3'-C-branched thymidine 5'-triphosphates: Synthesis, application, and coupling with near-infrared oxazine fluorescent dye

Article abstract of DOI:10.1055/s-2000-6388

The synthesis of the novel class of 3'-C-modified thymidine nucleotides is described. The aldehyde 1 was converted into the derivatives 5, 7, and 10 that differ in the length of the carbon chain. One-pot phosphorylation gave the corresponding triphosphate

Full text of DOI:10.1055/s-2000-6388

PAPER
707
3¢-C-Branched Thymidine 5´-Triphosphates: Synthesis, Application, and
Coupling with Near-Infrared Oxazine Fluorescent Dye
Thomas Schoetzau,a Ulrich Koert,a Joachim W. Engels*b
aFachbereich Chemie der Humboldt-Universität, Hessische Str. 1-2, 10115 Berlin, Germany
bInstitut für Organische Chemie, Johann Wolfgang Goethe-Universität, Marie-Curie-Str. 11, 60439 Frankfurt/Main, Germany
Received 24 September 1999; revised 8 December 1999
phonium salt and the base NaN(TMS)2. With an excess of
the 4-carboxybutyltriphenylphosphonium bromide
(3 equivalents) and the NaN(TMS)2 (6 equivalents) to the
Abstract: The synthesis of the novel class of 3¢-C-modified thymi-
dine nucleotides is described. The aldehyde 1 was converted into
the derivatives 5, 7, and 10 that differ in the length of the carbon
chain. One-pot phosphorylation gave the corresponding triphos-
nucleoside 1, only the desired product was obtained in
phates 11, 12, 13. The 3¢-C-(2-azidoethyl)-3¢-deoxythymidine 5¢- good yield (68%). NOESY-NMR spectra show a cross
triphosphate 13 was reduced by triphenylphosphine to the amino
nucleotide 14. All amino nucleotides have been dye-labelled at the
aliphatic amino group with the oxazine dye JA242. All triphos-
phates were tested as terminators in DNA-sequencing reactions.
peak between both of the newly formed olefinic protons.
The coupling constant is 10.9 Hz, indicating the formation
of the (Z)-isomer of 2.
For further carbon-chain elongation, the TSTU-method7
Key words: 3¢-C-modified nucleoside, dideoxynucleoside 5¢-triph-
osphates, dye labelling, radical reaction
was used. Therefore the free carboxyl group of 2 was ac-
tivated with N,N,N¢,N¢-tetramethyl-O-succinimidouroni-
um tetrafluoroborate (TSTU) in DMF/dioxane. The
activation was monitored by TLC and was completed af-
ter 20 min. The carboxylic acid 2 reacted with ethylenedi-
amine to form compound 3 in 43% yield after silica gel
chromatography. The free amino group of 3 was protected
with trifluoroacetic anhydride in CH2Cl2 and triethy-
lamine and analyzed by 19F NMR. The NMR shift for the
trifluoroacetyl group at -76.30 ppm indicated the suc-
cessful reaction. Product 4 was deprotected with 1 M
Bu4NF in THF affording 5 in 82% yield.
The use
of labelled
2¢,3¢-dideoxynucleoside
5¢-triphosphates1 as terminators in DNA-sequencing has
found wide acceptance, and is the method of choice for
the determination of single-stranded DNA sequences. In
recent years, much effort has been made in developing se-
quencing protocols, which are faster and deliver results
with improved accuracy. The labelling of each of the four
2¢,3¢-dideoxynucleoside 5¢-triphosphates with different
dyes and the use of capillary electrophoresis methods of-
fers the possibility to measure the fluorescence decay
time2 with pulsed lasers and therefore allow the identifica-
tion of each nucleotide in short time. The formation of
3¢-dye-labelled 2¢,3¢-dideoxynucleoside 5¢-triphosphates
via heteroatoms has been reported.3 However, the
problem of cleavage of 3¢-ester and -amide modified nu-
cleoside 5¢-triphosphates by DNA polymerases4 cannot be
excluded during sequencing reaction and thus a class of
terminators with more suitable properties is required.
Following our concept5 of 3¢-modified sequencing termi-
nators derived from sugar substitutions the synthesis
of 3¢-C-modified nucleoside 5¢-triphosphates labelled
with oxazine derivative JA242, as fluorescent dye, will be
described (Scheme 1 and 2).
The transformation of the carboxylic acid 2 into 6 with
70% yield followed the same procedure, i.e. condensation
step with TSTU followed by reaction with N-trifluoro-
acetyl-1.6-diaminohexane8 in DMF/dioxane/diisopropyl
ethylamine (DIPEA). The shift for the trifluoroacetyl
group at -76.21 ppm in the 19F NMR spectrum again in-
dicated the successful reaction. The product 6 was depro-
tected with 1M Bu4NF in THF affording 7 in 97% yield.
The synthetic route for the preparation of the shortest link-
er containing one C2-unit was elaborated by reduction of
the aldehyde 1 by NaBH4 affording the 5¢-O-TBDPS-3¢-
C-(2-hydroxyethyl)-3¢-deoxythymidine 8 with a yield of
95%. The next step was the displacement of the 2-hydroxy
group by the azide moiety. The azido group should act as
an in situ protecting group of the amino function in the
triphosphate synthesis. Instead of two nucleophilic substi-
tutions under vigorous reaction conditions,6c we used the
Mitsunobu reaction9 and achieved very high yields. Under
mild conditions, nucleoside 8 reacted with triphenylphos-
phine, hydrazoic acid, and diethyl azodicarboxylate
(DEAD). The standard work-up method was difficult as
nucleoside 9 and the formed triphenylphosphine oxide
have the same Rf value in CHCl3/CH3OH (9:1). To over-
come this problem, we changed the procedure Compound
9 was purified by silica gel column chromatography using
hexane and a slow gradient of EtOAc, affording 94% of
The aldehyde 1 as starting material was synthesized from
thymidine via a radical reaction involving regioselective
addition of a C-centered radical to allyltributyltin as de-
scribed in the literature.6 The overall yield over four steps
was 28%.
For elongation of the carbon chain we used the Wittig re-
action for the C=C bond. The aldehyde 1 reacted with
4-carboxybutyltriphenylphosphonium bromide in THF
resulting in the formation of 2. The protection of the base
on N3-position is not necessary in this reaction step. The
ylide which reacted with 1 was generated from the phos-
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T. Schoetzau et al.
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Reagents and conditions: (a) 4-carboxybutyltriphenylphosphonium bromide, NaN(TMS)2, THF, 30 min, r.t.; (b) (i) TSTU, DIPEA, DMF,
dioxane, 20 min, r.t., (ii) ethylenediamine, 10 min, r.t.; (c) (CF3CO)2O, Et3N, CH2Cl2, 1 h, 0 °C; (d) TBAF, THF, 1 h, r.t.; (e) (i). TSTU, DI-
PEA, DMF, dioxane, 20 min, r.t., (ii) CF3C(O)NH(CH2)6NH2, 10 min, r.t.; (f) TBAF, THF, 1 h, r.t.; (g) NaBH4, MeOH, 2 h, r.t.; (h) Ph3P,
DEAD, HN3, benzene, r.t.; (i) TBAF, THF, 1 h, r.t.; (j-l) (i) 2-chloro-4H-1,3,2-benzodioxaphosphorin-4-one, dioxane, pyridine, 15 min, r.t.,
(ii) tributylammonium pyrophosphate, DMF, Bu3N, 20 min, r.t., (iii) I2, pyridine/H2O (98:2), 20 min, r.t.; (m) (i) Ph3P, dioxane/H2O (3:1), 24
h, r.t., (ii) 25% aq NH3, 2 h, r.t.
Scheme 1
5¢-O-TBDPS-3¢-C-(2-azidoethyl)-3¢-deoxythymidine 9. oxane/water (3:1) and further treatment with 25% aque-
1
The structure of 9 was determined by H and 13C NMR ous ammonia giving compound 14 (yield: 95%). After
spectroscopy, and IR spectra. Compound 9 was deprotect- purification on DEAE-Sephadex A25, lyophilisation and
ed with 1 M Bu4NF in THF yielding 10 in 98% yield.
precipitation the products were obtained as sodium salts.
The identity of the products 11-14 was determined by
1H, 13C and 31P NMR spectroscopy as well as by mass
spectra.
The method of Ludwig et al.10 was applied for the prepa-
ration of the nucleoside 5¢-triphosphates 11, 12, 13. Ac-
cording to the original method, the nucleoside was
allowed to react with salicoyl chlorophosphite in dioxane/ The JA242-labelled nucleotide 5¢-triphosphates were gen-
pyridine, and the intermediate formed was treated in situ erated by reaction of 1-(3-carboxypropyl)-11-ethyl-
with bis(tributylammonium) pyrophosphate in DMF. The 3,4,8,9,10,11-hexahydro-8,10,10-trimethyl-2H-dipyrido-
oxidation was carried out in a freshly prepared solution of [3,2-b: 2¢,3¢-i]phenoxazinium inner salt (JA242) with the
I2 in pyridine/water (98:2). For deprotection 25% aqueous nucleotide. In the first step, the carboxyl group of JA242
ammonia was used. The yields of the nucleoside 5¢-triph- was activated with TSTU in DMF/dioxane/DIPEA. The
osphates 11, 12, 13 were between 32% and 63%. Subse- reaction was complete within 20 min, and the activated
quently, the azido group of compound 13 was converted dye could be detected by TLC. The reaction was contin-
into the corresponding amino group by reduction with a ued by the addition of a 0.2 M aqueous solution of the nu-
10-fold excess of triphenylphosphine11 in a mixture of di- cleoside 5¢-triphosphate. The free amino group of the
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Synthesis, Application, and Coupling with Near-Infrared Oxazine Fluorescent Dye
709
nucleotide reacted with the activated carboxyl group of ica gel chromatography. To separate the conjugates from
the dye under formation of the corresponding amide. The the free dye, the formation of the sodium salt of com-
purification of the 3¢-dye labelled nucleoside 5¢-triphos- pounds 15, 16 and 17 was required in the next step. Puri-
phates comprises of three steps. However, this procedure fication on Mono Q-HPLC and desalting gave the desired
has proven to be most suitable for the purification of dye- products in 5-8% yield.
labelled triphosphates and thus, in the first step the dye-
linker-nucleotide-conjugates were subjected to RP-18 sil-
Using the polymerase Thermo Sequenase the incorpora-
tion of compounds 11, 12, and 14-17 at different concen-
Reagents and conditions: (a) TSTU, DMF, dioxane, H2O, DIPEA, 20 min, r.t., 20 min for activation, and then 48 h in the dark, r.t.
Scheme 2
trations into the DNA for sequencing failed, only
unspecific band patterns were obtained and could not be
correlated with the standard dTTP pattern.
Table Properties of 3’-C-Dye-Labelled Thymidine 5’-Triphos-
phates 15−17 Prepared
Moreover, we found that the compounds 11, 12, and
Com- Calcd for [M+H]+
pound
Found
tR/minb labs-max lem-max
c
c
14-17 are no substrates for terminal deoxynucleotidyl
transferase (Tdt). We assume that the change of the con-
formation (3¢-endo-typ) in the 3¢-C-branched nucleotide
and the increasing steric hindrance of 3¢-substituents dur-
ing the DNA-polymerisation are the reasons for their fail-
ure as terminators in the DNA sequencing reaction.
[M+H]+a
15
16
17
C46H64N7O16P3:
1064.37
1064.43 10.13
1120.45 10.68
939.33 9.55
668
668
669
684
683
683
C50H72N7O16P3:
1120.43
C39H53N6O15P3:
939.29
All commercial reagents were used without further purification.
The aldehyde 1 as starting material was synthesized as reported in
the literature.6b,c Column chromatography was performed using
Merck Kieselgel 60 (70-230 mesh), and TLC silica 60 F254 plates
(Merck). Ion-exchange chromatography was performed using
a MALDI TOF Mass Spectral Analyses.
b Ion exchange column (Mono Q), flow rate 1.0 mL/min, A: 20 mM
NaOAc, pH 6.5, 20 % CH3CN, B: 20 mM NaOAc, pH 6.5, 1 M NaCl,
20 % CH3CN, from 0 to 60 % in 60 min.
-
DEAE-Sephadex A25 (HCO3 and Cl- form) from Pharmacia. IR
c Measured in H2O.
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710
T. Schoetzau et al.
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spectra were obtained on a IR-408 (Shimadzu, Japan), and 1600 se-
ries (Perkin Elmer, USA). HPLC was performed using pump-sys-
tem LC 1110, System Organiser LC 1431, UV-Detectors GAT-
PHD 601, GATZ LCD 502 (Gamma Analysen Technik, Germany),
Mono Q (Pharmacia). UV/VIS-spectra were obtained on a Spekord
UV 160 A (Shimadzu, Japan). NMR spectra were obtained on an
DPX 300 and AMX 600 spectrometer (Bruker Physics AG, USA).
Coupling constants (J) are given in Hz, and all chemical shifts are
relative to an internal standard of TMS (1H and 13C) or with 85%
phosphoric acid as an external standard (31P). Mass spectra were ob-
tained on a FAB-MS (SSQ 7000), EI-MS (MAT 90), and MALDI-
TOF-MS (Voyager DE-STR).
(C3¢), 39.66 (C2¢), 41.84, 42.08 (2 x CH2), 65.74 (C5¢), 86.54 (C4¢),
87.66 (C1¢), 111.70 (C5), 128.97 (olefin), 132.41 (olefin), 129.39-
137.11 (12C, Ar), 137.96 (C6), 152.82 (C2), 166.88 (C4), 176.95
(C=O).
MALDI-TOF-MS: m/z calc for (M+H)+ 633.35. Found: 633.53.
5¢-O-TBDPS-3¢-C-[N-[N-(2-trifluoracetylaminoethyl)]-6-car-
bamoylhex-2(Z)-enyl]-3¢-deoxythymidine (4)
The nucleoside 3 (0.20 g, 0.32 mmol) was dissolved in CH2Cl2
(5 mL) and Et3N (1.3 mL), cooled in an ice bath, and trifluoroacetic
anhydride (0.053 mL, 0.38 mmol) was added dropwise. The solu-
tion was stirred for 1 h in the ice bath, evaporated to dryness, and
purified by column chromatography on silica gel with CHCl3. The
product 4 (0.22 mg, 95%) was obtained as a solid white foam.
1H NMR (300 MHz, CDCl3): d = 1.02 (s, 9H, C(CH3)3), 1.52 (s, 3H,
C5-CH3), 1.62 (q, 2H, J = 7.5, CH2), 1.83-2.22 (m, 8H, H-2¢,
3 x CH2), 2.35 (m, 1H, H-3¢), 3.38 (br s, 4H, 2 x NCH2), 3.71 (m,
2H, H-4¢, H-5¢a), 3.96 (dd, 1H, J5¢a,5¢b = -11.5, J5¢b,4¢ = 2.0, H-5¢b),
5.26 (m, 1H, olefin), 5.36 (m, 1H, olefin), 6.02 (t, 1H, J = 6.0, H-1¢),
7.29-7.60 (m, 11H, ArH, H-6), 8.31 (br s, 1H, NH).
13C NMR (75 MHz, CDCl3): d = 12.03 (C5-CH3), 19.32 (-C(CH3)3),
25.34, 26.86 (2 x CH2), 26.92 (-C(CH3)3), 35.71, 37.60 (2 x CH2),
38.29 (C3¢), 38.34 (C2¢), 41.18, 43.35 (2 x CH2), 64.66 (C5¢), 85.07
(C4¢), 85.59 (C1¢), 110.76 (C5), 113.94 (-CF3), 127.01 (olefin),
129.94 (olefin), 127.83-135.66 (12C, Ar), 135.46 (C6), 150.83
(C2), 157.54 (COCF3), 164.65 (C4), 175.06 (C=O).
5¢-O-TBDPS-3¢-C-(6-carboxyhex-2(Z)-enyl)-3¢-deoxythymi-
dine (2)
(4-Carboxybutyl)triphenylphosphonium
bromide
(2.62 g,
5.92 mmol) was dissolved in dry THF (23 mL). The vessel was
closed with a septum, and a low pressure of Ar was maintained. Into
the vessel, 1 M NaN(TMS)2 (12.82 mL, 12.82 mmol) in THF was
injected through a septum. The formation of the orange-red ylide
was finished after 15 min. The aldehyde 1 (1.0 g, 1.97 mmol), dis-
solved in dry THF (2 mL), was then injected into the well-stirred so-
lution of the ylide. After 15 min, the reaction mixture was poured
into CHCl3 (500 mL) and extracted with 5% HCl (500 mL), and
brine (500 mL). The organic layer was dried (Na2SO4). Removal of
the solvent in vacuo afforded an oily residue, which was purified by
column chromatography on silica gel with CHCl3/MeOH (0-10%
MeOH). The product 2 (0.79 g, 68%) was obtained as a solid white
foam.
19F NMR (282 MHz, CDCl3): d = -76.30.
1H NMR (300 MHz, CDCl3): d = 1.02 (s, 9H, C(CH3)3), 1.53 (s, 3H,
C5-CH3), 1.61 (q, 2H, J = 7.2, CH2), 1.91-2.10 (m, 6H, H-2¢,
2 x CH2), 2.24 (t, 2H, J = 7.2, CH2), 2.32 (m, 1H, H-3¢), 3.69 (m,
2H, H-4¢, H-5¢a), 3.95 (dd, 1H, J5¢a,5¢b = -12.5, J5¢b,4¢ = 3.2, H-5¢b),
5.23 (m, 1H, olefin), 5.30 (m, 1H, olefin), 6.05 (t, 1H, J = 5.3, H-1¢),
7.28-7.61 (m, 11H, ArH, H-6), 10.03 (br s, 1H, NH).
13C NMR (75 MHz, CDCl3): d = 11.96 (C5-CH3), 19.29 (-C(CH3)3),
26.48 (CH2), 26.91 (-C(CH3)3), 29.48, 30.51, 33.23 (3 x CH2), 37.47
(C3¢), 38.56 (C2¢), 63.79 (C5¢), 84.72 (C4¢), 85.67 (C1¢), 110.42
(C5), 127.02 (olefin), 130.88 (olefin), 127.75-135.39 (12C, Ar),
135.75 (C6), 150.57 (C2), 164.67 (C4), 177.63 (C=O).
3¢-C-[N-[N-(2-Trifluoracetylaminoethyl)]-6-carbamoylhex-
2(Z)-enyl]-3¢-deoxythymidine (5)
The nucleoside 4 (0.20 mg, 0.27 mmol) was dissolved in dry THF
(2 mL). A solution of 1 M Bu4NF (TBAF) (0.30 mL, 0.30 mmol) in
THF was added. The reaction was completed after 1 h. The solvent
was evaporated in vacuo. The residue was dissolved in CHCl3
(50 mL) and extracted twice with H2O (50 mL). The organic layer
was dried (NaSO4), and the solvent was removed under reduced
pressure. The resulting syrup was chromatographed over silica gel
with CHCl3. The compound 5 (0.11 mg, 82%) was obtained as a sol-
id colorless product.
1H NMR (300 MHz, CD3OD): d = 1.77 (s, 3H, C5-CH3), 1.88-2.19
(m, 10H, H-2¢, 4 x CH2), 2.22 (m, 1H, H-3¢), 3.28 (m, 4H, 2 x
NCH2), 3.63 (m, 2H, H-4¢, H-5¢a), 3.81 (dd, 1H, J5¢a,5¢b = -13.6,
MALDI-TOF-MS: m/z calc for (M+H)+ 591.29. Found: 591.02.
5¢-O-TBDPS-3¢-C-[N-(2-aminoethyl)-6-carbamoylhex-2(Z)-
enyl]-3¢-deoxythymidine (3)
J
5¢b,4¢ = 3.4, H-5¢b), 5.34 (m, 2H, olefin), 5.95 (dd, 1H, J1¢,2¢a = 4.0,
J1¢,2¢b = 6.0, H-1¢), 7.90 (s, 1H, H-6).
A mixture of 2 (0.47 g, 0.79 mmol), diisopropyl ethyl amine
(0.40 mL, 2.3 mmol), and N,N,N¢,N¢-tetramethyl-O-succinimidou-
ronium tetrafluoroborate (TSTU, 0.29 mg, 0.95 mmol) was dis-
solved in dioxane (1.5 mL) and DMF (1.5 mL). The activation of
the free carboxylic acid 2 was completed after 20 min and could be
observed on TLC. Freshly distilled ethylenediamine (0.21 mL, 3.17
mmol) was added. The reaction was completed at r.t. after 10 min.
The resulting suspension was evaporated to dryness, dissolved in
CHCl3 (100 mL), and extracted with sat. NaHCO3 (100 mL). The
organic phase was separated, dried (Na2SO4), and evaporated again
to dryness. The resulting residue was purified by column chroma-
tography on silica gel with CHCl3/MeOH (0-15% MeOH). The
product 3 (0.22 mg, 43%) was obtained as a colorless oil.
13C NMR (75 MHz. CD3OD): d = 12.93 (C5-CH3), 25.18, 27.00,
33.41, 36.86 (4 x CH2), 38.71 (C3¢), 39.68 (C2¢), 40.16, 40.94 (2 x
CH2), 62.86 (C5¢), 86.75 (C4¢), 88.21 (C1¢), 111.17 (C5), 116.24
(CF3), 128.95 (olefin), 132.28 (olefin), 138.88 (C6), 152.75 (C2),
158.00 (-COCF3), 166.95 (C4), 176.93 (C=O).
19F NMR (282 MHz, CD3OD): d = -77.72.
HRMS (FAB-MS): m/z calc for C21H29F3N4O6 (M+H)+ 491.2117.
Found: 491.2115.
5¢-O-TBDPS-3¢-C-[N-[N-(6-trifluoroacetylaminohexyl)]-6-car-
bamoylhex-2(Z)-enyl]-3¢-deoxythymidine (6)
1H NMR (300 MHz, CDCl3): d = 0.98 (s, 9H, C(CH3)3), 1.42 (s, 3H,
C5-CH3), 1.55 (q, 2H, J = 7.4, CH2), 1.90-2.14 (m, 8H, H-2¢, 3 x
CH2), 2.37 (m, 1H, H-3¢), 2.72 (t, 2H, J = 6.2, NCH2), 3.20 (t, 2H,
J = 6.2, NCH2), 3.71 (m, 2H, H-4¢, H-5¢a), 3.95 (dd, 1H, J5¢a,5¢b = -
11.5, J5¢b,4¢ = 2.0, H-5¢b), 5.33 (m, 2H, olefin), 5.99 (dd, 1H,
The nucleoside 2 (0.26 g, 0.44 mmol), diisopropyl ethyl amine
(0.24 mL, 1.41 mmol), and TSTU (0.16 g, 0.53 mmol) were dis-
solved in dioxane (0.82 mL) and DMF (0.82 mL). The activation of
the free carboxylic acid 2 was completed within 20 min. The N-tri-
fluoracetyl-1,6-diaminohexane (0.11 g, 0.58 mmol) was added at
r.t. and stirred for 30 min. The solvent was evaporated in vacuo. The
residue was dissolved in 50 mL CHCl3, and extracted with aq
NaHCO3 (50 mL). The organic layer was seperated, dried (Na2SO4),
and the solvent was removed under reduced pressure. The resulting
J
1¢,2¢a = 6.6, J1¢,2¢b = 4.7, H-1¢), 7.25-7.77 (m, 11H, ArH, H-6).
13C NMR (75 MHz, CDCl3): d = 12.84 (C5-CH3), 20.73 (-C(CH3)3),
27.71 (CH2), 28.03 (-C(CH3)3), 28.31, 33.54, 36.92 (3 x CH2), 39.44
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Synthesis, Application, and Coupling with Near-Infrared Oxazine Fluorescent Dye
711
syrup was chromatographed over silica gel with CHCl3. The prod-
uct 6 (0.16 g, 70%) was obtained as a solid colorless foam.
1H NMR (300 MHz, CDCl3): d = 1.03 (s, 9H, C(CH3)3), 1.53 (s, 3H,
C5-CH3), 1.15-1.74 (m, 10H, 5 x CH2), 1.91-2.14 (m, 8H, H-2¢,
6 x CH2), 2.37 (m, 1H, H-3¢), 3.16 (t, 2H, J = 6.5, NCH2), 3.26 (t,
2H, J = 6.4, NCH2), 3.67 (m, 2H, H-4¢, H-5¢a), 3.96 (dd, 1H,
13C NMR (75 MHz, CDCl3): d = 12.12 (C5-CH3), 19.28 (-C(CH3)3),
26.91 (-C(CH3)3), 34.50 (C3¢), 34.62 (CH2), 38.98 (C2¢), 60.82
(CH2), 63.61 (C5¢), 84.94 (C4¢), 86.29 (C1¢), 110.30 (C5), 127.76-
135.48 (12C, Ar), 135.30 (C6), 150.56 (C2), 164.23 (C4).
HRMS (FAB-MS): m/z calc for C28H36N2O5Si (M+H)+ 509.2472.
Found: 509.2465.
J
5¢b,5¢a = -10.0, J5¢b,4¢ = 2.0, H-5¢b), 5.25 (m, 1H, olefin), 5.36 (m,
1H, olefin), 6.04 (t, 1H, J = 6.0, H-1¢), 7.31-7.61 (m, 11H, ArH, H-
6), 8.67 (s, 1H, NH).
5¢-O-TBDPS-3¢-C-(2-azidoethyl)-3¢-deoxythymidine (9)
The nucleoside 8 (0.37 g, 0.72 mmol), Ph3P (0.23 g, 0.87 mmol),
and DEAD (0.12 mL, 0.80 mmol) were dissolved in dry benzene
(4.6 mL). A solution of the 1.28 M HN3 (1.7 mL, 2.18 mmol) in ben-
zene was then added dropwise. The reaction was completed at r.t. as
soon as the colour of the solution changed from red to slight yellow.
The solvent was carefully removed under reduced pressure. The
residue was dissolved in CHCl3 (50 mL), and extracted with brine
(50 mL). The organic phase was dried (Na2SO4), and the solvent
was removed in vacuo. The residue was purified by flash chroma-
tography using hexane/EtOAc (0-50% EtOAc) as eluent. The prod-
uct 9 (0.36 g, 94%) was obtained as a solid white foam.
13C NMR (75 MHz, CDCl3): d = 12.12 (C5-CH3), 19.41 (-C(CH3)3),
25.69, 25.75, 26.91 (3 x CH2), 27.01 (-C(CH3)3), 28.60, 29.49,
30.05, 31.96, 35.99 (5 x CH2), 38.04 (C3¢), 38.16 (C2¢), 38.83,
39.39 (2 x CH2), 64.37 (C5¢), 84.91 (C4¢), 85.68 (C1¢), 110.79 (C5),
115.25 (-CF3), 130.10 (olefin), 131.46 (olefin), 126.92-135.54
(12C, Ar), 135.35 (C6), 150.33 (C2), 157.58 (-COCF3), 163.79
(C4), 172.95 (C=O).
19F NMR (282 MHz, CDCl3): d = -76.21.
MALDI-TOF-MS: m/z calc for (M+H)+ 785.39. Found: 785.57.
IR (film): n = 2099 cm-1 (N3).
3¢-C-[N-[N-(6-Trifluoracetylaminohexyl)]-6-carbamoylhex-
2(Z)-enyl]-3¢-deoxythymidine (7)
1H NMR (300 MHz, CDCl3): d = 1.03 (s, 9H, C(CH3)3), 1.45 (m,
1H, C3¢- CH2a), 1.51 (s, 3H, C5-CH3), 1.65 (m, 1H, C3¢- CH2b),
2.04-2.40 (m, 3H, H-2¢, H-3¢), 3.22 (m, 2H, CH2N3), 3.67 (m, 2H,
H-4¢, H-5¢a), 3.98 (dd, 1H, J5¢b,5¢a = -11.7, J5¢b,4¢ = 2.3, H-5¢b), 6.04
(dd, 1H, J1¢,2¢a = 7.2, J1¢,2¢b = 4.1, H-1¢), 7.31-7.62 (m, 11H, ArH, H-
6), 9.69 (br s, 1H, NH).
13C NMR (75 MHz, CDCl3): d = 12.63 (C5-CH3), 19.77 (-C(CH3)3),
27.39 (-C(CH3)3), 31.66 (CH2), 35.46 (C3¢), 39.13 (C2¢), 50.21
(CH2N3), 63.81 (C5¢), 85.18 (C4¢), 86.27 (C1¢), 111.12 (C5),
128.24-135.93 (12C, Ar), 135.77 (C6), 150.93 (C2), 164.58 (C4).
The nucleoside 6 (0.15 g, 0.19 mmol) was dissolved in dry THF
(2 mL). A solution of 1 M Bu4NF (0.21 mL, 0.21 mmol) in THF was
added. The reaction was completed after 1 h. The solvent was evap-
orated in vacuo. The residue was dissolved in CHCl3 (50 mL) and
extracted twice with H2O (50 mL). The organic layer was dried
(NaSO4), and the solvent was removed under reduced pressure. The
resulting syrup was chromatographed over silica gel with CHCl3.
The product 7 (0.10 g, 87%) was obtained as a solid colorless prod-
uct.
1H NMR (300 MHz, CD3OD): d = 1.24-1.58 (m, 10H, 5 x CH2),
1.77 (s, 3H, C5-CH3), 1.92-2.27 (m, 9H, H-2¢, H-3¢, 3 x CH2), 3.06
(t, 2H, J = 6.9, NCH2), 3.17 (t, 2H, J = 7.2, NCH2), 3.63 (m, 2H, H-
4¢, H-5¢a), 3.79 (dd, 1H, J5¢b,5¢a = -9.9, J5¢b,4¢ = 3.9, H-5’b), 5.36 (m,
2H, olefin), 6.04 (dd, 2H, J1¢,2¢a = 6.4, J1¢,2¢b = 3.8, H-1’), 7.79 (s, 1H,
H-6).
13C NMR (75 MHz, CD3OD): d = 12.93 (C5-CH3), 25.17, 27.37,
27.83, 27.92, 28.20, 30.73, 33.43, 36.96 (8 x CH2), 40.19 (C3¢),
40.22 (C2¢), 40.65, 41.03 (2 x CH2), 62.82 (C5¢), 86.74 (C4¢), 88.22
(C1¢), 111.15 (C5), 116.08 (-CF3), 128.89 (olefin), 132.31 (olefin),
138.87 (C6), 152.73 (C2), 159.30 (-COCF3), 166.95 (C4), 176.22
(C=O).
MALDI-TOF-MS: m/z calc for (M+H)+ 534.25. Found: 534.33.
3¢-C-(2-Azidoethyl)-3¢-deoxythymidine (10)
The nucleoside 9 (0.36 g, 0.66 mmol) was dissolved in dry THF
(3 mL). A solution of 1 M Bu4NF (0.74 mL, 0.74 mmol) in THF was
added. The reaction was completed after 1 h. The solvent was evap-
orated in vacuo. The residue was dissolved in CHCl3 (50 mL) and
extracted twice with H2O (50 mL). The organic layer was separated,
and the solvent was removed under reduced pressure. The resulting
syrup was chromatographed over silica gel with CHCl3. The prod-
uct 10 (0.19 g, 98%) was obtained as a solid colorless product.
IR (film): n = 2104 cm-1 (N3).
1H NMR (300 MHz, CDCl3): d = 1.59-1.80 (m, 2H, CH2), 1.83 (s,
3H, C5-CH3), 2.01-2.39 (m, 3H, H-2¢, H-3¢), 3.30 (t, 2H, J = 6.8,
CH2N3), 3.69 (m, 2H, H-4¢, H-5¢a), 3.95 (dd, 1H, J5¢b,5¢a = -13.4,
19F NMR (282 MHz, CD3OD): d = -77.72.
HRMS (FAB-MS): m/z calc for C25H37F3N4O6 (M+H)+ 547.2734.
Found: 547.2743.
J
5¢b,4¢ = 3.4, H-5¢b), 6.05 (dd, 1H, J1¢,2¢a = 3.0, J1¢,2¢b = 7.5, H-1¢), 7.55
(s, 1H, H-6).
5¢-O-TBDPS-3¢-C-(2-hydroxyethyl)-3¢-deoxythymidine (8)
The aldehyde 1 (1.0 g, 1.97 mmol) was dissolved in dry MeOH
(20 mL). Within 20 min, NaBH4 (0.23 g, 5.92 mmol) was added to
the mixture. The reaction was stirred at r.t. for 2 h. The reaction was
quenched by adding H2O (1 mL), the solvent was evaporated at re-
duced pressure, taken up in CHCl3 (200 mL), and extracted with
brine (200 mL). The organic layer was then evaporated, and the res-
idue was purified by flash chromatography using CHCl3/MeOH
(0-10% MeOH) as eluent. The product 8 (0.95 g, 95%) was ob-
tained as a solid white foam.
1H NMR (300 MHz, CDCl3): d = 1.01 (s, 9H, C(CH3)3), 1.45 (m,
1H, C3¢- CH2a), 1.51 (s, 3H, C5-CH3), 1.58 (m, 1H, C3¢- CH2b),
2.01-2.38 (m, 3H, H-2¢, H-3¢), 3.60 (m, 4H, H-4¢, H-5¢a, CH2OH),
3.97 (dd, 1H, J5¢b,5¢a = -11.7, J5¢b,5¢4¢ = 2.6, H-5¢b), 6.02 (dd, 1H,
13C NMR (75 MHz, CDCl3): d = 12.52 (C5-CH3), 31.15 (CH2),
34.67 (C3¢), 38.81 (C2¢), 49.88 (CH2), 61.56 (C5¢), 85.17 (C4¢),
86.17 (C1¢), 110.49 (C5), 136.29 (C6), 150.37 (C2), 163.97 (C4).
HRMS (EI-MS): m/z calc for C12H17N5O4 (M-H)- 294.1202.
Found: 294.1220.
3¢-C-Branched 3¢-deoxythymidine 5¢-triphosphates 11, 12, 13;
General Procedure
Nucleosides 5, 7 and 10 (100 µmol) were dissolved in anhyd pyri-
dine (5 mL) and evaporated to dryness in vacuo. The residue was
dried (P4O10) overnight, and during all following steps, a low pres-
sure of Ar was maintained. Into the vessel containing 5, 7, 10,
through a septum, was injected anhyd pyridine (100 µL) followed
by anhyd dioxane (300 µL). Freshly prepared 1 M 2-chloro-4H-
1,3,2-benzodioxaphosphorin-4-one (salicoyl chlorophosphite) in
anhyd dioxane (110 µL, 110 µmol) was then injected into the well-
J
1¢,2¢a = 3.8, J1¢,2¢b = 6.8, H-1¢), 7.29-7.62 (m, 11H, ArH, H-6), 9.69
(br s, 1H, NH).
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712
T. Schoetzau et al.
PAPER
stirred solution of the 3¢-C-modified 2¢,3¢-dideoxyribonucleoside.
After 15 min, a mixture of 0.5 M tributylammonium pyrophosphate
in anhyd DMF (300 µL) and Bu3N (100 µL) was quickly injected.
A solution of 1% I2 in pyridine/H2O (98:2, 2 mL) was added after
20 min and the excess of I2 destroyed by adding a 5% aq NaHSO3
(1 mL). In the case of 5 and 7, the solutions were evaporated and the
residue dissolved in concd NH3 (3 mL). The mixtures were stirred
at r.t. for 6 h, then evaporated, and the residues were dissolved in
H2O. The aq triphosphate solutions obtained from 5, 7 or 10 were
H-5¢a), 3.99 (m, 1H, H-5¢b), 5.83 (dd, 1H, J1¢,2¢a = 3.0, J1¢,2¢b = 7.6,
H-1¢), 7.43 (s, 1H, H-6).
13C NMR (75 MHz, D2O): d = 12.02 (C5-CH3), 30.41 (CH2), 35.52
(C3’), 37.66 (C2¢), 49.99 (CH2), 65.81 (C5¢), 85.04 (C4¢), 85.14
(C1¢), 111.52 (C5), 137.95 (C6), 152.18 (C2), 166.90 (C4).
31P NMR (121 MHz, D2O): d = -10.53 (d, a-P), -21.61 (t, b-P),
-5.57 (d, g-P).
MALDI-TOF-MS: m/z calc for (M-H)+ 534.02. Found: 533.71.
3¢-C-(2-Aminoethyl)-3¢-deoxythymidine 5¢-triphosphate (14)
-
applied on a DEAE Sephadex A-25 column (2.5 x 25 cm, HCO3 )
and eluted with a linear gradient of TEAB (0.05-1 M). After evap-
oration and repeated coevaporation with EtOH, the products were
precipitated from methanolic triphosphate soln (0.1 mM, 5 mL) by
NaClO4 (7.5 mmol) in anhyd acetone (25 mL) as Na+ salts. Yields
30-60%.
3¢-C-(2-Azidoethyl)-3'-deoxythymidine
5'-triphosphate
13
(23 µmol) was dissolved in a mixture (4.6 mL) of dioxane/H2O (3:1,
v/v). Ph3P (230 µmol) was then added, and the solution was stirred
at r.t. for 24 h. After all starting material had reacted, a 25% aq NH3
(2 mL) was added, and the mixture was stirred at r.t. for an addition-
al 2 h. The solution was evaporated, and the residue was dissolved
in H2O. The solution was poured onto a DEAE Sephadex A-25 col-
3¢-C-[N-[N-(2-Aminoethyl)]-6-carbamoylhex-2(Z)-enyl]-3¢-des-
oxythymidine 5’-triphosphate (11)
Obtained from nucleoside 5 as a colorless Na+ salt (47 mg, 63%).
-
umn (2.5 x 25 cm, HCO3 ) and eluted with a linear gradient of
TEAB (0.05-1 M). The product 14 was precipitated from methan-
olic triphosphate solution (0.1 mM, 5 mL) by dry NaClO4 (7.5
mmol) in anhyd acetone (25 mL) as the colorless Na+ salt (15 mg,
95%).
UV (H2O): lmax = 267 nm.
1H NMR (300 MHz, D2O): d = 1.54 (q, 2H, J = 6.7, CH2), 1.77 (s,
3H, C5-CH3), 1.85-2.30 (m, 9H, H-2¢, H-3¢, 3 x CH2), 2.98 (t, 2H,
J = 5.8, NCH2), 3.35 (t, 2H, J = 5.8, NCH2), 3.86 (m, 1H, H-4¢),
4.01 (m, 1H, H-5¢a), 4.17 (m, 1H, H-5¢b), 5.37 (m, 2H, olefin), 5.98
(dd, 1H, J1¢,2¢a = 3.4, J1¢,2¢b = 6.6, H-1¢), 7.61 (s, 1H, H-6).
13C NMR (75 MHz, D2O): d = 12.07 (C5-CH3), 25.20, 26.38, 28.73,
35.45 (4 x CH2), 37.37 (C2¢), 37.59 (C3¢), 37.97, 39.24 (2 x CH2),
66.11 (C5¢), 85.50 (C4¢), 85.58 (C1¢), 111.45 (C5), 127.78 (olefin),
131.63 (olefin), 137.92 (C6), 152.17 (C2), 167.27 (C4), 178.11
(C = O).
UV (H2O): lmax = 267 nm.
1H NMR (300 MHz, D2O): d = 1.63 (m, 1H, C3¢-CH2a), 1.87 (s, 3H,
C5-CH3), 2.07-2.53 (m, 4H, H-2¢, H-3¢, C3¢-CH2b), 3.02 (t, 2H,
J = 5.7, CH2NH2), 3.94 (d, 1H, J = 9.0, H-5¢a), 4.22 (m, 2H, H-4¢,
H-5¢b), 6.09 (d, 1H, J = 6.8, H-1¢), 7.79 (s, 1H, H-6).
13C NMR (75 MHz, D2O): d = 12.06 (C5-CH3), 28.71 (CH2), 34.05
(C3¢), 37.73 (C2¢), 38.26 (CH2), 64.40 (C5¢), 85.09 (C4¢), 85.35
(C1¢), 111.37 (C5), 138.04 (C6), 152.08 (C2), 167.21 (C4).
31P NMR (121 MHz, D2O): d = -9.67 (d, a-P), -20.32 (t, b-P),
-4.63 (d, g-P).
MALDI-TOF-MS: m/z calc for (M-H)+ 508.03. Found: 508.39.
31P NMR (121 MHZ, D2O): d = -10.07 (d, a-P), -20.84 (t, b-P),
-4.90 (d, g-P).
MALDI-TOF-MS: m/z calc for (M+H)+ 635.19. Found: 635.02.
3¢-C-[N-[N-(6-Aminohexyl)]-6-carbamoylhex-2(Z)-enyl]-3¢-des-
oxythymidine 5¢-triphosphate (12)
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
(JA242) Modified 3¢-C-Branched Deoxythymidine 5¢-Triphos-
phates 15-17; General Procedure
Obtained from nucleoside 7 as a colorless Na+ salt (60 mg, 60%).
UV (H2O): lmax = 267 nm.
1H NMR (300 MHz, D2O): d = 1.29 (m, 4H, 2 x CH2), 1.44 (q, 2H,
J = 6.3, CH2), 1.61 (m, 4H, 2 x CH2), 1.87 (s, 3H, C5-CH3), 1.94-
2.43 (m, 9H, H-2¢, H-3¢, 3 x CH2), 2.91 (t, 2H, J = 6.7, NCH2), 3.10
(t, 2H, J = 7.5, NCH2), 3.95 (m, 1H, H-4¢), 4.09 (m, 1H, H-5¢a),
4.25 (m, 1H, H-5¢b), 5.45 (m, 2H, olefin), 6.08 (dd, 1H, J1¢,2¢a = 3.7,
To a stirred solution of JA242 (10 mg, 22.4 µmol), DIPEA (23 µL,
134µmol), and TSTU (6 mg, 22.4 µL) in dioxane (224 µL) and
DMF (224 µL) one of the nucleotides 11, 12 or 14 (22.4 µmol) in
H2O (112 µL) was added. The mixture was stirred in the dark at r.t.
for 24 h. After evaporation, the mixture was applied to a column
(XK 16, Pharmacia) of RP-18 and eluted with 0.1 M TEAAc with
MeCN as gradient (0-30%). The first dye-fraction was collected,
and then the products were precipitated as Na+ salts to remove the
free dye. Next, the compound was purified by anion-exchange
HPLC (Mono Q) using 20 mM NaOAc and 20% CH3CN with NaCl
(1 M) as gradient. To remove the buffer salt an additional RP-18
column was applied.
J1¢,2¢b = 6.4, H-1¢), 7.70 (s, 1H, H-6).
13C NMR (75 MHz, D2O): d = 12.22 (C5-CH3), 25.54, 25.62, 25.90,
26.30, 27.05, 28.48, 29.06, 35.55 (8 x CH2), 37.51 (C2¢), 37.96
(C3¢), 39.47, 39.71 (2 x CH2), 66.35 (C5¢), 85.56 (C4¢), 85.70 (C1¢),
111.56 (C5), 127.75 (olefin), 131.61 (olefin), 137.81 (C6), 152.98
(C2), 168.32 (C4), 176.97 (C = O).
31P NMR (121 MHz, D2O): d = -10.10 (d, a-P), -21.08 (t, b-P),
-5.13 (d, g-P).
MALDI-TOF-MS: m/z calc for (M+H)+ 691.19. Found: 691.09.
Acknowledgement
We thank Ms. B. Meinelt for excellent technical assistance, Ms. R.
Wartbichler (Roche Diagnostics GmbH) for the DNA sequencing,
Dr. M. Sauer (Universität Heidelberg) for the determination of the
quantum yields, and Dr. M. Bartoszek (Institut für Angewandte
Chemie, Berlin-Adlershof) for performing the mass spectra. Fluo-
rescent dye was kindly supplied by Prof. Dr. K. H. Drexhage, Uni-
versity Siegen, Germany. This research was supported by the
Bundesministerium für Bildung, Wissenschaft, Forschung, und
Technologie (BEO 0311088).
3¢-C-(2-Azidoethyl)-3¢-desoxythymidine 5¢-triphosphate (13)
Obtained from nucleoside 10 as a colorless Na+ salt (40 mg, 32%).
UV (H2O): lmax = 267 nm.
IR (film): n = 2050 cm-1 (N3).
1H NMR (300 MHz D2O): d = 0.94 (m, 1H, C3¢- CH2a), 1.31 (m,
1H, C3¢- CH2b), 1.61 (s, 3H, C5-CH3), 1.90-2.07 (m, 3H, H-2¢, H-
3¢), 3.10 (t, 2H, J = 6.8, CH2N3), 3.71 (m, 1H, H-4¢), 3.84 (m, 1H,
Synthesis 2000, No. 5, 707–713 ISSN 0039-7881 © Thieme Stuttgart · New York

PAPER
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Article Identifier:
1437-210X,E;2000,0,05,0707,0713,ftx,en;H07699SS.pdf
Wojczewski, C.; Faulstich, K.; Engels, J. W. Nucleosides.
Synthesis 2000, No. 5, 707–713 ISSN 0039-7881 © Thieme Stuttgart · New York