Synthesis of γ-labeled nucleoside 5′-triphosphates using click chemistry

Article abstract of DOI:10.1039/c3cc48937j

Real-time enzymatic studies are gaining importance as their chemical and technical instrumentation improves. Here we report the efficient synthesis of γ-alkyne modified triphosphate amidates that are converted into a variety of γ-fluorophore labeled triph

Full text of DOI:10.1039/c3cc48937j

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Synthesis of c-labeled nucleoside 50-triphosphates
using click chemistry†
Cite this:DOI: 10.1039/c3cc48937j
S. Serdjukow, F. Kink, B. Steigenberger, M. Tomas-Gamasa and T. Carell*
´
Received 22nd November 2013,
Accepted 19th December 2013
DOI: 10.1039/c3cc48937j
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Real-time enzymatic studies are gaining importance as their a straightforward synthesis of g-labeled nucleotides. Herein we
chemical and technical instrumentation improves. Here we report report a practical method for the preparation of nucleoside
the efficient synthesis of c-alkyne modified triphosphate amidates triphosphate analogs carrying an alkyne group linked to the
that are converted into a variety of c-fluorophore labeled triphos- g-phosphate. The attachment of a fluorophore group can effi-
phates by Cu(I) catalyzed alkyne/azide click reactions. The syn- ciently be performed via the Cu(I) catalyzed click reaction
thesized triphosphates are incorporated into DNA by DNA between this alkyne triphosphate and a fluorophore azide in
polymerases.
aqueous mixtures. The generality of our two-step method is
demonstrated by applying it to all eight (deoxy)nucleotides using
With the advancement of technical instrumentation, real-time four fluorophore azides from different dye classes (rhodamine,
and single molecule enzymatic studies are becoming feasible fluorescein, BODIPY and coumarin). Finally, we show that these
tools.1 At the centre of these technologies are chemical probes fluorescently labeled triphosphates are accepted as substrates by
that are for example modified substrates or cofactors needed DNA polymerases in primer extension experiments.
for the enzyme of interest. To enable direct monitoring of the
Starting point of the synthesis were the commercially avail-
reaction, these molecules are usually equipped with fluoro- able deoxyribonucleoside triphosphates (dATP, dCTP, dGTP
phores at positions of the substrates that barely interfere with and dTTP) and ribonucleoside triphosphates (ATP, CTP, GTP
enzyme binding or activity.2 Recently, an ATP probe containing and UTP) in their sodium salt form. In contrast to existing
a FRET donor–acceptor pair was reported which was used to methods for the g-modification of triphosphates, it was not
observe ubiquitin activation in real-time.3 Finally, single mole- necessary to prepare the tetrabutylammonium salt of the nucleo-
cule real-time (SMRT) DNA sequencing has become a powerful tides or to use dry solvents.5,7 Instead, the triphosphates were
application of these technologies. SMRT sequencing in princi- directly converted into the alkyne-carrying g-phosphoramidates
ple allows to obtain sequence information of non-canonical 1–4 and 5–8 using commercially available 1-aminobut-3-yne and
bases such as 5-methyl-dC and its oxidized derivatives in 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide hydrochloride
addition to the canonical bases (dA, dC, dG and dT).4,5 This (EDCꢀHCl) as the coupling reagent (Scheme 1). When performing
method and other DNA based processes require deoxynucleo- the reaction in a water/DMF (1 : 1) mixture at pH = 7.5, the
side triphosphates with different fluorophores linked to the
respective g-phosphate, which are released when the nucleoside
triphosphate is consumed, e.g. by attachment to the growing
primer strand.5 The synthesis of these labeled compounds
involves a multistep protocol which furnishes low overall yields
and requires anhydrous conditions, which interfere with the
solubility of the triphosphates.6 Consequently, there is a need for
¨
¨
Department of Chemistry, Ludwig-Maximilians Universitat Munchen,
Butenandtstraße 5-13, 81377 Munich, Germany.
E-mail: thomas.carell@cup.uni-muenchen.de
† Electronic supplementary information (ESI) available: Experimental procedures
for 1–8, a–b, d and click reactions, characterization data for compounds, HPLC
profiles, NMR spectra and primer extension conditions. See DOI: 10.1039/
c3cc48937j
Scheme 1 Synthesis of alkyne nucleotides labeled at the g-phosphate.
HPLC-purification yielded the labeled triphosphates 1–8 as their tris-
triethylammonium salts.
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Table 1 Click reactions overview
Yielda
g-alkyne labeled triphosphates were obtained as their tris-
triethylammonium salts (Scheme 1) with isolated yields from
70% to 86%. RP-HPLC analysis of aliquots from the crude
reaction mixture proved that the process itself proceeded with
conversion yields of Z90% in most cases (S1–S3, ESI†). Carry-
ing out the reaction at lower or higher pH values or using other
coupling reagents like carbonyldiimidazole afforded lower
yields. This is in accordance with existing similar protocols,
to which our present procedure showed an almost 2–4-fold
improvement in yield.7,8 Moreover, we illustrate the generality
of the method, as it was successfully applied to all major
natural nucleotides without significant decreases in yield. It
is also noteworthy that no protecting reagents are needed.
The coupling reaction takes place with complete selectivity at
the g-phosphate unit as has been proved by 31P–1H HMBC NMR
spectroscopy (S6, ESI†).
Chemical
formula [M–H]ꢁ
MS calc. MS found
Entry g-NTP Azide (%)
m/z
m/z
ꢁ
ꢁ
ꢁ
ꢁ
1
2
3
4
5
6
7
8
dATP
dCTP d
dGTP d
dTTP
dTTP
dTTP
dTTP
ATP
ATP
CTP
GTP
UTP
d
Z90
Z90
Z90
Z90
C43H48N10O20P3
C42H48N8O21P3
C43H48N10O21P3
1117.2265 1117.2221
1093.2152 1093.2110
1133.2214 1133.2168
1108.2149 1108.2113
735.0624 735.0620
d
a
b
c
a
d
d
d
d
C43H49N7O22P3ꢁ
Z90, 70b C23H26N6O16P3
ꢁ
Z90
Z90
C44H60BF2N9O18P3 1144.3336 1144.3344
C42H49N9O17P3ꢁ
ꢁ
1044.2465 1044.2438
760.0688 760.0687
1133.2214 1133.2224
1109.2101 1109.2122
1149.2122 1149.2163
1110.1942 1110.1913
Z90, 77b C23H25N9O15P3
ꢁ
ꢁ
ꢁ
ꢁ
9
Z90
Z90
Z90
Z90
C43H48N10O21P3
C42H48N8O22P3
C43H48N10O22P3
10
11
12
C42H47N7O23P3
a
b
Conversion yield determined by analytical RP-HPLC. Isolated yield
after RP-HPLC-purification.
To exemplify its generality we reacted the alkyne containing
nucleotide 4 with the four different dye azides a–d (Scheme 2).
Reaction progress was monitored using analytical RP-HPLC
proving conversion yields of Z90% for the click products
4a–4d (Table 1, entries 4–7) in the presence of only 1.5 equiva-
lents of fluorophore azide. Moreover, no Cu(I)-stabilizing ligand
like TBTA or THPTA was necessary to achieve good yields in a
reaction time of 1–4 h.12 Due to the high cost of fluorophore
dyes, especially the ones that are routinely used in SMRT
sequencing,6a we simply synthesized three easy accessible
azides (a, b, and d).
The general applicability of the protocol is demonstrated
by the treatment of all nucleotide phosphoramidates (1–8)
with fluorophore azide d in a 400 nmol setup. Again, analytical
RP-HPLC indicated conversion yields of Z90% for the click
products 1d–8d (S4, ESI† and Table 1, entries 1–4 and 9–12).
All click products were characterized by HPLC high resolution
ESI-MS to analyze the purity and prove the formation of the
correct product.
We next attempted the coupling of the fluorophore group
by using the Cu(I) catalyzed reaction between alkynes and
azides.9,10 Thus, we first optimized the cycloaddition protocol
of the already prepared alkyne nucleotide 4 towards fluorophore-
containing azides. In order to find the initial click conditions we
took advantage of the specific properties of coumarin azide a
(Scheme 2), which starts to fluoresce upon formation of the
triazole and moreover is easily synthesized.11 We observed that
the best yields were obtained when we used a freshly prepared
Cu(I) catalyst by in situ reduction of CuSO4 with sodium ascor-
bate in a water/THF mixture from 0–25 1C. Under these condi-
tions, the triazole products 4a and 5a were synthesized in 70%
and 77% isolated yield, respectively (Table 1, entries 5 and 8).
Full characterization includes 1H, 13C and 31P NMR spectroscopy
(S7, ESI†). Through the adjustment of the THF content according
to the dye azide solubility, we could successfully extend this
method to other fluorophores.
Having established the procedures for both reaction steps,
we investigated whether the synthesis could be carried out in a
one-pot fashion directly from the commercially available dTTP
(Scheme 3). Without isolating the alkyne intermediate by means
of time-consuming HPLC-purification, the click procedure was
successfully performed. For the reaction of dTTP and the cou-
marin azide a, this one-pot process provided the labeled nucleo-
tide 4a in 60% overall yield.
In order to investigate the acceptance of our dye-labeled
nucleotides by DNA polymerases, dTTP labeled with four
Scheme 2 Synthesis of fluorophore g-labeled nucleoside 50-triphosphates
using Cu(I) catalyzed Huisgen-cycloadditions. HPLC-purification afforded
the g-fluorophore triphosphates as their tris-triethylammonium salts.
Scheme 3 One-pot synthesis of g-coumarin dTTP 4a from dTTP.
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In summary we present a short and high-yielding proce-
dure for the preparation of g-fluorophore labeled nucleoside
50-triphosphates. The data show that the triazole containing
building blocks synthesized by the click reaction are well
accepted by polymerases. Potential application could be an
improved synthesis of substrates for SMRT sequencing.
Furthermore, real-time studies as well as labeling experiments
involving triphosphate consuming enzymes could benefit from
our results.
We thank the Alexander von Humboldt Foundation for a
postdoctoral fellowship to M. T.-G. Funding for this research
was obtained from the Volkswagen Foundation and the DFG
(SFB749, TP4 and SFB1032, TPA5).
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Fig. 1 (A) Sequence of the DNA template for the primer extension
experiments using the Klenow fragment (exoꢁ), 0.5 mM template and
200 mM nucleotides. The nucleotides are partly replaced by (B) different
dye-labeled dTTPs (4a–4d) instead of dTTP (lanes 3–6) or by (C)
fluorescein-labeled deoxynucleotides (1d–4d) for the natural dNTPs (lanes
3–6). In both gels a primer (lane 1) and a control using all natural
deoxynucleotides (lane 2) are given for comparison.
different fluorophore azides 4a–4d was incorporated by primer
extensions. To this end, the natural dTTP was exchanged by
HPLC-purified labeled dTTP derivatives 4a–4d (lanes 3–6 in
Fig. 1B). In an analogous fashion, natural dNTPs were replaced
by fluorescein-labeled dNTPs 1d–4d (lanes 3–6 in Fig. 1C). As
shown in Fig. 1, all seven dye-labeled triphosphate analogs were
accepted as substrates under standard primer extension condi-
tions to give the fully extended DNA strand. Only fluorescein-
labeled dTTP 4d seemed to be a somewhat more difficult substrate
for the DNA polymerase Klenow fragment (exoꢁ) (lane 5, Fig. 1C),
since a fully elongated primer is not exclusively generated. From
the data we conclude that the triazole linked dye-labeled nucleo-
side triphosphate amidates presented here could be useful sub-
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consuming enzymes in real-time. To minimize undesired sub-
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12 For the exact reaction conditions see ESI,† pp. 19–20.
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