved in H2O (166 ml). At 378, 50 ml 5 Â
reaction buffer (ˆ 1  final concentration), 25 ml of 2.5 mm CoCl2, 4 ml of 10 mm dye-labeled nucleoside
triphosphate soln. (21 or 22, resp.), and 125 units of terminal deoxynucleotidyl transferase (5 ml) were added.
After 2 h, no unlabeled oligodeoxyribonucleotide remained.
Sequencing Reactions with Nucleotides 22, 23, or 24. Sequencing with ThermoSequenase and Chimeric DNA
Polymerase. The optimal nucleotide ratio for sequencing reactions with ThermoSequenase and chimeric DNA
polymerase was 1 :100 (1.5 mm ddNTP :150 mm dNTP each). The unlabeled ddNTPs in the termination mixes
were replaced by 1.5 mm, 15 mm, or 150 mm 22, 23, or 24. A template/primer/enzyme mix containing 0.2 mg of
M13mp18 DNA (100 fmol), 2 ml of unlabeled or labeled M13/pUC sequencing primer, 2 ml 10 Â reaction buffer,
2 ml of ThermoSequenase or chimeric DNA polymerase (4 U/ml), and deionized H2O to a total volume of 17.5 ml
was prepared. Of each termination mix, a sample (4 ml) was dispensed into a test tube, and the template/primer/
enzyme mixture (3.5 ml) was added. Cycle sequencing was performed in 30 cycles: at 958 for 30 s (denaturating),
at 558 for 30 s (annealing), at 728 for 60 s (elongation). To remove the excessive dye terminators in the
nucleotide 22 and 24 reaction, 92 ml of H2O, 10 ml of 3m NaOAc, and 250 ml of abs. EtOH (r.t.) were added to
each tube. The samples were centrifuged at 15000 rpm for 15 min, washed with 300 ml of fresh 70% EtOH/H2O,
and the pellets of dye-labeled DNA were dried. Each pellet was diluted in 6 ml of stop soln. and denaturated by
heating for 2 min at 978, and 2 ml of each mixture was loaded in an appropriate well of a 6% denaturating
polyacrylamide sequencing gel. Sequencing was performed using a modified automated LiCor-4000-L
sequencer.
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Received May 21, 1999