Nucleotides part LXXX: Synthesis of 3'-O fluorescence labeled thymidine derivatives and their 5'-o-triphosphates

Article abstract of DOI:10.1080/15257770.2011.578089

A new labeling technique attaching a fluorescent pteridine derivative (3, 5) via a linker onto the 3'-OH group of 5'-O-dimethoxytritylthymidine (7) was developed to lead to the conjugates 8 and 11. After detritylation to give 9 and 12, the final conversion into the corresponding 5'-triphosphates (13, 14), which were isolated as sodium salts, was performed by known methods. Copyright Taylor and Francis Group, LLC.

Full text of DOI:10.1080/15257770.2011.578089

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Nucleotides Part LXXX: Synthesis of
3′-O Fluorescence Labeled Thymidine
Derivatives and Their 5′-O-Triphosphates
Tamara I. Prykota a & Wolfgang Pfleiderer a
a Fachbereich Chemie , Universität Konstanz , Konstanz , Germany
Published online: 02 Sep 2011.
To cite this article: Tamara I. Prykota & Wolfgang Pfleiderer (2011) Nucleotides Part LXXX: Synthesis
of 3′-O Fluorescence Labeled Thymidine Derivatives and Their 5′-O-Triphosphates, Nucleosides,
Nucleotides and Nucleic Acids, 30:7-8, 544-551, DOI: 10.1080/15257770.2011.578089
To link to this article: http://dx.doi.org/10.1080/15257770.2011.578089
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Nucleosides, Nucleotides and Nucleic Acids, 30:544–551, 2011
Copyright ꢀC Taylor and Francis Group, LLC
ISSN: 1525-7770 print / 1532-2335 online
DOI: 10.1080/15257770.2011.578089
NUCLEOTIDES PART LXXX: SYNTHESIS OF 3ꢀ-O FLUORESCENCE
LABELED THYMIDINE DERIVATIVES AND THEIR
5ꢀ-O-TRIPHOSPHATES
Tamara I. Prykota and Wolfgang Pfleiderer
Fachbereich Chemie, Universita¨t Konstanz, Konstanz, Germany
A new labeling technique attaching a fluorescent pteridine derivative (3, 5) via a linker onto
2
the 3ꢁ-OH group of 5ꢁ-O-dimethoxytritylthymidine (7) was developed to lead to the conjugates 8 and
11. After detritylation to give 9 and 12, the final conversion into the corresponding 5ꢁ-triphosphates
(13, 14), which were isolated as sodium salts, was performed by known methods.
Keywords Isoxanthopterin derivatives; fluorescent marker; thymidine conjugates; nu-
cleoside 5ꢁ-triphosphates
INTRODUCTION
Nucleoside 5ꢁ-triphosphates with fluorescent labels can be efficient tools
for structural and functional investigations of oligonucleotides and nucleic
acids.[1] Over the last few decades, some approaches to syntheses of 2ꢁ-
deoxynucleoside 5ꢁ-triphosphates bearing at the 3ꢁ-position acyloxy groups
with fluorescent markers have been described.[2–5] A large variety of labels
have been introduced in various positions of the nucleoside[6–8] for spe-
cific reasons. We concentrated our interest on pteridine derivatives, which
show strong fluorescence, high quantum yields, and high extinctions in the
region of 360 nm. The aim of these investigations was the synthesis of mod-
ified thymidine 5ꢁ-triphosphates bearing at the 3ꢁ-OH group via a C6- and
a triethoxy-carbonyl linker the 8-(3,6-dimethylisoxanthopterinyl) residue as
the fluorescing marker.
SYNTHESIS
Starting from 2-amino-6-chloro-3-methyl-5-nitro-4(3H)pyrimidone,[9] re-
action with 6-aminohexanoic acid and 2-[2-(2-aminoethoxy)ethoxy]ethanol,
Received 17 March 2011; accepted 1 April 2011.
Part of the special issue in honor of Vladimir Efimov.
Address correspondence to Wolfgang Pfleiderer, Fachbereich Chemie, Universita¨t Konstanz, Post-
fach 5560, 78457 Konstanz, Germany. E-mail: wolfgang.pfleiderer@uni-konstanz.de
544

Nucleotides Part LXXX
545
respectively, led in good yields to 6-(2-amino-3-methyl-5-nitro-4-oxo-3,4-
dihydropyrimidin-6-ylamino)hexanoic acid (1) and 2-amino-6-{2-[2-(2-
hydroxyethoxy)ethoxy]ethylamino}-3-methyl-5-nitropyrimidin-4(3H)-one
(2). Catalytic reduction of 1 with Pd/C under hydrogen atmosphere to the
corresponding 5-amino derivative led on condensation with ethyl pyruvate to
a mixture of 6-(2-amino-3,6-dimethyl-4,7-dioxo-3,4-dihydropteridin-8(7H)-
yl)hexanoic acid (3) and 6-(2-amino-1,8-dimethyl-6-oxo-1,6-dihydropurin-
9-yl)hexanoic acid (4), which were separated by column chromatography
to give yields of 50% and 23%, respectively. Similar hydrogenation of
2 and subsequent reaction with ethyl pyruvate gave 2-amino-8-{2-[2-(3-
hydroxyethoxy)ethoxy]ethyl}-3,6-dimethyl-pteridin-4,7-(3H,8H)-dione (5)
in 44% yield. Coupling of 3 with 5ꢁ-dimethoxytritylthymidine (7)[10]
afforded activation of the carboxylgroup by ethyl choroformate to the
mixed anhydride leading to the conjugate 8 at 0◦C in 50% yield. As a
side-product ethyl 6-(2-amino-3,6-dimethyl-4,7-dioxo-3,4-dihydropteridin-
8(7H)-yl)hexanoate (6) was isolated in 20% yield. The analogous conjugate
11 resulted from the reaction between 5 and 5ꢁ-O-dimethoxytrityl-3ꢁ-O-
(4-nitrophenyl-oxycarbonyl)thymidine (10) in pyridine in the presence
of DMAP at room temperature in 73% yield. Treatment of 8 and 11,
respectively, in 80% acetic acid led under detritylation in 81% to 9 and 12.
In the final step, the 5ꢁ-triphosphates 13 and 14 were prepared by a slightly
modified Ludwig’s one-pot procedure[11] working in pyridine at −10◦C with
two equivalents of POCl3. The intermediary dichlorophosphate was then
treated with bis-(tri-n-butylammonium) pyrophosphate and after quenching
with triethylammonium bi-arbonate and DEAE-cellulose chromatography
the tetra(triethylammonium) salts were isolated and then converted into
the tetrasodium salts[12] 13 and 14, respectively (Figure 1).
EXPERIMENTAL
General
Products were dried under high vacuum. All solvents used were of anh.
grade. TLC: precoated silica gel (SiO2) thin-layer sheets 60 F254 (Merck,
Germany) and cellulose thin-layer sheets F 1440 LS254 (Schleicher &
Schu¨ll, Germany). m.p.: Bu¨chi-B-545 melting-point apparatus; uncorrected.
UV/VIS: Perkin-Elmer Lambda 5; λmax (log ε) in nm. 1H-NMR: Bruker AC
250; 31P-NMR: Jeol JMN-GX 400; δ in ppm rel. to Me4Si or CDCl3 as inter-
nal standard, J in Hz. Elemental analyses were performed by the Analytical
Laboratory of the Department of Chemistry, Konstanz University.
6-(2-Amino-3-methyl-5-nitro-4-oxo-3,4-dihydropyrimidin-6-
ylamino)hexanoic Acid (1)
A suspension of 2-amino-6-chloro-3-methyl-5-nitropyrimidin-4-one[9]
(5.75 g, 28 mmol) and 6-amino hexanoic acid (8.11 g, 62 mmol) in EtOH

546
T. I. Prykota and W. Pfleiderer
FIGURE 1 Reaction scheme.
(200 ml) was refluxed for 5 hours. After cooling the solid was collected
and dried at 80◦C to give 7.56 g (90%) of 1 as colorless crystals. f.p. 229◦C.
UV (MeOH): 216 (4.29), 230 (sh 4.13), 298 (sh 3.68), 331 (4.03). 1H-NMR
(DMSO-d6): 11.87 (bs, COOH); 9.33 (t, HN -CH2); 7.76 (bs, NH2); 3.47 (m,
HN-CH2); 3.18 (s, N-CH3), 3.50 (m, CH2); 1.55 (m, 4H, 2 x CH2); 1.33 (m,
CH2). Anal. Calc. for C11H17N5O5 (299.3): C 44.15, H 5.73, N 23.40; Found:
C 44.27, H 5.78, N 23.21.

Nucleotides Part LXXX
547
2-Amino-6-{2-[2-(2-hydroxyethoxy)ethoxy]ethylamino}-3-methyl-
5-nitropyrimidin-4(3H)-one (2)
A
mixture of 2-amino-6-chloro-3-methyl-5-nitropyrimidin-4-one[9]
(10.2 g, 0.05 mol) 8-aminoethoxyethoxyethanol (7.5 g, 0.05 mol) and
triethylamine (10 ml) in EtOH (150 ml) was refluxed for 30 minutes.
The hot solution was filtered from little precepitate and the filtrate stored
overnight in the icebox. The solid was collected and dried at 60◦C to give
13.1 g (83%) of 2 as colorless crystals. f.p.125–127◦C. UV (MeOH): 215
1
(4.30), 236 (sh 4.11), 284 (sh 3.58), 330 (4.07). H-NMR (DMSO-d6):
9.38 (t, HN -CH2); 7.88 (bs, NH2), 4.52 (t, OH); 3.65 (m, N-CH2); 3.60
(m, CH2), 3.55 (m, 4H, 2 x CH2); 3.50 (m, CH2); 3.42 (m, CH2); 3.18
(s, N-CH3).
Anal. Calc. for C11H19N5O6 (317.3): C 41.64, H 6.04, N 22.07; Found: C
41.56, H 5.99, N 21.95.
6-(2-Amino-3,6-dimethyl-4,7-dioxo-3,4-dihydropteridin-8(7H)-
yl)hexanoic Acid (3) and 6-(2-Amino-1,8-dimethyl-6-oxo-
1,6-dihydropurin-9-yl)hexanoic Acid (4)
A suspension of compound 1 (0.9 g. 3 mmol) and Pd/C (90 mg, 10%) in
H2O/MeOH (100 ml, 1:1) was shaken for 36 hours under H2-atmosphere.
The catalyst was filtered off, washed with hot H2O (100 ml) and hot MeOH
(100 ml). The combined filtrates were concentrated to 100 ml and after
addition of ethyl pyruvate (0.6 g, 5 mmol) and AcOH (2 ml) refluxed for
5 hours. It was evaporated, coevaporated with MeOH (5 × 10 ml). The
residue was treated by flash column chromatography on silica-gel (25 g, with
CH2Cl2/MeOH (100:5, 400 ml), (100:7.5, 400 ml) and (100:10, 400 ml). The
first product fractions yielded after evaporation 0.48 g (50%) of 3 as yellow
foam. Recrystallization from MeOH gave brownish crystals. f.p.. 232◦C. UV
1
(MeOH): 217 (4.56), 293 (4.01), 340 (4.15). H-NMR (DMSO-d6): 12.01
(bs, COOH); 7.63 (bs, 2H, NH2); 4.07 (t, N-CH2); 3.29 (s, N-CH3); 2.24 (s,
C-CH3); 2.20 (t, CH2); 1.53 (m, 4H, 2 x CH2); 1.29 (m, CH2).
Anal. calc. for C14H19N5O4 (321.3): C 52.33, H 5.96, N 21.79; Found: C
51.66, H 6.02, N 21.64.
The second product fraction gave after evaporation and treatment with
MeOH 0.2 g (23%) of 4 as colorless foam. UV (MeOH): 205 (4.26), 255
1
(4.08), 264 (sh 4.00). H-NMR (DMSO-d6): 12.05 (bs, COOH); 6.91 (bs,
2H, NH2); 3.85 (t, N-CH2); 3.28 (s, N-CH3); 2.32 (s, C-CH3); 2.19 (t, CH2);
1.70–1.40 (m, 4H, 2 x CH2); 1.24 (m, CH2).
Anal. calc. for C13H19N5O3 x H2O (311.3): C 50.15, H 6.80, N 22.49;
Found: C 50.86, H 6.34, N 22.67.

548
T. I. Prykota and W. Pfleiderer
2-Amino-8-{2-[2-(3-hydroxyethoxy)ethoxy]ethyl}-3,6-
dimethylpteridin-4,7-(3H,8H)-dione (5)
A suspension of 2 (3.17 g, 0.01 mol) and Pd/C (0.3 g, 10%) in MeOH
(100 ml) was shaken under H2-atmosphere for 20 hours. MeOH/HCl
(5 ml, 10%) was added, the catalyst filtered off and the filtrate concentrated
to 50 ml. After addition of ethyl pyruvate (2 ml) the mixture was stirred
at room temperature for 18 hours. The solution was treated with charcoal,
filtered and evaporated. The residue was recrystallized from EtOH to give
1.5 g (44%) of 5 as yellow crystals. f.p.. 170◦C.
1
UV (MeOH): 216 (4.55), 293 (3.99), 340 (4.11). H-NMR (DMSO-d6):
7.67 (bs, NH2), 4.53 (t, OH); 4.31 (t, N-CH2); 3.61 (t, CH2), 3.55 (t] CH2);
3.48 (m, 4H, 2 x CH2); 3.38 (t, CH2); 3.31 (s, N-CH3), 2.26 (s, C-CH3).
Anal. Calc. for C14H21N5O5 x H2O (357.4): C 47.05, H 6.48, N 19.59;
Found: C 46.87, H 6.15, N 19.43.
5ꢀ-O-Dimethoxytrityl-3ꢀ-O-[6-(2-amino-3,6-dimethyl-4,7-dioxo-
3,4,7,8-tetrahydropteridin-8-yl)hexanoyl]thymidine (8) and Ethyl
6-(2-amino-3,6-dimethyl-4,7-dioxo-3,4,7,8-tetrahydro-pteridin-8-
yl)hexanoate (6)
To a cooled (ice bath) stirred suspension of hexanoic acid 3 (0.321
g, 1 mmol) in dry CH2Cl2 (5 ml) triethylamine (0.101 mg, 1 mmol) and
ethyl chloroformate (0.108 mg, 1 mmol) were added. The mixture was
stirred for 20 minutes, then 5ꢁ-O-dimethoxytritylthymidine (7)[10] (0.817
g, 1.5 mmol) and after 5 minutes DMAP (24 mg, 0.02 mmol) were added.
After stirring at 0◦C for 1 hour the mixture was diluted with CH2Cl2 (30
ml), washed with aqueous NaHCO3, the organic layer separated, dried over
Na2SO4, and evaporated. The residue was purified by flash column chro-
matography on silica-gel (30 g) with CH2Cl2 (100 ml), CH2Cl2/MeOH 100:1
(100 ml), CH2Cl2/MeOH 100:2 (200 ml), CH2Cl2/MeOH 100:3 (300 ml),
CH2Cl2/MeOH 100:4 (400 ml) and CH2Cl2/MeOH 100:5 (500 ml). The
product fractions were collected to give starting material 7 (0.4 g), com-
pound 8 (0.425 g, 50%) and ethyl 6-(2-amino-3,6-dimethyl-4,7-dioxo-3,4,7,8-
tetrahydropteridin-8-yl)hexanoate (6) 70 mg (20%).
Compound 8: UV (MeOH): 204 (4.86), 211 (sh 4.79), 229 (sh 4.53), 251
1
(sh 4.16), 289 (sh 4.03), 340 (4.13). H-NMR (DMSO-d6): 11.38 (s, NH);
7.61 (bs, NH2); 7.52 (s, H-6); 7.40–7.20 (m, 9 H, arom. H); 6.88 (d, 4H, o to
OCH3); 6.20 (t, H-C(1ꢁ)); 5.29 (m, H-C(3ꢁ)); 4.06 (m, 3H, H-C(4ꢁ), N-CH2);
3.72 (2, 6H, 2 x OCH3); 3.32 (m, 3H, H-C(5ꢁ), N-CH3); 3.21 (m, H-C(5ꢁꢁ));
2.45 (m, H-C(2ꢁ)); 2.31 (m, 3H, H-C(2ꢁꢁ), CH2); 2.23 (s, C-CH3); 1.55 (m,
4H, 2 x CH2); 1.41 (s, C-CH3); 1.12 (m, H-C(2ꢁ)).
Anal. Calc. for C45H49N7O10 × 1.5 H2O (874.9): C 61.78, H 6.00, N 11.21;
Found: C 61.57, H 5.87, N 11.30.

Nucleotides Part LXXX
549
Compound 6: UV (MeOH): 217 (4.55), 293 (3.9), 341 (4.13). 1H-NMR
(CDCl3): 5.77 (s, 2H, NH2); 4.15 (m, 4H, N-CH2, OCH2CH3); 3.51 (s, N-
CH3); 2.47 (s, C-CH3); 2.32 (t, CH2); 1.73 (m, 4H, 2 x CH2); 1.42 (m, CH2)
1.26 (t, OCH2CH3).
Anal. calc. for C16H23N5O4 (349.4): C 55.00, H 6.64, N 20.04; Found: C
54.78, H 7.19, N 19.81.
3ꢀ-O-[6-(2-Amino-3,6-dimethyl-4,7-dioxo-3,4,7,8-
tetrahydropteridin-8-yl)]hexanoyl-thymidine (9)
A solution of compound 8 (0.425 g, 0.5 mmol) in 80% AcOH (5 ml)
was stirred at room temperature for 1 hour. Evaporation and coevapora-
tion with MeOH (4 × 10 ml) and flash chromatography (FC) (SiO2) with
CH2Cl2/MeOH (100:2, 100 ml), (100:4, 100 ml), (100:6, 100 ml), (100:7,
100 ml), (100:8, 100 ml) gave in the product fraction 0.22 g (81%) of 9 as a
colorless foam. UV (MeOH): 216 (4.58), 258 (4.08), 290 (3.99), 340 (4.11).
1H-NMR (DMSO-d6): 11.35 (s, NH); 7.72 (s, H-6); 7.63 (bs, NH2); 6.20 (dd,
H-C(1ꢁ)); 5.21 (m, 2H, H-C(3ꢁ), 5ꢁ-OH); 4.11 (m, N-CH2); 3.93 (m, H-C(4ꢁ));
3.60 (m, 3H, H-C(5ꢁ,5ꢁꢁ)); 3.29 (s, N-CH3); 2.35 (t, CH2); 2.23 (m, 5H, H-C(2ꢁ,
2ꢁꢁ), C-CH3); 1.77 (s, CH3 Thy); 1.59 (m, 4H, 2 x CH2); 1.30 (m, CH2).
Anal. Calc. for C24H31N7O8 × 1.5 H2O (572.6): C 50.34, H 5.98, N 17.12;
Found: C 50.50, H 5.86, N 17.03.
5ꢀ-O-Dimethoxytrityl-3ꢀ-O-(4-nitrophenoxycarbonyl)thymidine (10)
The nucleoside 7 (1.09 g, 2 mmol) was coevaporated with dry pyridine
(3 × 5 ml) and then dissolved in dry pyridine (5.0 ml) and dry CH2Cl2 (5 ml).
After addition of 4-nitrophenyl chloroformate (0.605 g, 3 mmol) the mixture
was stirred at room temperature for 24 hours. The solvent were evaporated,
the residue dissolved in CH2Cl2 (30 ml), washed with aqueous NaHCO3
solution, the organic layer separated, dried over Na2SO4, evaporated, and
coevaporated with toluene (3 × 5 ml). The residue was purified by FC (40
g SiO2) with n-hexane/EtOAc 4:1 (200 ml); 2:1 (300 ml), 1:1 (300 ml) and
EtOAc (300 ml). Evaporation of the product fraction 1.0 g (70%) of 10 as
1
colorless foam. UV (MeOH): 203 (4.88), 234 (4.43), 266 (4,32). H-NMR
(CDCl3): 8.54 (s, HN); 8.29 (m, 2H, o to NO2); 7.62 (s, H-C(6)); 7.40–7.20
(m, 11H, arom. H); 6.84 (d, 4H, o to OCH3); 6.51 (dd, H-C(1ꢁ)); 5.44 (d,
H-C(3ꢁ)); 4.31 (t, H-C(4ꢁ)); 3.79 (s, 6H, 2 x OMe); 3.59 (dd, H-C5ꢁ)); 3.47
(dd, H-C(5ꢁꢁ)); 2.60 (m, H-C(2ꢁ)); 2.51 (m, H-C(2ꢁꢁ)); 1.41 (s, C-CH3).
5ꢀ-O-Dimethoxytrityl-3ꢀ-O-[9-(2-amino-3,6-dimethyl-4.7-dioxo-
3,4,7,8-tetrahydropteridin-8-yl)
ethoxyethoxyethoxycarbonyl]thymidine (11)
The nucleoside 10 (0.74 g, 1.04 mmol) was coevaporated with dry pyri-
dine (3 × 5 ml), then dissolved in dry pyridine (10 ml) and after addition of

550
T. I. Prykota and W. Pfleiderer
compound 5 (0.27 g, 0.8 mmol) and DMAP (0.122 g, 1 mmol) the mixture
was stirred at room temperature for 24 hours. The work-up is analogous to
8 to give on evaporation of the product fraction 0.53 g (73%) of 11 as a
yellowish foam. UV (MeOH): 203 (4.90), 229 (sh 4.55), 252 (sh 4.19), 275
1
(sh 4.16), 340 (4.12). H-NMR (DMSO-d6): 11.40 (s, NH); 7.68 (bs, NH2);
7.51 (s, H-6); 7.40–7.20 (m, 9 H, arom. DMTr); 6.88 (d, 4H, o to OCH3);
6.19 (t, H-C(1ꢁ)); 5.22 (m, H-C(3ꢁ)); 4.28 (t, N-CH2); 4.13 (m, 3H, H-C(4ꢁ),
O-CH2); 3.72 (2, 6H, 2 x OCH3); 3.55 (m, 8H, 4 x CH2); 3.29 (m, 4H, H-
C(5ꢁ), N-CH3); 3.20 (m, H-C(5ꢁꢁ)); 2.43 (m, H-C(2ꢁ,2ꢁꢁ)); 2.23 (s, C(6)-CH3);
1.41 (s, C(5)-CH3).
Anal. Calc. for C46H51N7O13 x H2O (927.9): C 59.54, H 5.76, N 10.56;
Found: C 59.68, H 5.72, N 10.54.
3ꢀ-O-[9-(2-Amino-3,6-dimethyl-4.7-dioxo-3,4,7,8-
tetrahydropteridin-8-yl)ethoxyethoxy-
ethoxycarbonyl]thymidine (12)
A solution of 11 (0.5 g, 0.55 mmol) in 80% AcOH (5 ml) was stirred at
room temperature for 1 hour. The work up was performed analogous to 9
by FC to give 0.27 g (81%) of 12 as a yellowish powder. UV (MeOH): 216
(4.61), 259 (4.09), 341 (4.11). 1H-NMR (DMSO-d6): 11.34 (s, NH); 6.71 (s,
H-C(6)); 6.68 (bs, NH2); 6.14 (t, H-C(1ꢁ)); 5.23 (t, 5ꢁ-OH); 5.13 (m, H-C(3ꢁ));
4.28 (t, N-CH2); 4.17 (t, O-CH2); 4.03 (m, H-C(4ꢁ)), 3.55 (m, 10H, 4 x CH2,
H-C(5ꢁ,5ꢁꢁ)); 3.29 (s, N-CH3); 2.30 (m, H-C(2ꢁ,2ꢁꢁ)); 2.24 (s, C(6)-CH3); 1.76
(s, C(5)-CH3).
Anal. Calc. for C24H31N7O8 × 0.5 H2O (616.6): C 48.70, H 5.56, N 15.89;
Found: C 48.41, H 5.84, N 15.37.
3ꢀ-O-[6-(2-Amino-3,6-dimethyl-4,7-dioxo-3,4,7,8-
tetrahydropteridin-8-yl)]hexanoyl-thymidine 5ꢀ-triphosphate
Tetrasodium Salt (13)
The conjugate 9 (57 mg, 0.1 mmol) was coevaporated with dry pyri-
dine (3 × 1 ml), then dissolved in dry pyridine (1 ml) and after cooling
to −10◦C under Ar-atmosphere POCl3 (20 µl, 0.2 mol) added. The mix-
ture was stirred for 10 minutes, warmed to 0◦C and under vigorous stirring
tributylamine (130 µl) and 0.5 M bis-(tri-n-butylammonium pyrophosphate
in anhydrous DMF (1 ml) added. After stirring for 30 minutes was 0.2 M
TEAB buffer (12.5 ml) slowly added and stirring continued for another 45
minutes. The resulting solution was applied on a DEAE cellulose column
in the bicarbonate form. The column was washed with H2O and then with
a linear gradient of aqueous tributylammonium bicarbonate (0.01–0.5 M).
The pooled triphosphate peak was evaporated and the residue coevaporated
with MeOH (5 × 5 ml). The final residue was dissolved in MeOH (0.5 ml)
and then under stirring a solution of 1 M NaI in acetone (0.5 ml) followed

Nucleotides Part LXXX
551
by acetone (2 ml) added. The precipitate was collected, washed with acetone
(4 × 1 ml), and dried overnight in vacuum over P4O10 to give 0.18 g (20%)
of 13 as a colorless powder. UV (MeOH): 216 (4.50), 258 (4.01), 340 (3.97).
1H-NMR (D2O): 7.62 (s, H-6); 5.97 (t, H-C(1ꢁ)); 5.30 (m, H-C(3ꢁ)); 4.20–3.95
(m, 5H, N-CH2, H-C(4ꢁ), H-C(5ꢁ,5ꢁ)); 3.28 (s, N-CH3); 2.33 (t, CH2); 2.22
(m, 5H, C-CH3, H-C(2ꢁ, 2ꢁꢁ)); 1.82 (s, CH3 Thy); 1.57 (m, 4H, 2 x CH2); 1.27
(m, CH2). 31P-NMR (D2O): -8.22 (d, γ P), -10.47 (d, αP), -21.48 (t, βP).
3ꢀ-O-[9-(2-Amino-3,6-dimethyl-4,7-dioxo-3,4,7,8-
tetrahydropteridin-8-yl)ethoxyethoxy-ethoxycarbonyl]thymidine
5ꢀ-triphosphate Tetrasodium Salt (14)
Analogous to 13 with 12 (62 mg, 0.1 mmol) to give 0.38 g (40%) of 14
1
as a colorless powder. UV (MeOH): 215 (4.49), 260 (4.02), 341 (4.00). H-
NMR (D2O): 7.62 (s, H-6); 6.04 (t, H-C(1ꢁ)); 5.27 (m, H-C(3ꢁ)); 4.35–4.05
(m, 7H, N-CH2, O-CH2, H-C(4ꢁ), H-C(5ꢁ,5ꢁ)); 3.75–3.50 (m, 8H, 4 x CH2);
3.30 (s, N-CH3); 2.52 (m, H-C(2ꢁ, 2ꢁꢁ)); 2.24 (s, C-CH3(6)); 1.81 (s, CH3 Thy).
31P-NMR (D2O): -9.69 (d, γ P), -10.54 (d, αP), -21.82 (t, βP).
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