Synthesis of deoxynucleoside triphosphates that include proline, urea, or sulfonamide groups and their polymerase incorporation into DNA

Article abstract of DOI:10.1002/chem.201201662

To expand the chemical array available for DNA sequences in the context of in vitro selection, I present herein the synthesis of five nucleoside triphosphate analogues containing side chains capable of organocatalysis. The synthesis involved the coupling

Full text of DOI:10.1002/chem.201201662

FULL PAPER
DOI: 10.1002/chem.201201662
Synthesis of Deoxynucleoside Triphosphates that Include Proline, Urea, or
Sulfonamide Groups and Their Polymerase Incorporation into DNA
Marcel Hollenstein*[a]
Abstract: To expand the chemical
array available for DNA sequences in
the context of in vitro selection, I pres-
ent herein the synthesis of five nucleo-
side triphosphate analogues containing
side chains capable of organocatalysis.
The synthesis involved the coupling of
l-proline-containing residues (dUtPTP
and dUcPTP), a dipeptide (dUFPTP), a
urea derivative (dUBpuTP), and a sulfa-
mide residue (dUBsTP) to a suitably
protected common intermediate, fol-
lowed by triphosphorylation. These
modified dNTPs were shown to be ex-
cellent substrates for the Vent (exoÀ)
and Pwo DNA polymerases, as well as
the Klenow fragment of E. coli DNA
polymerase I, although they were only
acceptable substrates for the 98Nm pol-
ymerase. All of the modified dNTPs,
with the exception of dUBpuTP, were
readily incorporated into DNA by the
polymerase chain reaction (PCR).
Modified oligonucleotides efficiently
served as templates for PCR for the re-
generation of unmodified DNA. Ther-
mal denaturation experiments showed
that these modifications are tolerated
in the major groove. Overall, these
heavily modified dNTPs are excellent
candidates for SELEX.
Keywords: DNA · nucleosides · oli-
gonucleotides
· organocatalysis ·
polymerase chain reaction
Introduction
The enzymatic polymerization of modified nucleoside tri-
phosphates (dNTPs) is an alternative, milder and more ver-
satile way to generate chemically altered nucleic acids.[7,13,14]
Indeed, dNTPs embellished with a variety of functional
groups, including amino acid or amino acid-like side
chains,[15–27] boronic acids,[28,29] thiols,[30,31] diamondoid-like
residues,[32] bile acids,[33] ligands for metal complexation,[34]
electrochemical tags[35,36] and even oligonucleotides,[37] have
been reported. The vast majority of the existing modified
dNTPs have found applications in DNA-tagging and -label-
ling, either by direct incorporation of suitably altered tri-
phosphates,[38–41] by post-modification (e.g., through click-
couplings)[42,43] or by tail-labelling of single-stranded pri-
mers.[44]
Consequently, modified dNTPs appear to be a very con-
venient platform to supplement the poor functional arsenal
of nucleic acids. Thus, it is baffling that only a few modified
dNTPs have been used in in vitro selection experiments to
generate aptamers and catalytic nucleic acids, including de-
oxyribozymes (DNAzymes).[45–47] Indeed, with the exception
of an elegantly generated RNA amide synthetase[48] and an
efficient Diels–Alder ribozyme,[49] most efforts were dedicat-
ed to the generation of nucleic acid enzymes catalyzing the
scission of ribophosphodiester linkages,[50] either in the pres-
ence[26,51,52] or in the absence of divalent metal cations
(M2+).[22,53–56]
The urge for chemical modification of nucleic acids originat-
ed from the development of the antisense and antigene
strategies and has never abated. Undeniably, chemically al-
tered nucleosides have found uses in a variety of applica-
tions ranging from chemical biology to nanotechnology.[1–6]
A common approach to the crafting of modified nucleic
acids is the automated solid-phase synthesis of oligonucleoti-
des, during which altered phosphoramidite precursors are
appended to the nascent chain. However, the nature of
these modifications is rather limited and often depends on
their resilience to the rather harsh conditions imposed by
solid-phase synthesis.[7] In addition, phosphoramidite build-
ing blocks (whether chemically modified or not) are incom-
patible with enzymatic amplification techniques, such as the
polymerase chain reaction (PCR), and thus cannot be ap-
plied in SELEX and related combinatorial methods of in
vitro selection.[8] Moreover, oligonucleotides can be modi-
fied by postsynthetic approaches, which circumvent the need
for individual phosphoramidite building blocks by using one
stable and convertible precursor,[9] but still require the de-
velopment and application of suitable and selective proto-
cols.[10–12]
Herein, I report the synthesis, and incorporation into
DNA, of nucleoside triphosphates adorned with functional-
ized side chains. These are of paramount importance in or-
ganocatalysis and increase the functional (and catalytic)
tools available for the crafting of DNAzymes with enhanced
and hitherto unknown activities (Figure 1).
[a] Dr. M. Hollenstein
Department of Chemistry & Biochemistry
University of Bern
Freiestrasse 3, 3012 Bern (Switzerland)
Fax : (+41)31-631-3422
E-mail: hollenstein@dcb.unibe.ch
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201201662.
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Figure 1. Chemical structures of dUtPTP (1), dUcPTP (2), dUFPTP (3), dUBpuTP (4), and dUBsTP (5).
In organocatalysis, the rates of reactions are accelerated
by supplementing the reaction mixture with substoichiomet-
ric amounts of an organic compound (without metal
atoms).[57] In this context, the natural amino acid l-proline is
one of the most prominent and successful organocatalysts
and mediates a vast array of reactions. The key structural
features of l-proline are the secondary amine, which can
form enamine or iminium ion intermediates, and the carbox-
ylic acid, which acts as a Brønsted acid in various reac-
tions.[58] Both of these catalytically essential functionalities
were maintained in the design of dUtPTP (5-trans-prolylall-
yl-dU) 1 and dUcPTP (5-cis-prolylallyl-dU) 2, in both of
which an l-proline residue was anchored on the nucleobase
through a linker arm grafted on the 4-position of the pyrro-
lidine ring, in either a trans or a cis fashion relative to the
carboxylic acid, respectively. Moreover, dUFPTP (5-phenyla-
lanineprolylallyl-dU) 3 was embellished with the l-proline-
containing a-dipeptide Phe–Pro, which is a known and effi-
cient organocatalyst, especially for asymmetric aldol reac-
tions.[59,60] Finally, a non-covalent approach to organocataly-
sis involves the use of low-molecular-weight compounds that
are able to form strong and distinct hydrogen-bond net-
works with functional groups of a suitable reaction part-
ner.[61] More particularly, examples of (thio)urea derivatives
flanked by acidifying and/or chiral directing groups are
abundant in the recent literature.[62–64] Sulfamides represent
a smaller subgenre of catalophores that adopt the same cat-
alytic mechanism as urea-based derivatives, that is, the si-
multaneous provision of two hydrogen bonds.[65] Based on
these facts, dUBpuTP (5-bis(trifluoromethyl)phenylureidoall-
yl-dU) 4 and dUBsTP (5-benzylsulfonylallylamino-dU) 5
were equipped with a urea and a sulfamide functional
group, respectively.
Results and Discussion
Synthesis of the proline-containing dNTPs 1, 2, and 3: All
of the chemically altered dNTPs considered herein, contain
modifications at the C5 position of deoxyuridine. Indeed, it
is well-known that the anchoring of functionalities at this
particular location causes only a minor perturbation in
Watson–Crick-base-pairing ability and these modified
dNTPs usually display a very high polymerase accept-
ance.[21,66,67] The synthetic route to these three modified
dNTPs was initiated by the elaboration of the common in-
termediate 11 (Scheme 1). The suitably protected 5-allylami-
no-2’-deoxyuridine 11 was obtained in a series of uneventful
steps starting from commercially available 5-iodo-2’-deoxy-
uridine 6. Briefly, after a Heck-type of coupling between 6
and N-allyltrifluoroacetamide,[68] the amine was deprotected
and an Fmoc protecting group was installed. Compound 9
was then converted into the fully protected nucleoside 10
and removal of the Fmoc protecting group resulted in for-
mation of 11 in 31% overall yield (see the Supporting Infor-
mation).
The synthesis of the proline-containing dNTPs 1 and 2
was driven by the generation of the prerequisite intermedi-
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Synthesis of Deoxynucleoside Triphosphates
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analogues 18 and 21 were then
detritylated and converted, by
application of the Ludwig–Eck-
stein protocol,[70] into the corre-
sponding fully protected tri-
phosphates 20 and 23 with
yields of 42 and 28%, respec-
tively. Initially, a Boc-tert-butyl
ester protecting-group strategy
had been selected to allow for a
facile and global acid-mediated
deprotection of the modified
dNTPs. However, the condi-
tions required for the full re-
moval of the tert-butyl ester
were found to be too harsh for
the triphosphate group, result-
ing in
a significant drop in
yields and a difficult HPLC sep-
aration from the corresponding
diphosphates (data not shown).
Thus, synthetic efforts turned to
a protecting-group strategy that
involved milder deprotection
conditions that are compatible
Scheme 1. Synthesis of intermediate 11. i) N-Allyltrifluoroacetamide, Na2ACTHNUTRGEN[UNG PdCl4], NaOAc buffer (pH 5.2),
DMF, 808C, 2 h, 70%; ii) NH3(aq), RT, 12 h; iii) N-(9-fluorenylmethoxycarbonyloxy)succinimide (FmocOSu),
iPr2NEt, H2O, THF, RT, 12 h, 60% (2 steps); iv) a) 4,4’-dimethoxytritylchloride (DMTrCl), pyridine, RT, 12 h,
b) Ac2O, pyridine, RT, 5 h, 98%; v) piperidine/DMF (1:4), RT, 1 h, 77%. TFA=trifluoro acetyl; Fmoc=9-flu-
orenylmethoxycarbonyl; DMTr=4,4’-dimethyoxytrityl.
ates 12 and 13, which were easily obtained from 4-trans-hy-
droxyproline methylester (see the Supporting Information).
To install the linker arm, the resulting 4-aminoproline deriv-
atives 12 and 13 were reacted with succinic anhydride
(Scheme 2) to give the carboxylic acids 14 and 16 in good
yields (73 and 77%, respectively).
with triphosphates. In addition, isolation of the fully protect-
ed dNTPs 20 and 23 was carried out to simplify the purifica-
tion of the rather polar dNTPs 1 and 2. Finally, removal of
the Fmoc groups, followed by ester hydrolysis,[69] gave the
proline-functionalized dNTPs 1 and 2, after isolation by
semi-preparative RP-HPLC.
Moreover, intermediate 11 could easily be converted into
the modified nucleoside 24 (Scheme 4) by standard EDC-
mediated amide-bond formation with the suitably protected
dipeptide ProFmocPhe-OH S4 (see the Supporting Informa-
tion) in good yield. After detritylation, compound 25 was
converted into dUFPTP (3) by application of the one-pot
four-step triphosphorylation method developed by Ludwig
and Eckstein.[70]
Synthesis of the H-bond-donor-containing dNTPs 4 and 5:
The modified dNTP 4 was obtained in three steps starting
from nucleoside 11 (Scheme 5). In the first step, the urea
functionality in intermediate 26 was installed by reaction of
the terminal amine residue of nucleoside 11 with 3,5-bis(tri-
fluoromethyl)phenyl isocyanate. Following removal of the
DMTr-masking group under acidic conditions, 27 was con-
verted into the corresponding triphosphate dUBpuTP (4) in
21% yield by application of the Ludwig–Eckstein protocol.
The formation of the sulfamide-functionalized dNTP 5
followed a similar pathway (Scheme 6). Indeed, the reaction
of nucleoside 11 and 2-hydroxyphenyl N-benzylsulfamate
28[65,71] led to the bifunctional sulfamide 29 in 72% yield.
After acidic treatment, derivative 30 was triphosphorylated
and semi-preparative RP-HPLC purification led to the isola-
tion of dUBsTP (5) in 36% yield.
Scheme 2. Synthesis of intermediates 15 and 17. i) Succinic anhydride,
CH2Cl2, RT, 3.5 h, 14: 73, 16: 77%; ii) a) trifluoroacetic acid, CH2Cl2, RT,
2 h; b) FmocCl, iPr2NEt, CH2Cl2, RT, 12 h, 15: 66, 17: 81%. Boc=tert-
butyloxycarbonyl.
After a protecting-group swap, from Boc to Fmoc, to
allow for mild deprotection of the modified dNTPs (see
below), the resulting compounds 15 and 17 were coupled to
nucleoside 11 under standard 1-ethyl-3-(3-dimethylamino-
propyl)carbodiimide (EDC)-mediated amide bond-forma-
tion conditions (Scheme 3). The proline-modified nucleoside
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M. Hollenstein et al.
Primer extension assays: Incor-
poration by polymerases into
newly synthesized DNA is one
prerequisite
for
modified
dNTPs to be compatible with in
vitro selection methods.[55] In
this context, I explored the ac-
ceptance of the triphosphates
1–5 by different polymerases in
primer extension reactions. A
variety of polymerases were
employed,
including
the
Klenow fragment of E. coli
DNA polymerase I (family A
polymerase) and the family B
polymerases from Pyrococcus
woesi (Pwo), Thermococcus li-
toralis [Vent (exoÀ)] and hyper-
thermophilic Archaea Thermo-
coccus sp. 98Nm. In addition,
three different sets of primer-
template sequence were used
(Table 1), each of which re-
quires the polymerases to incor-
porate two or three modified
dNTPs at different locations
Scheme 3. Synthesis of modified dNTPs 1 and 2. i) EDC·HCl, hydroxybenzotriazole (HOBt), N-methylmalei-
mide (NMM), CH2Cl2, RT, 12 h, 18: 88%, 21: quantitative; ii) 2,4-dichlorophenylacetic acid (DCAA; 1%),
CH2Cl2, RT, 40 min, 19: 93, 22: 99%; iii) a) 2-chloro-1,3,2-benzodioxaphosphorin-4-one, pyridine, dioxane, RT,
45 min, b) (nBu3NH)2H2P2O7, DMF, nBu3N, RT, 40 min, c) I2, pyridine, H2O, RT, 30 min, 20: 42; 23: 28%
(3 steps); iv) a) piperidine, DMF, RT, 30 min, b) NaOH (0.46m), 08C, 5 min, 1: 12; 2: 28% (over 2 steps).
(i.e., standing-start (template T1), running-start (template
T2) and incorporation of three neighboring modifications
(template T3)).
Table 1. Sequences of the primer and templates used in the primer ex-
tension reactions. Underlined Aꢁs indicate the position at which the poly-
merases need to insert the modified dNTPs.
P1
T1
T2
T3
5’-TGTAAAACGACGGCCAGT-3’
5’-TACGGAGCTGAACTGGCCGTCGTTTTACA-3’
5’-TACGGAGCTGCACTGGCCGTCGTTTTACA-3’
5’-TCCGGAAATGCACTGGCCGTCGTTTTACA-3’
Scheme 4. Synthesis of triphosphate 3. i) ProFmocPhe-OH S4, EDC·HCl,
HOBt, NMM, CH2Cl2, RT, 12 h, 58%; ii) DCAA (1%), CH2Cl2, RT,
40 min, 81%; iii) a) 2-chloro-1,3,2-benzodioxaphosphorin-4-one, pyridine,
dioxane, RT, 45 min, b) (nBu3NH)2H2P2O7, DMF, nBu3N, RT, 40 min,
c) I2, pyridine, H2O, RT, 30 min, d) NH3(aq), RT, 1 h, 15% (4 steps).
The polymerase Vent (exoÀ) was revealed to be the most
proficient because efficient incorporation of all of the modi-
fied dNTPs in all of the sequence contexts was observed
(Figure 2). Indeed, in all cases only full-length products
were observed after incubation at 728C for only 15 min, al-
though, as expected, all bands showed slightly slower elec-
trophoretic mobilities than the natural control.
The Pwo and 98Nm polymerases were equally efficient at
incorporating all of the modified dNTPs in the P1–T1
system (Figures S6 and S7 in the Supporting Information).
Moreover, the Pwo polymerase produced full extension
products when using both templates T2 and T3. Surprisingly,
the 98Nm polymerase stalled with most of the dNTPs, lead-
ing to shorter fragments and more diffuse product bands
(see Figure S7 in the Supporting Information). Finally, the
Klenow fragment showed a high propensity to incorporate
the modified triphosphates in all three primer–template sys-
Scheme 5. Synthesis of triphosphate 4. i) 3,5-Bis(trifluoromethyl)phenyl
isocyanate, NEt3, CH2Cl2, RT, 18 h, 84%; ii) DCAA (1%), CH2Cl2, RT,
40 min, 88%; iii) a) 2-chloro-1,3,2-benzodioxaphosphorin-4-one, pyridine,
dioxane, RT, 45 min, b) (nBu3NH)2H2P2O7, DMF, nBu3N, RT, 40 min,
c) I2, pyridine, H2O, RT, 30 min, d) NH3(aq), RT, 1 h, 21% (4 steps).
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Synthesis of Deoxynucleoside Triphosphates
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98Nm), increasing the concen-
tration of dNTPs (500 mm) and/
or supplementing the reaction
mixture with Mg2+ had no
impact on the substrate ability
of dUBpuTP (4; see Figure S9 in
the Supporting Information).
Finally, increasing the extension
time to 3 min also had no effect
on the PCR uptake of dUBpuTP
(4; data not shown). This lack
of polymerase acceptance might
be due to the slow uptake ki-
netics of dUBpuTP (4), which
will affect the amplification in
PCR experiments, but have
little effect on the outcome of
the primer extension reactions.
Scheme 6. Synthesis of triphosphate 5. i) NEt3, MeCN, reflux, 20 h, 72%; ii) DCAA (1%), CH2Cl2, RT, 40 min,
90%; iii) a) 2-chloro-1,3,2-benzodioxaphosphorin-4-one, pyridine, dioxane, RT, 45 min, b) (nBu3NH)2H2P2O7,
DMF, nBu3N, RT, 40 min, c) I2, pyridine, H2O, RT, 30 min, d) NH3(aq), RT, 1 h, 36% (4 steps). Bn=benzyl.
In
a different experiment,
modified oligonucleotides were
shown to serve as templates in
PCR experiments for the regen-
eration of natural DNA tem-
plates (see Scheme S3 and Fig-
ures S10 and S11 in the Sup-
porting Information), which is
another prerequisite for the use
of these modified dNTPs in se-
lection experiments.[55] Indeed,
the regeneration of unmodified
oligonucleotides by using modi-
fied ssDNAs in PCR is crucial
Figure 2. Gel images (polyacrylamide gel electrophoresis (PAGE) 20%) of primer extension reactions with
Vent (exoÀ): A) Primer P1 and template T1; B) Primer P1 and template T2; C) Primer P1 and template T3.
Lane 1: positive control with natural dNTPs; lane 2: dUBsTP (5); lane 3: dUBpuTP (4); lane 4: dUFPTP (3);
lane 5: dUcPTP (2); lane 6: dUtPTP (1); lane 7: 32P-radiolabelled primer. Reactions in lanes 2–6 included dATP,
dCTP, and dGTP.
tems investigated giving full-length modified oligonucleoti-
des (see Figure S8 in the Supporting Information).
Polymerase chain reactions (PCRs): The compatibility of
the modified dNTPs 1–5 for in vitro selection methods was
then further assessed by gauging their ability to act as sub-
strates in polymerase chain reactions. In this context, PCR
experiments were conducted by using a 98-mer template[33]
flanked by 20 and 25 nt primers and Vent (exoÀ) as the poly-
merase. All of the modified dNTPs, with the exception of
dUBpuTP (4), were found to be good substrates under these
conditions and led to amplified full-length products
(Figure 3). Moreover, the proline-containing dNTPs 1–3 per-
formed the best with strong bands corresponding to the full-
length 98-mer product being observed, with an efficiency
that matches even that of the natural dNTPs (Figure 3,
lanes 6–8). On the other hand, dUBsTP (5) led to a slightly
less efficient PCR, as shown by the fainter and slightly
blurry band, but shorter products were also not observed in
this case (Figure 3, lane 4). Surprisingly, even though
dUBpuTP (4) is a good substrate for all of the polymerases
utilized in the primer extension reactions, no amplification
could be detected (Figure 3, lane 5). Moreover, changing
Vent (exoÀ) for the other polymerases (Pwo, Klenow and
Figure 3. Agarose gel (2%) stained with ethidium bromide, showing the
PCR products with the 98-mer template T4. Lane 1: ladder; lane 2: posi-
tive control with natural dNTPs; lane 3: dCTP, dGTP, dATP; lane 4:
same as lane 3 including dUBsTP (5); lane 5: same as lane 3 including
dUBpuTP (4); lane 6: same as lane 3 including dUFPTP (3); lane 7: same
as lane 3 including dUcPTP (2); lane 8: same as lane 3 including dUtPTP
(1).
for in vitro selection because these natural DNAs will serve
as templates in primer extension reactions in subsequent
rounds of selection.[22,23,26,51–56] Consequently, a 5’-phosphory-
lated 79-mer template that has been used in the context of
highly functionalized DNAs,[19] was annealed with a comple-
mentary 19-mer primer and the resulting duplex was extend-
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M. Hollenstein et al.
ed by using the polymerase Vent (exoÀ) in conjunction with
the various modified dNTPs. PAGE analysis of the resulting
extension reaction products (by using a 32P-radiolabelled
primer) further confirmed the excellent polymerase uptake
of all of the modified dNTPs because only full-length prod-
ucts were observed (Figure S10 in the Supporting Informa-
tion). The 5’-phosphorylated template of the double-strand-
ed mDNA/DNA hybrids was removed by a l-exonuclease
digest. The resulting single-stranded modified DNAs then
served as templates in PCR experiments by using natural
dNTPs to regenerate unmodified templates (Figure S11 and
Scheme S3 in the Supporting Information). As observed
previously, all but the dUBpu-containing template were am-
plified and led to the full-length 79-mer amplicons. Finally,
the modified dNTPs have not yet been used in conjunction
with modified templates for the generation of modified oli-
gonucleotides through PCR.
intermediate and modified 4-aminoproline derivatives fol-
lowed by triphosphorylation led to the formation of dUtPTP
(1) and dUcPTP (2), both of which are adorned with the pro-
teinogenic amino acid l-proline with both the carboxylic
acid and the secondary amine available for enamine-based
catalysis in water.[72] Coupling of the suitably protected a-di-
peptide Phe–Pro with intermediate 11 allowed for the syn-
thesis of triphosphate dUFPTP (3). Finally, the allylamino
moiety is an expedient synthetic handle for the introduction
of urea residues, as in dUBpuTP (4), or sulfamide units, as in
dUBsTP (5).
All of these modified dNTPs were found to be excellent
substrates for three different DNA polymerases (Vent
(exoÀ), Pwo, and the Klenow fragment of E. coli DNA poly-
merase I) in various primer-extension reactions. Only the
98Nm polymerase was found to be more reluctant to accept
these modified dNTPs as substrates and proved to be very
dependent on the sequence context. Moreover, four of these
triphosphates were found to be efficient in PCR amplifica-
tions, with only the dUBpuTP (4) being resistant to polymer-
ase uptake under PCR conditions. In addition, modified
ssDNAs resulting from primer-extension reactions could
serve as templates in PCR experiments and be converted
into natural DNA amplicons. Again, the modified oligonu-
cleotide obtained with dUBpuTP seemed to be reluctant to
function as a template in this context. Moreover, thermal
denaturation experiments showed that the modified nucleo-
tides had a marginal impact (DTm range of +1.7 to À2.88C
per modification) on the stability of the duplexes, hinting at
good acceptance of these side chains in the major groove.
This study further underscores the high tolerance of DNA
polymerases for modifications anchored at the C5-position
of pyrimidine nucleobases.[23,26] Application of these modi-
fied triphosphates in the context of in vitro selection could
generate DNAzymes capable of organocatalysis in water
without the addition of an organic co-solvent, a task which
still remains challenging.[58,73–75] This could open up new re-
search avenues blending organo- and biocatalysis and fur-
ther highlight the synthetic usefulness of DNAzymes.[47,51]
Thermal denaturation of duplexes: Next, to assess the effect
of the modifications in general, and the strong H-bond-do-
nating moieties in particular, on the stability of the duplexes,
large-scale primer-extension reactions were carried out. The
UV–melting curves of the obtained triple-modified dsDNAs
were then measured under standard saline buffer conditions
and the Tm data are summarized in Table 2.
Table 2. Melting temperatures of DNA duplexes.[a]
Template T1
Template T3
[b]
[b]
Tm [8C]
DTm
Tm [8C]
DTm
T
66.5
63.2
62.6
61.1
58.1
59.0
–
66.5
71.7
65.3
65.4
65.4
64.4
–
UtP
UcP
UFP
1
2
3
4
5
À1.1
À1.3
À1.8
À2.8
À2.5
+1.7
À0.4
À0.4
À0.4
À0.7
UBpu
UBs
[a] Duplex (0.7 mm), NaH2PO4 (10 mm), NaCl (150 mm), pH 7.0. Estimat-
ed error in Tm = Æ0.58C. [b] DTm =(Tm,unmodÀTm,mod)/nmo
.
All of the modifications caused only a slight destabiliza-
tion of the duplex (DTm in the range of À1.1 to À2.88C per
modification) when spread out within the sequence (tem-
plate T1). In addition, three consecutive modifications (tem-
plate T3) had only a marginal effect on the Tm values, with
1 even slightly stabilizing the duplex (+1.78C/modification).
This indicates that neither the proline-containing dNTPs nor
the strong H-bond-donating motifs interfere with duplex for-
mation and are well-accommodated in the major groove.
Experimental Section
Synthesis of nucleosides
General method for amide coupling with nucleoside 11: Nucleoside 11
(1 equiv) was dissolved in dry CH2Cl2 (3.5 mL/0.1 g 11). N-methylmor-
pholine (1.3 equiv) and the corresponding amino acid (1.2 equiv) were
then added to the reaction mixture, which was cooled to 08C. HOBt
(1.1 equiv) and EDC·HCl (1.1 equiv) were then added in turn and the re-
action mixture was allowed to warm to room temperature and stirred for
12 h. The reaction mixture was combined with a saturated solution of am-
monium chloride (10 mL) and extracted with CH2Cl2 (3ꢂ20 mL). The
combined organic layers were then washed with a saturated solution of
sodium bicarbonate (1ꢂ40 mL), dried over MgSO4 and the solvent re-
moved under reduced pressure. The residue was purified by flash column
chromatography.
Conclusion
I have developed a robust synthetic pathway for heavily
modified nucleoside triphosphate analogues containing side
chains that are of crucial importance in organocatalysis. The
synthetic route involves a suitably protected 5-allylamino-2’-
deoxyuridine intermediate that is readily accessible in a se-
quence of trivial steps. Amide-bond formation between this
General method for detritylation: The nucleoside analogue was dissolved
in dry CH2Cl2 (10 mL) and dichloroacetic acid (0.1%, 0.1 mL) was
added. The orange-coloured reaction mixture was stirred at room tem-
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Synthesis of Deoxynucleoside Triphosphates
FULL PAPER
perature for 40 min. MeOH (3 mL) was added and the solution allowed
to stir for another 5 min at room temperature. Following removal of the
solvent under reduced pressure, the residue was purified by flash column
chromatography.
48.2, 49.2, 53.1, 53.5, 57.6, 58.3, 68.0, 120.2, 125.2, 127.3, 128.0, 141.6,
143.9, 144.1, 144.2, 154.9, 171.6, 175.1 ppm; HRMS: m/z calcd for
C25H27O7N2+: 467.1813 [M+H]+; found: 467.1800.
5-[(2S,4R)-N-9-Fluorenylmethoxycarbonyl-2-methoxycarbonyl-4-(3’-carb-
amidopropanoyl)aminopyrrolidineallyl]-5’-O-(4,4’-dimethoxytrityl)-3’-O-
acetyl-2’-deoxyuridine (18): The reaction was carried out with 11 (0.15 g,
0.24 mmol) and 15 (0.12 g, 0.28 mmol) by application of the general cou-
pling method. The residue was purified by flash column chromatography
on silica gel by eluting with CH2Cl2/MeOH (96:4) to give 18 as a white
foam (0.23 g, 88%). Rf =0.44 (CH2Cl2/MeOH 95:5); 1H NMR (400 MHz,
CDCl3): d=2.04 (s, 3H), 2.15–2.22 (m, 4H), 2.24–2.44 (m, 4H), 3.38–3.48
(m, 2H), 3.49 (dd, J=2.4, 10.4 Hz, 2H), [3.51 (s), 3.61 (s), 3H], 3.76 (s,
6H), 4.10 (d, J=2.0 Hz, 2H), 4.22–4.38 (m, 2H), 4.41–4.45 (m, 2H),
5.37–5.40 (m, 2H), 5.44–5.49 (m, 1H), 6.14 (dt, J=6.2, 15.6 Hz, 1H), 6.40
(td, J=2.7, 5.7 Hz, 1H), 6.81 (d, J=8.4 Hz, 4H), 7.24–7.38 (m, 13H),
7.53–7.56 (m, 2H), 7.72 (d, J=7.2 Hz, 2H), 7.79 ppm (s, 1H); 13C NMR
(101 MHz, [D6]DMSO): d=21.2, 29.9, 31.4, 32.0, 35.8, 36.9, 38.6, 41.9,
47.4, 47.7, 48.7, 51.9, 52.7, 55.6, 57.8, 58.2, 67.9, 75.3, 84.4, 84.8, 87.3,
112.4, 113.7, 120.2, 122.6, 125.2, 125.4, 127.9, 128.3, 128.4, 130.3, 135.6,
136.6, 141.5, 144.0, 144.2, 144.4, 149.5, 154.5, 155.0, 159.0, 161.9, 170.6,
171.9, 172.6 ppm; HRMS: m/z calcd for C60H61O14N5Na+: 1098.4107
[M+Na]+; found: 1098.4092.
4-{[(3R,5S)-1-(tert-Butoxycarbonyl)-5-(methoxycarbonyl)pyrrolidin-3-
yl]amino}-4-oxobutanoic acid (14): 4-trans-Amino proline 12 (0.9 g,
3.7 mmol) was dissolved in dry CH2Cl2 (20 mL) and succinic anhydride
(0.37 g, 3.7 mmol) was added. The reaction mixture was stirred at 258C
for 2 h and the solvent was removed under reduced pressure. The residue
was purified by flash column chromatography by eluting with CH2Cl2/
MeOH (95:5) to give 14 as
a white foam (0.93 g, 73%). Rf =0.40
(CH2Cl2/MeOH 9:1); 1H NMR (400 MHz, CDCl3): d=[1.38 (s), 1.42 (s),
9H], [2.08–2.18 (m), 2.33–2.43 (m), 2H], 2.48–2.57 (m, 2H), 2.60–2.78
(m, 2H), [3.52 (d, J=11.6 Hz), 3.63 (dd, J=5.6, 11.4 Hz), 2H], 3.72 (s,
3H), [4.28 (t, J=8.0 Hz), 4.36–4.38 (m), 1H], 4.38–4.55 ppm (m, 1H);
13C NMR (101 MHz, CDCl3): d=28.5, 28.6, 29.1, 30.9, 36.6, 37.2, 48.8,
51.5, 52.5, 52.7, 52.9, 57.5, 57.9, 81.8, 155.1, 156.0, 171.5, 171.9, 172.8,
173.1, 176.0 ppm; HRMS: m/z calcd for C15H25O7N2+: 345.1656 [M+H]+;
found: 345.1650.
4-{[(3S,5S)-1-(tert-Butoxycarbonyl)-5-(methoxycarbonyl)pyrrolidin-3-yl]-
amino}-4-oxobutanoic acid (16): This compound was prepared in the
same way as 14 starting from 4-cis-amino proline 13 (1.35 g, 5.5 mmol).
The residue was purified by flash column chromatography on silica gel
by eluting with CH2Cl2/MeOH (95:5) to give 16 as a white foam (0.14 g,
77%). Rf =0.46 (CH2Cl2/MeOH 9:1); 1H NMR (400 MHz, CDCl3): d=
[1.40 (s), 1.44 (s), 9H], 1.93 (t, J=13.6 Hz, 1H), 2.45–2.51 (m, 3H), 2.65–
2.73 (m, 2H), [3.45 (d, J=12.0 Hz), 3.58 (d, J=12.0 Hz), 1H], 3.56 (d,
J=4.0 Hz, 1H), [3.75 (s), 3.77 (s), 3H], [4.25 (d, J=8.8 Hz), 4.34 (d, J=
9.2 Hz), 1H], 4.60–4.63 ppm (m, 1H); 13C NMR (101 MHz, CDCl3): d=
28.4, 28.6, 29.8, 31.0, 35.8, 36.7, 48.3, 49.2, 52.8, 53.1, 53.7, 57.9, 81.1,
171.8, 175.5 ppm; HRMS: m/z calcd for C15H25O7N2+: 345.1656 [M+H]+;
found: 345.1664.
5-[(2S,4R)-N-9-Fluorenylmethoxycarbonyl-2-methoxycarbonyl-4-(3’-carb-
amidopropanoyl)aminopyrrolidineallyl]-3’-O-acetyl-2’-deoxyuridine (19):
The reaction was carried out with 18 (0.28 g, 0.26 mmol) by application
of the general detritylation method. The residue was purified by flash
column chromatography on silica gel by eluting with CH2Cl2/MeOH
(gradual gradient from 100:0 to 94:6) to give 19 as a white solid (0.19 g,
93%). Rf =0.36 (CH2Cl2/MeOH 9:1); 1H NMR (300 MHz, [D6]DMSO):
d=2.06 (s, 3H), 2.09–2.30 (m, 1H), 2.35–2.50 (m, 6H), 3.22 (dd, J=5.3,
10.8 Hz, 1H), 3.30 (dd, J=5.1, 10.4 Hz, 1H), 3.60–3.75 (m, 2H), 3.65 (s,
3H), 3.75–3.77 (m, 2H), 4.02 (d, J=2.0 Hz, 1H), 4.25–4.31 (m, 3H),
4.40–4.44 (m, 1H), 5.22–5.24 (m, 1H), 6.14–6.19 (m, 2H), 6.37–6.44 (m,
1H), 7.32–7.36 (m, 2H), 7.42 (dt, J=5.5, 7.4 Hz, 1H), 7.61–7.64 (m, 2H),
7.90 (dd, J=5.6, 7.4 Hz, 2H), 8.02 (s, 1H), 8.19 ppm (dd, J=10.2,
13.6 Hz, 1H); 13C NMR (101 MHz, [D6]DMSO): d=20.8, 30.5, 30.6, 39.3,
46.5, 46.6, 47.8, 51.3, 51.7, 52.2, 57.1, 57.6, 61.3, 66.8, 74.6, 84.2, 84.8,
110.7, 120.1, 122.2, 124.9, 125.1, 127.1, 127.7, 137.0, 140.7, 143.7, 149.5,
153.5, 153.9, 162.0, 170.0, 170.9, 171.5, 172.3, 172.4 ppm; HRMS: m/z
calcd for C39H44O12N5+: 774.2981 [M+H]+; found: 774.2983.
4-{[(3R,5S)-1-{[(9H-Fluoren-9-yl)methoxy]carbonyl}-5-(methoxycarbo-
nyl)pyrrolidin-3-yl]amino}-4-oxobutanoic acid (15): The Boc-protected
derivative 14 (0.5 g, 1.45 mmol) was dissolved in dry CH2Cl2 (6 mL). Tri-
fluoroacetic acid (5 mL) was added and the resulting reaction mixture
was stirred at 258C for 2 h. TLC analysis (CH2Cl2/MeOH, 95:5) revealed
complete consumption of the starting material and the solvent was then
removed in vacuo. The oily residue was then co-evaporated with CHCl3
(3ꢂ5 mL) and dried under vacuum for 2 h. After dissolution in dry
CH2Cl2 (35 mL) and cooling to 08C, iPrNEt2 (0.55 mL, 3.2 mmol) and
FmocCl (0.42 g, 1.6 mmol) were added in turn. The solution was allowed
to warm to room temperature and stirred for 12 h. The solvent was re-
moved under reduced pressure and then dissolved in CH2Cl2 (50 mL).
After washing with saturated KHSO4 (1ꢂ50 mL), drying over MgSO4
and removal of the solvent in vacuo, the residue was purified by flash
column chromatography by eluting with CH2Cl2/MeOH (gradient from
95:5 to 9:1) to give 15 as a white foam (0.44 g, 66%). Rf =0.19 (CH2Cl2/
MeOH 95:5); 1H NMR (400 MHz, CDCl3): d=2.02–2.20 (m, 2H), 2.45–
2.48 (m, 2H), 2.64–2.70 (m, 2H), [3.48 (dd, J=3.2, 12.4 Hz), 3.56 (dd, J=
3.0, 10.0 Hz), 1H], 3.69–3.74 (m, 1H), [3.63 (s), 3.73 (s), 3H], [4.11–4.14
(m), 4.20–4.23 (m), 1H], 4.26–4.32 (m, 1H), 4.40–4.48 (m, 2H), 4.55–4.58
(m, 1H), 7.24–7.29 (m, 2H), 7.36–7.40 (m, 2H), 7.49–7.56 (m, 2H),
7.74 ppm (d, J=7.6 Hz, 2H); 13C NMR (101 MHz, CDCl3): d=29.1, 30.9,
36.3, 37.3, 47.3, 47.4, 48.4, 49.0, 52.1, 52.6, 52.7, 52.8, 57.5, 57.9, 68.2,
120.3, 125.0, 125.2, 125.3, 127.3, 128.1, 141.6, 171.6, 172.0, 172.4, 172.5,
175.8, 175.9 ppm; HRMS: m/z calcd for C25H27O7N2+: 467.1813 [M+H]+;
found: 467.1810.
5-[(2S,4S)-N-9-Fluorenylmethoxycarbonyl-2-methoxycarbonyl-4-(3’-carb-
amidopropanoyl)aminopyrrolidineallyl]-5’-O-(4,4’-dimethoxytrityl)-3’-O-
acetyl-2’-deoxyuridine (21): The reaction was carried out with 11 (0.15 g,
0.24 mmol) and 17 (0.13 g, 0.29 mmol) by application of the general cou-
pling method. The residue was purified by flash column chromatography
on silica gel by eluting with CH2Cl2/MeOH (96:4) to give 21 as a white
foam (0.26 g, quant). Rf =0.46 (CH2Cl2/MeOH 95:5); 1H NMR
(300 MHz, CDCl3): d=2.05 (s, 3H), 2.25–2.35 (m, 2H), 2.37–2.47 (m,
6H), 3.37 (dd, J=3.0, 9.0 Hz, 1H), 3.45–3.53 (m, 3H), 3.64 (s, 3H), 3.70
(t, J=4.7 Hz, 2H), 3.77 (s, 6H), 4.12 (d, J=2.7 Hz, 2H), 4.22 (t, J=
7.4 Hz, 1H), 4.36–4.54 (m, 3H), 5.37–5.41 (m, 1H), 6.17 (dt, J=7.0,
15.9 Hz, 1H), 6.41 (dd, J=4.8, 8.7 Hz, 1H), 6.82 (d, J=8.7 Hz, 4H),
7.24–7.38 (m, 13H), 7.50–7.58 (m, 2H), 7.73 (d, J=7.5 Hz, 2H), 7.83 ppm
(s, 1H); 13C NMR (101 MHz, CDCl3): d=21.2, 31.2, 31.7, 38.6, 46.6, 47.4,
52.9, 53.0, 55.5, 55.6, 63.8, 67.2, 68.0, 75.3, 84.4, 84.8, 87.3, 112.5, 113.7,
120.2, 125.2, 127.3, 128.0, 128.4, 130.3, 135.6, 136.5, 141.5, 141.2, 144.4,
149.5, 159.0, 161.6, 170.6, 171.4, 171.6 ppm; HRMS: m/z calcd for
C60H61O14N5Na+: 1098.4107 [M+Na]+; found: 1098.4095.
4-{[(3S,5S)-1-{[(9H-Fluoren-9-yl)methoxy]carbonyl}-5-(methoxycarbo-
nyl)pyrrolidin-3-yl]amino}-4-oxobutanoic acid (17): This compound was
prepared in the same way as 15 starting from 16 (0.6 g, 1.7 mmol). The
residue was purified by flash column chromatography on silica gel by
eluting with CH2Cl2/MeOH (95:5) to give 17 as a white foam (0.64 g,
81%). Rf =0.19 (CH2Cl2/MeOH 95:5); 1H NMR (300 MHz, CDCl3): d=
2.42–2.47 (m, 3H), 2.52–2.67 (m, 2H), 3.47–3.52 (m, 1H), 3.63–3.76 (m,
1H), 3.76 (s, 3H), 4.18–4.24 (m, 2H), 4.40 (d, J=6.9 Hz, 2H), 4.61–4.65
(m, 1H), 7.24–7.38 (m, 4H), 7.55 (q, J=7.8 Hz, 2H), 7.75 ppm (d, J=
7.5 Hz, 2H); 13C NMR (75.5 MHz, CDCl3): d=29.6, 31.0, 35.8, 36.9, 47.4,
5-[(2S,4S)-N-9-Fluorenylmethoxycarbonyl-2-methoxycarbonyl-4-(3’-carb-
amidopropanoyl)aminopyrrolidineallyl]-3’-O-acetyl-2’-deoxyuridine (22):
The reaction was carried out with 21 (0.22 g, 0.20 mmol) by application
of the general detritylation method. The residue was purified by flash
column chromatography on silica gel by eluting with CH2Cl2/MeOH
(gradual gradient from 100:0 to 95:5) to give 22 as a slightly pale yellow
solid (0.16 g, 99%). Rf =0.40 (CH2Cl2/MeOH 9:1); 1H NMR (300 MHz,
[D6]DMSO): d=2.06 (s, 3H), 2.28–2.33 (m, 6H), 2.50–2.56 (m, 2H),
3.16–3.22 (m, 1H), 3.38 (q, J=7.0 Hz, 2H), 3.57–3.61 (m, 1H), 3.64 (s,
3H), 3.69–3.75 (m, 4H), 4.01 (d, J=1.8 Hz, 1H), 4.15 (t, J=6.0 Hz, 1H),
Chem. Eur. J. 2012, 00, 0 – 0
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M. Hollenstein et al.
4.26–4.30 (m, 3H), 5.99 (brs, 2H), 6.11–6.20 (m, 2H), 6.42 (dt, J=5.9,
15.9 Hz, 1H), 7.32–7.45 (m, 4H), 7.58–7.66 (m, 2H), 7.90 (d, J=6.5 Hz,
2H), 8.03 ppm (s, 1H); 13C NMR (75.5 MHz, [D6]DMSO): d=20.8, 30.4,
30.6, 36.9, 46.5, 51.9, 52.1, 57.3, 57.5, 61.2, 66.7, 74.6, 84.2, 84.8, 110.7,
122.0, 123.4 (q, J=272.8 Hz, CF3), 127.5, 130.55 (q, J=32.5 Hz, CCF3),
136.9, 142.7, 149.5, 154.7, 162.0, 165.7, 170.0 ppm; 19F NMR (376.5 MHz,
CDCl3): d=À61.7 ppm; HRMS: m/z calcd for C23H23O7N4F6+: 581.1465
[M+H]+; found: 581.1467; UV (MeOH): lmax =291, 240 nm (e291 =8190,
e239 =11660mÀ1 cmÀ1).
120.1, 122.2, 124.8, 127.1, 127.7, 137.0, 140.7, 143.7, 149.5, 153.5, 153.8,
+
162.0, 170.0, 170.9, 171.6 ppm; HRMS: m/z calcd for C39H44O12N5
:
5-[3-(N-Benzylsulfamoyl)aminoallylamino]-5’-O-(4,4’-dimethoxytrityl)-3’-
O-acetyl-2’-deoxyuridine (29): Nucleoside 11 (0.15 g, 0.24 mmol) was dis-
solved in dry CH3CN (5 mL). NEt3 (0.07 mL, 0.53 mmol) and sulfamate
28[65,71] (0.07 g, 0.26 mmol) were added and the resulting mixture was
heated at reflux for 20 h. TLC analysis (CH2Cl2/MeOH 95:5; Rf(starting
774.2981 [M+H]+; found: 774.2982.
5-[3-(ProFmocPhe)allylamido]-5’-O-(4,4’-dimethoxytrityl)-3’-O-acetyl-2’-de-
oxyuridine (24): The reaction was carried out with 11 (0.15 g, 0.24 mmol)
and ProFmocPhe-OH S4 (0.13 g, 0.26 mmol) by application of the general
coupling method. The residue was purified by flash column chromatogra-
phy on silica gel by eluting with EtOAc/n-hexane (4:1) to give 24 as a
material)=0.31, RfACTHUNRGTNEUNG(product)=0.57) revealed the complete disappearance
of the starting material. The solvent was then removed under reduced
pressure. The residue was purified by flash column chromatography by
eluting with CH2Cl2/MeOH (98:2) to give 29 as a white foam (0.14 g,
72%). 1H NMR (300 MHz, CDCl3): d=2.04 (s, 3H), 2.36–2.51 (m, 2H),
3.17–3.27 (m, 2H), 3.42 (ddd, J=2.3, 9.6, 18.9 Hz, 2H), 3.72 (s, 6H), 4.11
(s, 2H), 4.22–4.26 (m, 1H), 4.66 (brs, 1H), 4.86 (brs, 1H), 5.13–5.27 (m,
1H), 5.40 (d, J=5.4 Hz, 1H), 6.30 (dt, J=6.6, 15.9 Hz, 1H), 6.39 (dd, J=
5.6, 8.6 Hz, 1H), 6.79 (d, J=8.7 Hz, 4H), 7.20–7.34 (m, 9H), 7.73 ppm (s,
1H); 13C NMR (75.5 MHz, CDCl3): d=21.1, 38.4, 45.8, 47.3, 55.5, 63.8,
75.3, 84.3, 84.8, 87.3, 112.1, 113.6, 127.3, 127.4, 128.2, 128.3, 128.4, 128.8,
130.2, 135.4, 135.6, 137.1, 137.2, 144.2, 149.7, 158.9, 162.2, 170.6 ppm;
HRMS: m/z calcd for C42H44O10N4NaS+: 819.2670 [M+Na]+; found:
819.2658.
1
white foam (0.15 g, 58%). Rf =0.36 (EtOAc 100%); H NMR (400 MHz,
CDCl3): d=1.93–1.97 (m, 2H), 1.99–2.06 (m, 2H), 2.07 (s, 3H), 2.32–2.42
(m, 2H), 2.89–3.01 (m, 1H), 3.14 (dd, J=15.0, 6.0 Hz, 1H), 3.28–3.43 (m,
6H), 3.72–3.76 (m, 2H), 3.75 (s, 6H), 4.07–4.21 (m, 3H), 4.34–4.38 (m,
2H), 4.57–4.60 (m, 1H), 5.26 (d, J=16.0 Hz, 1H), 5.39–5.41 (m, 1H),
6.20–6.28 (m, 1H), 6.35 (apparent t, J=6.8 Hz, 1H), 6.80 (d, J=8.0 Hz,
4H), 7.07–7.38 (m, 18H), 7.54 (d, J=7.2 Hz, 2H), 7.76 (d, J=7.2 Hz,
2H), 7.95 ppm (s, 1H); 13C NMR (101 MHz, CDCl3): d=21.2, 24.6, 29.1,
29.9, 31.1, 38.3, 42.3, 47.4, 53.9, 55.5, 61.2, 63.9, 68.0, 75.4, 84.3, 84.8, 87.3,
113.6, 120.3, 125.3, 128.3, 128.4, 129.3, 130.2, 130.3, 135.4, 136.7, 141.6,
+
149.4, 159.0, 161.2, 170.6 ppm; HRMS: m/z calcd for C64H64O12N5
:
1094.4546 [M+H]+; found: 1094.4533.
5-[3-(ProFmocPhe)allylamido]-3’-O-acetyl-2’-deoxyuridine (25): The reac-
tion was carried out with 24 (0.12 g, 0.11 mmol) by application of the
general detritylation method. The residue was purified by flash column
chromatography on silica gel by eluting with CH2Cl2/MeOH (gradual gra-
dient from 100:0 to 95:5) to give 25 as a white solid (0.07 g, 81%). Rf =
0.47 (CH2Cl2/MeOH 95:5); 1H NMR (300 MHz, CDCl3): d=1.81–1.90
(m, 2H), 1.99–2.06 (m, 2H), 2.05 (s, 3H), 2.24–2.37 (m, 2H), 3.06–3.21
(m, 2H), 3.31–3.40 (m, 1H), 3.52 (t, J=5.6 Hz, 1H), 3.72–3.92 (m, 2H),
4.02–4.23 (m, 4H), 4.32 (t, J=6.2 Hz, 1H), 4.63–4.79 (m, 1H), 5.30–5.34
(m, 1H), 5.94–6.03 (m, 1H), 6.34 (apparent t, J=6.5 Hz, 1H), 6.39–6.50
(m, 1H), 7.08–7.43 (m, 11H), 7.69 (d, J=7.5 Hz, 2H), 8.15 ppm (s, 1H).
13C NMR (75.5 MHz, CDCl3): d=21.2, 24.6, 29.9, 37.0, 38.5, 41.2, 46.1,
47.3, 54.4, 55.4, 65.6, 68.2, 76.8, 85.8, 112.0, 113.4, 120.2, 120.3, 127.3,
128.9, 129.2, 136.4, 138.3, 143.7, 149.7, 162.3, 167.5, 170.8, 173.2 ppm;
HRMS: m/z calcd for C43H46O10N5+: 792.3239 [M+H]+; found: 792.3242.
5-[3-(N-Benzylsulfamoyl)aminoallylamino]-3’-O-acetyl-2’-deoxyuridine
(30): Compound 30 was obtained by application of the general detrityla-
tion method starting from 29 (0.13 g, 0.16 mmol). The residue was puri-
fied by flash column chromatography on silica gel by eluting with
CH2Cl2/MeOH (gradual gradient from 100:0 to 95:5) to give 30 as a
white solid (0.073 g, 90%). Rf =0.19 (CH2Cl2/MeOH 95:5); 1H NMR
(300 MHz, [D6]DMSO): d=2.07 (s, 3H), 2.28–2.33 (m, 2H), 3.53 (t, J=
5.7 Hz, 2H), 3.63–3.66 (m, 2H), 4.01–4.03 (m, 3H), 5.23–5.29 (m, 2H),
6.19–6.25 (m, 1H), 6.49 (dt, J=6.1, 16.0 Hz, 1H), 7.14 (t, J=6.0 Hz, 1H),
7.26–7.37 (m, 4H), 8.04 (s, 1H), 11.49 ppm (s, 1H); 13C NMR (75.5 MHz,
[D6]DMSO): d=8.6, 20.8, 36.9, 44.8, 45.6, 45.8, 61.3, 74.6, 84.2, 84.8,
110.6, 123.0, 126.3, 126.9, 127.7, 128.1, 137.2, 138.4, 149.5, 162.0,
170.0 ppm; HRMS: m/z calcd for C21H27O8N4S+: 495.1544 [M+H]+;
found: 495.1554; UV (MeOH): lmax =291, 239 nm (e291 =7330, e239
=
10340mÀ1 cmÀ1).
Triphosphorylation of nucleosides
5-[3,5-Bis(trifluoromethylphenyl)ureidoallylamido]-5’-O-(4,4’-dimethoxy-
trityl)-3’-O-acetyl-2’-deoxyuridine (26): Amine 11 (0.15 g, 0.24 mmol) was
dissolved in dry CH2Cl2 (4 mL). NEt3 (0.1 mL, 0.72 mmol) and 3,5-bis(tri-
fluoromethyl)phenyl isocyanate (0.045 mL, 0.26 mmol) were added in
turn. The resulting reaction mixture was allowed to stir at room tempera-
ture for 18 h. After removal of the solvent under reduced pressure, the
residue was purified by flash column chromatography by eluting with
CH2Cl2/MeOH (98:2) to give 26 as a pale yellow solid (0.18 g, 84%).
Rf =0.48 (CH2Cl2/MeOH 95:5); 1H NMR (300 MHz, CDCl3): d=2.05 (s,
3H), 2.40–2.49 (m, 2H), 3.36–3.54 (m, 4H), 3.75 (s, 6H), 4.11 (d, J=Hz,
1H), 4.39–4.44 (m, 1H), 5.36 (d, J=3.0 Hz, 1H), 6.04 (dt, J=7.5,
15.0 Hz, 1H), 6.41 (dd, J=6.0, 8.1 Hz, 1H), 6.82 (d, J=4.8 Hz, 4H),
7.24–7.37 (m, 14H), 7.75 (s, 2H), 7.89 ppm (s, 1H); 13C NMR (101 MHz,
CDCl3): d=20.9, 38.5, 41.9, 46.0, 55.4, 63.5, 74.9, 84.2, 84.3, 87.1, 112.3,
113.5, 113.6, 118.0, 120.8 (q, J=273.5 Hz, CF3), 122.0, 128.1, 130.1, 132.2
(q, J=32.3 Hz, CCF3), 132.4, 135.4, 136.3, 140.8, 144.3, 153.9, 158.7, 162.0,
170.4 ppm; 19F NMR (376.5 MHz, CDCl3): d=À63.0 ppm; HRMS: m/z
calcd for C44H40O9N4F6Na+: 905.2592 [M+Na]+; found: 905.2578.
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 dissolving 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 equiv). The reaction mix-
ture was allowed to stir at room temperature for 45 min. A solution of
tributylammonium pyrophosphate (1.3 equiv) in dry DMF (170 mL) and
tributylamine (58 mL) was then added and the resulting solution was stir-
red at room temperature for 40 min. Iodine (1.6 equiv) in pyridine
(980 mL) and water (20 mL) was then added and the resulting dark solu-
tion 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 (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 ap-
propriate fractions were freeze-dried and co-evaporated several times
from water.
5-[3,5-Bis(trifluoromethylphenyl)ureidoallylamido]-3’-O-acetyl-2’-deoxyur-
idine (27): The reaction was carried out by application of the general de-
tritylation method with 26 (0.17 g, 0.19 mmol). The residue was purified
by flash column chromatography on silica gel by eluting with CH2Cl2/
MeOH (gradual gradient from 100:0 to 95:5) to give 27 as a white solid
(0.097 g, 88%). Rf =0.34 (CH2Cl2/MeOH 95:5); 1H NMR (400 MHz,
[D6]DMSO): d=2.06 (s, 3H), 2.23–2.35 (m, 2H), 3.65 (d, J=4.0 Hz, 2H),
3.82 (t, J=5.4 Hz, 2H), 4.02 (d, J=2.0 Hz, 1H), 5.20–5.26 (m, 2H), 6.17–
6.22 (m, 2H), 6.48 (dt, J=5.8, 16.0 Hz, 1H), 7.54 (s, 1H), 8.00 (s, 1H),
8.10 (s, 2H), 9.38 (brs, 1H), 11.47 ppm (s, 1H); 13C NMR (75.5 MHz,
[D6]DMSO): d=20.8, 36.8, 61.3, 69.3, 74.6, 84.2, 84.8, 110.7, 113.3, 117.1,
HPLC of triphosphates: The purification of the triphosphates was carried
out on a Phenomenex Jupiter semi-preparative RP-HPLC column (5m
C18 300 ꢃ) at a flow rate of 3.5 mLminÀ1 and the eluents contained trie-
thylammonium bicarbonate (TEAB; 50 mm) in H2O (eluent I) and
CH3CN/H2O 1:1 (eluent II). The various HPLC programs used are high-
lighted in Table 3.
Protected triphosphate 20: This compound was prepared according to the
general procedure starting with derivative 19 (0.11 g, 0.142 mmol). RP-
HPLC purification by using program A (Rt =35.9 min) afforded 20 as its
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Synthesis of Deoxynucleoside Triphosphates
FULL PAPER
Table 3. HPLC programs used for the purification of the dNTPs.
Modified dUTP 3 (dUFPTP): This compound was prepared according to
the general procedure starting with derivative 25 (40 mg, 0.05 mmol). De-
protection was carried out by incubation in aqueous NH3 (10 mL) for 2 h
at room temperature. RP-HPLC purification by using program B (Rt =
35.8 min) afforded 3 as its triethylammonium salt (white solid, 8.4 mg,
17%). 1H NMR (300 MHz, D2O): d=1.25 (t, 18H, J=7.7 Hz), 1.66–1.70
(m, 2H), 1.75–1.92 (m, 2H), 2.39–2.44 (m, 2H), 2.88–3.09 (m, 2H), 3.19
(q, J=7.0 Hz, 12H), 3.79 (d, J=6.0 Hz, 2H), 4.22–4.31 (m, 4H), 6.22–
6.27 (m, 2H), 6.29 (t, J=7.5 Hz, 1H), 7.28–7.39 (m, 5H), 8.07 ppm (s,
1H); 31P NMR (121.4 MHz, D2O): d=À22.21 (t, J=20.6 Hz, Pb), À11.44
(d, J=19.4 Hz, Pa), À6.16 ppm (d, J=20.6 Hz, Pg); MS (MALDIÀ): m/z
calcd for C26H35N5O16P3À: 766.13 [MÀH]À; found: 765.64; UV: lmax =291,
240 nm.
Time
[min]
Program A
[%II]
Program B
[%II]
Time
[min]
Program C
ACHTUNGTNER[NUNG %II]
G
R
G
U
0
7
0
0
0
0
0
7
0
0
49
51
54
100
100
0
50
100
0
29
40
42
45
47
15
40
100
100
0
triethylammonium salt (white solid, 78.0 mg, 42%). 1H NMR (300 MHz,
D2O): d=1.27 (t, J=7.5 Hz, 27H), 2.15 (s, 3H), 2.24–2.29 (m, 2H), 2.45–
2.63 (m, 6H), 3.20 (q, J=7.2 Hz, 18H), 3.37–3.41 (m, 1H), 3.74 (s, 3H),
3.89 (d, J=6.0 Hz, 2H), 4.19–4.25 (m, 4H), 4.53–4.57 (m, 1H), 4.76–4.80
(m, 3H), 5.35–5.45 (m, 1H), 6.29–6.46 (m, 2H), 7.36–7.51 (m, 4H), 7.61–
7.67 (m, 2H), 7.85–7.92 (m, 2H), 7.85 ppm (s, 1H); 31P NMR
(121.4 MHz, D2O): d=À22.47 (t, J=20.0 Hz, Pb), À11.59 (d, J=19.4 Hz,
Pa), À6.40 ppm (d, J=20.6 Hz, Pg); MS (MALDIÀ): m/z calcd for
C39H45N5O21P3À: 1012.18 [MÀH]À; found: 1011.59.
Protected triphosphate 23: This compound was prepared according to the
general procedure starting with derivative 22 (66 mg, 0.085 mmol). RP-
HPLC purification by using program A (Rt =35.8 min) afforded 23 as its
triethylammonium salt (white solid, 32.1 mg, 28%). 1H NMR (300 MHz,
D2O): d=1.28 (t, J=7.4 Hz, 27H), 2.12 (s, 3H), 2.22–2.33 (m, 2H), 2.47–
2.61 (m, 6H), 3.17 (q, J=7.6 Hz, 18H), 3.39–3.42 (m, 1H), 3.72 (s, 3H),
3.82–3.87 (m, 2H), 4.18–4.32 (m, 4H), 4.53–4.57 (m, 1H), 4.76–4.80 (m,
3H), 5.35–5.45 (m, 1H), 6.14–6.38 (m, 3H), 7.32–7.41 (m, 4H), 7.49 (d,
J=9.0 Hz, 2H), 7.73 (d, J=9.0 Hz, 2H), 7.84 ppm (s, 1H); 31P NMR
(121.4 MHz, D2O): d=À23.19 (t, J=20.0 Hz, Pb), À11.64 (d, J=20.6 Hz,
Pa), À10.58 ppm (d, J=20.6 Hz, Pg); MS (MALDIÀ): m/z calcd for
C39H45N5O21P3À: 1012.18 [MÀH]À; found: 1011.61.
Modified dUTP 4 (dUBpuTP): Triphosphate 4 was synthesized according
to the general phosphorylation procedure starting from 27 (30 mg,
0.05 mmol). Deprotection was carried out by incubation in aqueous NH3
(10 mL) for 1 h at room temperature. RP-HPLC purification by using
program B (Rt =35.6 min) afforded 4 as its triethylammonium salt (white
solid, 12 mg, 21%). 1H NMR (300 MHz, D2O): d=1.28 (t, J=7.4 Hz,
27H), 2.40–2.42 (m, 2H), 3.06–3.19 (m, 2H), 3.18 (q, J=7.0 Hz, 18H),
3.99 (d, J=3.0 Hz, 2H), 4.23–4.26 (m, 3H), 4.67–4.71 (m, 1H), 6.34–6.53
(m, 3H), 7.72 (s, 1H), 7.95 (s, 2H), 8.02 ppm (s, 1H); 31P NMR
(121.4 MHz, D2O): d=À22.85 (t, J=19.4 Hz, Pb), À11.35 (d, J=19.4 Hz,
Pa), À10.02 ppm (d, J=21.8 Hz, Pg); 19F NMR (376.5 MHz, D2O): d=
À
À62.8 ppm; MS (MALDIÀ): m/z calcd for C21H22F6N4O15P3
: 777.02
[MÀH]À; found: 776.41; UV: lmax =290, 248 nm.
Modified dUTP 5 (dUBsTP): Triphosphate 5 was synthesized according to
the general phosphorylation procedure starting from 30 (30 mg,
0.06 mmol). Deprotection was carried out by incubation in aqueous NH3
(10 mL) for 1.5 h at room temperature. RP-HPLC purification by using
program B (Rt =28.0 min) afforded 5 as its triethylammonium salt (white
solid, 22 mg, 36% yield). 1H NMR (300 MHz, D2O): d=1.27 (t, J=
7.3 Hz, 27H), 2.38 (dd, J=5.1, 6.6 Hz, 2H), 3.05–3.16 (m, 2H), 3.19 (q,
J=7.4 Hz, 18H), 3.73 (d, J=6.0 Hz, 2H), 4.18–4.22 (m, 3H), 4.66–4.68
(m, 1H), 6.28–6.46 (m, 3H), 7.21–7.36 (m, 5H), 7.85 ppm (s, 1H);
31P NMR (121.4 MHz, D2O): d=À23.23 (t, J=20.0 Hz, Pb), À11.57 (d,
J=19.4 Hz, Pa), À10.71 ppm (d, J=19.8 Hz, Pg); MS (MALDIÀ): m/z
Modified dUTP 1 (dUtPTP): Triphosphate 20 (78 mg, 0.077 mmol) was
dissolved in dry DMF (200 mL) and piperidine (50 mL) was added. The
resulting solution was allowed to stir at room temperature for 30 min.
The solvent was then removed under pressure (the water bath of the
rotary evaporator should be kept below 308C) and the residue dissolved
in H2O (420 mL). After cooling to 08C, NaOH (95 mL, 2.5m; 0.46m final
concentration) was added and the reaction stirred at 08C for 5 min. The
mixture was then neutralized by adding NaH2PO4 buffer (230 mL; 1m,
pH 1.9) and further diluted by addition of H2O (3 mL). The solution was
then washed with EtOAc (2ꢂ4 mL) to remove the cleaved protecting
groups. The solvent was then removed in vacuo and the residue purified
by RP-HPLC using program C (Rt =26.4 min). dUtPTP 1 was obtained as
its triethyl ammonium salt (white solid, 7.7 mg, 12% yield). 1H NMR
(300 MHz, D2O): d=1.29 (t, J=7.4 Hz, 27H), 1.97–2.12 (m, 1H), 2.41 (t,
J=6.3 Hz, 2H), 2.58 (t, J=5.4 Hz, 3H), 2.67–2.74 (m, 1H), 2.99–3.11 (m,
2H), 3.21 (q, J=7.3 Hz, 18H), 3.38–3.45 (m, 1H), 3.62–3.69 (m, 2H),
3.92 (d, J=5.1 Hz, 2H), 4.12–4.23 (m, 3H), 4.42–4.51 (m, 1H), 4.67–4.70
(m, 1H), 6.30–6.49 (m, 3H), 7.96 ppm (s, 1H); 31P NMR (121.4 MHz,
D2O): d=À22.96 (t, J=20.0 Hz, Pb), À11.54 (d, J=20.6 Hz, Pa),
À10.59 ppm (d, J=19.4 Hz, Pg); MS (MALDIÀ): m/z calcd for
C21H31N5O18P3À: 734.09 [MÀH]À; found: 733.64; UV: lmax =291, 240 nm.
calcd for C19H26N4O16P3SÀ: 691.03 [MÀH]À; found: 690.55; UV: lmax
=
291, 242 nm.
Primer extension reactions: The 5’-[32P]-labelled primer P1 (ca. 1 pmol)
was annealed to the appropriate template (6 pmol) in the presence of the
1ꢂ enzyme buffer (provided by the supplier of the DNA polymerase) by
heating to 958C and then gradually cooling to room temperature. Single-
stranded binding protein (SSB; 0.2 U), thermostable inorganic pyrophos-
phatase (0.3 U), dithiothreitol (DTT; 1 mL of 100 mm solution), and DNA
polymerase (1 U) were added to the annealed oligonucleotides as a cock-
tail at 48C. The importance of SSB in the reaction mixture was not inves-
tigated, but it can be omitted at longer incubation times (>1 h) and
higher reaction temperatures (>608C).[16] Finally, dNTPs (final concen-
tration of 50 mm) were added for a total reaction volume of 20 mL. Fol-
lowing incubation at the optimal temperature for the enzyme, the reac-
tions were quenched by adding stop solution (20 mL; formamide (70%),
ethylenediaminetetraacetic acid (EDTA; 50 mm), bromophenol (0.1%),
xylene cyanol (0.1%)). The reaction mixtures were subjected to gel elec-
trophoresis in denaturing polyacrylamide gel (20%) containing trisbo-
rate-EDTA (TBE) 1ꢂ buffer (pH 8) and urea (7m). Visualization was
performed by phosphorimaging.
Modified dUTP 2 (dUcPTP): This triphosphate was synthesized in the
same way as compound 1 starting from 23 (30 mg, 0.03 mmol). RP-HPLC
purification by using program C (Rt =26.7 min) afforded 2 as its triethy-
lammonium salt (white solid, 10.3 mg, 28%). 1H NMR (300 MHz, D2O):
d=1.28 (t, J=7.6 Hz, 27H), 2.39–2.43 (m, 2H), 2.55–2.59 (m, 4H), 2.67–
2.74 (m, 1H), 2.86–3.03 (m, 1H), 3.20 (q, J=7.3 Hz, 18H), 3.24–3.38 (m,
1H), 3.62–3.69 (m, 2H), 3.91 (d, J=5.1 Hz, 2H), 4.16–4.23 (m, 3H),
4.42–4.51 (m, 1H), 4.68–4.70 (m, 1H), 6.29–6.49 (m, 3H), 7.96 ppm (s,
1H); 31P NMR (121.4 MHz, D2O): d=À23.02 (t, J=19.4 Hz, Pb), À11.54
(d, J=19.4 Hz, Pa), À9.85 ppm (d, J=19.4 Hz, Pg); MS (MALDIÀ): m/z
calcd for C21H31N5O18P3À: 734.09 [MÀH]À; found: 733.83; UV: lmax =292,
240 nm.
Polymerase chain reactions: The PCR reaction mixtures (20 mL total)
contained both primers (400 nm each; P2 and P3, see the Supporting In-
formation for the sequences), template T4 (25 nm), dNTPs (200 mm; natu-
ral and modified), and Vent (exoÀ) (1 U) in the reaction buffer (Thermo-
pol buffer) provided by the manufacturer. The PCRs were carried out in
a LightCycler 2.0 from Roche and 30 cycles were performed. Each cycle
included denaturation at 948C for 1 min, annealing for 1 min at 558C,
and extension for 1.5 min at 728C. A final extension step of 5 min at
728C was included. All PCR products were analysed by agarose gels
(2%) in 1ꢂ TBE buffer, containing ethidium bromide. The gels were vi-
sualized by phosphorimager analysis.
Chem. Eur. J. 2012, 00, 0 – 0
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M. Hollenstein et al.
Thermal denaturation experiments: The oligonucleotides for the thermal
denaturation experiments were obtained by large scale primer extension
reactions.[33,35] Briefly, primer P1 (1 nmol) and template (T1 or T3;
1 nmol) were annealed by heating at 958C and slowly cooling to room
temperature over 1 h. Thermopol buffer 10ꢂ, dNTPs (200 mm final con-
centration) and Vent (exoÀ) (1 U) were added to the annealed duplex.
H2O was added to reach a final volume of 500 mL and the reaction mix-
tures were incubated at 728C for 1 h. The reaction mixtures were then
evaporated to dryness in a speed-vac and re-dissolved in H2O (100 mL).
The primer-extension products were then purified by using the QIAquick
Nucleotide Removal Kit Protocol (Qiagen). The purified duplexes were
then diluted with H2O and 2ꢂ buffer to reach a final volume of 700 mL of
1ꢂ buffer (NaH2PO4 (10 mm), NaCl (150 mm), pH 7.0). Prior to the ther-
mal denaturation experiment, the concentration of the duplexes was de-
termined by UV/Vis (0.7 mm). UV–melting curves were recorded on a
Varian Cary 100 Bio UV/Vis spectrophotometer. Absorbances were
monitored at 260 nm and the heating rate was set to 0.58CminÀ1. A cool-
ing–heating–cooling cycle in the temperature range 20–908C was applied.
Tm values were obtained from the derivative curves by using the Varian
WinUV software. To avoid evaporation, the sample solutions were cov-
ered with a layer of dimethylpolysiloxane.
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Synthesis of Deoxynucleoside Triphosphates
FULL PAPER
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Received: May 11, 2012
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Published online: && &&, 0000
Chem. Eur. J. 2012, 00, 0 – 0
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M. Hollenstein et al.
New kids on the block: Modified
DNA
nucleoside triphosphates containing
side chains that could promote organo-
catalysis were synthesized. These
modified dNTPs were shown to be
fully compatible with in vitro selection
methods because they are substrates
for polymerases in both primer exten-
sion reactions and the polymerase
chain reaction.
M. Hollenstein* ............... &&&& — &&&&
Synthesis of Deoxynucleoside Triphos-
phates that Include Proline, Urea, or
Sulfonamide Groups and Their Poly-
merase Incorporation into DNA
Five base-modified nucleoside triphosphates
(dNTPs)……adorned with functionalities that play a
central role in organocatalysis (i.e., proline, urea
and sulfamide groups) were synthesized. In addition,
these dNTPs were shown to be compatible with
methods of in vitro selection since they are good
substrates for polymerases, both in primer extension
reactions and the polymerase chain reaction (PCR).
Thus these modified dNTPs could supplement the
chemical collection of DNA enzymes when used in
selection experiments. For more information, see
the Full Paper by M. Hollenstein on page && ff.
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ÝÝ
These are not the final page numbers!