Deoxynucleoside triphosphates bearing histamine, carboxylic acid, and hydroxyl residues-synthesis and biochemical characterization

Article abstract of DOI:10.1039/c3ob40842f

Modified nucleoside triphosphates (dA^HsTP, dU^POHTP, and dC^ValTP) bearing imidazole, hydroxyl, and carboxylic acid residues connected to the purine and pyrimidine bases through alkyne linkers were prepared. These modified d

Full text of DOI:10.1039/c3ob40842f

Organic &
Biomolecular Chemistry
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PAPER
Deoxynucleoside triphosphates bearing histamine,
carboxylic acid, and hydroxyl residues – synthesis and
biochemical characterization†
Cite this: Org. Biomol. Chem., 2013, 11,
5162
Marcel Hollenstein*
Modified nucleoside triphosphates (dAHsTP, dUPOHTP, and dCValTP) bearing imidazole, hydroxyl, and carb-
oxylic acid residues connected to the purine and pyrimidine bases through alkyne linkers were prepared.
These modified dN*TPs were excellent substrates for various DNA polymerases in primer extension reac-
tions. Moreover, the combined use of terminal deoxynucleotidyl transferase (TdT) and the modified
dNTPs led to efficient tailing reactions that rival those of natural counterparts. Finally, the triphosphates
were tolerated by polymerases under PCR conditions, and the ensuing modified oligonucleotides served
as templates for the regeneration of unmodified DNA. Thus, these modified dN*TPs are fully compatible
with in vitro selection methods and can be used to develop artificial peptidases based on DNA.
Received 24th April 2013,
Accepted 5th June 2013
DOI: 10.1039/c3ob40842f
www.rsc.org/obc
transition metal-based proteases achieve cleavage with mul-
Introduction
tiple turnover12 and with some site selectivity,8,13 they suffer
The selective scission of the amide bonds of proteins and pep- from severe drawbacks such as side-reactions, low kcat values,
tides is of crucial importance for numerous biochemical and and in general perform rather poorly when compared to
biotechnological applications1 and plays a decisive role in protein enzymes.
physiological processes.2,3 Considering the inertness of the
DNA enzymes or DNAzymes have emerged as a new and
peptide bond (t1/2 nonenz ∼ 500 years),4,5 proficient proteolytic prominent class of biomolecular catalysts, and have been
enzymes with high catalytic efficiencies (kcat/KM) need to be selected to accelerate an increasing number of chemical
used in order to degrade proteins or peptides into smaller frag- processes.14–18 Indeed, application of SELEX and related com-
ments or for promoting site-specific cleavage. However, in binatorial methods of in vitro selection19,20 resulted in the
terms of practical applications, proteolytic enzymes are not identification of nucleic acid enzymes catalyzing an abun-
always applicable since they often lead to the generation of dance of reactions ranging from the formation of C–C,21–24
rather short fragmentation products, lack sequence-specificity, C–N,25 C–S,26 and P–O,27,28 bonds to the scission of ribo-
and cleave at multiple sites. Thus, artificial proteases based on phosphodiester linkages.29,30 Surprisingly, the scission of amide
synthetic reagents that could favor the selective cleavage of linkages is a reaction that has eluded catalytic nucleic acids so
proteins under mild conditions are highly sought after. In this far.31 Indeed, in an early effort towards this goal, Joyce et al.
context, transition metal complexes were suggested as prime initially reported on the selection of a ribozyme capable of
candidates to serve as biomimetic chemical proteases since cleaving a substrate containing an embedded 3′-NH–C(O)–
they are often more effective than organic residues at promot- CH2-5′ linkage32 but later realized that the reaction proceeded
ing the scission of inert bonds.1,6,7 Moreover, metal centers through a different mechanism leading to scission of a phos-
are often combined with reagents such as β-cyclodextrins and phodiester bond adjacent to the target site rather than the
porphyrins that facilitate the recognition and the binding to intended amide bond.33 Similarly, Silverman et al. set out to
the target site and thus reach appreciable catalytic efficiencies select a DNAzyme capable of the selective hydrolysis of amide
for the scission of amide linkages in peptides and even bonds of a tripeptide sequence,34 which culminated in the ser-
proteins.8–11 However, even though some of these artificial, endipitous discovery of a class of very potent DNA-cleaving
DNAzymes.35,36
Recently, blending of the chemical functionalities found in
Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3,
the active sites of enzymes together with tools of in vitro
CH-3012 Bern, Switzerland. E-mail: hollenstein@dcb.unibe.ch;
selection resulted in the identification of potent DNA-
Fax: (+41) 31 631 3422; Tel: (+41) 31 631 4372
zymes.37,38 In particular, this approach allowed for the devel-
†Electronic supplementary information (ESI)
available. See DOI:
opment of highly functionalized DNA-based RNase A mimics
10.1039/c3ob40842f
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Fig. 1 Chemical structures of dAHsTP 1, dUPOHTP 2, and dCValTP 3.
Scheme 1 Synthesis of modified dAHsTP 1. Reagents and conditions: (i)
DMTrCl, pyridine, rt, 15 h, 75%; (ii) NaOH, MeOH, H2O, rt, 12 h, 84%; (iii) S2,
EDC, HOBt, NMM, DMF, rt, 12 h, 64%; (iv) Ac2O, DMAP, NEt3, pyridine, 0 °C,
2.5 h, 86%; (v) DCAA, CH2Cl2, rt, 40 min, quant.; (iv) 1. 2-chloro-1,3,2-benzodioxa-
phosphorin-4-one, pyridine, dioxane, rt, 45 min; 2. (nBu3NH)2H2P2O7, DMF,
nBu3N, rt, 45 min; 3. I2, pyridine, H2O, rt, 30 min; 4. NH3(aq.), rt, 1.5 h, 17% (4
steps).
that operate either in the presence of cofactors39–41 or in an
M2+-independent regime.42–47 Moreover, all the functional
groups encompassed in these modified DNAzymes seem to act
in synergy and in a cooperative manner, one of the funda-
ments in the design of de novo enzyme mimics and catalysts in
general.48 Finally, this methodology is applicable to other reac-
tions than the cleavage of ribophosphodiester bonds25,49 pro-
vided that the functional groups can easily be introduced into
DNA, i.e. that the modified nucleoside triphosphates (dN*TPs)
are compatible with methods of in vitro selection.
In this context, the enzymatic polymerization of dN*TPs
has advanced as a versatile platform for the inclusion of func-
tional groups into nucleic acids.50 Indeed, dN*TPs have been
used to introduce a myriad of functionalities including amino
acids,51–57 boronic acids,58,59 ligands for transition metals,60–65
thiols,66–68 diamondoid-like residues,69 bile acids,70 side chains
capable of organocatalysis,71 and even oligonucleotides.72
Herein, I report on the synthesis and biochemical charac-
terization of modified dN*TPs bearing the side chains found
in the active site of serine proteases.31,73,74 Indeed, dAHsTP
(7-propargylamido-histamine-dA) 1, dUPOHTP (5-pentynol-dU) 2,
and dCValTP (5-valeric acid-dC) 3 (Fig. 1) are equipped with
imidazole, hydroxyl, and carboxylic acid residues, respectively,
that are reminiscent of the side chains of the amino acids His,
Ser, and Asp that form the catalytic triad of serine proteases.
These modified dN*TPs are good substrates for a variety of
DNA polymerases in the context of primer extension reactions.
In addition, when used in conjunction with the terminal deoxy-
nucleotidyl transferase (TdT), efficient tailing reactions that
rival those of the natural counterparts could be observed.
Finally, these modified dN*TPs were readily incorporated into
DNA by the polymerase chain reaction (PCR), further under-
scoring their compatibility with in vitro selection techniques.
modifications can easily be introduced via palladium-
catalyzed coupling reactions) and because of their minimal
disturbance and impact on substrate acceptance and duplex
stability.53,71,75–77
The adenosine analogue 1 adorned with a histamine
residue was obtained in a 6-step sequence that started with the
DMTr-protection of the known propargylamino modified
7-deaza-2′-deoxyadenosine 4 (Scheme 1).53,75,78 After removal
of the TFA masking group, nucleoside 6 was converted into
derivative 7 in 64% yield under standard 1-ethyl-3-(3-dimethyl-
aminopropyl)carbodiimide (EDC)-mediated amide bond-for-
mation conditions using the suitably protected carboxylic acid
S2 (ESI†). The resulting intermediate 7 was then 3′-O-acetylated
under standard conditions and in good yield (84%). Following
a global acid-mediated deprotection step (quantitative yield),
nucleoside analogue 9 could be converted into the corres-
ponding triphosphate dAHsTP 1 in acceptable yields (17%) by
application of the one-pot four-step triphosphorylation
method developed by Ludwig and Eckstein.79
The synthetic path leading to dUPOHTP 2 commenced with
the Sonogashira cross-coupling reaction of the DMTr-protected
deoxyuridine analogue 11 (which was easily obtained by trityl-
ation of the commercially available 5-iodo-2′-deoxyuridine 10,
ESI†) and free 4-pentyn-1-ol as outlined in Scheme 2. After an
uneventful acetylation–detritylation sequence, precursor 14
could be converted to the corresponding triphosphate 2 by
application of the Ludwig and Eckstein conditions and was
obtained in 14% yield after a thorough RP-HPLC purification
step.79
The synthesis of the modified dC*TP analogue 3 proceeded
in an analogous manner as highlighted in Scheme 3. Indeed,
commercially available 5-iodo-2′-deoxycytidine 15 was con-
verted into the ester functionalized nucleoside 16 in good
yield (90%) using a Sonogashira cross-coupling reaction with
4-pentynoic acid methyl ester.80 The free 5′-hydroxyl residue of
Results and discussion
Synthesis of the modified nucleoside triphosphates
The nucleoside analogues considered herein all bear modifi-
cations either at the C5 position of the pyrimidine nucleo-
base or at the N7 of 7-deaza-2′-deoxyadenosine. Indeed, these
anchoring sites were chosen for synthetic purposes (the
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Scheme 2 Synthesis of modified dUPOHTP 2. Reagents and conditions: (i)
DMTrCl, pyridine, rt, 12 h, 93%; (ii) 4-pentyn-1-ol, Pd(PPh3)4, CuI, NEt3, DMF, rt,
12 h, 91%; (iii) Ac2O, DMAP, NEt3, pyridine, rt, 12 h, 86%; (iv) DCAA, CH2Cl2, rt,
40 min, 87%; (v) 1. 2-chloro-1,3,2-benzodioxaphosphorin-4-one, pyridine,
dioxane, rt, 45 min; 2. (nBu3NH)2H2P2O7, DMF, nBu3N, rt, 45 min; 3. I2, pyridine,
H2O, rt, 30 min; 4. NH3(aq.), rt, 1.5 h, 14% (4 steps).
Fig. 2 Gel image (PAGE 15%) of primer extension reactions with primer P1
and template T1 using Vent (exo−) DNA polymerase. Lane 1: dAHsTP 1; lane 2:
dUPOHTP 2; lane 3: dCValTP 3; lane 4: dAHsTP 1 and dCValTP 3; lane 5: dAHsTP 1
and dUPOHTP 2; lane 6: dUPOHTP 2 and dCValTP 3; lane 7: dAHsTP 1, dUPOHTP 2,
and dCValTP 3; lane 8: natural dNTPs; lane 9: natural dNTPs without dATP; lane
10: natural dNTPs without dTTP; lane 11: natural dNTPs without dCTP; lane 12:
primer P1.
acceptance of the modified dN*TPs 1–3 with different DNA
Scheme 3 Synthesis of triphosphate dCValTP 3. Reagents and conditions: polymerases under primer extension reaction conditions
(i) 4-pentynoic acid methyl ester, Pd(PPh3)4, CuI, NEt3, DMF, rt, 12 h, 90%;
(Fig. 2 and ESI†). Therefore, a variety of DNA polymerases
(ii) DMTrCl, DMAP, NEt3, pyridine, rt, 18 h, 45%; (iii) Ac2O, DMAP, NEt3, pyridine,
(Vent (exo−), Pwo, 9°Nm, and the Klenow fragment of E. coli
0 °C, 1 h, 61%; (iv) DCAA, CH2Cl2, rt, 40 min, 92%; (v) 1. 2-chloro-1,3,2-benzodi-
DNA polymerase I) were used in conjunction with the 98-mer
oxaphosphorin-4-one, pyridine, dioxane, rt, 45 min; 2. (nBu3NH)2H2P2O7, DMF,
long template T1 and the 25-mer primer P1 (see ESI† for the
sequence compositions).70,71 The modified dN*TPs were then
investigated as to whether they could act as surrogates for their
natural counterparts, either as lone modifications (lanes 1–3,
Fig. 2), as combinations of two modified and two natural
dNTPs (lanes 4–6, Fig. 2), or in conjunction with the lone
natural dGTP (lane 7, Fig. 2). Vent (exo−) was revealed to be
highly proficient at extending primer P1 to full length since no
faster running bands were apparent in all the combinations
nBu3N, rt, 45 min; 3. I2, pyridine, H2O, rt, 30 min; 4. NaOH, H2O, 0 °C, 5 min,
9% (4 steps).
16 was then protected with a 4,4′-dimethoxytrityl group in
moderate yields. The ensuing protected nucleoside 17 was
then acetylated yielding a mixture of the desired 3′-O-mono-
acetylated 18 (61%) and diacetylated product S4 (ESI†). Unfortu-
67
nately, all attempts at N-4-deacetylating S4 using ZnBr2 only
that were investigated. Moreover, 16 dAHs 1 and 21 dUPOH
2
led to detritylation (data not shown). Finally, following
removal of the DMTr masking group under acidic conditions,
intermediate 19 could be converted to the corresponding tri-
phosphate 3 in 9% yield (4 steps), again by application of the
Ludwig–Eckstein protocol.79
that were incorporated in the nascent chain led to a significant
and expected gel retardation as compared to the natural
control sequence (compare lanes 1 and 2 with lane 8, Fig. 2).
Surprisingly, the inclusion of 18 dCVal 3 had little impact on
the gel mobility of the resulting modified DNA (lane 3 vs. lane
8, Fig. 2).
Primer extension reactions
An important prerequisite for modified dN*TPs to be appli-
cable in selection experiments is that they serve as substrates
for DNA polymerases in primer extension reactions.71 Further-
Furthermore, besides Vent (exo−), all the other DNA poly-
merases that were tested in primer extension reactions showed
very high substrate tolerance for all the dN*TPs and led to fully
more, not only do primer extension reactions constitute the extended products (Fig. S1–S3, ESI†). Interestingly, the Klenow
fragment, a prominent member of the family A polymerases,
first step of an in vitro selection experiment, but they represent
was as efficient at incorporating the modified dN*TPs as the
the main tool for gauging the enzymatic recognition of modi-
fied dN*TPs in general. Consequently, I explored the substrate more tolerant family B polymerases (Fig. S2, ESI†).53,81,82
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TdT-mediated polymerization
The terminal deoxynucleotidyl transferase (TdT) is a Co2+-
polydisperse longer-sized oligonucleotides. Furthermore, at a
concentration of 50 μM several tens of both the dAHs and dA
nucleotides were appended. Moreover, dUPOHTP 2 (lanes 2 in
Fig. 3) led to the best tailing efficiencies amongst the pyrimi-
dine triphosphates, a tendency that is opposite to that
observed with natural dNTPs (vide infra). Indeed, the TdT-
mediated homopolymerization was rather ineffective at 10 μM
since only 2–5 additional dUPOH units were appended, but at
higher concentrations (>50 μM) only highly modified slow-
running products could be observed. Unlike its natural
counterpart, dCValTP 3 stifles the TdT and only a few
additional residues (∼3 to 15 dCValMPs) are added, even at
200 μM (lanes 3 in Fig. 3).
dependent family
X DNA polymerase that catalyzes the
random and template-independent polymerization of nucleo-
side triphosphates at the 3′-OH termini of single-stranded oligo-
nucleotides.83,84 Recently, TdT-mediated reactions using
modified dN*TPs have been employed for the generation of
3′-fluorescently labelled oligonucleotides,85,86 to confer exo-
nuclease stability to specific sequences,87 and to synthesize
ssDNAs containing stretches of nucleotides bearing redox
active tags.88 However, the tailing reaction was never used for
the incorporation of nucleotides bearing side chains that
potentially could confer catalytic activity to the ensuing modi-
fied ssDNA. Consequently, the modified dN*TPs 1–3 were
assayed for their capacity to act as substrates in TdT-mediated
tailing reactions. Thus, the 5′-32P-labelled 15 nt long primer P3
(ESI†) was incubated at 37 °C for 60 min in the presence of
TdT and varying concentrations of the modified dN*TPs
(Fig. 3).
Polymerase chain reactions (PCR)
An additional and important part of the biochemical charac-
terization of modified dN*TPs is the assessment of their sub-
strate acceptance by DNA polymerases under PCR conditions.
Consequently, the dN*TPs 1–3 were evaluated for their capacity
to amplify the 98-mer template T170,71 flanked by a 20 nt long
forward and a 25 nt reverse primer using four different DNA
polymerases (Fig. 4 and ESI†).
The 7-substituted 7-deaza-deoxyadenosine derivative dAHsTP
1 was revealed to be the best substrate for TdT, since
the tailing efficiencies are better than those of its natural
counterpart (compare lanes 1 and 4, Fig. 3). Indeed, at a con-
centration as low as 10 μM, mainly 7–15 additional dAHs
nucleotides were incorporated along with some longer tailed
sequences, while the natural dATP led to the appendage
of a rather narrow 4–17 nucleotide distribution with little
Surprisingly, unlike what had been observed for primer
extension reactions, under standard PCR conditions (2 mM
Mg2+, 200 μM dNTPs, 30 PCR cycles), only the Vent (exo−) DNA
polymerase was capable of faithfully amplifying the template,
albeit with a slightly reduced efficiency as compared to the
control experiment with natural dNTPs (lane 2 versus lane 8,
Fig. 4). Indeed, the 9°Nm polymerase led to a marked smearing
pattern (lane 3), the Pwo polymerase only partially accepted
the dN*TPs under these conditions and yielded a mixture of a
full length product and a shorter product (lane 4), and no
amplicon could be observed in the case of the Klenow frag-
ment (lane 5). However, when the dNTP concentration was
raised from 200 to 500 μM, an efficient amplification could be
observed with Vent (exo−) (lane 6). Furthermore, additional
Mg2+ (lane 7) had no positive impact on the amplification
efficiency. Good amplifications were observed as well when
only one of the natural dNTPs was substituted with a modified
one (data not shown).
Fig. 4 Agarose gel (2%) stained with ethidium bromide, showing the PCR pro-
ducts with the 98-mer template T1, a dNTP mixture composed of dAHsTP 1,
dUPOHTP 2, dCValTP 3, and natural dGTP and different enzymes and conditions.
Lane 1: ladder; lane 2: Vent (exo−); lane 3: 9°Nm polymerase; lane 4: Pwo poly-
merase; lane 5: Klenow fragment of E. coli DNA polymerase I; lane 6: Vent
(exo−) with 500 μM dN*TPs; lane 7: Vent (exo−) with 500 μM dN*TPs and 7 mM
Mg2+; lane 8: natural dNTPs and Vent (exo−).
Fig. 3 Gel image (PAGE 15%) of the TdT-catalyzed extension reactions. Lane 1:
dAHsTP 1; lane 2: dUPOHTP 2; lane 3: dCValTP 3; lane 4: dATP; lane 5: dTTP; lane
6: dCTP; lane 7: primer P3.
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In terms of in vitro selections, it is of primordial impor- more markedly, dATP.90 The additional negative charge on
tance that single-stranded modified DNAs can be transformed dCValTP might account for its limited substrate acceptance by
into wild-type oligonucleotides that can serve as templates for TdT.
subsequent rounds of selection.30,44 Thus, in a different PCR
Finally, these modified dN*TPs were shown to be good sub-
experiment a 5′-phosphorylated 79-mer long template T2 strates for the Vent (exo−) DNA polymerase under PCR con-
(ESI†) was amplified using the 19- and 20-mer primers P4 and ditions. However, unlike what was observed for primer
P5 in the presence of Vent (exo−) and 500 μM dNTPs. The extension reactions, only optimized conditions and the sole
resulting modified dsDNA was then λ-exonuclease digested, Vent (exo−) DNA polymerase led to clean and robust amplifica-
and used as a template for a subsequent PCR using natural tions. The resulting modified oligonucleotides could serve as a
dNTPs, the same set of primers, and Vent (exo−) (Fig. S5, template to regenerate natural DNA, which is an important
ESI†).71 This experiment clearly underscores the compatibility prerequisite for the use of these dN*TPs in selection experi-
of the modified dN*TPs 1–3 with methods of in vitro selection ments. Moreover, PCR amplification using the modified
since a modified oligonucleotide could serve as a template to dN*TPs and the subsequent conversion into natural DNA was
regenerate natural DNA (Fig. S5, ESI†).
shown to proceed with high fidelity.
Finally, in order to assess the fidelity of the PCR amplifica-
Thus, these three nucleoside triphosphate analogues,
tion with the modified dN*TPs 1–3, a sequencing experiment equipped with functionalities that are reminiscent of the resi-
based on the dideoxynucleoside triphosphate (ddNTP) method dues found in the active site of serine proteases, could be
developed by Sanger et al. was carried out (Fig. S6 and S7, shown to be fully compatible with methods of in vitro selec-
respectively, ESI†).52,89 Briefly, the 5′-phosphorylated template tion. Consequently, their simultaneous polymerization will
T2 and primer P4 were used in a primer extension reaction considerably increase the chemical landscape that can be
with Vent (exo−). The unmodified template was then λ-exo- covered in the course of a selection experiment for the gene-
nuclease digested, and the resulting modified ssDNA was used ration of DNAzymes that can promote the catalytic scission of
as a template for PCR with each one of the 5′-biotinylated amide bond linkages, which is a long-standing goal in the
primers P4B and P5B. Following immobilization of the field of nucleic acid enzymes. Such an artificial peptidase
duplexes on streptavidin coated magnetic beads, the unbioti- would be an invaluable synthetic and biochemical tool, and
nylated oligonucleotides were eluted with hydroxide washes selection for such an enzyme mimic is currently under way.
and then sequenced. Comparison of the sequencing patterns
of both the forward and reverse directions (Fig. S6 and S7,
ESI†) clearly reveals that no loss of sequence information
occurs upon converting a heavily modified ssDNA into its
General method for the Sonogashira coupling reactions
complementary natural sequence, and that the fidelity of PCR
Experimental part
The nucleoside was dissolved in dry DMF (5 mL mmol−1). CuI
with the modified dN*TPs is high.
(0.1 eq.) and NEt3 (2 eq.) were then added and the reaction
mixture was stirred for 10 min at room temperature. The
alkyne (4 eq.) and Pd(PPh3)4 (0.05 eq.) were then added in
turn. The reaction mixture was cooled down to −78 °C and
Conclusions
thoroughly degassed. After stirring at room temperature over-
night, the DMF was removed under reduced pressure. The
blackened crude product was then dissolved in CH2Cl2 (50 mL)
and washed with brine (50 mL). Following drying (MgSO4) and
removal of the solvent in vacuo, the residue was purified by
flash column chromatography.
Three modified dN*TPs adorned with side-chains reminiscent
of the Ser, His, and Asp residues found in the active site of
serine proteases could easily be accessed in 5 or 6 steps start-
ing from known and/or commercially available precursors. The
functionalities were anchored on the C5 of pyrimidines and
the N-7 of 7-deaza-adenosine in order to minimize the destabi-
lization of duplexes and disruption of Watson–Crick base
pairing, and concomitantly, to maximize the substrate accep-
General method for detritylation
tance. All of the modified dN*TPs were found to be excellent The nucleoside analogue was dissolved in dry CH2Cl2 (10 mL)
substrates for all four DNA polymerases, including the Klenow and dichloroacetic acid (0.1%, 0.1 mL) was added. The orange-
fragment of DNA polymerase I, in primer extension reactions. coloured reaction mixture was stirred at room temperature for
It is noteworthy that the polymerases readily accept and toler- 40 min. MeOH (3 mL) was added and the solution was stirred
ate the presence of the negative charge of the carboxylic acid for another 5 min at room temperature. Following removal of
of dCValTP 3 and allow for the incorporation of up to 3 con- the solvent under reduced pressure, the residue was purified
secutive dCVal residues.
by flash column chromatography.
Moreover, the substrate acceptance for TdT is heavily
Synthesis of 7-[3-(trifluoroacetamido)-propynyl]-5′-O-(4,4′-di-
dependent on the nature and position of the substituent and methoxytrityl)-7-deaza-2′-deoxyadenosine (5). The nucleoside
followed the order dAHsTP > dUPOHTP > dCValTP. This trend is analogue 4 (0.35 g, 0.9 mmol) was co-evaporated twice with dry
opposite to what is observed for natural dNTPs, since TdT uti- pyridine (5 mL). After dissolving 4 in dry pyridine (5 mL),
lizes dGTP and dCTP much more efficiently than dTTP and DMTrCl (0.36 g, 1.1 mmol) was added and the resulting
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solution was stirred at rt for 15 h. After completion of the reac- 31.9, 39.8, 40.9, 55.5, 64.0, 72.3, 83.4, 85.5, 86.8, 88.1, 95.9,
tion, 5 mL of MeOH were added and the mixture was stirred at 103.8, 113.5, 118.8, 125.2, 128.1, 128.2, 128.4, 129.8, 130.3,
rt for 5 min. After removal of the solvent under reduced 131.3, 134.6, 136.1, 138.8, 139.2, 142.9, 144.8, 149.9, 153.3,
pressure, the residue was purified by flash column chromato- 157.7, 158.7, 159.3, 172.1, 172.9. HR MS: m/z: calcd for
graphy (CH2Cl2–MeOH 95 : 5), yielding 5 as a pale yellow foam C64H63O8N8 ([M + H]+): 1071.4763, found: 1071.4750.
(0.46 g, 75%). Rf: 0.46 (CH2Cl2–MeOH 9 : 1 +1% NEt3). 1H-NMR
Synthesis of 7-[N-4-(methoxytrityl)-1H-imidazol-5-yl-ethyl-
(400 MHz, CDCl3): δ 2.41–2.52 (m, 2H), 3.37 (d, 2H, J = 4.4 Hz), amino-3-(carbamido)-propynyl]-5′-O-(4,4′-dimethoxytrityl)-3′-O-
3.76 (s, 6H), 4.05 (q, 1H, J = 4.0 Hz), 4.35–4.39 (m, 2H), 4.58 (q, acetyl-7-deaza-2′-deoxyadenosine (8). Compound 7 (0.39 g,
1H, J = 4.9 Hz), 5.64 (br s, 2H), 6.62 (t, 1H, J = 6.2 Hz), 6.81 (dd, 0.36 mmol) was dissolved in dry pyridine (3 mL) and the
4H, J = 2.0, 8.8 Hz), 7.20–7.32 (m, 7H), 7.39–7.46 (m, 3H), 8.25 resulting solution was cooled down to 0 °C. DMAP (0.011 g,
(s, 1H). 13C-NMR (101 MHz, CDCl3): δ 30.9, 41.2, 53.6, 63.7, 0.09 mmol), NEt3 (0.1 mL, 0.73 mmol), and acetic anhydride
72.1, 78.3, 83.6, 85.3, 85.7, 86.9, 94.9, 103.6, 113.6, 126.7, (0.09 mL, 0.91 mmol) were then added in turn. The resulting
127.2, 128.2, 130.3, 136.2, 136.3, 144.4, 149.8, 153.3, 157.5, reaction mixture was then stirred at 0 °C for 2.5 h, at which
158.7. 19F-NMR (376.5 MHz, CDCl3): δ −75.67. HR MS: m/z: stage MeOH (5 mL) was added and the mixture stirred for an
calcd for C37H35O6N5F3 ([M + H]+): 702.2534, found: 702.2524.
additional 5 min. The solvent was then removed under
Synthesis of 7-[3-amino-propynyl]-5′-O-(4,4′-dimethoxytrityl)- reduced pressure and the residue was purified by flash column
7-deaza-2′-deoxyadenosine (6). Nucleoside 5 (0.95 g, 1.4 mmol) chromatography (CH2Cl2–MeOH 96 : 4). The fully protected
was dissolved in MeOH (8 mL). A solution of NaOH (0.16 g, analogue 8 was obtained as a white foam (0.35 g, 86%). Rf:
4.1 mmol) in H2O (0.78 mL) was then added and the resulting 0.63 (CH2Cl2–MeOH 95 : 5 +1% NEt3). 1H-NMR (400 MHz,
mixture was stirred at rt for 12 h. The solvent was removed CDCl3): δ 2.06 (s, 3H), 2.45–2.53 (m, 5H), 2.56–2.62 (m, 1H),
under reduced pressure and the residue dissolved in 50 mL of 2.65 (t, 2H, J = 6.0 Hz), 3.35 (t, 2H, J = 3.2 Hz), 3.46 (q, 2H, J =
CH2Cl2. After washing with brine (1 × 50 mL) and extracting 6.0 Hz), 3.76 (s, 6H), 3.78 (s, 3H), 4.12 (d, 2H, J = 5.6 Hz), 4.16
the aqueous phase with CH2Cl2 (2 × 50 mL), the combined (q, 1H, J = 3.2 Hz), 5.37–5.40 (m, 1H), 5.87 (br s, 2H), 6.55 (d,
organic layers were dried (MgSO4) and the solvent removed 1H, J = 1.2 Hz), 6.62 (q, 1H, J = 4.7 Hz), 6.78–6.83 (m, 6H), 7.01
in vacuo. Purification by flash column chromatography (CH2Cl2– (d, 2H, J = 8.8 Hz), 7.07–7.11 (m, 4H), 7.17–7.22 (m, 2H),
MeOH 9 : 1) yielded 6 as a white foam (0.69 g, 84%). Rf: 0.33 7.23–7.26 (m, 3H), 7.27–7.31 (m, 10H), 7.37–7.40 (m, 2H), 8.18
1
(CH2Cl2–MeOH 9 : 1). H-NMR (400 MHz, CDCl3): δ 2.29–2.51 (s, 1H). 13C-NMR (101 MHz, CDCl3): δ 21.3, 27.6, 30.5, 31.9,
(m, 2H), 3.30–3.40 (m, 2H), 3.64 (s, 2H), 3.77 (s, 6H), 4.03 (q, 38.4, 39.8, 55.5, 64.0, 75.3, 83.4, 83.7, 96.3, 103.8, 113.5, 118.8,
1H, J = 3.1 Hz), 4.56 (q, 1H, J = 4.9 Hz), 5.57 (br s, 2H), 6.62 (t, 124.9, 127.14, 128.1, 128.2, 128.3, 128.5, 129.8, 130.3, 130.4,
1H, J = 6.4 Hz), 6.81 (d, 4H, J = 8.7 Hz), 7.17–7.32 (m, 8H), 131.3, 134.6, 135.9, 136.0, 138.9, 139.3, 142.9, 144.7, 150.2,
7.38–7.43 (m, 2H), 8.22 (s, 1H). 13C-NMR (101 MHz, CDCl3): 153.5, 157.7, 158.8, 159.3, 170.6, 172.1, 172.9. HR MS: m/z:
δ 32.6, 40.9, 55.5, 64.0, 72.6, 76.0, 83.5, 85.5, 86.8, 92.7, 96.2, calcd for C66H65O9N8 ([M + H]+): 1113.4869, found: 1113.4872.
103.7, 113.5, 125.6, 127.1, 128.1, 128.4, 130.2, 130.3, 136.0,
144.8, 149.9, 153.2, 157.5, 158.8. HR MS: m/z: calcd for propynyl]-3′-O-acetyl-7-deaza-2′-deoxyadenosine (9). The reac-
C35H36O5N5 ([M + H]+): 606.2711, found: 606.2704.
tion was carried out with 8 (0.34 g, 0.3 mmol) by application of
Synthesis of 7-[1H-imidazol-5-yl-ethylamino-3-(carbamido)-
Synthesis of 7-[N-4-(methoxytrityl)-1H-imidazol-5-yl-ethyl- the general detritylation method. The residue was purified by
amino-3-(carbamido)-propynyl]-5′-O-(4,4′-dimethoxytrityl)-7- flash column chromatography on silica gel by eluting with
deaza-2′-deoxyadenosine (7). Analogue 6 (0.35 g, 0.58 mmol) CH2Cl2–MeOH 9 : 1 to give 9 as a white foam (0.17 g, quant.).
was dissolved in dry DMF (10 mL). N-Methylmorpholine Rf: 0.16 (CH2Cl2–MeOH 9 : 1). 1H-NMR (400 MHz, DMSO-d6):
(0.13 mL, 1.2 mmol) and derivative S2 (0.34 g, 0.69 mmol) δ 2.09 (s, 3H), 2.30–2.38 (m, 5H), 2.45–2.50 (m, 2H), 2.60 (t,
were then added and the resulting solution was cooled down 2H, J = 7.4 Hz), 2.65–2.72 (m, 1H), 3.23 (q, 2H, J = 6.8 Hz), 3.60
to 0 °C. At this stage, HOBt (0.086 g, 0.64 mmol) and EDC·HCl (d, 2H, J = 1.6 Hz), 4.02 (q, 1H, J = 1.9 Hz), 4.09 (d, 2H, J =
(0.12 g, 0.64 mmol) were added in turn. After allowing the reac- 5.2 Hz), 5.31 (d, 1H, J = 6.0 Hz), 6.46 (dd, 1H, J = 5.6, 9.2 Hz),
tion mixture to warm up to room temperature, the solution 6.75 (br s, 1H), 7.50 (s, 1H), 7.69 (s, 1H), 7.88 (t, 1H, J =
was stirred at rt for 12 h. The solvent was removed under 3.6 Hz), 8.10 (s, 1H), 8.44 (t, 1H, J = 5.2 Hz). 13C-NMR
reduced pressure and the crude mixture was purified by flash (101 MHz, DMSO-d6): δ 20.9, 29.2, 30.5, 30.6, 36.9, 54.9, 61.7,
column chromatography by eluting with a gradual gradient of 74.8, 75.1, 83.3, 84.8, 89.3, 95.0, 102.3, 125.7, 134.5, 149.3,
CH2Cl2–MeOH from 95 : 5 to 9 : 1. The histamine modified 152.7, 157.5, 170.0, 171.0, 171.6. HR MS: m/z: calcd for
derivative 7 was obtained as a white foam (0.40 g, 64%). C25H31O6N8 ([M + H]+): 539.2361, found: 539.2375.
Rf: 0.55 (CH2Cl2–MeOH 9 : 1). 1H-NMR (400 MHz, CDCl3):
Synthesis of 5-[5-hydroxy-pentynyl]-5′-O-(4,4′-dimethoxy-
δ 2.39–2.49 (m, 7H), 2.64 (t, 2H, J = 6.4 Hz), 3.30–3.36 (m, 2H), trityl)-2′-deoxyuridine (12). The reaction was carried out with
3.45 (q, 2H, J = 6.2 Hz), 3.75 (s, 6H), 3.77 (s, 3H), 4.03 (q, 1H, 11 (0.6 g, 0.9 mmol) by application of the general method for
J = 3.7 Hz), 4.12 (d, 1H, J = 5.2 Hz), 5.55 (br s, 2H), 6.55 (d, 1H, Sonogashira coupling reactions. However, slightly larger quan-
J = 0.8 Hz), 6.59 (t, 1H, J = 6.4 Hz), 6.77–6.83 (m, 7H), 7.00–7.02 tities of CuI (0.4 eq.) and Pd(PPh3)4 (0.2 eq.) were used. The
(m, 3H), 7.07–7.13 (m, 2H), 7.18–7.32 (m, 12H), 7.35–7.40 (m, crude product was purified by flash column chromatography
3H), 8.18 (s, 1H). 13C-NMR (101 MHz, CDCl3): δ 27.6, 30.5, on silica gel (gradual gradient from CH2Cl2–MeOH 100 : 0 +1%
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NEt3 to 95 : 5) to give 12 as a white foam (0.51 g, 91%). Rf: 0.32 chromatography on silica gel (gradient from CH2Cl2–MeOH
(CH2Cl2–MeOH 95 : 5 +1% NEt3). 1H-NMR (400 MHz, CDCl3): 96 : 4 to 9 : 1). Rf: 0.20 (CH2Cl2–MeOH 9 : 1). 1H-NMR
δ 1.43–1.49 (m, 2H), 2.19 (t, 2H, J = 6.8 Hz), 2.23–2.28 (m, 1H), (400 MHz, DMSO-d6): δ 1.93–2.00 (m, 1H), 2.10–2.16 (m, 1H),
2.48 (ddd, 1H, J = 2.8, 5.6, 13.6 Hz), 3.28 (dd, 1H, J = 3.4, 2.60–2.67 (m, 4H), 3.52–3.61 (m, 2H), 3.62 (s, 3H), 3.78 (q, 1H.
10.6 Hz), 3.40 (dd, 1H, J = 3.0, 10.6 Hz), 3.47 (t, 2H, J = 6.0 Hz), J = 3.3 Hz), 4.17–4.22 (m, 1H), 5.04 (t, 1H, J = 5.2 Hz), 5.18 (d,
3.77 (s, 6H), 4.06 (q, 1H, J = 2.9 Hz), 4.49–4.53 (m, 1H), 6.31 1H, J = 4.0 Hz), 6.10 (t, 1H, J = 6.6 Hz), 6.68 (br s, 1H), 7.71 (br
(dd, 1H, J = 5.8, 7.4 Hz), 6.82 (d, 4H, J = 9.2 Hz), 7.24–7.33 s, 1H), 8.06 (s, 1H). 13C-NMR (101 MHz, DMSO-d6): δ 15.0,
(m, 7H), 7.41 (d, 2H, J = 7.2 Hz), 8.00 (s, 1H). 13C-NMR 32.4, 45.7, 51.9, 61.0, 70.2, 72.5, 85.3, 87.4, 90.0, 94.3, 143.5,
(101 MHz, CDCl3): δ 16.3, 30.9, 41.7, 55.5, 61.5, 63.7, 71.5, 153.5, 164.4, 172.2. HR MS: m/z: calcd for C15H20O6N3
72.5, 85.8, 86.7, 87.2, 95.0, 101.1, 113.6, 127.2, 128.2, 128.3, ([M + H]+): 338.1347, found: 338.1344.
130.1, 130.2, 135.8, 135.9, 141.7, 144.7, 149.4, 158.8, 162.2. HR
MS: m/z: calcd for C35H37O8N2 ([M + H]+): 613.2544, found: dimethoxytrityl)-2′-deoxycytidine (17). Derivative 16 (0.27 g,
613.2537. 0.81 mmol) was dissolved in pyridine (4 mL). DMTrCl (0.33 g,
Synthesis of 5-[5-(pent-1-ynylic acid methyl ester)]-5′-O-(4,4′-
Synthesis of 5-[5-acetoxy-pentynyl]-5′-O-(4,4′-dimethoxy- 1 mmol), NEt3 (0.22 mL, 1.6 mmol), and DMAP (0.024 g,
trityl)-3′-O-acetyl-2′-deoxyuridine (13). Compound 12 (0.3 g, 0.2 mmol) were then added to the solution and stirred for 18 h
0.49 mmol) was dissolved in pyridine (3 mL) and the solution at rt. MeOH (2 mL) was added and the yellow solution was
cooled down to 0 °C. NEt3 (0.31 mL, 2.2 mmol), acetic anhy- stirred for an additional 5 min. After removal of the solvent
dride (0.12 mL, 1.2 mmol), and DMAP (0.015 g, 0.12 mmol) under reduced pressure, the residue was dissolved in CH2Cl2
were then added in turn and the reaction mixture was stirred (50 mL) and washed with NaHCO3 (satd., 1 × 50 mL). The
at rt for 5 h. MeOH (3 mL) was then added and after an aqueous phase was extracted with CH2Cl2 (2 × 50 mL) and the
additional 5 min of stirring, the solvent was removed under combined organic layers were dried (MgSO4). After removal of
reduced pressure and the residue purified by flash column the solvent in vacuo, the residue was purified by flash column
chromatography on silica (EtOAc–hexanes 1 : 1). The fully pro- chromatography on silica gel by eluting with CH2Cl2–MeOH
tected compound 13 was obtained as a white foam (0.30 g, 96 : 4 to give 17 as a pale yellow solid (0.23 g, 45%). Rf: 0.45
86%). Rf: 0.34 (CH2Cl2–MeOH 95 : 5 +1% NEt3). 1H-NMR (CH2Cl2–MeOH 9 : 1 +1% NEt3). 1H-NMR (400 MHz, CDCl3):
(400 MHz, CDCl3): δ 1.34–1.41 (m, 2H), 1.91 (s. 3H), 1.99 (s, δ 2.08–2.15 (m, 1H), 2.25–2.31 (m, 2H), 2.33–2.39 (m, 2H),
3H), 2.06 (t, 2H, J = 7.0 Hz), 2.25–2.33 (m, 1H), 2.43–2.48 (m, 2.63–2.68 (m, 1H), 3.20–3.28 (m, 2H), 3.58 (s, 3H), 3.69 (s, 6H),
1H), 3.28–3.37 (m, 2H), 3.70 (s, 6H), 3.81 (t, 2H, J = 6.4 Hz), 4.06 (q, 1H, J = 3.3 Hz), 4.40–4.43 (m, 1H), 5.89 (br s, 1H), 6.24
4.05 (d, 1H, J = 1.6 Hz), 5.30 (d, 1H, J = 6.0 Hz), 6.25 (dd, 1H, (t, 1H, J = 6.4 Hz), 6.42 (br s, 1H), 6.73 (dd, 4H, J = 1.2, 8.8 Hz),
J = 5.6, 8.8 Hz), 6.75 (d, 4H, J = 8.8 Hz), 7.11–7.27 (m, 7H), 7.09–7.20 (m, 3H), 7.23–7.27 (m, 4H), 7.35 (d, 2H, J = 7.2 Hz),
7.32–7.36 (m, 2H), 7.98 (s, 1H). 13C-NMR (101 MHz, CDCl3): 8.01 (s, 1H). 13C-NMR (101 MHz, CDCl3): δ 15.5, 33.0, 46.2,
δ 16.5, 21.1, 21.2, 27.6, 29.9, 38.8, 55.4, 63.3, 63.8, 71.3, 75.2, 52.1, 55.4, 63.8, 72.3, 72.5, 86.6, 87.0, 87.1, 91.4, 94.5, 113.5,
84.7, 85.3, 87.5, 94.1, 101.4, 113.6, 127.2, 128.1, 128.3, 130.2, 127.1, 128.1, 128.2, 130.1, 130.2, 135.9, 136.0, 143.4, 144.8,
135.6, 135.7, 136.2, 141.7, 144.6, 149.4, 149.7, 158.9, 161.7, 154.9, 158.8, 165.2, 172.7. HR MS: m/z: calcd for C36H38O8N3
170.5, 171.1. HR MS: m/z: calcd for C39H41O10N2 ([M + H]+): ([M + H]+): 640.2653, found: 640.2647.
697.2756, found: 697.2763.
Synthesis of 5-[5-(pent-1-ynylic acid methyl ester)]-5′-O-(4,4′-
Synthesis of 5-[5-acetoxy-pentynyl]-3′-O-acetyl-2′-deoxy- dimethoxytrityl)-3′-O-acetyl-2′-deoxycytidine (18). The DMTr-
uridine (14). The reaction was carried out with 13 (0.43 g, protected nucleoside 17 (0.35 g, 0.55 mmol) was dissolved in
0.6 mmol) by application of the general detritylation method. dry pyridine (3 mL) and the solution was cooled down to 0 °C.
The residue was purified by flash column chromatography on DMAP (0.017 g, 0.14 mmol) and NEt3 (0.15 mL, 1.1 mmol)
silica gel by eluting with CH2Cl2–MeOH 9 : 1 to give 9 as a pale were then added. Acetic anhydride (0.13 mL, 1.4 mmol) was
yellow solid (0.21 g, 87%). Rf: 0.16 (CH2Cl2–MeOH 9 : 1). then added dropwise to the solution. The reaction was stirred
1H-NMR (400 MHz, DMSO-d6): δ 1.76–1.83 (m, 2H), 2.01 (s, at 0 °C. TLC analysis (CH2Cl2–MeOH 95 : 5 +1% NEt3, Rf (start-
3H), 2.06 (s, 3H), 2.25–2.32 (m, 2H), 2.45 (t, 2H, J = 7.2 Hz), ing material): 0.38; Rf (18): 0.5; Rf (S2): 0.86) revealed the com-
3.63 (d, 2H, J = 2.8 Hz), 4.01 (q, 1H, J = 2.8 Hz), 4.09 (t, 2H, J = plete disappearance of the starting material. MeOH (3 mL) was
6.4 Hz), 5.20–5.22 (m, 1H), 6.14 (apparent t, 1H, J = 7.2 Hz), then added and the solution was stirred for an additional
8.13 (s, 1H), 11.72 (s, 1H). 13C-NMR (101 MHz, DMSO-d6): 5 min at 0 °C. The solvents were removed under reduced
δ 15.6, 20.7, 20.8, 27.3, 37.1, 61.1, 62.7, 63.4, 73.1, 74.6, 84.4, pressure and the crude product was purified by flash column
84.9, 92.3, 99.2, 142.6, 149.5, 161.6, 165.5, 170.0, 170.4. HR chromatography on silica gel (gradient from CH2Cl2–MeOH
MS: m/z: calcd for C18H23O8N2 ([M + H]+): 395.1449, found: 98 : 2 to 95 : 5), yielding 18 (0.23 g, 61%) and S4 (0.12 g, 30%)
395.1441.
both as white foams. Characterization for 18: 1H-NMR
Synthesis of 5-[5-(pent-1-ynylic acid methyl ester)]-2′-deoxy- (400 MHz, CDCl3): δ 2.04 (s, 3H), 2.20–2.28 (m, 1H), 2.35–2.40
cytidine (16). Nucleoside analogue 16 (pale yellow solid, (m, 2H), 2.43–2.48 (m, 2H), 2.69 (ddd, 1H, J = 1.5, 5.5, 14.1
0.52 g, 90%) was obtained by application of the general Hz), 3.06–3.12 (m, 1H), 3.31–3.39 (m, 2H), 3.66 (s, 3H), 3.77 (s,
method for Sonogashira coupling reactions starting from 15 6H), 4.16 (q, 1H, J = 2.5 Hz), 5.34 (apparent d, 1H, J = 6.0 Hz),
(0.6 g, 1.7 mmol) and after purification by flash column 6.10 (br s, 1H), 6.20 (br s, 1H), 6.31 (dd, 1H, J = 5.4, 8.2 Hz),
5168 | Org. Biomol. Chem., 2013, 11, 5162–5172
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6.81 (dd, 4H, J = 1.8, 9.0 Hz), 7.16–7.33 (m, 8H), 7.40 (apparent
d, 1H, J = 7.2 Hz), 8.07 (s, 1H). 13C-NMR (101 MHz, CDCl3):
δ 15.6, 21.2, 33.0, 39.7, 52.2, 55.5, 63.8, 72.2, 75.4, 84.6, 86.8,
87.3, 91.6, 94.8, 113.5, 127.4, 128.1, 128.2, 130.1, 130.2, 135.7,
135.8, 143.2, 144.7, 154.3, 158.9, 164.9, 170.6, 172.7. HR MS:
m/z: calcd for C38H40O9N3 ([M + H]+): 682.2759, found:
682.2750.
Table 1 HPLC programs used for the purification of the dNTPs
Time (min)
Program A (% II)
Program B (% II)
Time (min)
0
8
0
0
0
0
0
8
31
33
35
37
50
52
54
56
50
100
100
0
17
80
80
0
Synthesis of 5-[5-(pent-1-ynylic acid methyl ester)]-3′-O-
acetyl-2′-deoxycytidine (19). The reaction was carried out with
18 (0.22 g, 0.3 mmol) by application of the general detritylation
method. The residue was purified by flash column chromato-
graphy on silica gel (gradient from CH2Cl2–MeOH 100 : 0 to
94 : 6), to give 9 as a white solid (0.11 g, 92%). Rf: 0.31
(CH2Cl2–MeOH 95 : 5 +% NEt3). 1H-NMR (400 MHz, DMSO-d6):
δ 2.06 (s, 3H), 2.11–2.19 (m, 1H), 2.25–2.32 (m, 1H), 2.63–2.67
(m, 4H), 3.56–3.60 (m, 1H), 3.64 (s, 3H), 4.01 (q, 1H, J =
2.8 Hz), 5.19 (apparent d, 2H, J = 6.0 Hz), 6.15 (dd, 1H, J = 5.8,
7.8 Hz), 6.83 (br s, 1H), 7.73 (br s, 1H), 8.06 (s, 1H). 13C-NMR
(101 MHz, DMSO-d6): δ 15.0, 20.8, 32.4, 37.8, 51.5, 61.2, 72.3,
74.7, 84.9, 85.2, 90.5, 94.5, 143.3, 153.5, 164.4, 170.0, 172.2.
HR MS: m/z: calcd for C17H22O7N3 ([M + H]+): 380.1452, found:
380.1443.
procedure starting from 9 (35 mg, 0.065 mmol). Deprotection
was carried out by incubation in aqueous NH3 (10 mL) at rt for
1.5 h. RP-HPLC purification (program A, Rt = 26.9 min) yielded
1 as its triethylammonium salt (pale yellow solid, 12 mg,
17%). 1H-NMR (400 MHz, D2O): δ 1.31 (t, 27 H, J = 7.2 Hz),
2.43–2.52 (m, 1H), 2.56–2.67 (m, 5H), 2.72 (t, 2H, J = 7.0 Hz),
3.00–3.05 (m, 1H), 3.06–3.13 (m, 2H), 3.22 (q, 18H, J = 7.3 Hz),
3.29–3.56 (m, 2H), 3.52 (q, 1H, J = 7.2 Hz), 4.21 (d, 2H, J = 4.0
Hz), 4.26–4.32 (m, 2H), 6.52 (t, 1H, J = 6.8 Hz), 7.02 (s, 1H),
7.74 (s, 1H), 8.50 (s, 1H). 13P-NMR (121.4 MHz, D2O): δ −5.18
(d, 1P, J = 18.5 Hz, Pγ), −5.56 (d, 1P, J = 19.4 Hz, Pα), −20.32
(t, 1P,
J =
17.3 Hz, Pβ). MS (MALDI−): m/z calcd for
C23H30N8O14P3−: 735.11 [M − H]−; found: 734.84; UV: λmax
=
Triphosphorylation of nucleosides
282, 240 nm.
General method for the triphosphorylation of nucleosides.
The nucleoside was co-evaporated twice with dry pyridine
(1 mL) and then dried under vacuum overnight. After dissol-
ving the nucleoside in dry pyridine (0.2 mL), dry dioxane
(0.4 mL) was added, followed by the addition of 2-chloro-1,3,2-
benzodioxaphosphorin-4-one (1.1 eq.). The reaction mixture
was stirred at room temperature for 45 min. A solution of tri-
butylammonium pyrophosphate (1.3 eq.) in dry DMF (170 μL)
and tributylamine (58 μL) was then added and the resulting
solution was stirred at room temperature for 45 min. Iodine
(1.6 eq.) in a mixture of pyridine (980 μL) and water (20 μL)
was then added and the resulting dark solution was stirred at
room temperature for 30 min. Excess iodine was then
quenched by addition of a solution of sodium thiosulfate
(10%) and the solvents were removed under reduced pressure
(water bath ≤30 °C). Water (5 mL) was then added and the
mixture was allowed to stand at room temperature for 30 min.
Following removal of the various protecting groups (see below)
and evaporation of the solvent, the crude triphosphates were
precipitated with NaClO4 in acetone (2%) and then purified by
RP-HPLC. The appropriate fractions were freeze-dried and co-
evaporated several times with water.
Modified dUTP 2 (dUPOHTP). Triphosphate 2 was syn-
thesized by application of the general triphosphorylation pro-
cedure starting from 14 (40 mg, 0.101 mmol). Deprotection
was carried out by incubation in aqueous NH3 (10 mL) at rt for
1.5 h. RP-HPLC purification (program A, Rt = 24.7 min) yielded
1 as its triethylammonium salt (white solid, 12 mg, 14%).
1H-NMR (300 MHz, D2O): δ 1.28 (t, 27H, J = 7.5 Hz), 1.82
(t, 2H, J = 6.6 Hz), 2.34–2.41 (m, 2H), 2.51 (t, 2H, J = 7.1 Hz),
2.96–3.11 (m, 2H), 3.20 (q, 18H, J = 7.3 Hz), 3.73 (t, 2H, J =
6.3 Hz), 4.14 (m, 3H), 4.59–4.68 (m,1H), 6.29 (t, 1H, J = 6.6 Hz),
8.05 (s, 1H). 13P-NMR (121.4 MHz, D2O): δ −10.27 (d, 1P, J =
23.2 Hz, Pγ), −11.34 (d, 1P, J = 20.5 Hz, Pα), −23.11 (t, 1P, J =
18.0 Hz, Pβ). MS (MALDI−): m/z calcd for C14H20N2O15P3
549.01 [M − H]−; found: 548.76; UV: λmax = 233, 292 nm.
:
−
Modified dCTP 3 (dCValTP). Analogue 3 was obtained by
application of the general triphosphorylation procedure start-
ing from 19 (45 mg, 0.119 mmol). Deprotection: the crude
reaction mixture was dissolved in H2O (1.69 mL). After cooling
to 0 °C, NaOH (380 μL, 2.5 M; 0.46 M final concentration) was
added and the reaction stirred at 0 °C for 5 min. The mixture
was then neutralized by adding NaH2PO4 buffer (925 μL; 1 M,
pH 1.9) and further diluted by addition of H2O (3 mL). The
solvent was then removed in vacuo and the residue purified
by RP-HPLC using program B (Rt = 21.8 min). dCValTP 3 was
obtained as its triethylammonium salt (white solid, 10 mg,
9%). 1H-NMR (300 MHz, D2O): δ 1.28 (t, 27H, J = 7.4 Hz),
2.25–2.35 (m, 1H), 2.38–2.45 (m, 1H), 2.49 (t, 2H, J = 6.9 Hz),
2.68 (t, 2H, J = 6.9 Hz), 2.95–3.01 (m, 1H), 3.03–3.11 (m, 2H),
3.20 (q, 18H, J = 7.3 Hz), 3.39 (q, 2H, J = 7.3 Hz), 4.20 (d,
2H, J = 4.5 Hz), 4.59 (t, 1H, J = 2.7 Hz), 6.27 (t, 1H, J = 6.8 Hz),
8.03 (s, 1H). 13P-NMR (121.4 MHz, D2O): δ −10.71 (d, 1P,
HPLC of triphosphates
The purification of the various triphosphates was carried out
on a Phenomenex Jupiter semi-preparative RP-HPLC column
(see ESI†) at a flow rate of 3.5 mL min−1, using a triethylammo-
nium bicarbonate (TEAB) buffer system (eluent I: 50 mM
TEAB, pH 7.7; eluent II: 50 mM TEAB in CH3CN–H2O 1 : 1),
and one of the programs highlighted in Table 1.
Modified dATP 1 (dAHsTP). This modified triphosphate was
obtained by application of the general triphosphorylation
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J = 14.6 Hz, Pγ), −11.27 (d, 1P, J = 19.3 Hz, Pα), −23.18 (d, Smith for a critical reading of the manuscript. M.H. thanks
1P,
J =
20.6 Hz, Pβ). MS (MALDI−): m/z calcd for Mr Luís ‘Lucho’ Lara for fruitful discussions.
C14H19N3O15P3−: 562.00 [M − H]−; found: 562.16; UV: λmax
=
209, 235, 293 nm.
Primer extension experiments. The 5′-32P-labelled primer P1
(ca. 1 pmol) and 10 pmol of primer P1 were annealed to appro-
priate template T1 (10 pmol) in the presence of 10× enzyme
buffer (provided by the supplier of the DNA polymerase) by
heating to 95 °C and then gradually cooling to room temp-
erature (over 30 min). The appropriate DNA polymerase (1 U) was
then added to the annealed oligonucleotides mixture at 4 °C.
Finally, dNTPs (final concentration of 100 μM) were added
for a total reaction volume of 20 μL. Following incubation
at the optimal temperature for the enzyme, the reactions
were quenched by adding stop solution (20 μL; formamide
(70%), ethylenediaminetetraacetic acid (EDTA; 50 mM),
bromophenol (0.1%), xylene cyanol (0.1%)). The reaction
mixtures were subjected to gel electrophoresis in denaturing
polyacrylamide gel (15%) containing trisborate-EDTA (TBE)
1× buffer (pH 8) and urea (7 M). Visualization was performed
by phosphorimaging.
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